thromboplastin and Inflammation

Worldwide COVID-19 pandemic has taken a huge toll of morbidity and mortality. In selected patients, classified as severe, the overwhelming inflammatory state imposed by this infection is accompanied by a hypercoagulable state, hallmarked by a unique pattern; a marked increase in D-dimer, out of proportion to other markers of coagulopathy. In this review, we turn a spotlight to this phenomenon, offering a unified conceptual model depicting the leading hypotheses of coagulopathy in COVID-19. The key players of the coagulation cascades accompanying the COVID-19 inflammation malfunction on virtually every level; tissue factor expression is amplified, physiological anti-coagulant pathways (anti-thrombin, protein C and S, and the inhibitor of the tissue factor pathway) are impaired and fibrinolysis is inhibited. Components of autoimmunity, the complement system amongst others, further contribute to the pathology. As data continue to gather, our model offers a pathophysiological overview of COVID-19 coagulopathy, defined by the resultant histopathology: either intra-vascular or extra-vascular. We hope this review will facilitate understanding and serve as a lead point to future therapeutic directives.

Topics: Blood Coagulation; COVID-19; Humans; Inflammation; Pandemics; Thromboplastin

The interplay between innate immunity and coagulation after infection or injury, termed immunothrombosis, is the primary cause of disseminated intravascular coagulation (DIC), a condition that occurs in sepsis. Thrombosis associated with DIC is the leading cause of death worldwide. Interest in immunothrombosis has grown because of COVID-19, the respiratory disease caused by SARS-CoV-2, which has been termed a syndrome of dysregulated immunothrombosis. As the relatively new field of immunothrombosis expands at a rapid pace, the focus of academic and pharmacological research has shifted from generating treatments targeted at the traditional 'waterfall' model of coagulation to therapies better directed towards immune components that drive coagulopathies. Immunothrombosis can be initiated in macrophages by cleavage of the non-canonical inflammasome which contains caspase-11. This leads to release of tissue factor (TF), a membrane glycoprotein receptor that forms a high-affinity complex with coagulation factor VII/VIIa to proteolytically activate factors IX to IXa and X to Xa, generating thrombin and leading to fibrin formation and platelet activation. The mechanism involves the post-translational activation of TF, termed decryption, and release of decrypted TF via caspase-11-mediated pyroptosis. During aberrant immunothrombosis, decryption of TF leads to thromboinflammation, sepsis, and DIC. Therefore, developing therapies to target pyroptosis have emerged as an attractive concept to counteract dysregulated immunothrombosis. In this review, we detail the three mechanisms of TF control: concurrent induction of TF, caspase-11, and NLRP3 (signal 1); TF decryption, which increases its procoagulant activity (signal 2); and accelerated release of TF into the intravascular space via pyroptosis (signal 3). In this way, decryption of TF is analogous to the two signals of NLRP3 inflammasome activation, whereby induction of pro-IL-1β and NLRP3 (signal 1) is followed by activation of NLRP3 (signal 2). We describe in detail TF decryption, which involves pathogen-induced alterations in the composition of the plasma membrane and modification of key cysteines on TF, particularly at the location of the critical, allosterically regulated disulfide bond of TF in its 219-residue extracellular domain. In addition, we speculate towards the importance of identifying new therapeutics to block immunothrombotic triggering of TF, which can involve inhibition of pyroptosis to limit TF r

Topics: Anticoagulants; COVID-19 Drug Treatment; Humans; Inflammation; Pyroptosis; SARS-CoV-2; Thromboinflammation; Thromboplastin; Thrombosis

COVID-19 is a highly contagious respiratory infection caused by the SARS-CoV-2 virus. The infected lung epithelial cells secrete a group of chemokines and cytokines, which triggers harmful cytokine storms and hyper-thrombotic responses. Recent studies have proposed that viral-induced senescence is responsible for cytokine release and inflammation in COVID-19 patients. However, it is unknown whether cellular senescence is commonly triggered after viral infection and how inflammation and thrombosis, hyper-activated in these patients, are functionally connected. To address these questions, we conducted a bioinformatics-based meta-analysis using single-cell and bulk RNA sequencing datasets obtained from human patient studies, animal models, and cell lines infected with SARS-CoV-2 and other respiratory viruses. We found that the senescence phenotype is robustly upregulated in most SARS-CoV-2-infected patients, especially in the infected lung epithelial cells. Notably, the upregulation of Tissue factor (F3), a key initiator of the extrinsic blood coagulation pathway, occurs concurrently with the upregulation of the senescence-associated secretory phenotype (SASP) factors. Furthermore, F3 levels are positively correlated with the senescence and hyper-coagulation gene signatures in COVID-19 patients. Together, these data demonstrate the prevalence of senescence in respiratory viral infection and suggest F3 as a critical link between inflammation, thrombosis, and senescence in these disease states.

Topics: Animals; COVID-19; Cytokines; Humans; Inflammation; SARS-CoV-2; Thromboplastin; Thrombosis

Endothelial cells (ECs) are vascular, nonconventional immune cells that play a major role in the systemic response after bacterial infection to limit its dissemination. Triggered by exposure to pathogens, microbial toxins, or endogenous danger signals, EC responses are polymorphous, heterogeneous, and multifaceted. During sepsis, ECs shift toward a proapoptotic, proinflammatory, proadhesive, and procoagulant phenotype. In addition, glycocalyx damage and vascular tone dysfunction impair microcirculatory blood flow, leading to organ injury and, potentially, life-threatening organ failure. This review aims to cover the current understanding of the EC adaptive or maladaptive response to acute inflammation or bacterial infection based on compelling recent basic research and therapeutic clinical trials targeting microvascular and endothelial alterations during septic shock.

Topics: Alarmins; Animals; Apoptosis; Blood Coagulation; Capillary Permeability; Cell Adhesion Molecules; Cytokines; Endothelial Cells; Endothelins; Endothelium, Vascular; Epoprostenol; Fibrinolysis; Glycocalyx; Humans; Inflammation; Microcirculation; Nitric Oxide; Pathogen-Associated Molecular Pattern Molecules; Phenotype; Platelet Adhesiveness; Sepsis; Thromboplastin; Vasoconstriction; Vasodilation

The hemostatic cascade is initiated by the transmembrane coagulation protein - tissue factor (TF) and eventuates in fibrin formation. Heparanase protein was demonstrated to directly enhance TF activity resulting in increased activation of the coagulation system. In addition, heparanase was found to increase hemostatic system activation via two other mechanisms: up-regulating TF expression in endothelial cells and releasing the protein tissue factor pathway inhibitor (TFPI) from the cell surface. Peptides derived from TFPI-2, a protein similar to TFPI, were shown to inhibit the TF/heparanase complex as well as attenuate sepsis and tumor growth. Increased heparanase procoagulant activity was observed in several clinical settings, including women using oral contraceptives, women at delivery, patients following orthopedic surgery and patients with diabetic foot, shift work female nurses, patients with lung cancer, retinal vein thrombosis and prosthetic heart valve thrombosis. Remarkably, the heparanase profile was significantly different across the tested groups. Inhibition of TF / heparanase interaction may represent a new target for attenuating coagulation, cancer and inflammation.

Topics: Blood Coagulation; Endothelial Cells; Glucuronidase; Humans; Inflammation; Neoplasms; Thromboplastin

In a stunningly short period of time, the unexpected coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has turned the unprepared world topsy-turvy. Although the rapidity with which the virus struck was indeed overwhelming, scientists throughout the world have been up to the task of deciphering the mechanisms by which SARS-CoV-2 induces the multisystem and multiorgan inflammatory responses that, collectively, contribute to the high mortality rate in affected individuals. In this issue of the JCI, Skendros and Mitsios et al. is one such team who report that the complement system plays a substantial role in creating the hyperinflammation and thrombotic microangiopathy that appear to contribute to the severity of COVID-19. In support of the hypothesis that the complement system along with neutrophils and platelets contributes to COVID-19, the authors present empirical evidence showing that treatment with the complement inhibitor compstatin Cp40 inhibited the expression of tissue factor in neutrophils. These results confirm that the complement axis plays a critical role and suggest that targeted therapy using complement inhibitors is a potential therapeutic option to treat COVID-19-induced inflammation.

Topics: Betacoronavirus; Blood Platelets; Complement Activation; Coronavirus Infections; COVID-19; Humans; Inflammation; Neutrophils; Pandemics; Peptides, Cyclic; Pneumonia, Viral; SARS-CoV-2; Severity of Illness Index; Thromboplastin; Thrombotic Microangiopathies

Intravascular infusion is the most popular route for therapeutic multipotent mesenchymal stromal/stem cell (MSC) delivery in hundreds of clinical trials. Meta-analysis has demonstrated that bone marrow MSC infusion is safe. It is not clear if this also applies to diverse new cell products derived from other sources, such as adipose and perinatal tissues. Different MSC products display varying levels of highly procoagulant tissue factor (TF) and may adversely trigger the instant blood-mediated inflammatory reaction (IBMIR). Suitable strategies for assessing and controlling hemocompatibility and optimized cell delivery are crucial for the development of safer and more effective MSC therapies.

Topics: Animals; Clinical Trials as Topic; Humans; Inflammation; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Thromboplastin

The tissue factor (TF) pathway plays a central role in hemostasis and thrombo-inflammatory diseases. Although structure-function relationships of the TF initiation complex are elucidated, new facets of the dynamic regulation of TF's activities in cells continue to emerge. Cellular pathways that render TF non-coagulant participate in signaling of distinct TF complexes with associated proteases through the protease-activated receptor (PAR) family of G protein-coupled receptors. Additional co-receptors, including the endothelial protein C receptor (EPCR) and integrins, confer signaling specificity by directing subcellular localization and trafficking. We here review how TF is switched between its role in coagulation and cell signaling through thiol-disulfide exchange reactions in the context of physiologically relevant lipid microdomains. Inflammatory mediators, including reactive oxygen species, activators of the inflammasome, and the complement cascade play pivotal roles in TF procoagulant activation on monocytes, macrophages and endothelial cells. We furthermore discuss how TF, intracellular ligands, co-receptors and associated proteases are integrated in PAR-dependent cell signaling pathways controlling innate immunity, cancer and metabolic inflammation. Knowledge of the precise interactions of TF in coagulation and cell signaling is important for understanding effects of new anticoagulants beyond thrombosis and identification of new applications of these drugs for potential additional therapeutic benefits.

Topics: Animals; Blood Coagulation; Endothelial Cells; Factor VIIa; Factor Xa; Humans; Inflammation; Myeloid Cells; Neoplasms; Receptor, PAR-2; Signal Transduction; Thromboplastin; Thrombosis

Tissue factor (TF) is a cell surface receptor for coagulation factor VII (fVII). The TF-activated fVII (fVIIa) complex is an essential initiator of the extrinsic blood coagulation process. Interactions between cancer cells and immune cells via coagulation factors and adhesion molecules can promote progression of cancer, including epithelial ovarian cancer (EOC). This process is not necessarily advantageous, as tumor tissues generally undergo hypoxia due to aberrant vasculature, followed by reduced access to plasma components such as coagulation factors. However, hypoxia can activate TF expression. Expression of fVII, intercellular adhesion molecule-1 (ICAM-1), and multiple pro-inflammatory cytokines can be synergistically induced in EOC cells in response to hypoxia along with serum deprivation. Thus, pro-inflammatory responses associated with the TF-fVIIa-ICAM-1 interaction are expected within hypoxic tissues. Tumor tissue consists of multiple components such as stromal cells, interstitial fluid, albumin, and other micro-factors such as proton and metal ions. These factors, together with metabolism reprogramming in response to hypoxia and followed by functional modification of TF, may contribute to coagulation factor-driven inflammatory responses in EOC tissues. The aim of this review was to describe potential coagulation factor-driven inflammatory responses in hypoxic EOC tissues. Arguments were extended to clinical issues targeting this characteristic tumor environment.

Topics: Animals; Blood Coagulation; Blood Coagulation Factors; Carcinoma, Ovarian Epithelial; Female; Humans; Hypoxia; Inflammation; Inflammation Mediators; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Signal Transduction; Thromboplastin

Sickle cell disease (SCD) is a hematologic disorder caused by a well-characterized point mutation in the β-globin gene. Abnormal polymerization of hemoglobin tetramers results in the formation of sickle red blood cells that leads to vascular occlusions, hemolytic anemia, vascular inflammation and cumulative, multiple organ damage. Ongoing activation of coagulation is another hallmark of SCD. Recent studies strongly suggested that hypercoagulation in SCD is not just a secondary event but contributes directly to the disease pathophysiology. In this article we summarize mechanisms leading to the activation of coagulation, review data indicating direct contribution of coagulation to the pathology of SCD and, we discuss the anticoagulation as a possible treatment strategy to attenuate the disease progression.

Topics: Anemia, Sickle Cell; Animals; Anticoagulants; beta-Globins; Blood Coagulation; Disease Progression; Endothelial Cells; Erythrocytes; Humans; Inflammation; Leukocytes; Mice; Quality of Life; Thrombin; Thromboplastin; Thrombosis; Vascular Diseases

Cancer is associated with an increased risk of venous thromboembolism (VTE); the exact mechanisms for the induction of VTE remain to be fully elucidated, but it is widely acknowledged that tissue factor (TF)-bearing microparticles (TF-MPs) may play a significant role. However, TF-MPs have yet to be accepted as a genuine biomarker for cancer-associated VTE, as the presence of elevated TF-MP levels is not always accompanied by thrombosis; interestingly, in certain cases, particularly in pancreatic cancer, VTE seems to be more likely in the context of acute inflammation. Although several potential mechanisms for the development of VTE in cancer have been postulated, this review explores the homeostatic disruption of TF-MPs, as the main reservoir of bloodborne TF, in the context of cancer and inflammation, and considers the abrogated responses of the activated endothelium and mononuclear phagocyte system in mediating this disruption.

Topics: Biomarkers; Blood Coagulation; Cell-Derived Microparticles; Female; Homeostasis; Humans; Inflammation; Male; Models, Biological; Neoplasms; Phagocytes; Risk Factors; Thromboplastin; Venous Thromboembolism

Due to its receptor activity for factor VII, tissue factor (TF) is primary initiator of the blood coagulation cascade and ensures rapid hemostasis in case of organ damage. Inflammatory cytokines, such as tumor necrosis factor-α or interleukins, strongly induce expression of both full-length TF as well as the alternatively spliced TF in endothelial and blood cells. Beyond its role in hemostasis, TF also has signaling activity and promotes pleiotropic inflammatory responses via protease-activated receptors in concert with other coagulation factors. Alteration of TF expression and TF alternative splicing provides an effective means to change the endothelial phenotype and modulate inflammatory responses of the vessel.

Topics: Alternative Splicing; Animals; Blood Coagulation; Gene Expression Regulation; Humans; Inflammation; Inflammation Mediators; Protein Conformation; Signal Transduction; Thromboplastin

Tissue factor is the primary initiator of the coagulation cascade. Formation of the TF:FVIIa complex activates both FX and FIX, with subsequent thrombin generation, fibrin deposition and activation of platelets. In addition to playing important role in hemostasis and thrombosis, TF and downstream coagulation proteases can mediate intracellular signaling via activation of protease-activated receptors (PARs). Maintaining hemostasis in the brain is of utmost importance: bleeding or thrombosis within this organ can lead to significant morbidity and mortality. Both TF and PARs are widely expressed within the CNS, with TF expressed predominantly by astrocytes and PARs expressed in multiple cell types including astrocytes, neurons, microglia and oligodendrocytes [1-4]. PARs activation can result in either neuronal survival or death and link the coagulation system with the inflammatory response. In this brief review we summarize the contribution of the coagulation system to brain hemostasis as well as to the pathophysiology of stroke and multiple sclerosis.

Topics: Animals; Astrocytes; Autoimmunity; Blood Coagulation; Brain; Hemorrhage; Hemostasis; Humans; Inflammation; Multiple Sclerosis; Stroke; Thromboplastin

Obesity is a major cause for a spectrum of metabolic syndrome-related diseases that include insulin resistance, type 2 diabetes, and steatosis of the liver. Inflammation elicited by macrophages and other immune cells contributes to the metabolic abnormalities in obesity. In addition, coagulation activation following tissue factor (TF) upregulation in adipose tissue is frequently found in obese patients and particularly associated with diabetic complications. Genetic and pharmacological evidence indicates that TF makes significant contributions to the development of the metabolic syndrome by signaling through G protein-coupled protease activated receptors (PARs). Adipocyte TF-PAR2 signaling contributes to diet-induced obesity by decreasing metabolism and energy expenditure, whereas hematopoietic TF-PAR2 signaling is a major cause for adipose tissue inflammation, hepatic steatosis and inflammation, as well as insulin resistance. In the liver of mice on a high fat diet, PAR2 signaling increases transcripts of key regulators of gluconeogenesis, lipogenesis and inflammatory cytokines. Increased markers of hepatic gluconeogenesis correlate with decreased activation of AMP-activated protein kinase (AMPK), a known regulator of these pathways and a target for PAR2 signaling. Clinical markers of a TF-induced prothrombotic state may thus indicate a risk in obese patient for developing complications of the metabolic syndrome.

Topics: Animals; Humans; Immunologic Factors; Inflammation; Metabolic Syndrome; Models, Immunological; Obesity; Receptor, PAR-2; Signal Transduction; Thromboplastin

Behçet disease (BD) is a rare multisystem, inflammatory disease of unknown etiology with vascular involvement and associated thrombogenicity. This review aims to describe the involvement of various mediators in endothelial cell damage and in the hypercoagulable state of BD. The scenario of the chronic inflammation present in BD shows an increased oxidative condition that contributes to endothelial cell damage and induces platelet, leukocyte, and endothelial cell activation through the release of proinflammatory cytokines and chemokines. These factors, together with the increased levels of homocysteine observed in BD patients, induce the endothelial cell expression of adhesion molecules (VCAM-1 and ICAM-1) and tissue factor; the release of cytokines, soluble CD40L (sCD40L), matrix metalloproteinase-9, and blood coagulation factor V; the inhibition of fibrinolysis; the disruption of nitric oxide metabolism; and the increase in platelet reactivity and lipid peroxidation. Endothelial cell dysfunction leads to a prothrombotic and antifibrinolytic phenotype in BD patients. Increased levels of homocysteine, fibrinogen, and plasminogen activator inhibitor type 1 seem to be involved in the procoagulant condition of this pathology that has been verified by end-point tests as well as by global coagulation tests.

Topics: Autoantibodies; Behcet Syndrome; Blood Coagulation Tests; CD40 Antigens; CD40 Ligand; Cell Adhesion Molecules; Cytokines; Endothelium, Vascular; Fibrinolysis; Humans; Hyperhomocysteinemia; Inflammation; Leukocytes; Lipid Peroxidation; Matrix Metalloproteinase 9; Models, Biological; Nitric Oxide; Platelet Activation; Reactive Oxygen Species; Thromboembolism; Thrombophilia; Thromboplastin

Coagulopathy is common in acute sepsis and may range from subclinical activation of blood coagulation (hypercoagulability), which may contribute to venous thromboembolism, to acute disseminated intravascular coagulation, characterized by widespread microvascular thrombosis and consumption of platelets and coagulation proteins, eventually causing bleeding. The key event underlying this life-threatening complication is the overwhelming inflammatory host response to the pathogen leading to the overexpression of inflammatory mediators. The latter, along with the microorganism and its derivatives drive the major changes responsible for massive thrombin formation and fibrin deposition: (1) aberrant expression of tissue factor mainly by monocytes-macrophages, (2) impairment of anticoagulant pathways, orchestrated by dysfunctional endothelial cells (ECs), and (3) suppression of fibrinolysis because of the overproduction of plasminogen activator inhibitor-1 by ECs and thrombin-mediated activation of thrombin-activatable fibrinolysis inhibitor. Neutrophils and other cells, upon activation or death, release nuclear materials (neutrophil extracellular traps and/or their components such as histones, DNA, lysosomal enzymes, and High Mobility Group Box-1), which have toxic, proinflammatory and prothrombotic properties thus contributing to clotting dysregulation. The ensuing microvascular thrombosis-ischemia significantly contributes to tissue injury and multiple organ dysfunction syndromes. These insights into the pathogenesis of sepsis-associated coagulopathy may have implications for the development of new diagnostic and therapeutic tools.

Topics: Animals; Disease Models, Animal; Disseminated Intravascular Coagulation; Endothelium, Vascular; Endotoxemia; Extracellular Traps; Fibrinolysis; Humans; Immunity, Innate; Inflammation; Inflammation Mediators; Macrophages; Models, Biological; Monocytes; Multiple Organ Failure; Neutrophils; Protein C; Sepsis; Thrombophilia; Thromboplastin; Thrombotic Microangiopathies

Venous thromboembolic disease often arises as a complication of another pathological condition and/or triggering event. Infectious diseases result from both the direct action of the pathogens themselves and their effect on the immune system. The resulting inflammatory process and the coagulation and fibrinolysis processes share common pathways, explaining why infection is associated with thrombosis. In this brief overview, besides certain chronic infectious diseases, we also consider some acute infections, as the mechanisms are likely to be similar, particularly in the initial infective stage or the more acute episodes of a chronic infection. The infectious agent can be viral, bacterial, fungal, or parasitic. However, the literature on the link between infections and venous thromboembolism (VTE) is uneven, favoring infections that are found in more developed countries where physicians have access to VTE diagnostic tools. Thus, large epidemiological studies in this field are restricted to a limited number of the common chronic infectious diseases such as tuberculosis, while for other infections, particularly parasitic and fungal infections, the link with VTE is only evoked in a few scattered case reports.

Topics: Cell-Derived Microparticles; Chronic Disease; Developed Countries; Developing Countries; Disease Susceptibility; Endothelium, Vascular; HIV Infections; Humans; Infections; Inflammation; Macrophages; Models, Biological; Risk; Thrombophilia; Thromboplastin; Tuberculosis; Venous Thromboembolism

The transition from the classic role of neutrophils as the first line of defense of the immune system to that of physiological regulators of intravascular thrombosis took nearly three decades to emerge. This transition needed time to unravel the complex links between the mechanisms that regulate hemostasis and inflammation and the innate immune response, multicellular and multifactorial processes that interact with each other. The present review is focused on the expression of tissue factor by neutrophils, a critical event in their inflammatory and thrombotic function.

Topics: Animals; Gene Expression Regulation; Humans; Inflammation; Neutrophils; Thromboplastin; Thrombosis

Tissue factor (TF), the cellular receptor and cofactor for factor VII/VIIa, initiates haemostasis and thrombosis. Initial tissue distribution studies suggested that TF was sequestered from the circulation and only present at perivascular sites. However, there is now clear evidence that TF also exists as a blood-borne form with critical contributions not only to arterial thrombosis following plaque rupture and to venous thrombosis following endothelial perturbation, but also to various other clotting abnormalities associated with trauma, infection, or cancer. Because thrombin generation, fibrin deposition, and platelet aggregation in the contexts of haemostasis, thrombosis, and pathogen defence frequently occur without TF de novo synthesis, considerable efforts are still directed to understanding the molecular events underlying the conversion of predominantly non-coagulant or cryptic TF on the surface of haematopoietic cells to a highly procoagulant molecule following cellular injury or stimulation. This article will review some of the still controversial mechanisms implicated in cellular TF activation or decryption with particular focus on the coordinated effects of outer leaflet phosphatidylserine exposure and thiol-disulfide exchange pathways involving protein disulfide isomerase (PDI). In this regard, our recent findings of ATP-triggered stimulation of the purinergic P2X7 receptor on myeloid and smooth muscle cells resulting in potent TF activation and shedding of procoagulant microparticles as well as of rapid monocyte TF decryption following antithymocyte globulin-dependent membrane complement fixation have delineated specific PDI-dependent pathways of cellular TF activation and thus illustrated additional and novel links in the coupling of inflammation and coagulation.

Topics: Animals; Antilymphocyte Serum; Blood Coagulation; Carboxy-Lyases; Complement Activation; Drug Synergism; Humans; Inflammation; Myeloid Cells; Myocytes, Smooth Muscle; Protein Disulfide-Isomerases; Receptors, Purinergic P2X7; Thromboplastin; Thrombosis

Sepsis, defined as a clinical syndrome brought about by an amplified and dysregulated inflammatory response to infections, is one of the leading causes of death worldwide. Despite persistent attempts to develop treatment strategies to manage sepsis in the clinical setting, the basic elements of treatment have not changed since the 1960s. As such, the development of effective therapies for reducing inflammatory reactions and end-organ dysfunction in critically ill patients with sepsis remains a global priority. Advances in understanding of the immune response to sepsis provide the opportunity to develop more effective pharmaceuticals. This article details current information on the modulation of the lipopolysaccharide (LPS) receptor complex with synthetic Lipid A mimetics. As the initial and most critical event in sepsis pathophysiology, the LPS receptor provides an attractive target for antisepsis agents. One of the well-studied approaches to sepsis therapy involves the use of derivatives of Lipid A, the membrane-anchor portion of an LPS, which is largely responsible for its endotoxic activity. This article describes the structural and conformational requirements influencing the ability of Lipid A analogues to compete with LPS for binding to the LPS receptor complex and to inhibit the induction of the signal transduction pathway by impairing LPS-initiated receptor dimerization.

Topics: Animals; Cytokines; Drug Design; Endotoxins; Humans; Immune System; Inflammation; Lipid A; Lipopolysaccharide Receptors; Lipopolysaccharides; Mice; Molecular Conformation; Phosphorylation; Protein Binding; Sepsis; Signal Transduction; Solubility; Thromboplastin; Tumor Necrosis Factor-alpha

Inflammation and oxidative stress play key roles in atherosclerotic plaque instability, and plaque rupture/erosion and subsequent thrombus formation constitute the principal mechanisms of total vessel occlusion and acute ST-elevation myocardial infarction (STEMI). Plaque disruption triggers the formation of initial platelet aggregates that grow in association with an increase in fibrin formation, leading to persistent coronary flow obstruction and blood coagulation. The fibrin network may trap large numbers of erythrocytes and inflammatory cells to form an erythrocyte-rich thrombus. In fact, previous clinical studies have shown that not only platelet-rich white thrombi, but also erythrocyte-rich red thrombi can be visualized using angioscopy in patients with acute coronary syndrome. Recently, the development of thrombus aspiration and distal protection devices has significantly improved the clinical outcomes of percutaneous intervention in STEMI patients and has enabled the evaluation of antemortem coronary artery thrombi. This is important because previous autopsy studies were unable to differentiate coronary thrombi responsible for myocardial ischemia from postmortem clots. Using frozen samples of aspirated thrombi and specific monoclonal antibodies, we investigated the cellular components of thrombi (platelets, erythrocytes, fibrin and inflammatory cells, such as myeloperoxidase-positive cells) and pathologically evaluated the relationships between erythrocyte-rich thrombi and inflammation, oxidative stress and clinical outcomes in STEMI patients. Therefore, this review article focuses on the efficacy of thrombus aspiration therapy and the components of aspirated intracoronary thrombi in STEMI patients and presents the results of recent studies regarding the relationship between the composition of aspirated intracoronary thrombi and clinical outcomes.

Topics: Cell-Derived Microparticles; Coronary Thrombosis; Erythrocytes; Humans; Inflammation; Myocardial Infarction; Oxidative Stress; Plaque, Atherosclerotic; Suction; Thrombectomy; Thromboplastin

Clinical and epidemiological studies support a connection between obesity and thrombosis, involving elevated expression of the prothrombotic molecules plasminogen activator inhibitor-1 and tissue factor (TF) and increased platelet activation. Cardiovascular diseases and metabolic syndrome-associated disorders, including obesity, insulin resistance, type 2 diabetes, and hepatic steatosis, involve inflammation elicited by infiltration and activation of immune cells, particularly macrophages, into adipose tissue. Although TF has been clearly linked to a procoagulant state in obesity, emerging genetic and pharmacologic evidence indicate that TF signaling via G protein-coupled protease-activated receptors (PAR2, PAR1) additionally drives multiple aspects of the metabolic syndrome. TF-PAR2 signaling in adipocytes contributes to diet-induced obesity by decreasing metabolism and energy expenditure, whereas TF-PAR2 signaling in hematopoietic and myeloid cells drives adipose tissue inflammation, hepatic steatosis, and insulin resistance. TF-initiated coagulation leading to thrombin-PAR1 signaling also contributes to diet-induced hepatic steatosis and inflammation in certain models. Thus, in obese patients, clinical markers of a prothrombotic state may indicate a risk for the development of complications of the metabolic syndrome. Furthermore, TF-induced signaling could provide new therapeutic targets for drug development at the intersection between obesity, inflammation, and thrombosis.

Topics: Adipocytes; Adipose Tissue; Animals; Cardiovascular Diseases; Gene Expression Regulation; Humans; Inflammation; Insulin Resistance; Macrophages; Metabolic Syndrome; Mice; Mice, Obese; Models, Biological; Obesity; Plasminogen Activator Inhibitor 1; Receptor, PAR-1; Receptor, PAR-2; Risk Factors; Signal Transduction; T-Lymphocytes, Regulatory; Thrombophilia; Thromboplastin; Thrombosis

Platelet activation is known to be associated with the release of a vast array of chemokines and proinflammatory lipids which induce pleiotropic effects on a wide variety of tissues and cells, including leukocytes. During thrombosis, the recruitment of leukocytes to activated platelets is considered an important step which not only links thrombosis to inflammatory responses but may also enhance procoagulant state. This phenomenon is highly regulated and influenced by precise mutual interactions between the cells at site of vascular injury and thrombi formation. Platelet-leukocyte interaction involves a variety of mediators including adhesion molecules, chemokines and chemoattractant molecules, shed proteins, various proinflammatory lipids and other materials. The current review addresses the detailed mechanisms underlying platelet-leukocyte crosstalk. This includes their adhesive interactions, transcellular metabolisms, induced tissue factor activity and neutrophil extracellular traps formation as well as the impacts of these phenomena in modulation of the proinflammatory and procoagulant functions in a reciprocal manner that enhances the physiological responses.

Topics: Animals; Blood Platelets; Cell Adhesion; Chemokines; Coagulants; Humans; Inflammation; Integrins; Leukocyte Rolling; Leukocytes; Lipids; Mice; Neutrophils; P-Selectin; Platelet Activation; Signal Transduction; Thromboplastin; Thrombosis

The interaction of coagulation factors with the perivascular environment affects the development of disease in ways that extend beyond their traditional roles in the acute hemostatic cascade. Key molecular players of the coagulation cascade like tissue factor, thrombin, and fibrinogen are epidemiologically and mechanistically linked with diseases with an inflammatory component. Moreover, the identification of novel molecular mechanisms linking coagulation and inflammation has highlighted factors of the coagulation cascade as new targets for therapeutic intervention in a wide range of inflammatory human diseases. In particular, a proinflammatory role for fibrinogen has been reported in vascular wall disease, stroke, spinal cord injury, brain trauma, multiple sclerosis, Alzheimer's disease, rheumatoid arthritis, bacterial infection, colitis, lung and kidney fibrosis, Duchenne muscular dystrophy, and several types of cancer. Genetic and pharmacologic studies have unraveled pivotal roles for fibrinogen in determining the extent of local or systemic inflammation. As cellular and molecular mechanisms for fibrinogen functions in tissues are identified, the role of fibrinogen is evolving from a marker of vascular rapture to a multi-faceted signaling molecule with a wide spectrum of functions that can tip the balance between hemostasis and thrombosis, coagulation and fibrosis, protection from infection and extensive inflammation, and eventually life and death. This review will discuss some of the main molecular links between coagulation and inflammation and will focus on the role of fibrinogen in inflammatory disease highlighting its unique structural properties, cellular targets, and signal transduction pathways that make it a potent proinflammatory mediator and a potential therapeutic target.

Topics: Alzheimer Disease; Animals; Arthritis, Rheumatoid; Bacterial Infections; Blood Coagulation; Brain Injuries; Colitis; Fibrinogen; Humans; Inflammation; Kidney Diseases; Multiple Sclerosis; Muscular Dystrophy, Duchenne; Neoplasms; Pulmonary Fibrosis; Spinal Cord Injuries; Stroke; Thrombin; Thromboplastin; Vascular Diseases

Activated platelets may adhere to leukocytes and form circulating mixed aggregates. The latter are considered a reliable marker of a prothrombotic state and are associated with several cardiovascular conditions. The molecular mechanisms responsible of this cellular interaction include a central role of platelet P-selectin and of P-selectin glycoprotein ligand-1 (PSGL-1), its counter receptor on leukocytes in a signaling cascade, resulting in the activation of the beta-2 integrin Mac-1 and in the firm adhesion between the two cell types. The interaction of P-selectin with PSGL-1 also induces upregulation of leukocyte tissue factor, biosynthesis of several cytokines and other inflammatory reactions, thereby contributing to the thrombotic progression. In this review the main determinants of mixed aggregate formation, the heritability component, the major pathological conditions associated with higher levels of mixed aggregates in the circulation will be discussed. Besides current anti-platelet or antithrombotic drugs, natural compounds, such as the polyphenols present in vegetable foods and red wine, have been tested for their inhibitory effect on mixed aggregate formation. The promising results shown by studies in vitro and in experimental animal models, remain to be carefully investigated in humans. Platelet-leukocyte aggregates provide a novel link between inflammation and thrombosis, two central processes in atherogenesis. A better understanding of the role of platelet-leukocyte interactions in athero-thrombosis will be instrumental for the progress of prevention and treatment of ischaemic cardiovascular disease.

Topics: Animals; Blood Platelets; Cell Adhesion Molecules; Cell Communication; Fibrinolytic Agents; Humans; Inflammation; Leukocytes; Platelet Activation; Platelet Adhesiveness; Thromboplastin; Thrombosis

The transmembrane receptor tissue factor is a prominent protein expressed at macrophages and smooth muscle cells within human atherosclerotic lesions. While many coagulation proteins are detectable in atherosclerosis, a locally active thrombin and fibrin generating molecular machinery may be instrumental in manipulating cellular functions involved in atherogenesis. These include inflammation, angiogenesis and cell proliferation. Indeed, many experimental studies in mice show a correlation between hypercoagulability and increased atherosclerosis. In mice, the amount of atherosclerosis and/or the plaque phenotype, appear to be modifiable by specific anticoagulant interventions. While attempts to vary tissue factor level in the vasculature does not directly reduce plaque burden, the overexpression of tissue factor pathway inhibitor attenuates thrombogenicity and neo intima formation in mice. Moreover, inhibition of factor Xa or thrombin with novel selective agents, including rivaroxaban and dabigatran, inhibits inflammation associated with atherosclerosis in apoE(-/-) mice. The potential to modify a complex chronic disease like atherosclerosis with novel selective anticoagulants merits further clinical study.

Topics: Animals; Atherosclerosis; Humans; Inflammation; Mice; Thrombin; Thromboplastin

The pathophysiology of acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS), is characterized by increased vascular and epithelial permeability, hypercoagulation and hypofibrinolysis, inflammation, and immune modulation. These detrimental changes are orchestrated by cross talk between a complex network of cells, mediators, and signaling pathways. A rapidly growing number of studies have reported the appearance of distinct populations of microparticles (MPs) in both the vascular and alveolar compartments in animal models of ALI/ARDS or respective patient populations, where they may serve as diagnostic and prognostic biomarkers. MPs are small cytosolic vesicles with an intact lipid bilayer that can be released by a variety of vascular, parenchymal, or blood cells and that contain membrane and cytosolic proteins, organelles, lipids, and RNA supplied from and characteristic for their respective parental cells. Owing to this endowment, MPs can effectively interact with other cell types via fusion, receptor-mediated interaction, uptake, or mediator release, thereby acting as intrinsic stimulators, modulators, or even attenuators in a variety of disease processes. This review summarizes current knowledge on the formation and potential functional role of different MPs in inflammatory diseases with a specific focus on ALI/ARDS. ALI has been associated with the formation of MPs from such diverse cellular origins as platelets, neutrophils, monocytes, lymphocytes, red blood cells, and endothelial and epithelial cells. Because of their considerable heterogeneity in terms of origin and functional properties, MPs may contribute via both harmful and beneficial effects to the characteristic pathological features of ALI/ARDS. A better understanding of the formation, function, and relevance of MPs may give rise to new promising therapeutic strategies to modulate coagulation, inflammation, endothelial function, and permeability either through removal or inhibition of "detrimental" MPs or through administration or stimulation of "favorable" MPs.

Topics: Acute Lung Injury; Biological Transport; Capillary Permeability; Cell Communication; Cell-Derived Microparticles; Endothelium; Immunomodulation; Inflammation; Respiratory Distress Syndrome; Thromboplastin; Thrombosis

The increasing understanding of trauma-induced coagulopathy has led to an expansion of treatment strategies in the acute management of trauma patients. The aim of this manuscript is to give a summary of current recommendations for the treatment of trauma-induced coagulopathy based on current literature and valid guidelines. Thetrauma-induced coagulopathyis an independentacutemultifactorial diseasewith significantimpact on the mortalityof severelyinjured patients. Largely responsible for the occurrence and severity of trauma-induced coagulopathy seems to be tissue trauma and shock-induced hypoperfusion. Coagulopathy is amplified by accompanying factors such as hypothermia or dilution. Diagnosis and therapy of deranged coagulation should start as soon as possible. Routinely tested coagulation parameters are of limited use to confirm diagnosis. Therapy follows the concept of "damage control resuscitation". Infusion of large volumes should be avoided and a mean arterial pressure of 65mmHg (in consideration of contraindications!) may be aimed.A specific protocol for massive transfusion should be introduced and continued.Acidaemia should be prevented and treated by appropriate shock therapy.Loss of body temperature should be prevented and treated. Hypocalcaemia <0.9 mmol/l should be avoided and may be treated. For actively bleeding patients, packed red blood cells (pRBC) may be given at haemoglobin<10g/dl(0,62mmol/l). If massive transfusion is performed using fresh frozen plasma (FFP), a ratio of FFP to pRBC of 1:2 to 1:1 should be achieved.For treatment of hyperfibrinolysis after severe trauma the use of tranexamic acid should be considered at an early stage. Fibrinogen should be substituted at levels <1,5g/l (4,41μmol/l). Prothrombin complex concentrates may be helpfull for treatment of diffuse bleeding or anticoagulativemedikation. In acute bleeding, platelets may be transfused at a platet count <100000/μl. For diffuse bleeding or thrombocytopathic patients desmopressin might be a therapeutic option.If a factor XIII (FXIII) measurement is not promptly available, a factor XIII blind-dose should be considered in severe ongoing bleeding. The use of recombinant activated coagulation factor VII (rFVIIa) be considered if major bleeding persists despite standard attempts to control bleeding and best practice use of blood components.

Topics: Acidosis; Antifibrinolytic Agents; Blood Coagulation Disorders; Blood Coagulation Factors; Blood Transfusion; Calcium; Evidence-Based Medicine; Hemodilution; Humans; Hypotension, Controlled; Hypothermia, Induced; Inflammation; Platelet Activation; Resuscitation; Thromboplastin; Wounds and Injuries

Tissue factor (TF) is a glycosylated transmembrane protein, expressed by endothelial and other cells. Its best known function is the initiation of thrombus formation, but TF also participates in inflammation processes. This article presents the established knowledge of TF expression in endothelial cells and current opinions on encrypted TF form and mechanisms of its activation.

Topics: Animals; Endothelial Cells; Endothelium, Vascular; Gene Expression Regulation; Humans; Inflammation; Models, Molecular; Thromboplastin; Thrombosis

Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are characterized by excessive intraalveolar fibrin deposition, driven, at least in part by inflammation. The imbalance between activation of coagulation and inhibition of fibrinolysis in patients with ALI/ARDS favors fibrin formation and appears to occur both systemically and in the lung and airspace. Tissue factor (TF), a key mediator of the activation of coagulation in the lung, has been implicated in the pathogenesis of ALI/ARDS. As such, there have been numerous investigations modulating TF activity in a variety of experimental systems in order to develop new therapeutic strategies for ALI/ARDS. This review will summarize current understanding of the role of TF and other proteins of the coagulation cascade as well the fibrinolysis pathway in the development of ALI/ARDS with an emphasis on the pathways that are potential therapeutic targets. These include the TF inhibitor pathway, the protein C pathway, antithrombin, heparin, and modulation of fibrinolysis through plasminogen activator- 1 (PAI-1) or plasminogen activators (PA). Although experimental studies show promising results, clinical trials to date have proven unsuccessful in improving patient outcomes. Modulation of coagulation and fibrinolysis has complex effects on both hemostasis and inflammatory pathways and further studies are needed to develop new treatment strategies for patients with ALI/ARDS.

Topics: Acute Lung Injury; Animals; Antithrombins; Blood Coagulation; Factor VIIa; Heparin; Humans; Inflammation; Protein C; Recombinant Proteins; Respiratory Distress Syndrome; Thromboplastin

Atherosclerosis is an inflammatory disease that involves the arterial wall and is characterised by the progressive accumulation of lipids in the vessel wall. The first step is the internalisation of lipids (LDL) in the intima with endothelial activation which enhances the permeability of the endothelial layer and the expression of cytokines/chemokines and adhesion molecules. These events increase LDL particles accumulation in the extracellular matrix where they aggregate/fuse, are retained by proteoglycans and become targets for oxidative and enzymatic modifications. In turn, retained pro-atherogenic LDLs enhance selective leukocyte recruitment and attachment to the endothelial layer inducing their transmigration across the endothelium into the intima. While smooth muscle cell numbers decline with the severity of plaque progression, monocytes differentiate into macrophages, a process associated with the upregulation of pattern recognition receptors including scavenger receptors and Toll-like receptors leading to foam cell formation. Foam cells release growth factors, cytokines, metalloproteinases and reactive oxygen species all of which perpetuate and amplify the vascular remodelling process. In addition, macrophages release tissue factor that, upon plaque rupture, contributes to thrombus formation. Smooth muscle cells exposed in eroded lesions are also able to internalise LDL through LRP-1 receptors acquiring a pro-thrombotic phenotype and releasing tissue factor. Platelets recognise ligands in the ruptured or eroded atherosclerotic plaque, initiate platelet activation and aggregation leading to thrombosis and to the clinical manifestation of the atherothrombotic disease. Additionally, platelets contribute to the local inflammatory response and may also participate in progenitor cell recruitment.

Topics: Animals; Atherosclerosis; Cell Communication; Endothelium, Vascular; Humans; Inflammation; Lipid Metabolism; Platelet Activation; Thromboplastin; Thrombosis

Acute thrombus formation on disrupted atherosclerotic plaques plays a key role during the onset of acute coronary syndromes. Lesion disruption facilitates the interaction between circulating blood and prothrombotic substances, such as tissue factor (TF) present within the atherosclerotic lesion. For a long period of time, vessel-wall TF has been considered the major determinant of thrombosis. However, this old dogma has been recently changed owing to the discovery of a different pool of TF that circulates in flowing blood (blood-borne TF). Several studies have shown that blood-borne TF circulates in different pools that are associated with selected blood cells, such as monocytes, granulocytes and platelets in cell-derived microparticles, and as a soluble protein generated by alternative splicing of its full-length mRNA. Recent studies have identified a hypercoagulable state associated with an increased circulating TF activity, leading to the concept of 'vulnerable blood'. Part of the blood-borne TF circulates in an 'inactive' form and it is required to be 'activated' to exert its thrombogenic potential. Certain pathological conditions, such as smoking, hyperlipidemia and diabetes, show a higher incidence of thrombotic complications. These conditions are also characterized by the presence of high levels of circulating TF activity. Recent evidence may also suggest that an increased circulating TF activity may potentiate the initial thrombogenic stimulus represented by vessel wall-associated TF, leading to the formation of larger and/or more stable thrombus, and thus more severe acute coronary syndromes. It has been reported that inflammation increases TF expression and activity by different cell types. On the other hand, TF upregulation may facilitate inflammation by enhancing intravascular fibrin deposition, formation of proinflammatory fragments of fibrin, and by generating coagulation proteases, including FVIIa, FXa and thrombin, that activate protease-activated receptors. Furthermore, the biology of TF is know known to be more complex than previously thought by the demonstration that this protein, apart from its known effects on blood coagulation, can also function as a signaling receptor.

Topics: Atherosclerosis; Humans; Inflammation; Thromboplastin; Thrombosis

Coagulation, innate immunity, angiogenesis, and lipid metabolism represent fundamental and interdependent biological systems. While tissue factor pathway inhibitor (TFPI) is the major physiological inhibitor of TF, its unique structure and endothelial expression allow multi-modal interactions with constituent molecules in each of these systems. We review emerging data describing roles for TFPI beyond simply opposing the action of TF, particularly with regard to the highly basic c-terminus of TFPI, and highlight potentially exciting new areas for future research.

Topics: Algorithms; Atherosclerosis; Blood Coagulation; Humans; Immunity, Innate; Inflammation; Lipids; Lipoproteins; Models, Biological; Neovascularization, Pathologic; Protein Structure, Tertiary; Thromboplastin

Microparticles are submicron vesicles shed from plasma membranes in response to cell activation, injury, and/or apoptosis. The measurement of the phospholipid content (mainly phosphatidylserine; PSer) of microparticles and the detection of proteins specific for the cells from which they are derived has allowed their quantification and characterization. Microparticles of various cellular origin (platelets, leukocytes, endothelial cells) are found in the plasma of healthy subjects, and their amount increases under pathological conditions. Endothelial microparticles (EMP) not only constitute an emerging marker of endothelial dysfunction, but are also considered to play a major biological role in inflammation, vascular injury, angiogenesis, and thrombosis. Although the mechanisms leading to their in vivo formation remain obscure, the release of EMP from cultured cells can be caused in vitro by a number of cytokines and apoptotic stimuli. Recent studies indicate that EMP are able to decrease nitric-oxide-dependent vasodilation, increase arterial stiffness, promote inflammation, and initiate thrombosis at their PSer-rich membrane, which highly co-expresses tissue factor. EMP are known to be elevated in acute coronary syndromes, in severe hypertension with end organ damage, and in thrombotic thrombocytopenic purpura, all conditions associated with endothelial injury and pro-thrombotic state. The release of EMP has also been associated with endothelial dysfunction of patients with multiple sclerosis and lupus anticoagulant. More recent studies have focused on the role of low shear stress leading to endothelial cell apoptosis and subsequent EMP release in end-stage renal disease. Improved knowledge of EMP composition, their biological effects, and the mechanisms leading to their clearance will probably open new therapeutic approaches in the treatment of atherothrombosis.

Topics: Animals; Apoptosis; Biomarkers; Blood Vessels; Cardiovascular Diseases; Cell-Derived Microparticles; Cytokines; Endothelial Cells; Humans; Inflammation; Kidney Failure, Chronic; Lupus Coagulation Inhibitor; Multiple Sclerosis; Neovascularization, Pathologic; Nitric Oxide; Phosphatidylserines; Shear Strength; Thromboplastin

The aim of this overview is to summarize the available data on the involvement of monocytes in the pathological processes related to the development of acute coronary syndromes and the recovery of damaged areas, the prevention of excessive inflammatory and procoagulant response, and the restoration of microcirculation (angiogenesis).

Topics: Acute Coronary Syndrome; Animals; Blood Platelets; Cell Communication; Chemokine CCL2; Coronary Artery Disease; Endothelial Cells; Humans; Inflammation; Lipoproteins, LDL; Monocytes; Stem Cells; Thromboplastin

Tissue factor (TF) is a member of the cytokine receptor superfamily and binds FVII/VIIa. The TF:FVIIa complex has both procoagulant and signaling activities. It functions in many biological processes, including hemostasis, thrombosis, inflammation, angiogenesis and tumor growth. Importantly, TF is essential for hemostasis. However, increased TF expression within atherosclerotic plaques and elevated levels of circulating TF-positive micro particles promote thrombosis. TF increases inflammation by enhancing intravascular fibrin deposition, by increasing the formation of pro-inflammatory fragments of fibrin and by generating coagulation proteases, including FVIIa, FXa and thrombin, that activate protease-activated receptors (PARs). In endotoxemia and sepsis, TF-dependent thrombin generation and activation of PAR1 on dendritic cells enhance inflammation. Finally, the TF:FVIIa complex contributes to tumor growth by activating PAR2.

Topics: Animals; Blood Coagulation Factors; Hemostasis; Humans; Inflammation; Neoplasms; Receptors, Proteinase-Activated; Thromboplastin; Thrombosis

Although, the main physiological role of monocytes is attributed to innate immunity (that is, phagocytosis) and the development of tissue macrophages and dendritic cells, the pathophysiological role of these goes far behind these (simplistic) limits. Indeed, monocytes constitute a major source of blood tissue factor, a key element of the extrinsic coagulation cascade. Monocytes actively bind to platelets, thus forming very prothrombotic monocyte-platelet aggregates. Additionally, these cells link inflammation and the procoagulant state observed in various prothrombotic conditions. However, monocytes are also crucial for successful thrombus recanalisation. In this article, we review the available data on potential mechanisms that link monocytes with thrombosis-related processes.

Topics: Animals; Antiphospholipid Syndrome; Blood Platelets; Cell Adhesion; Cells, Cultured; Coronary Disease; Cytokines; Female; Humans; Inflammation; Male; Mice; Models, Biological; Monocytes; Neovascularization, Physiologic; Platelet Aggregation; Rabbits; Risk Factors; Swine; Thrombophilia; Thromboplastin; Thrombosis

Sepsis is a systemic response to infection, and symptoms are produced by host defense systems rather than by the invading pathogens. Amongst the most prominent features of sepsis, contributing significantly to its outcome, is activation of coagulation with concurrent down-regulation of anticoagulant systems and fibrinolysis. Inflammation-induced coagulation on its turn contributes to inflammation. Another important feature of sepsis, associated with key symptoms such as hypovolemia and hypotension, is endothelial dysfunction. Under normal conditions, the endothelium provides for an anticoagulant surface, a property that is lost in sepsis. In this review, data about the interplay between inflammation and coagulation in sepsis are summarized with a special focus on the influence of the endothelium on inflammation-induced coagulation and vice versa. Possible procoagulant properties of the endothelium are described, such as expression of tissue factor (TF) and von Willebrand factor and interaction with platelets. Possible procoagulant roles of microparticles, circulating endothelial cells and endothelial apoptosis, are also discussed. Moreover, the important roles of the endothelium in down-regulating the anticoagulants TF pathway inhibitor, antithrombin, and the protein C (PC) system and inhibition of fibrinolysis are discussed. The influence of coagulation on its turn on inflammation and the endothelium is described with a special focus on protease-activated receptors (PARs). We conclude that the relationship between endothelium and coagulation in sepsis is tight and that further research is needed, for example, to better understand the role of activated PC signaling via PAR-1, the role of the endothelial PC receptor herein, and the role of the glycocalyx.

Topics: Anticoagulants; Blood Coagulation; Blood Platelets; Endothelium, Vascular; Fibrinolysis; Heparin; Humans; Inflammation; Models, Biological; Sepsis; Thromboplastin; von Willebrand Factor

Malaria remains a highly prevalent disease in more than 90 countries and accounts for at least 1 million deaths every year. Plasmodium falciparum infection is often associated with a procoagulant tonus characterized by thrombocytopenia and activation of the coagulation cascade and fibrinolytic system; however, bleeding and hemorrhage are uncommon events, suggesting that a compensated state of blood coagulation activation occurs in malaria. This article (i) reviews the literature related to blood coagulation and malaria in a historic perspective, (ii) describes basic mechanisms of coagulation, anticoagulation, and fibrinolysis, (iii) explains the laboratory changes in acute and compensated disseminated intravascular coagulation (DIC), (iv) discusses the implications of tissue factor (TF) expression in the endothelium of P. falciparum infected patients, and (v) emphasizes the procoagulant role of parasitized red blood cells (RBCs) and activated platelets in the pathogenesis of malaria. This article also presents the Tissue Factor Model (TFM) for malaria pathogenesis, which places TF as the interface between sequestration, endothelial cell (EC) activation, blood coagulation disorder, and inflammation often associated with the disease. The relevance of the coagulation-inflammation cycle for the multiorgan dysfunction and coma is discussed in the context of malaria pathogenesis.

Topics: Animals; Blood Platelets; Coma; Disseminated Intravascular Coagulation; Endothelium, Vascular; Erythrocytes; Fibrinolysis; Humans; Inflammation; Malaria, Falciparum; Plasmodium falciparum; Platelet Activation; Thrombocytopenia; Thromboplastin

Tissue factor (TF), is a cellular receptor that binds the ligand factor VII/VIIa to initiate the blood coagulation cascade. In addition to its role as the initiator of the hemostatic cascade, TF is known to be involved in angiogenesis via an interaction with factor VIIa and protease-activated receptor-2 (PAR-2). In this article we review previous studies from our laboratory demonstrating that the pattern and level of TF expression is altered in multiple cell types derived from eutopic and ectopic endometrium from women with endometriosis compared with normal endometrium. We posit that the inflammatory environment that occurs in ectopic and eutopic endometrium from patients with disease results in high TF expression that in turn, signals via PAR-2 to further produce inflammatory cytokine or chemokine production and macrophage recruitment. Thus, our studies suggest that TF might be an ideal target for therapeutic intervention in endometriosis.

Topics: Animals; Cell Proliferation; Cytokines; Endometriosis; Endometrium; Female; Gene Expression Regulation; Humans; Immunohistochemistry; Infertility, Female; Inflammation; Mice; Models, Biological; Neovascularization, Pathologic; Thromboplastin

Adenosine is an endogenous purine nucleoside that, following its release into the extracellular space, binds to specific adenosine receptors expressed on the cell surface. Adenosine appears in the extracellular space under metabolically stressful conditions, which are associated with ischemia, inflammation, and cell damage. There are 4 types of adenosine receptors (A(1), A(2A), A(2B) and A(3)) and all adenosine receptors are members of the G protein-coupled family of receptors. Adenosine receptors are expressed on monocytes and macrophages and through these receptors adenosine modulates monocyte and macrophage function. Since monocytes and macrophages are activated by the same danger signals that cause accumulation of extracellular adenosine, adenosine receptors expressed on macrophages represent a sensor system that provide monocytes and macrophages with information about the stressful environment. Adenosine receptors, thus, allow monocytes and macrophages to fine-tune their responses to stressful stimuli. Here, we review the consequences of adenosine receptor activation on monocyte/macrophage function. We will detail the effect of stimulating the various adenosine receptor subtypes on macrophage differentiation/proliferation, phagocytosis, and tissue factor (TF) expression. We will also summarize our knowledge of how adenosine impacts the production of extracellular mediators secreted by monocytes and macrophages in response to toll-like receptor (TLR) ligands and other inflammatory stimuli. Specifically, we will delineate how adenosine affects the production of superoxide, nitric oxide (NO), tumor necrosis factor-alpha, interleukin (IL)-12, IL-10, and vascular endothelial growth factor (VEGF). A deeper insight into the regulation of monocyte and macrophage function by adenosine receptors should assist in developing new therapies for inflammatory diseases.

Topics: Adenosine; Animals; Cell Differentiation; Cell Proliferation; Cytokines; Humans; Inflammation; Macrophages; Nitric Oxide; Phagocytosis; Receptors, Purinergic P1; Respiratory Burst; Thromboplastin; Vascular Endothelial Growth Factor A

Atherothrombotic vascular disease is a complex disorder in which inflammation and coagulation play a pivotal role. Rupture of high-risk, vulnerable plaques with the subsequent tissue factor (TF) exposure is responsible for coronary thrombosis, the main cause of unstable angina, acute myocardial infarction, and sudden cardiac death. Tissue factor (TF), the key initiator of coagulation is an important modulator of inflammation. TF is widely expressed in atherosclerotic plaques and found in macrophages, smooth muscle cells, extracellular matrix and acellular lipid-rich core. TF expression can be induced by various stimulants such as C-reactive protein, oxLDL, hyperglycemia and adipocytokines. The blood-born TF encrypted on the circulating microparticles derived from vascular cells is a marker of vascular injury and a source of procoagulant activity. Another form of TF, called alternatively spliced has been recently identified in human and murine. It is soluble, circulates in plasma and initiates coagulation and thrombus propagation. Evidence indicates that elevated levels of blood-borne or circulating TF has been associated with metabolic syndrome, type 2 diabetes and cardiovascular risk factors and is a candidate biomarker for future cardiovascular events. Therapeutic strategies have been developed to specifically interfere with TF activity in the treatment of cardiovascular disease.

Topics: Coronary Artery Disease; Coronary Thrombosis; Diabetes Mellitus; Endothelium, Vascular; Humans; Hyperglycemia; Inflammation; Obesity; Thromboplastin

Greater understanding of the cellular interactions associated with tissue factor (TF), activated factor (F) VII and TF-FVIIa complexes is likely to provide considerable clinical benefit. This article reviews current knowledge on the function and regulation of TF and its role in a range of biological processes, including hemostasis, thrombosis and inflammation.

Topics: Blood Coagulation; Factor VIIa; Hemostasis; Humans; Inflammation; Lipoproteins; Models, Molecular; Multiprotein Complexes; Protein Processing, Post-Translational; Signal Transduction; Solubility; Thromboplastin; Thrombosis

A series of coordinated enzymatic reactions takes place in the body whenever blood clots. The major physiological initiator of these reactions is a membrane-bound glycoprotein known as tissue factor (TF), which is normally separated from the bloodstream by the vascular endothelium. Bleeding, caused by injury or tissue damage, activates a complex enzyme cascade as TF becomes exposed to the bloodstream. In disease states, leukocytes or the vascular endothelium may abnormally express TF to cause intravascular coagulation. The blood-coagulation cascade is also relevant to diseases such as hemophilia, in which patients are deficient in blood proteins necessary for clotting, and is linked to vascular diseases such as heart attack and stroke, in which clotting can lead to the occlusion of blood vessels. Coagulation is also activated in inflammation and cancer. In this article, we discuss characteristics of TF and review its role in inflammation and cancer.

Topics: Biomarkers; Blood Coagulation; Humans; Inflammation; Neoplasms; Thromboplastin

Hemostatic serine proteinases-thrombin, Factor VIIa, Factor Xa, play the central role in blood coagulation and thrombosis. Activation of coagulation and generation of active proteinases is initiated by tissue factor (TF) that is expressed by cells of the innate immune system and endothelial cells after tissue damage and cell activation induced by trauma, infection, hypoxia and other cell injury. Coagulation and inflammation are the essential part of the defensive host response. These processes have several connecting points account for the associate and/or the interaction between coagulation and inflammation pathways. The first link between these processes is endothelium, which after damage expresses the adhesive proteins (vWF,P-selectin), inductors and receptors, involved in both coagulation and inflammation. The second link is platelets, which stored in and after activation release proteins with procoagulant and proinflammatory properties. The third link is the serine proteinases, which produced for blood coagulation and activate via its specifical receptors--PARs (proteinase activated receptors) the cells of both coagulation and inflammation system thereby controlling these processes. The generation of these proteinases is initiated by tissue factor (TF) which triggers blood coagulation at sites of tissue injury by selective binding of FVIIa. TF/VIIa complexes with substrate--FX that is activated to FXa. TF/VIIa/Xa can activate both the inflammatory responses of endothelial and other cells and also blood coagulation through stimulation of thrombin generation. This review summarizes the latest data on the blood coagulation activation that include generation of active surface for coagulation, generation of hemostatic serine proteinases and its role as signalling molecules that via PARs and other receptors involved in regulation and control of the interaction of blood coagulation and inflammation and illustrates the potential for therapeutic intervention.

Topics: Animals; Anticoagulants; Blood Coagulation; Blood Platelets; Cell Adhesion; Endothelium, Vascular; Factor VIIa; Factor Xa; Hemostasis; Humans; Inflammation; Models, Biological; P-Selectin; Peptide Hydrolases; Protein Binding; Receptors, Proteinase-Activated; Serine Endopeptidases; Signal Transduction; Thrombin; Thromboplastin

Tissue factor (TF) plays a role in thrombogenesis. TF initiates blood coagulation resulting in the generation of protease coagulant mediators (FVIIa, FXa, and FIIa) and fibrin production. TF hypercoagulablility directly contributes to thrombus formation resulting from the major events of fibrin deposition and FIIa-induced platelet activation/aggregation. In addition, blood coagulation indirectly promotes thrombogenicity via the coagulation-inflammation cycle in which TF plays a diverging and converging role. As the consequence of coagulation-dependent inflammation in which protease-activated receptor (PAR) mediates the coagulant signaling to elicit cytokines, selectins, and growth factors, such inflammation facilitates thrombosis by platelet aggregation and leukocyte recruitment. As TF hypercoagulability concerned, anti-thrombotic strategies involve the prevention by anticoagulation and PAR antagonism. Anticoagulants block the direct and indirect thrombotic contributions, while PAR antagonists arrest coagulation-dependent inflammation. With respect to both thrombosis and inflammation being cardiovascular risk factors, such strategies offer diverse benefits to cardioprotection.

Topics: Animals; Blood Coagulation; Blood Vessels; Cardiovascular Diseases; Factor VIIa; Factor Xa; Fibrin; Humans; Inflammation; Models, Biological; Myocardium; Peptides; Platelet Aggregation; Prothrombin; Receptors, Proteinase-Activated; Thromboplastin; Thrombosis

The complex of the cell surface receptor tissue factor (TF) and its ligand coagulation factor VIIa (FVIIa) is the primary initiator of the coagulation cascade. It is now clear the TF-initiated coagulation pathway also plays major nonhemostatic roles in inflammation, tumor growth, and angiogenesis. Direct or indirect cell signaling by TF-FVIIa or downstream coagulation proteases is an essential part of these nonhemostatic functions. The TF-FVIIa complex activates protease-activated receptor 2 and thus regulates gene transcription and protein translation, cell proliferation and survival, or cell motility and integrin activation. In this review, we relate our current understanding of direct TF signaling pathways to the emerging roles of TF in (patho)physiology.

Topics: Animals; Cell Movement; Cell Proliferation; Factor VIIa; Gene Expression Regulation; Humans; Inflammation; Integrins; Ligands; Neoplasms; Neovascularization, Pathologic; Neovascularization, Physiologic; Protein Biosynthesis; Receptor, PAR-2; Signal Transduction; Thromboplastin

In the pathogenesis of vascular disease, inflammation and coagulation play a pivotal role. Increasing evidence points to an extensive cross-talk between these two systems, whereby inflammation not only leads to activation of coagulation, but coagulation also considerably affects inflammatory activity. Tissue factor (TF) plays an important role at the crossroad of coagulation and inflammation, as the principal initiator of coagulation and an important modulator of inflammation. Proinflammatory cytokines can induce TF expression on mononuclear cells and endothelial cells and thereby commence pathways that lead to thrombin generation. Simultaneously, TF may bind to cellular receptors, which may affect the production and release of inflammatory mediators. There is increasing experimental evidence that TF inhibition may have beneficial effects in disease states in which the combination of coagulation and inflammation plays a prominent role.

Topics: Blood Coagulation; Cytokines; Humans; Infections; Inflammation; Signal Transduction; Thrombin; Thromboplastin; Vascular Diseases

Inflammation and thrombosis are key events in the long-lasting sequence of atherosclerotic plaque initiation, plaque growth, and eventual onset of complications leading to clinically manifest disease. Recent cellular and molecular studies have indicated that inside the plaque tissue complex, proinflammatory and prothrombotic mechanisms are intimately associated, and tissue factor (TF) is one of the main proteins that may link both processes. It is therefore not surprising that TF expression appeared to be a prominent feature in various types of vulnerable atherosclerotic plaques (i.e., lesions that specifically predispose to the onset of symptomatic atherosclerotic disease).

Topics: Animals; Atherosclerosis; Gene Expression Regulation; Humans; Inflammation; Thromboplastin; Thrombosis

Tissue factor (TF) performs an essential role in the blood clotting system by activating the extrinsic coagulation pathway following vascular injury. In addition to this physiological hemostatic role for wound repair, TF also plays pivotal roles in organ dysfunction in trauma patients by triggering pathological disseminated thrombosis and inflammation. Constitutively expressed TF in subendothelial cells is released into the circulation following trauma and can be detected as slightly elevated TF levels in the plasma. Liberation of constitutive TF into the blood and inducible tissue factor expression on monocytes and the other cells may synergistically increase plasma TF levels to higher values at the early stage of posttrauma, especially in patients with disseminated intravascular coagulation (DIC) in association with sustained systemic inflammatory response syndrome. Marked TF generation not adequately balanced by physiological coagulation inhibitors such as tissue factor pathway inhibitor in posttrauma DIC patients has been observed. Based on these pieces of evidence, it has now been accepted that combined activation of TF-dependent coagulation inadequately regulated by anticoagulant mechanisms and inflammation may synergistically play important roles in the pathogenesis of posttrauma multiple organ dysfunction syndrome.

Topics: Blood Coagulation; Disseminated Intravascular Coagulation; Gene Expression Regulation; Humans; Inflammation; Multiple Organ Failure; Thromboplastin; Wounds and Injuries

Tissue factor plays an essential role in the initiation of coagulation in vivo. In severe conditions, including sepsis and acute lung injury, increased expression of tissue factor may induce disseminated intravascular coagulation and fibrin deposition in organs, which are believed to have a determining impact on patient outcome. Tissue factor also acts as a signaling receptor and is involved in the systemic inflammatory response, as in cancer progression and atherosclerosis. Interventions aiming at limiting tissue factor activities have been evaluated in multiple experimental studies and the observed results have supported the potential benefits for coagulation disorders, inflammation, and survival. The effects of the main physiological inhibitor of tissue factor, tissue factor pathway inhibitor, have been evaluated in two large clinical trials in sepsis. Even though they are not associated with an improved outcome, the observed data support further clinical studies.

Topics: Animals; Blood Coagulation; Clinical Trials as Topic; Disease Models, Animal; Disseminated Intravascular Coagulation; Fibrin; Humans; Inflammation; Lipoproteins; Lung; Lung Injury; Sepsis; Signal Transduction; Thromboplastin

Thrombosis is associated with atherosclerosis, sepsis, cancer, and numerous other inflammatory diseases. Complications of thrombosis, such as myocardial infarction, stroke, and venous thromboembolism, contribute significantly to morbidity and mortality. Susceptibility to thrombosis is conferred by both genetic and environmental factors. Tissue factor is the primary cellular initiator of blood coagulation and is a major contributor to thrombosis. In this review, we discuss the association between various polymorphisms and the risk for thrombosis.

Topics: Cytokines; Genetic Predisposition to Disease; Humans; Inflammation; Lipopolysaccharide Receptors; Polymorphism, Genetic; Thromboplastin; Thrombosis

Plaque disruption and subsequent thrombus formation play a critical role in the clinical manifestations of atherothrombosis. Vulnerable lesions are characterized by the existence of core rich in lipid, macrophages and tissue factor (TF). Plaque disruption facilitates the interaction between flowing blood with the inner components (TF) of disrupted atherosclerotic lesions triggering the coagulation cascade. TF, thrombin, platelets, fibrin and inflammatory cells are involved in this process of acute thrombus formation. This pathologic process is significantly accelerated by several "cardiovascular risk factors" such as diabetes, smoking, dyslipemia, etc. We will review on the role of TF, plaque cell apoptosis and blood thrombogenicity acting as a thread of inflammatory and prothrombotic mediators. We will also review the role of activated platelets as source for pro-inflammatory cytokines and enunciation of thrombotic process. Overall, we will try to emphasize the most recent understanding of the concepts involved in the interaction between inflammation and coagulation within the setting of atherothrombotic disease.

Topics: Animals; Apoptosis; Atherosclerosis; Humans; Inflammation; Thrombin; Thromboplastin; Thrombosis

Recognition of the link between coagulation activation and inflammation has led to the hypothesis that anticoagulants may be effective in the treatment of septic patients by altering the inflammatory response. However, only limited methodologies exist that can be used in human volunteers to mimic the physiologic alterations observed in critically ill patients. The human endotoxemia model represents a model of inflammation-induced tissue factor triggered coagulation activation. As it permits elucidation of a key player in this proinflammatory and procoagulant response, it serves as a useful tool to investigate novel therapeutics in a standardized setting. The aim of this review is to focus on coagulation interventions in the human endotoxemia model.

Topics: Anticoagulants; Blood Coagulation; Cytokines; Endotoxemia; Hemostasis; Humans; Inflammation; Lipopolysaccharides; Models, Biological; Thromboplastin

Topics: Apoptosis; Arteries; Arteriosclerosis; Blood Coagulation; Blood Platelets; Humans; Inflammation; Thrombin; Thromboplastin; Thrombosis; Venous Thrombosis

In multicellular organisms, apoptosis and subsequent microparticle shedding play a key role in homeostasis. Having long been considered as << cellular dust >>, microparticles released in biological fluids upon cell activation or apoptosis appear as multifunctional bioeffectors involved in the modulation of key functions including immunity, inflammation, hemostasis and thrombosis, angiogenesis. MP constitute reliable markers of vascular damage, accessible to biological detection whilst the cells they originate from remain sequestered in tissues or are promptly submitted to phagocytosis.. MP modulate biological functions of target cells through the transfer of cytoplasmic content, lipids and membrane receptors. The pharmacological modulation of circulating levels of microparticles could be of particular interest in thrombotic or inflammatory diseases, cancer or hemophilia.. MP can now be viewed not only as a hallmark of cell damage but also as a true biological tool.

Topics: Adult; Apoptosis; Biomarkers; Cell Communication; Cell Membrane; Cytoskeleton; Female; Hemostasis; Homeostasis; Humans; Immunity; Inflammation; Male; Microbodies; Particle Size; Phagocytosis; Phenotype; Pregnancy; Selectins; Thromboplastin; Thrombosis

Vessel wall endothelial damage initiates a local inflammatory response, which promotes a prothrombotic state driven by tissue factor, adhesion molecules, and pro-inflammatory cytokines. Understanding how natural inflammatory mechanisms promote a procoagulant state, may lead to the development of new pharmacological interventions targeted at thrombosis.

Topics: Animals; Cell Adhesion Molecules; Humans; Inflammation; Inflammation Mediators; Thromboplastin; Thrombosis

Overexpression and exposition of tissue factor (TF) in atherosclerotic plaques and/or arterial thrombi are critical events in atherothrombosis. TF, the receptor for factor VII (FVII) and activated factor VII (FVIIa), is the principal initiator of blood coagulation and induces thrombin generation leading to fibrin formation and platelet activation. TF also plays a major role in cell migration and angiogenesis. TF activity is downregulated by Tissue Factor Pathway Inhibitor (TFPI), a Kunitz-type inhibitor, which forms a neutralizing complex with TF, FVIIa and activated factor X. In physiological conditions, TF is absent from vascular cells which come into contact with flowing blood and is present as an inactive pool in fibroblasts and smooth muscle cells (SMC). In contrast, TF is widely expressed in atherosclerotic plaques and is found in macrophages, SMCs, and foam-cells and also in extracellular matrix and acellular lipid-rich core. TF expression is up-regulated by inflammatory cytokines and oxidized lipids. Plaque thrombogenicity is directly correlated to their TF content. After fibrous cap disruption, TF is exposed on plaque surface and triggers thrombus formation leading to arterial lumen occlusion and/or downstream embolization. In coronary and carotid plaques, TF content was found to be higher in plaques from symptomatic than asymptomatic patients. Soluble forms of TF and microparticles of monocyte and platelet origin, and bearing TF, constitute "blood-born TF". The contribution of this TF pool to arterial thrombosis is still under discussion. TF pathway is a target for new therapeutic agents that can decrease TF activity, such as active site-inactivated factor VIIa, recombinant TFPI and antibodies against TF or peptides interfering with TF-FVIIa complex activity.

Topics: Arteriosclerosis; Cardiovascular Diseases; Cell Movement; Fibroblasts; Hemostatics; Humans; Inflammation; Muscle, Smooth; Neovascularization, Pathologic; Thromboplastin

The role of tissue factor (TF) in inflammation is mediated by blood coagulation. TF initiates the extrinsic blood coagulation that proceeds as an extracellular signaling cascade by a series of active serine proteases: FVIIa, FXa, and thrombin (FIIa) for fibrin clot production in the presence of phospholipids and Ca2+. TF upregulation resulting from its enhanced exposure to clotting factor FVII/FVIIa often manifests not only hypercoagulable but also inflammatory state. Coagulant mediators (FVIIa, FXa, and FIIa) are proinflammatory, which are largely transmitted by protease-activated receptors (PAR) to elicit inflammation including the expression of tissue necrosis factor, interleukins, adhesion molecules (MCP-1, ICAM-1, VCAM-1, selectins, etc.), and growth factors (VEGF, PDGF, bFGF, etc.). In addition, fibrin, and its fragments are also able to promote inflammation. In the event of TF hypercoagulability accompanied by the elevations in clotting signals including fibrin overproduction, the inflammatory consequence could be enormous. Antagonism to coagulation-dependent inflammation includes (1) TF downregulation, (2) anti-coagulation, and (3) PAR blockade. TF downregulation and anti-coagulation prevent and limit the proceeding of coagulation cascade in the generation of proinflammatory coagulant signals, while PAR antagonists block the transmission of such signals. These approaches are of significance in interrupting the coagulation-inflammation cycle in contribution to not only anti-inflammation but also anti-thrombosis for cardioprotection.

Topics: Anticoagulants; Blood Coagulation; Calcium; Cell Adhesion Molecules; Extracellular Signal-Regulated MAP Kinases; Fibrin; Humans; Inflammation; Interleukins; Phospholipids; Receptors, Proteinase-Activated; Serine Endopeptidases; Thromboplastin; Tumor Necrosis Factor-alpha

The primary importance of tissue factor (TF) in blood coagulation and thrombus propagation has been recognized for many years. Nevertheless, our view about the origin of TF activity, necessary for normal hemostasis and found in pathologic conditions, needs to be revised in the light of recent observations. Pioneering work by Yale Nemerson's group showed that circulating TF on microparticles (MPs), could promote thrombus growth. The origin and characteristics of this 'blood-borne' TF are targets of intense research as well as intense debate. Surprising observations now implicate the adhesion receptor P-selectin (P-sel), known for its role in inflammation, in these MPs' generation. P-sel, translocated from granules to the cell surfaces of activated platelets and endothelial cells, was recently found to play multiple roles in hemostasis. Expressed on endothelium, it can mediate platelet rolling. Signaling by P-sel through its receptor on leukocytes, P-selectin glycoprotein ligand 1 (PSGL-1), induces the generation of TF-positive, highly procoagulant MPs. In addition, P-sel on activated platelets helps to recruit these MPs specifically to thrombi. In this review, we discuss the roles of P-sel and TF-positive MPs and highlight strategies to modulate hemostasis by modulating the P-sel, TF, coagulation triad.

Topics: Animals; Blood Coagulation; Blood Coagulation Tests; Coagulants; Hemostasis; Humans; Inflammation; Leukocytes; Membrane Glycoproteins; Mice; Microscopy, Electron; Models, Biological; P-Selectin; Protein Transport; Thromboplastin; Time Factors; Umbilical Veins

The development of atherosclerotic disease results from the interaction between environment and genetic make up. A key factor in atherogenesis is the oxidative modification of lipids, which is involved in the recruitment of mononuclear leukocytes to the arterial intima--a process regulated by several groups of adhesion molecules and cytokines. Activated leukocytes, as well as endothelial mitochondria, can produce reactive oxygen species (ROS) that are associated with endothelial dysfunction, a cause of reduced nitric oxide (NO) bioactivity and further ROS production. Peroxisome proliferator-activated receptors (PPAR) and liver X receptors (LXR) are nuclear receptors significantly involved in the control of lipid metabolism, inflammation and insulin sensitivity. Also, an emerging role has been suggested for G protein coupled receptors and for the small Ras and Rho GTPases in the regulation of the expression of endothelial NO synthase (eNOS) and of tissue factor, which are involved in thrombus formation and modulation of vascular tone. Further, the interactions among eNOS, cholesterol, oxidated LDL and caveola membranes are probably involved in some molecular changes observed in vascular diseases. Despite the relevance of oxidative processes in atherogenesis, anti-oxidants have failed to significantly improve atherosclerosis (ATS) prevention, while statins have proved to be the most successful drugs.

Topics: Animals; Arteries; Atherosclerosis; Cell Adhesion; Cytokines; DNA-Binding Proteins; Humans; Inflammation; Insulin; Leukocytes, Mononuclear; Lipids; Liver X Receptors; Mitochondria; Nitric Oxide; Nitric Oxide Synthase Type III; Orphan Nuclear Receptors; Oxygen; Peroxisome Proliferator-Activated Receptors; Reactive Oxygen Species; Receptors, Cytoplasmic and Nuclear; Receptors, G-Protein-Coupled; Thromboplastin

Tissue factor (TF) is a transmembrane protein that, in complex with factor VIIa (FVIIa), initiates coagulation. It also influences various other physiological and pathological events, such as inflammation, and negatively influences survival during sepsis. TF resembles a conserved class of pro-inflammatory cytokine receptors and activates a set of pro-inflammatory intracellular signal transduction routes. Interestingly, whereas the homology of TF to the class II cytokine receptors is reflected in a similar type of signal transduction, the mechanism by which the signal is transduced across the membrane differs greatly. This review discusses the role of TF and its ligand FVIIa in inflammation, sepsis, and signal transduction, and describes the way in which these processes interact.

Topics: Animals; Cattle; Disseminated Intravascular Coagulation; Factor VIIa; Gene Expression Regulation; Guinea Pigs; Humans; Inflammation; Mice; Protein-Tyrosine Kinases; Rabbits; Rats; Receptors, Cytokine; Sepsis; Sequence Homology; Signal Transduction; Thromboplastin; Trans-Activators

The adhesion receptor P-selectin has long been known to support leukocyte rolling and emigration at sites of inflammation. Recently, P-selectin was also revealed to be a key molecule in hemostasis and thrombosis, mediating platelet rolling, generating procoagulant microparticles containing active tissue factor and enhancing fibrin deposition. Elevated levels of plasma P-selectin are indicative of thrombotic disorders and predictive of future cardiovascular events. Because the interaction between P-selectin and its receptor P-selectin glycoprotein ligand-1 (PSGL-1) represents an important mechanism by which P-selectin induces the formation of procoagulant microparticles and recruits the microparticles to thrombi, anti-thrombotic strategies are currently aimed at inhibiting this interaction. Recent developments also suggest that the procoagulant potential of P-selectin could be used to treat coagulation disorders such as hemophilia A.

Topics: Animals; Blood Coagulation Disorders; Cardiovascular Diseases; Cell Adhesion; Fibrin; Humans; Inflammation; Leukocytes; Membrane Glycoproteins; Mice; Models, Biological; P-Selectin; Phenotype; Protein Binding; Protein Structure, Tertiary; Thromboplastin; Thrombosis

Crucial advances in our understanding of the pathogenesis of atherothrombosis, defined as atherosclerosis and its thrombotic complications, have been achieved during the past two decades. The historical hypothesis of pathogenesis ("lipid accumulation") has evolved to integrate several factors contributing to the initiation and evolution of this complex disease. Endothelial dysfunction is considered to be the earliest event in atherogenesis. Inflammation and apoptosis play critical roles in its progression and onset. Tissue factor is postulated to be a central actor in determining plaque thrombogenicity. A hyperreactive state of the blood ("vulnerable blood") may be responsible for one-third of all the acute coronary syndromes. This review will discuss emerging concepts in the pathogenesis of and therapeutic approaches to atherothrombotic disease.

Topics: Acute Disease; Apoptosis; Arteriosclerosis; Endothelium; Humans; Inflammation; Risk Factors; Thromboplastin; Thrombosis

Recent studies have revealed a close association of the blood coagulation system with inflammation and immune reactions. The products of the cascade reaction of blood coagulation can work as inflammatory mediators or immune modulators, and, vice versa, some inflammatory or immune stimuli are linked to induction of blood coagulation. First, tissue factor (the blood coagulation initiator), the monocyte/macrophage tissue factor expression regulatory factors associated with inflammation and immune reactions, and the assembly of coagulation factors on leukocytes were reviewed. Second, evidence of leukocyte tissue factor expression and subsequent fibrin deposition were demonstrated at sites of infection or allergic reactions, using immunohistochemical staining. Third, the progress in the investigation of thrombin was reviewed from the viewpoint of its effects on inflammation (vascular permeability enhancement, leukocyte chemotaxis, chemical mediator release, etc.) and immune reactions (T-cell proliferation, cytokine production, etc.). The evidence presented here indicates a cross-talk between blood coagulation and inflammatory and immune reactions, suggesting that the products of the clotting reaction (e.g., thrombin) in lesions are real-time markers of inflammatory diseases.

Topics: Biomarkers; Blood Coagulation; Fibrin; Humans; Inflammation; Inflammation Mediators; Thrombin; Thromboplastin

As early as the nineteenth century, it was recognized that tissues exhibit clot-promoting activity. The realization that a single protein was responsible for this procoagulant effect led to the discovery of tissue factor (TF), but for many years it was thought that TF activity was restricted to the activation of an auxiliary pathway that had little biological relevance. Research over recent decades has greatly improved our understanding of TF function, and today the protein is recognized to be the primary biological initiator of the coagulation cascade. Furthermore, it is now clear that TF is a major player in several other physiological processes. We present recent data suggesting that TF is vital not only for effective haemostasis, but also for angiogenesis and tumour metastasis, for embryonic development, and for induction of a pro-inflammatory response. These advances in our knowledge of TF function will improve our understanding of several pathophysiological conditions, and may expand the therapeutic options available for their treatment.

Topics: Animals; Embryonic Development; Hemostasis; Humans; Inflammation; Neoplasm Metastasis; Neovascularization, Pathologic; Thromboplastin

Tissue factor (TF) contributes to a variety of biological processes by generating coagulation proteases and by the activation of protease activated receptors (PARs). Studies, using low TF mice, have provided us with novel insights into the role of TF in tissue-specific haemostasis. Low TF mice exhibit defects in the uterus, placenta, heart and lung, which are all tissues that normally express high levels of TF. We propose that these observed defects are primarily due to a reduction in the level of TF to below a critical threshold that is required to maintain adequate haemostasis. Nevertheless, a reduction in TF:FVIIa-dependent signaling may also reduce cell survival and/or compromise the integrity of the vasculature in these organs. Low TF mice are also a useful tool to study the role of TF and the coagulation cascade in other processes, such as thrombosis, inflammation and angiogenesis.

Topics: Animals; Female; Hemostasis; Inflammation; Mice; Neovascularization, Pathologic; Pregnancy; Thromboplastin; Thrombosis

Venous thromboembolism (VTE), manifested as either deep venous thrombosis (DVT) or pulmonary embolism (PE), is an extremely common medical problem, occurring either in isolation or as a complication of other diseases or procedures. Yet, despite its frequency, much remains to be learned regarding the pathogenic mechanisms that initiate VTE, about tailoring its treatment to the individual with her/his specific set of risk factors for recurrence, and about its medical management when associated with specific disease entities, such as cancer. These three topics are addressed in this chapter. In Section I, Drs. López and Conde discuss the mechanisms by which venous thrombi may be initiated on the vessel wall in the absence of anatomically overt vessel wall injury. The authors propose a model whereby tissue factor (TF)-bearing microvesicles that arise from cells of monocyte/macrophage lineage can fuse with activated endothelial cells in regions of vessel activation or inflammation and initiate blood coagulation. Key components of this model include docking of the microvesicles to the stimulated endothelium through P-selectin glycoprotein ligand-1 on their surfaces binding to either P-selectin or E-selectin on the endothelium, and the role of hypoxia during blood stasis in initiating local endothelial activation. Elevations in the levels of TF-bearing microvesicles associated with inflammatory conditions would help to explain the increased risk of thrombosis associated with infections and inflammatory states such as inflammatory bowel disease. In Section II, Dr. Clive Kearon discusses the risk factors for recurrent thrombosis and strategies for determining length of therapy and tailoring specific therapies through risk stratification. Those patients who experience VTE in association with a major reversible risk factor such as surgery are much less likely to experience a recurrence when anticoagulation is discontinued than are patients with a persistent risk factor, such as thrombophilia or cancer unresponsive to therapy. Those with a minor reversible risk factor, such as prolonged air travel, have an intermediate risk of recurrence after discontinuance of anticoagulant therapy. The author provides an algorithm for using risk assessment as a means of determining the length and type of therapy to be used to minimize the rate of recurrence while simultaneously diminishing the risk of bleeding associated with anticoagulation. In Section III, Dr. Agnes Lee updates th

Topics: Heparin, Low-Molecular-Weight; Humans; Hypoxia; Inflammation; Neoplasms; Prognosis; Pulmonary Embolism; Risk Factors; Thromboplastin; Thrombosis; Venous Thrombosis

The interaction between cell-surface tissue factor (TF) and the plasma coagulation factor VII (FVII) initiates the coagulation network that leads to the generation of thrombin and the formation of a fibrin clot. Thrombin also activates cellular protease activated receptors (PARs) through which it activates components of the inflammatory pathway. TF is expressed constitutively by cardiomyocytes and evidence from mice transgenic for a human TF mini-gene that express very low levels of human TF suggests that the TF-FVII interaction is critical for haemostasis within the heart. Pathological contact between TF and FVII may occur in the heart during ischaemia-reperfusion (I-R) injury and this may lead to activation of coagulation and thrombin generation. Evidence from animal models now suggests that thrombin is an important mediator of inflammation in I-R injury. The coagulation pathway therefore represents a novel therapeutic target for intervention in the prevention of I-R injury.

Topics: Animals; Factor VII; Humans; Inflammation; Mice; Models, Biological; Myocardium; Myocytes, Cardiac; Reperfusion Injury; Thrombin; Thromboplastin; Transgenes

Cellular signaling by proteases of the blood coagulation cascade through members of the protease-activated receptor (PAR) family can profoundly impact on the inflammatory balance in sepsis. The coagulation initiation reaction on tissue factor expressing cells signals through PAR1 and PAR2, leading to enhanced inflammation. The anticoagulant protein C pathway has potent anti-inflammatory effects, and activated protein C signals through PAR1 upon binding to the endothelial protein C receptor. Activation of the coagulation cascade and the downstream endothelial cell localized anticoagulant pathway thus have opposing effects on systemic inflammation. This dichotomy is of relevance for the interpretation of preclinical and clinical data that document nonuniform responses to anticoagulant strategies in sepsis therapy.

Topics: Anticoagulants; Blood Coagulation; Endopeptidases; Endothelium, Vascular; Humans; Inflammation; Protein C; Signal Transduction; Systemic Inflammatory Response Syndrome; Thromboplastin

To present and discuss the rationale and the results of clinical trials using supplementation with physiologic anticoagulants (Tissue Factor Pathway Inhibitor (TFPI), AntiThrombin (AT), and Protein C (PC) in patients with severe sepsis.. An early activation of the coagulation cascade occurs in severe sepsis. TFPI, AT, and PC are major inhibitors of the coagulation cascade, and additionally modulate inflammatory and vascular reactions. They are consumed or inhibited in the sepsis pathologic process. Therapeutic supplementation with these inhibitors could improve the sepsis-induced organ failures and mortality.. Randomized controlled studies were recently completed. No effect on the mortality rate could be documented after treatment with recombinant TFPI. AT concentrates neither improve mortality, but a biological interaction with heparin therapy could have biased the study results. Treatment with recombinant activated PC (alpha-drotrecogin) was associated with a significant reduction in the mortality rate of severely ill patients and received recently the approval from FDA and EC authorities in this indication. An increase in the rate of hemorrhagic adverse effects has been observed with these compounds, justifying a strict observance of contraindications and of patients selection. PROSPECTIVE: Additional studies are needed to give confirmation of the positive effects of activated PC supplementation in less severely ill patients, children and specific clinical situations. The effects of new anticoagulant compounds are currently evaluated in preclinical studies.

Topics: Anticoagulants; Antithrombins; Blood Coagulation Disorders; Drug Evaluation, Preclinical; Drug Interactions; Drug Monitoring; Heparin; Humans; Inflammation; Multiple Organ Failure; Patient Selection; Protein C; Randomized Controlled Trials as Topic; Recombinant Proteins; Sepsis; Severity of Illness Index; Thromboplastin; Treatment Outcome

Topics: Biomarkers; Dehydroepiandrosterone; Humans; Hydrocortisone; Inflammation; Respiration, Artificial; Sepsis; Severe Acute Respiratory Syndrome; Thromboplastin; United Kingdom

Tissue factor is a cellular receptor for plasma clotting factor VII. In health, tissue factor is constitutively expressed in many cells, such as fibroblasts and keratinocytes, but is absent in vascular cells that come in contact with blood. However, tissue factor is induced in vascular cells in certain pathophysiological conditions. Thus, vessel wall injury or a disease condition allows blood to come in contact with tissue factor, resulting in factor VII binding to tissue factor. Once native factor VII complexed with tissue factor is converted to the enzyme factor VIIa, the complex triggers the clotting cascade that ultimately leads to fibrin formation. In addition to its established role in coagulation, molecular links between factor VIIa/tissue factor and various biological processes, such as development, inflammation, and tumor metastasis, are also evident. Recent studies suggest that factor VIIa/tissue factor affects various cellular processes by inducing intracellular signaling. Emerging evidence suggest that factor VIIa/tissue factor participates in cell signaling by two distinct mechanisms: proteolysis-independent signaling via the cytoplasmic domain of tissue factor, and proteolysis-dependent signaling, which is independent of tissue factor's cytoplasmic tail. In proteolysis-independent signaling, filamin 1 is recruited to tissue factor upon its ligation, and this probably provides an essential intracellular link in transmitting signals from tissue factor. In proteolysis-dependent signaling, factor VIIa/tissue factor activates one or more protease-activated receptors, which couple to G proteins, to impact multiple signaling pathways. In this chapter, we review various nonhemostatic functions attributed to factor VIIa and tissue factor, describe signaling mechanisms initiated upon factor VIIa binding to tissue factor, and discuss how factor VIIa/tissue factor-induced signaling could contribute to various pathophysiological processes. The relationship between increased clotting and manifestation of various diseases is well recognized. Although aberrant activation of the coagulation pathway is primarily the consequence of a disease, activation of the coagulation pathway during the disease process, in turn, could contribute to pathogenesis of the disease. Further, recent transgenic studies in mouse suggest that the coagulation system also plays a role in embryogenesis and development. Then the question arises, how do proteins involved in the clotting

Topics: Animals; Blood Coagulation; Factor VIIa; Humans; Inflammation; Neoplasm Metastasis; Neovascularization, Pathologic; Signal Transduction; Thromboplastin

Thrombus formation on a disrupted atherosclerotic plaque is a threatening event that leads to vessel occlusion and acute ischemia. In this current perspective, we present evidence for apoptosis as a major determinant of the thrombogenicity of the plaque lipid core and a potential contributor to plaque erosion and associated thrombosis. Moreover, apoptosis may directly affect blood thrombogenicity through the release of apoptotic cells and microparticles into the bloodstream.

Topics: Angina, Unstable; Animals; Apoptosis; Arteriosclerosis; Blood Coagulation; Embolism; Humans; Inflammation; Myocardial Infarction; Phosphatidylserines; Stroke; Thromboplastin; Thrombosis

Review of primate studies of Escherichia coli sepsis and endotoxemia with a reexamination of the rationale for diagnosis and treatment of these multistage disorders.. Animal research and intensive care units in a university medical school.. Cyanocephalus baboons (E. coli) and normal human subjects (endotoxin).. Baboon studies: anti-tissue factor, protein C, endothelial protein C receptor, and anti-tumor necrosis factor antibodies, and active site inhibited factor recombinant VIIa and factor Xa.. This review concerns the primate microvascular endothelial response to inflammatory and hemostatic stress. Studies of the impact of inflammatory and hemostatic stress on this microvasculature have fallen into four categories. First, studies of pure hemostatic stress using factor Xa phospholipid vesicles showed that blockade of protein C as well as protein C plus tissue plasminogen activator produced a severe but transient consumptive and a lethal thrombotic coagulopathy, respectively. These studies showed that the protein C and fibrinolytic systems can work in tandem to regulate even a severe response if the endothelium is not rendered dysfunctional by metabolic or inflammatory factors. Second, studies of compensated (nonlethal) inflammatory stress using E. coli or endotoxin in baboon and human subjects showed that even under minimal stress in which there is no evidence of overt disseminated intravascular coagulation, injury of the endothelium and activation of neutrophils and hemostatic factors are closely associated. This showed that molecular markers of hemostatic activity could be used to detect microvascular endothelial stress (nonovert disseminated intravascular coagulation) in patients who are compensated but at risk. These studies also showed that the compensated response to inflammatory stress could exhibit two stages, each with its unique inflammatory and hemostatic response signature. The first is driven by vasoactive peptides, cytokines, and thrombin, followed 12 to 14 hrs later by a second stage driven by C-reactive protein/complement complexes, tissue factor, and plasminogen activator inhibitor 1 secondary to oxidative stress after reperfusion. Third, studies of uncompensated (lethal) inflammatory stress using E. coli showed that irreversible thrombosis of the microvasculature was not a link in the lethal chain of events even though inhibition of components of the protein C network (protein C and endothelial protein C receptor) converted compensated responses to sublethal E. coli into uncompensated lethal responses. Fourth, these studies also showed that there were variants of the lethal response ranging from capillary leak and shock to recurrent sustained inflammatory disorders. We believe that each of these variants arises from their sublethal counterparts, depending on underlying or modulating host factors operating at the time of challenge. Such underlying conditions range from preexisting microvascular ischemia, reperfusion, and oxidati

Topics: Animals; Cytokines; Disease Models, Animal; Endothelium, Vascular; Endotoxemia; Escherichia coli Infections; Factor VIIa; Factor Xa; Hemostasis; Homeostasis; Humans; Inflammation; Microcirculation; Papio; Protein C; Sepsis; Thrombin; Thromboplastin; Tumor Necrosis Factor-alpha

To review the preclinical and clinical evidence that provides the therapeutic rationale for recombinant human tissue factor pathway inhibitor (rTFPI) as a novel treatment for human sepsis.. A summary of published English-language literature regarding preclinical studies and limited information published about three phase II clinical studies for the evaluation of rTFPI safety in sepsis patients.. Tissue factor pathway inhibitor, the physiologic inhibitor of the tissue factor pathway, interrupts activation of coagulation at multiple steps, including tissue factor VIIa activity, Xa activity, prothrombinase complex, and thrombin generation. Recombinant human TFPI exhibits anticoagulant and anti-inflammatory activities in animal models and humans with sepsis. These activities appear to have an important therapeutic role in protecting the microvasculature from injury and preventing multiple organ failure in sepsis.. Tissue factor pathway inhibitor is a potent inhibitor of clotting in the microvasculature, which is thought to protect organs from injury. Recombinant TFPI improved survival of septic animals in multiple models. Recent phase II results suggest that rTFPI is well tolerated, and they show a trend toward reduction in 28-day all-cause mortality in rTFPI-treated patients; in addition, rTFPI demonstrated significant reduction in thrombin generation. These results suggest that a powered study is indicated to further evaluate rTFPI utility for the adjunctive management of severe sepsis.

Topics: Animals; Anticoagulants; Blood Coagulation Disorders; Disease Models, Animal; Drug Evaluation, Preclinical; Fibrinolytic Agents; Humans; Inflammation; Lipoproteins; Sepsis; Survival Analysis; Thromboplastin; Treatment Outcome

Because of its unique position at the convergence point of the intrinsic (contact) and extrinsic (tissue factor/factor VIIa) pathways in the coagulation system, coagulation factor Xa (FXa) has been a theoretically interesting therapeutic target for antithrombotic drugs for many years. More recently, the discovery of naturally occurring FXa inhibitors, such as tick anticoagulant peptide and antistasin, has helped substantiate FXa as a desirable target by demonstrating the efficacy and potential safety advantages of FXa inhibition over conventional antithrombotic therapy. These discoveries led to the design and development of many small-molecule inhibitors of FXa, which have provided potent and selective tools for evaluating the potential role of FXa in various diseases. In addition, these advances have been instrumental in defining the biology of FXa and have aided in the discovery of specific receptors and intracellular signaling pathways for FXa that may be important in the progression of, or the response to, various diseases.

Topics: Animals; Cell Adhesion; Clinical Trials as Topic; Coronary Restenosis; Cysteine Endopeptidases; Cytokines; Factor Xa; Factor Xa Inhibitors; Graft Occlusion, Vascular; Humans; Hyperplasia; Inflammation; MAP Kinase Signaling System; Neoplasm Proteins; Neoplasms; Platelet-Derived Growth Factor; Receptor, PAR-2; Receptors, Platelet-Derived Growth Factor; Receptors, Thrombin; Sepsis; Thromboplastin; Up-Regulation

Tissue factor (TF) is a 47-kDa transmembrane protein, involved in the onset of coagulation. However, it also influences pathophysiological processes such as inflammation and tumor angiogenesis. Although the molecular mechanisms responsible for these phenomena remain unclear, it is thought that they are brought about by the action of intracellular signaling, resulting in gene transcription and subsequent protein synthesis. In this review we focus on FVIIa/TF-induced intracellular signaling and its possible role in physiology.

Topics: Angiogenesis Inducing Agents; Blood Coagulation; Humans; Inflammation; Signal Transduction; Thromboplastin

Tissue factor, a 47 kDa membrane glycoprotein, lies at the basis of the extrinsic pathway of the coagulation cascade. Interaction of TF with factor VIIa results in the formation of fibrin from fibrinogen, thereby inducing the formation of a blood clot. In addition to this well-established role in blood coagulation, TF is associated with various other physiological processes such as sepsis, inflammation, angiogenesis, metastasis and atherosclerosis. The molecular basis of the latter events is slowly emerging. It has become clear that TF-FVIIa interaction elicits a variety of intracellular signalling events that may be implicated in these actions. These events include the sequential activation of Src-like kinases, MAP kinases, small GTPases and calcium signalling. How this progress in the understanding of TF associated signal transduction may generate answers as to the mechanism through which TF exerts it pleiotropic effects will be focus of this review.

Topics: Animals; Arteriosclerosis; Blood Coagulation; Calcium Signaling; Cytokines; Disseminated Intravascular Coagulation; Factor VIIa; Fibroblasts; Gene Expression Regulation; GTP Phosphohydrolases; Humans; Inflammation; Lipoproteins; Lipoproteins, LDL; MAP Kinase Signaling System; Mice; Mice, Knockout; Muscle, Smooth, Vascular; Neoplasm Metastasis; Neoplasms; Neovascularization, Physiologic; Papio; Phagocytes; Sepsis; Signal Transduction; src-Family Kinases; Thromboplastin

Accumulating evidence suggests that protease receptors and their cognate protease ligands play important roles in cell-signaling events that regulate cell adhesion and migration in inflammation as well as tumor invasion and metastasis. Tissue factor (TF), the cell surface receptor for the serine protease VIIa and the initiator of the coagulation pathways, supports metastatic implantation by activating extracellular, protease-dependent signaling pathways and by intracellular links to the actin cytoskeleton. The adhesion of TF-expressing tumor cells can be mediated by interactions of the receptor-protease complex with specific matrix-associated inhibitors, suggesting a novel bridging mechanism by which proteases participate in migratory functions of cells.

Topics: Humans; Inflammation; Neoplasm Metastasis; Serine Endopeptidases; Signal Transduction; Thromboplastin

Recent studies have focused on a myriad of mechanisms by which inflammation can potentiate blood clotting. Inflammatory mediators like endotoxin and tissue necrosis factor (TNF)-alpha can cause the expression of tissue factor on monocytes and, possibly, endothelium, thereby initiating the coagulation cascade. Activation of the complement system can lead to exposure of membrane surfaces capable of amplifying the initial tissue factor stimulus by facilitating the assembly of the factor VIIIa-factor IXa and the factor Xa-factor Va complexes. Inflammatory mediators, particularly interleukin-6, can also increase the levels of fibrinogen, an acute-phase reactant. In addition, the inflammatory mediators can elevate the levels of plasminogen activator inhibitor, thus suppressing the fibrinolytic system. These studies alone, however, do not prove that inflammation can trigger clinically relevant thrombus formation in vivo. For instance, TNF-alpha has been studied in cancer patients as a potential cure for cancer, and even though these patients are hypercoaguable, thrombosis was not commonly observed as a side effect of the near-lethal doses of TNF-alpha that were administered. Based on primate studies, inflammatory mediators like TNF-alpha can promote clot deposition effectively only if there is reduced flow and inhibition of the natural anticoagulant pathways. The requirement for multiple simultaneous injurious events probably explains why inflammation alone is not observed as a major cause of thrombosis.

Topics: Animals; Blood Coagulation; Blood Coagulation Factors; Complement Activation; Cytokines; Enzyme Activation; Humans; Inflammation; Inflammation Mediators; Membrane Lipids; Models, Biological; Oxidation-Reduction; Oxidative Stress; Phospholipids; Primates; Protein C; Thrombomodulin; Thrombophilia; Thromboplastin; Tumor Necrosis Factor-alpha

Clinical trials of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors or statin therapy have demonstrated that baseline or treated low-density lipoprotein (LDL) cholesterol levels are only weakly associated with net coronary angiographic change or cardiovascular events. The beneficial effects of statins on clinical events may involve nonlipid mechanisms that modify endothelial function, inflammatory responses, plaque stability, and thrombus formation. Experimental animal models suggest that statins may foster stability through a reduction in macrophages and cholesterol ester content and an increase in volume of collagen and smooth muscle cells. The thrombotic sequelae caused by plaque disruption is mitigated by statins through inhibition of platelet aggregation and maintenance of a favorable balance between prothrombotic and fibrinolytic mechanisms. These nonlipid properties of statins may help to explain the early and significant cardiovascular event reduction reported in several clinical trials of statin therapy.

Topics: Animals; Antithrombins; Cardiovascular Diseases; Cholesterol, LDL; Clinical Trials as Topic; Coronary Disease; Endothelium, Vascular; Fibrinogen; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypolipidemic Agents; Inflammation; Lovastatin; Muscle, Smooth, Vascular; Platelet Aggregation; Pravastatin; Simvastatin; Thromboplastin; Thrombosis

Under normal conditions, TFPI expression is restricted to megakaryocytes and the endothelium of the microvasculature. It is not synthesized by normal hepatocytes or by the endothelium of larger vessels. In contrast, endothelium and peripheral blood cells do not express tissue factor under normal conditions. Expression of tissue factor under normal physiologic conditions is widespread and is localized in areas which are physically separated from the circulating blood. During an inflammatory response, circulating monocytes have been shown to express tissue factor, whereas in general, expression of tissue factor has not been observed in the endothelium. Adherent monocytes/macrophages express both tissue factor and TFPI under pathologic conditions. Whether or not circulating peripheral blood monocytes under inflammatory conditions express TFPI is not known.

Topics: Animals; Blood Cells; Cells, Cultured; Cytokines; Endothelium, Vascular; Gene Expression Regulation; Genes; Humans; Inflammation; Lipoproteins; Liver; Macrophages; Megakaryocytes; Monocytes; Organ Specificity; Protein Conformation; Thromboplastin

Inflammation and the cytokines clearly affect the coagulation system. Less clear are the specific influences of the coagulation system on inflammation. In this chapter only some of the coagulation systems affected by the cytokines are discussed, and the influences on the fibrinolytic system, which is also downregulated by selected cytokines are not mentioned; see Schneiderman and Loskutoff (1991) for a brief review. The major focus is on possible models by which inflammation and coagulation are linked, and examples where in vitro studies have led to correct in vivo predictions and where the results remain ambiguous. Finally, this chapter is clearly a biased perspective with the primary emphasis on the components and pathways with which the author has personal experience.

Topics: Animals; Blood Coagulation Factors; Cell Adhesion Molecules; Cells, Cultured; Cytokines; Disseminated Intravascular Coagulation; Endothelium, Vascular; Humans; Inflammation; Models, Biological; Neoplasms; Papio; Protein C; Shock, Septic; Thrombomodulin; Thromboplastin; Thrombosis

Blood coagulation is initiated in response to vessel damage in order to preserve the integrity of the mammalian vascular system. The coagulation cascade can also be initiated by mediators of the inflammatory response, and fibrin deposition has been noted in a variety of pathological states. The cascade of coagulation zymogen activations which leads to clot formation is initiated by exposure of flowing blood to tissue factor (TF), the cellular receptor and cofactor for factor VII (FVII). FVII binds to the receptor in a 1:1 stoichiometric complex and is rapidly activated. FVIIa undergoes an active site transition upon binding TF in the presence of calcium which enhances the fundamental properties of the enzyme. This results in rapid autocatalytic activation of FVII to VIIa thereby amplifying the response by generating more TF-VIIa complexes. The TF-VIIa activates both FIX and FX. Further FXa generation by the IXa-VIIIa-Ca(2+)-phospholipid complex is required to sustain the coagulation mechanism, since the TF-VIIa complex is rapidly inactivated. Structure and function studies have identified a number of regions on both TF and FVII involved in this interaction. It is clear, however, that the molecular structures of TF, FVII and the TF-VII complex will have to be solved before we fully understand this complex interaction. The activity of the TF-VIIa complex is controlled by two inhibitors:tissue factor pathway inhibitor (TFPI) and antithrombin III (AT-III). TFPI circulates in plasma, is associated with vascular cell surface and is released from platelets following stimulation by thrombin. TFPI requires the formation of an active TF-VIIa complex and FXa generation before inhibition can occur. Similarly, AT-III which is unable to inhibit circulating FVIIa requires the formation of the TF-VIIa complex. TFPI prevents further participation of TF in the coagulation process by forming a stable quaternary complex, TF-VIIa-Xa-TFPI. In contrast, the AT-III-VIIa complex is thought to dissociate from TF allowing it to interact with additional FVII-VIIa. TFPI has been considered the primary regulator of TF-VIIa activity during haemostasis. Whether AT-III in the presence of glycosaminoglycans on cell surfaces expressing TF can function as an auxiliary second physiological regulator is not known.

Topics: Antithrombin III; Blood Coagulation; Blood Coagulation Factors; Endothelium, Vascular; Factor VII; Fibrin; Humans; Inflammation; Lipoproteins; Models, Molecular; Protein Binding; Protein Conformation; Thromboplastin

Topics: Animals; Bacteremia; Biomarkers; Disseminated Intravascular Coagulation; Dogs; Edema; Escherichia coli Infections; Inflammation; Papio; Protein C; Rats; Shock, Septic; Thromboplastin; Tumor Necrosis Factor-alpha

Tissue factor (TF) is an integral membrane glycoprotein which functions as an initiator of coagulation. Furthermore, it is probably the principal biological initiator of this essential hemostatic process. This article reviews the studies which form the basis for these assertions. The work on TF is traced from the 19th century discovery of the thromboplastic activity of tissues to the recent purification of the protein from bovine and human tissues and the isolation cDNA clones coding from human TF. The features of TF structure and function which tailor it to the role of initiator of the coagulation cascade are considered. For example, cell-surface TF and factor VII, the plasma serine proteases zymogen, form a proteolytic complex without prior proteolysis of either component. In addition, a kinetic model for the molecular mechanism of TF-initiated clotting is reviewed. The factors which control the expression of TF procoagulant activity by cultured cells are examined in light of the hypothesized role of TF in normal hemostasis. Also, the potential pathological consequences of aberrant TF expression, i.e., thrombosis and hemorrhage, are explored.

Topics: Animals; Base Sequence; Blood Coagulation; Cells, Cultured; DNA, Recombinant; Enzyme Activation; Genes; Inflammation; Kinetics; Molecular Sequence Data; Neoplasms; Species Specificity; Thromboplastin

Mononuclear phagocytes, a specialized cell lineage comprising bone-marrow precursors, blood monocytes and tissue macrophages, can interact with blood coagulation mechanisms with resulting thrombus formation or extravascular fibrin accumulation. Such procoagulant activity is usually activation dependent and requires interaction of the cells with immune or nonimmune stimuli. In the former case (e.g., alloantigens, soluble protein antigens) collaboration of mononuclear phagocytes with T lymphocytes is necessary and is mediated by cell-to-cell contact or lymphokines. Prototype of a direct acting stimulus is bacterial lipopolysaccharide. Mononuclear phagocyte procoagulant activity is expressed in the form of cell membrane-bound or released factors which display molecular heterogeneity. They include the initiator of the extrinsic clotting pathway, tissue factor, known clotting proteases such as factors V and VII, and novel proteolytic enzymes including prothrombinase and a factor X activator. Mononuclear phagocyte procoagulants are pathogenetically involved in generalized disorders with intravascular coagulation and thromboembolic phenomena. These disorders, exemplified by the Shwartzman reaction and possibly by paraneoplastic thromboembolism, are initiated by blood monocytes. Extravascular fibrin deposition can be initiated by tissue-infiltrating monocytes and macrophages in disease states such as acute renal allograft failure and solid tumours.

Topics: Animals; Blood Coagulation Factors; Cell Communication; Disseminated Intravascular Coagulation; Graft Rejection; Humans; Hypersensitivity, Delayed; Inflammation; Macrophages; Neoplasms; T-Lymphocytes; Thromboplastin

From the preceding exposition it is now clear that the regulation of monocyte/macrophage PCA is dependent upon a complex network of interacting pathways, some of which amplify the response of the monocyte/macrophage, while others inhibit. In all probability many more will emerge. The construct illustrated in Figure 3, therefore, is a simplified view of the two major stimulatory pathways: the T cell-dependent pathway, activated by immune recognition and mediated by lymphokine(s); and the T cell-independent pathway, activated by direct perturbation of monocytes by such stimuli as LPS. At least 2 or 3 different PCAs can be expressed by monocyte/macrophages from different species, depending upon the anatomic site of the origin of the cell and the types of stimuli imposed. Inhibition of PCA expression is accomplished by at least one set of regulatory lipoproteins, and other inhibitory loops may be found. The result of these multiple interactions is the deposition of fibrin on the cell surface or in the surrounding milieu. It is our belief that this close relationship between coagulation reactions and inflammatory reactions, resulting in fibrin deposition, represents a fundamental host defense designed to delimit the inflammatory response. Nevertheless, the precise role of monocyte procoagulants in vivo remains unclear. A number of potential mechanisms exist for activation of coagulation in both inflammatory and neoplastic disorders, and the finding of enhanced monocyte procoagulant activity by no means establishes its importance in physiologic or, pathosphysiologic responses in vivo. Further studies, possibly with agents capable of specific inhibition of monocyte procoagulants in vivo, will be necessary to define the precise importance of these procoagulants in clinical disorders.

Topics: Animals; Anti-Inflammatory Agents; Antigens; Blood Coagulation; Blood Coagulation Factors; Cell Line; Factor V; Factor VII; Factor X; Factor Xa; Fibrin; Fibrin Fibrinogen Degradation Products; Guinea Pigs; Humans; Hypersensitivity, Delayed; Immunologic Deficiency Syndromes; Infections; Inflammation; Lipopolysaccharides; Macrophages; Mice; Monocytes; Neoplasms; Neutrophils; Rabbits; Rats; T-Lymphocytes; Thromboembolism; Thromboplastin; Warfarin

Tissue factor is an ubiquitous phospholipid-protein complex, which triggers blood coagulation through the so-called extrinsic pathway. Reactions initiated by tissue factor bypass many of the early stages of coagulation (contact phase) and involve factors VII, X, V, II and fibrinogen but also factor IX (and VIII) as it was recently demonstrated. So, it appears that tissue factor has a key-role in the haemostasic process as it has been suggested by the mildness or the absence of haemorrhagic syndrome in contact factors deficiencies. Tissue factor activity has been found in many types of cells, especially in white bloods cells. Experimental studies have demonstrated the presence of tissue factor activity in polymorphonuclears, lymphocytes, monocytes (or macrophages). This activity is enhanced by gram-negative endotoxin stimulation, inflammation, cell mediated immunologic phenomena or malignancy. These data are in good agreement with a wild range of features observed in human pathology: fibrin deposits in inflammatory lesions, disseminated intravascular coagulation (DIC) during the course of gram-negative septicemias or acute promyelocytic leukemias, local thrombi at the early phase of graft rejection. The protective effect of a phospholipase C against DIC induced in rats by tissue factor infusion suggests in the future, a specific therapy would be possible in man that, in the frequent clinical conditions involving clotting activation through tissue factor pathway.

Topics: Animals; Blood Coagulation; Blood Coagulation Factors; Disseminated Intravascular Coagulation; Fibrin; Graft Rejection; Humans; Inflammation; Leukemia; Leukocytes; Rabbits; Sepsis; Thromboplastin

The macrophage is the characteristic cell type in chronic inflammatory reactions, in the rheumatoid synovium, as in other sites. When macrophages are activated, considerable synthesis of enzymes and other proteins occurs. Macrophages can be activated by (i) products of activated lymphocytes, (ii) immune complexes and (iii) the complement cleavage product C3b. Among the many consequences of macrophage activation are (i) secretion of hydrolytic enzymes, (ii) cleavage of C3 into C3a, which is cytolytic, and C3b, (iii) production of tissue thromboplastin, a powerful procoagulant, and (iv) formation of polyamine oxidase, which in the presence of appropriate substrates generates factors that lyse or limit the proliferation of tumour cells, lymphocytes and micro-organisms. The relevance of these observations to the pathogenesis of chronic inflammatory reactions is discussed.

Topics: Animals; Cells, Cultured; Chronic Disease; Colony-Stimulating Factors; Complement System Proteins; Erythrocyte Aggregation; Guinea Pigs; Humans; In Vitro Techniques; Inflammation; Interferons; Macrophages; Mice; Neoplasms; Plasminogen Activators; Prostaglandins; Pyrogens; T-Lymphocytes; Thromboplastin

Topics: Angioedema; Blood Coagulation; Complement C1 Inactivator Proteins; Factor XII; Fibrinolysin; Humans; Inflammation; Kallikreins; Kinins; Pain; Thromboplastin

Topics: Adrenal Cortex Hormones; Amines; Anaphylaxis; Animals; Anti-Inflammatory Agents; Brain; Carbohydrate Metabolism; Carbohydrates; Desensitization, Immunologic; Dextrans; Dogs; Energy Transfer; Enzymes; Glucose; Histamine H1 Antagonists; Histamine Release; Hot Temperature; Inflammation; Macromolecular Substances; Mast Cells; Molecular Biology; p-Methoxy-N-methylphenethylamine; Peptides; Pharmacology; Polymyxins; Rats; Reserpine; Species Specificity; Surface-Active Agents; Tachyphylaxis; Thromboplastin; Tissue Extracts

Thrombo-inflammation is central to COVID-19-associated coagulopathy. TF (tissue factor), a driver of disordered coagulation and inflammation in viral infections, may be a therapeutic target in COVID-19. The safety and efficacy of the novel TF inhibitor rNAPc2 (recombinant nematode anticoagulation protein c2) in COVID-19 are unknown.. ASPEN-COVID-19 was an international, randomized, open-label, active comparator clinical trial with blinded end point adjudication. Hospitalized patients with COVID-19 and elevated D-dimer levels were randomized 1:1:2 to lower or higher dose rNAPc2 on days 1, 3, and 5 followed by heparin on day 8 or to heparin per local standard of care. In comparisons of the pooled rNAPc2 versus heparin groups, the primary safety end point was major or nonmajor clinically relevant International Society of Thrombosis and Haemostasis bleeding through day 8. The primary efficacy end point was proportional change in D-dimer concentration from baseline to day 8, or discharge if before day 8. Patients were followed for 30 days.. rNAPc2 treatment in hospitalized patients with COVID-19 was well tolerated without excess bleeding or serious adverse events but did not significantly reduce D-dimer more than heparin at day 8.. URL: https://www.. gov; Unique identifier: NCT04655586.

Topics: Anticoagulants; Antifibrinolytic Agents; Blood Coagulation Disorders; COVID-19; Female; Fibrin Fibrinogen Degradation Products; Hemorrhage; Heparin; Humans; Inflammation; Male; Middle Aged; Thromboplastin; Venous Thromboembolism

Aging is associated with a vasoconstrictive, pro-coagulant, and pro-inflammatory profile of arteries and a decline in the bioavailability of the endothelium-derived molecule nitric oxide. Dietary nitrate elicits vasodilatory, anti-coagulant and anti-inflammatory effects in younger individuals, but little is known about whether these benefits are evident in older adults. We investigated the effects of 140 mL of nitrate-rich (HI-NI; containing 12.9 mmol nitrate) versus nitrate-depleted beetroot juice (LO-NI; containing ≤0.04 mmol nitrate) on blood pressure, blood coagulation, vascular inflammation markers, plasma nitrate and nitrite before, and 3 h and 6 h after ingestion in healthy older adults (five males, seven females, mean age: 64 years, age range: 57-71 years) in a randomized, placebo-controlled, crossover study. Plasma nitrate and nitrite increased 3 and 6 h after HI-NI ingestion (

Topics: Aged; Aging; Beta vulgaris; Biomarkers; Blood Platelets; Blood Pressure; Cardiovascular Diseases; CD11b Antigen; Cross-Over Studies; Diet; Double-Blind Method; Female; Fruit and Vegetable Juices; Granulocytes; Hemostasis; Humans; Inflammation; Male; Middle Aged; Monocytes; Nitrates; Nitrites; P-Selectin; Plant Roots; Prothrombin; Thromboplastin; Waist Circumference

Although enteral nutrition (EN) is provided to most mechanically ventilated patients, the effect of specific feeding strategies on circulating markers of coagulation and inflammation is unknown.. Markers of inflammation (tumor necrosis factor [TNF]-α, interleukin [IL]-1β, interferon [IFN]-γ, IL-6, IL-8, IL-10, IL-12) and coagulation (tissue factor [TF], plasminogen activator inhibitor-1) were measured at baseline (n = 185) and 6 days (n = 103) in mechanically ventilated intensive care unit patients enrolled in a randomized controlled study of trophic vs full-energy feeds to test the hypothesis that trophic enteral feeds would be associated with decreases in markers of inflammation and coagulation compared to full-energy feeds.. There were no differences in any of the biomarkers measured at day 6 between patients who were randomized to receive trophic feeds compared to full-energy feeds. However, TF levels decreased modestly in patients from baseline to day 6 in the trophic feeding group (343.3 vs 247.8 pg/mL, P = .061) but increased slightly in the full-calorie group (314.3 vs 331.8 pg/mL). Lower levels of TF at day 6 were associated with a lower mortality, and patients who died had increasing TF levels between days 0 and 6 (median increase of 39.7) compared to decreasing TF levels in patients who lived (median decrease of 95.0, P = .033).. EN strategy in critically ill patients with acute respiratory failure does not significantly modify inflammation and coagulation by day 6, but trophic feeds may have some modest effects in attenuating inflammation and coagulation.

Topics: Adult; Aged; Biomarkers; Blood Coagulation; Critical Illness; Energy Intake; Enteral Nutrition; Female; Humans; Inflammation; Inflammation Mediators; Intensive Care Units; Male; Middle Aged; Plasminogen Activator Inhibitor 1; Respiration; Respiration, Artificial; Respiratory Insufficiency; Thromboplastin

Thrombin has a role not only in the coagulation process but also in inflammatory responses. Oral direct thrombin inhibitors (DTIs) are currently being evaluated in patients with thromboembolic diseases.. To investigate whether an oral DTI affects markers for platelet and inflammatory activity after myocardial infarction (MI).. A total of 518 patients with MI were randomly assigned to ximelagatran treatment (four different dose groups) in combination with aspirin, or aspirin alone for 6 months. The levels of soluble (s) P-selectin, soluble tissue factor, C-reactive protein (CRP), interleukin (IL)-10 and IL-18 were analysed in serial blood samples.. sP-selectin concentration increased after 1 week and persisted at an elevated level for 6 months in all study groups (P < 0.001). In the two highest ximelagatran dose groups, there was a reduced increase in sP-selectin compared to treatment with lower doses of ximelagatran and aspirin alone (P = 0.01 and P = 0.002, respectively). IL-18 levels did not change in the aspirin alone treatment group. By contrast, there was an elevation in IL-18 level in the lower and higher ximelagatran dose groups after 6 months (P = 0.006 and P < 0.001, respectively). Ximelagatran increased IL-10 levels (P = 0.002) and reduced the decrease in CRP levels after 6 months compared to treatment with aspirin alone (P = 0.002).. A persistent elevation of platelet activity is found in patients with a recent MI after the cessation of acute antithrombotic treatment, and the addition of an oral DTI at higher doses decreases the activity. By contrast, long-term treatment with a DTI increases the levels of several markers of inflammation. Further studies with prolonged exposure of oral DTIs are needed for evaluation of the effect on inflammatory processes and to determine whether these agents influence clinical outcomes.

Topics: Administration, Oral; Aged; Aged, 80 and over; Antithrombins; Aspirin; Azetidines; Benzylamines; Biomarkers; Blood Platelets; C-Reactive Protein; Comorbidity; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Therapy, Combination; Female; Humans; Inflammation; Interleukin-10; Interleukin-18; Male; Middle Aged; Myocardial Infarction; P-Selectin; Platelet Activation; Risk Factors; Thromboplastin; Time Factors; Treatment Outcome

Traumatic brain injury (TBI) initiates interrelated inflammatory and coagulation cascades characterized by wide-spread cellular activation, induction of leukocyte and endothelial cell adhesion molecules and release of soluble pro/antiinflammatory cytokines and thrombotic mediators. Resuscitative care is focused on optimizing cerebral perfusion and reducing secondary injury processes. Hypertonic saline is an effective osmotherapeutic agent for the treatment of intracranial hypertension and has immunomodulatory properties that may confer neuroprotection. This study examined the impact of hypertonic fluids on inflammatory/coagulation cascades in isolated head injury.. Using a prospective, randomized controlled trial we investigated the impact of prehospital resuscitation of severe TBI (GCS < 8) patients using 7.5% hypertonic saline in combination with 6% dextran-70 (HSD) vs 0.9% normal saline (NS), on selected cellular and soluble inflammatory/coagulation markers. Serial blood samples were drawn from 65 patients (30 HSD, 35 NS) at the time of hospital admission and at 12, 24, and 48-h post-resuscitation. Flow cytometry was used to analyze leukocyte cell-surface adhesion (CD62L, CD11b) and degranulation (CD63, CD66b) molecules. Circulating concentrations of soluble (s)L- and sE-selectins (sL-, sE-selectins), vascular and intercellular adhesion molecules (sVCAM-1, sICAM-1), pro/antiinflammatory cytokines [tumor necrosis factor (TNF)-alpha and interleukin (IL-10)], tissue factor (sTF), thrombomodulin (sTM) and D-dimers (D-D) were assessed by enzyme immunoassay. Twenty-five healthy subjects were studied as a control group.. TBI provoked marked alterations in a majority of the inflammatory/coagulation markers assessed in all patients. Relative to control, NS patients showed up to a 2-fold higher surface expression of CD62L, CD11b and CD66b on polymorphonuclear neutrophils (PMNs) and monocytes that persisted for 48-h. HSD blunted the expression of these cell-surface activation/adhesion molecules at all time-points to levels approaching control values. Admission concentrations of endothelial-derived sVCAM-1 and sE-selectin were generally reduced in HSD patients. Circulating sL-selectin levels were significantly elevated at 12 and 48, but not 24 h post-resuscitation with HSD. TNF-alpha and IL-10 levels were elevated above control throughout the study period in all patients, but were reduced in HSD patients. Plasma sTF and D-D levels were also significantly lower in HSD patients, whereas sTM levels remained at control levels.. These findings support an important modulatory role of HSD resuscitation in attenuating the upregulation of leukocyte/endothelial cell proinflammatory/prothrombotic mediators, which may help ameliorate secondary brain injury after TBI.. NCT00878631.

Topics: Adult; Analysis of Variance; Antigens, CD; Blood Coagulation; Brain Injuries; Cytokines; Dextrans; Enzyme-Linked Immunosorbent Assay; Female; Fibrin Fibrinogen Degradation Products; Glasgow Coma Scale; Humans; Inflammation; Leukocytes; Male; Middle Aged; Resuscitation; Saline Solution, Hypertonic; Statistics, Nonparametric; Thrombomodulin; Thromboplastin; Time Factors; Vascular Cell Adhesion Molecule-1

Selectins mediate the adhesion of leukocytes to activated endothelial cells and activated platelets. In addition to these cell-to-cell interactions, they influence the fibrin content and size of venous thrombi in different animal models. However, the exact role of selectins in human endotoxemia still remains unclear. We aimed to investigate the effect of selectin inhibition in lipopolysaccharide (LPS)-induced tissue factor (TF)-dependent activation of coagulation in a well-standardized model of human endotoxemia. To explore whether selectin blockade attenuates LPS-induced coagulation in humans, we performed a randomized, double-bind placebo-controlled crossover trial in 16 healthy male volunteers. All subjects received 2 ng/kg of LPS and, 10 min thereafter, a 15-min infusion of either 30 mg/kg of the pan-selectin antagonist bimosiamose or equal volumes of placebo in random order, with a washout period of 6 weeks between both periods. Treatment with bimosiamose had no significant effect on LPS-induced TF expression, as quantified by TF mRNA levels, or on LPS-induced coagulation response, reflected by increases in plasma thrombin-antithrombin (TAT) complexes and prothrombin fragment (F1 + 2) levels. Furthermore, bimosiamose did not affect the LPS-dependent changes in leukocyte subpopulations or the increase in platelet-leukocyte aggregates, as determined in the level of CD41+ monocytes. Finally, neither the LPS-induced release of tumor necrosis factor, interleukin 6, leukocyte expression of CD11b, nor intercellular adhesion molecule 1 were affected by administration of bimosiamose. The pan-selectin antagonist bimosiamose does not attenuate TF-triggered coagulation or inflammation in human endotoxemia. This indicates a minor influence of this selectin antagonist in this model. In addition, infusion of bimosiamose was safe and well tolerated in human endotoxemia.

Topics: Adolescent; Adult; Blood Coagulation; Blood Platelets; Cell Adhesion Molecules; Cross-Over Studies; Double-Blind Method; Endotoxemia; Flow Cytometry; Hemodynamics; Hexanes; Humans; Inflammation; Interleukin-6; Leukocytes; Lipopolysaccharides; Male; Mannose; Platelet Membrane Glycoprotein IIb; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Selectins; Thromboplastin; Time Factors; Tumor Necrosis Factor-alpha

Various lung diseases are associated with local activation of coagulation and concurrent inhibition of fibrinolysis. Although salmeterol, a beta2-adrenoceptor agonist with profound bronchodilatory properties, has been studied extensively, the effects of this compound on the pulmonary hemostatic balance are not elucidated.. A single-blinded, placebo-controlled study.. University hospital and laboratory.. A total of 32 human volunteers.. Subjects inhaled 100 microg of salmeterol or placebo (t = -30 mins) followed by 100 microg of lipopolysaccharide (LPS) or normal saline (t = 0 mins; n = 8 per group).. Measurements were performed in bronchoalveolar lavage fluid obtained 6 hrs postchallenge. Inhalation of LPS enhanced pulmonary coagulation as determined by an increase in the concentrations of thrombin-antithrombin complexes, factor VIIa, and soluble tissue factor in bronchoalveolar lavage fluid (all p < .05 vs. saline). LPS concurrently inhibited pulmonary fibrinolysis, as reflected by a decrease in bronchoalveolar lavage fluid plasminogen activator activity together with an increase in plasminogen activator inhibitor type 1 (both p < .05 vs. saline). Moreover, LPS inhalation was associated with a suppression of the anticoagulant protein C pathway, as indicated by an increase in soluble thrombomodulin and decreases in protein C and activated protein C levels in bronchoalveolar lavage fluid (all p < .05 vs. saline). Salmeterol, either with or without LPS inhalation, enhanced fibrinolysis (plasminogen activator activity and tissue-type and urokinase-type plasminogen activator levels) but did not influence LPS-induced changes in coagulation or the protein C pathway.. Salmeterol has profibrinolytic properties in the normal lung and when applied in a model of sterile pulmonary inflammation.

Topics: Administration, Inhalation; Adrenergic beta-Agonists; Adult; Albuterol; Analysis of Variance; Bronchoalveolar Lavage Fluid; Bronchodilator Agents; Factor VIIa; Fibrinolysis; Humans; Inflammation; Lipopolysaccharides; Lung; Lung Diseases; Macrophages, Alveolar; Male; Plasminogen Activator Inhibitor 1; Plasminogen Activators; Protein C; Salmeterol Xinafoate; Single-Blind Method; Thrombomodulin; Thromboplastin; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator

Beyond lipid lowering, various antiinflammatory properties have been ascribed to statins. Moreover, in vitro studies have suggested the presence of anticoagulant effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, as lipopolysaccharide (LPS)-induced monocyte tissue factor (TF) was suppressed. In this study, we examined the role of statins in experimental endotoxemia on inflammatory and procoagulant responses in vivo.. In this double-blind, placebo-controlled, parallel-group study, 20 healthy, male subjects were randomized to receive either simvastatin (80 mg/d) or placebo for 4 days before intravenous administration of LPS (20 IU/kg IV). Plasma high-sensitive C-reactive protein (hsCRP), monocyte chemoattractant protein (MCP-1), sCD40L, sCD40, and prothrombin fragment F1+2 (F1.2) were determined by ELISAs at baseline and at 4 and 8 hours after LPS administration. Monocyte TF expression and monocyte-platelet aggregates were measured by whole-blood flow cytometry over the same time course. The increases in hsCRP and MCP-1, both known inducers of TF, were significantly suppressed by statin treatment after LPS challenge. Statin premedication blunted the increase of monocyte TF expression in response to LPS. In parallel, endotoxin-induced formation of F1.2 was significantly reduced by simvastatin after 4 and 8 hours. LPS infusion affected neither the formation and activation of monocyte-platelet aggregates nor plasma levels of sCD40 and sCD40L.. Simvastatin suppresses the inflammatory response to endotoxin and blunts monocyte TF expression but does not affect platelet activation.

Topics: Adult; Biomarkers; Endotoxins; Gene Expression Regulation; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Inflammation; Male; Monocytes; Platelet Activation; Premedication; Simvastatin; Thrombophilia; Thromboplastin

Pneumonia is frequently associated with changes in coagulation and fibrinolysis in the bronchoalveolar space. To determine the effect of lipopolysaccharide (LPS) on the hemostatic balance in the human lung, six healthy subjects inhaled nebulized LPS or saline in a randomized cross-over study and bronchoalveolar lavage fluid was obtained six hours thereafter. LPS induced soluble tissue factor and thrombin-antithrombin complexes and inhibited plasminogen activator activity in BALF. Additionally plasminogen activator inhibitor type 1 production was upregulated after LPS inhalation. LPS also elicited local activation of neutrophils (release of elastase, myeloperoxidase and bactericidal/permeability increasing protein) and secretion of interleukin (IL)-6 and IL-8. Inhalation of LPS by healthy humans reproduces major features of the procoagulant response to inflammatory and infectious lung diseases and may be used as a novel model to evaluate pathogenetic mechanisms and new interventions.

Topics: Administration, Inhalation; Adult; Antithrombin III; Blood Coagulation; Bronchoalveolar Lavage Fluid; Cross-Over Studies; Cytokines; Fibrinolysis; Humans; Inflammation; Inflammation Mediators; Lipopolysaccharides; Lung; Male; Models, Biological; Neutrophils; Peptide Hydrolases; Plasminogen Activator Inhibitor 1; Thromboplastin

Elevated homocysteine levels are associated with an increased cardiovascular disease (CVD) risk, but the underlying mechanism is still unclear. High homocysteine might affect the endothelium, and consequently lead to impaired haemostasis. In a randomized placebo controlled trial among 276 older adults with plasma total homocysteine concentrations above 13 mM at screening, we investigated the effect of homocysteine lowering by folic acid supplementation (0.8 mg/day) for 1 year on markers of endothelial function (von Willebrand factor), coagulation (tissue factor, factor VIIa, fragments 1+2), and fibrinolysis (fibrin degradation products, tissue-type plasminogen activator), and inflammation (C-reactive protein). Despite a 24% reduction in plasma homocysteine concentration and four-fold increase in serum folate concentration in the folic acid group compared to the placebo group, there was no clear change in any of the haemostasis markers, nor CRP. Although homocysteine is associated with vascular disease risk in the general population, marked lowering of slightly elevated homocysteine concentrations by one-year folic acid supplementation does not influence haemostasis markers.

Topics: Aged; C-Reactive Protein; Cardiovascular Diseases; Dietary Supplements; Factor VIIa; Female; Fibrin; Folic Acid; Hemostasis; Homocysteine; Humans; Inflammation; Male; Middle Aged; Placebos; Risk; Thromboplastin; Time Factors; von Willebrand Factor

The protein C pathway serves as a modulating system with both anti-inflammatory and anticoagulant properties and is intimately involved in the pathophysiology of inflammation and sepsis. Treatment with recombinant human activated protein C (rhAPC) can reduce the mortality of severe sepsis. We investigated whether an elevation of plasma protein C levels to supra-normal levels by infusion of a protein C zymogen concentrate has an effect on coagulation, protein C activation or inflammation in a human endotoxemia model. Eleven healthy male volunteers were enrolled in a double-blind, placebo-controlled two-way cross-over trial. Ten minutes after infusion of 2ng/kg endotoxin each volunteer received either placebo or a plasma-derived protein C zymogen concentrate (Ceprotin, Baxter) (150 U/kg as a slow bolus infusion followed by 30 U/kg/h continuous infusion until 4 hours after LPS-infusion). Protein C antigen and activity increased 4- to 5-fold after infusion of the concentrate. APC was generated during endotoxin-induced inflammation in the placebo (1.6 fold increase) and the protein C period (4.0-fold increase). The increase of APC levels correlated with the TNF-alpha and IL-6 release in both periods (r = 0.65-0.68; p < 0.05) and paralleled the protein C antigen and activity levels in the period with supranormal protein C levels. Supra normal protein C levels resulted in slightly, although non-significant, lower tissue factor mRNA expression and thrombin generation (TAT, F1+2). Systemic inflammation (TNF-alpha, IL-6) was not influenced by protein C zymogen concentrate administration. Infusion of protein C zymogen was safe and no adverse effects occurred. The increase of protein C levels several fold above the normal range resulted in a proportional increase of the APC levels, but had no major anticoagulant, anti-inflammatory or profibrinolytic effects. Low grade endotoxemia itself induces significant protein C activation, which correlates with the TNF release.

Topics: Adult; Blood Coagulation; Cross-Over Studies; Double-Blind Method; Endotoxemia; Endotoxins; Fibrinolysin; Fibrinolysis; Humans; Inflammation; Infusions, Intravenous; Interleukin-6; Male; Monocytes; Platelet Activation; Protein C; RNA, Messenger; Thrombin; Thromboplastin; Tumor Necrosis Factor-alpha

Statins appear to have beneficial effects on fibrous cap stabilisation but their effects on plaque thrombogenicity have not been reported. To evaluate the thrombogenicity of human carotid plaques before and after atorvastatin treatment, 59 patients with bilateral carotid stenosis eligible for two-step carotid endoarterectomy (CEA) were randomly assigned to atorvastatin, 20 mg/day, or placebo. Histological and immunohistochemical analyses, Tissue Factor (TF), Tissue Factor Pathway Inhibitor (TFPI) antigens (Ag) and TF activity were determined in endoarterectomy specimens obtained at baseline and after treatment. Mean TFAg and TFPIAg levels from plaques removed at the first CEA were 55 +/- 56 and 32 +/- 26 pg/mg. After placebo, TFAg and TFPIAg content was higher in the second than the first CEA. Plaques removed at the second CEA from atorvastatin-treated patients had a lower macrophage content than plaques at the first CEA. TFAg and TFPIAg levels, and TF activity in plaques after atorvastatin treatment were lower (respectively 29, 18% and 56%) than after placebo. These findings indicate that atorvastatin reduce the inflammatory/thrombotic phenotype of carotid plaque, suggesting that these drugs may indeed have a beneficial effect on cerebrovascular events.

Topics: Aged; Anticholesteremic Agents; Atorvastatin; Carotid Artery Diseases; Double-Blind Method; Female; Heptanoic Acids; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Inflammation; Lipoproteins; Macrophages; Male; Middle Aged; Prospective Studies; Pyrroles; Thromboplastin; Thrombosis

Coumarin derivatives are still widely used for prophylaxis of thromboembolic events and therefore represent important comparator substances for new anticoagulants. Measurement of the efficacy of such novel compounds in a human coagulation model with adequate biomarkers could be useful for early-phase clinical drug development. To evaluate the applicability of a well-established model of tissue factor-dependent coagulation for defining anticoagulant potency, we investigated the effects of acenocoumarol in experimental human endotoxemia.. In a randomized, controlled, 2-by-2 factorial design, healthy volunteers received an infusion of 2 ng/kg endotoxin or placebo after 18 days of pretreatment with acenocoumarol or placebo. Prothrombin fragment 1+2 (F(1+2)), soluble fibrin, and D-dimer were used as markers of thrombin and fibrin formation.. As expected, pretreatment with acenocoumarol decreased vitamin K-dependent coagulation factors, but it also decreased spontaneous thrombin formation. Acenocoumarol inhibited endotoxin-induced thrombin generation as measured by F(1+2) levels: endotoxin infusion increased F(1+2) levels 8-fold-from 0.5 to 4.1 nmol/L-in the placebo group, whereas peak F(1+2) levels reached only 1.0 nmol/L in subjects after acenocoumarol pretreatment. This inhibition was also reflected in decreased formation of soluble fibrin and decreased D-dimer levels, showing that depletion of endogenous coagulation factors limits the propagation of nonovert disseminated intravascular coagulation.. Human endotoxemia is a suitable tool for measurement of the efficacy of oral anticoagulants and therefore may become a valuable addition for expeditious pharmacodynamic characterization of lead compounds with anticoagulant potency.

Topics: Acenocoumarol; Adult; Analysis of Variance; Anticoagulants; Biomarkers; Blood Coagulation Tests; Confidence Intervals; Dimerization; Double-Blind Method; Endotoxemia; Factor VIIa; Fibrin Fibrinogen Degradation Products; Humans; Inflammation; Infusions, Intravenous; Lipopolysaccharides; Male; Peptide Fragments; Pilot Projects; Platelet Count; Prothrombin; Solubility; Statistics, Nonparametric; Thromboplastin

Inflammatory mediators were shown to exert procoagulant effects on cultured human endothelial cells (EC). In the present study the effect of intradermal application of tumor necrosis factor-alpha (TNF-alpha) on the expression of factors involved in regulation of coagulation at the EC surface, i.e. tissue factor (TF), thrombomodulin (TM) and tissue factor pathway inhibitor (TFPI) was studied in humans in vivo. The endothelial expression of these factors was evaluated immunohistochemically in biopsies taken after intradermal application of 5000 U TNF-alpha in 8 healthy volunteers. After 6 and 22 h biopsies were taken from the injection sites. At TNF-alpha injected sites typical inflammatory changes. e.g. EC upregulation of adhesion molecules and accumulation of leukocytes were detected. In parallel we could document EC expression of TF, downregulation of TM and depletion of tissue factor pathway inhibitor (TFPI) in inflamed areas. Early depletion of endothelial IkappaB alpha at the site of inflammation after application of TNF-alpha points to an activation of the NF-kappaB pathway. Our data suggest that, as shown in in vitro experiments, TNF-alpha activates the NF-kappaB pathway and induces specific procoagulant changes of EC due to expression of TF, down-regulation of TM and depletion of TFPI in vivo in humans. This procoagulant shift in the haemostatic balance on the cell surface, caused by TNF-alpha-induced inflammation, is likely to contribute to thrombosis associated with tissue inflammation in humans.

Topics: Adolescent; Adult; Anticoagulants; Biopsy; Blood Coagulation Factors; Double-Blind Method; Endothelium, Vascular; Hemostasis; Humans; Immunohistochemistry; Inflammation; Injections, Intradermal; Lipoproteins; Male; NF-kappa B; Skin; Thrombomodulin; Thromboplastin; Tumor Necrosis Factor-alpha; von Willebrand Factor

The procoagulant protein tissue factor (TF) has been implicated in thromboembolic complications associated with advanced atherosclerosis. In this study, we investigated whether TF expression in high-grade stenoses of the internal carotid artery (ICA) is associated with clinical features of plaque destabilization and addressed the relationship between TF expression and plaque inflammation.. In 36 consecutive patients undergoing surgery for high-grade ICA stenosis, clinical evidence of plaque instability was provided by the recent occurrence of ischemic symptoms attributable to the stenosis and the detection of cerebral microembolism by means of transcranial Doppler ultrasound monitoring of the ipsilateral middle cerebral artery. Endarterectomy specimens were stained immunocytochemically for TF expression as well as macrophage (CD68) and T cell (CD3) infiltration.. Morphologically, TF immunoreactivity was codistributed with plaque inflammation and predominantly localized to CD68+ macrophages. Accordingly, statistical analysis revealed a significant association of TF expression with plaque infiltration by macrophages (P<0.0001) and T cells (P=0.013). Plaques extensively stained for TF (median of TF+ total section area >40% in semiquantitative assessment) were more frequent in symptomatic (12/27) than in asymptomatic patients (1/9). Conversely, plaques exhibiting little TF expression (median of TF+ section area <20%) were more frequent in asymptomatic (3/9) than in symptomatic (1/27) patients (P=0.016). Likewise, we found a highly significant association of TF expression with the occurrence of cerebral microembolism (P=0.008).. Induction of TF at sites of plaque inflammation may play an important role in the destabilization of high-grade ICA stenosis.

Topics: Carotid Artery, Internal; Carotid Stenosis; Disease Progression; Endarterectomy, Carotid; Humans; Immunohistochemistry; Inflammation; Intracranial Embolism; Ischemic Attack, Transient; Macrophages; Prospective Studies; Severity of Illness Index; T-Lymphocytes; Thromboplastin; Ultrasonography, Doppler, Transcranial

Excessive inflammation-associated coagulation is a feature of infectious diseases, occurring in such conditions as bacterial sepsis and COVID-19. It can lead to disseminated intravascular coagulation, one of the leading causes of mortality worldwide. Recently, type I interferon (IFN) signaling has been shown to be required for tissue factor (TF; gene name F3) release from macrophages, a critical initiator of coagulation, providing an important mechanistic link between innate immunity and coagulation. The mechanism of release involves type I IFN-induced caspase-11 which promotes macrophage pyroptosis. Here we find that F3 is a type I IFN-stimulated gene. Furthermore, F3 induction by lipopolysaccharide (LPS) is inhibited by the anti-inflammatory agents dimethyl fumarate (DMF) and 4-octyl itaconate (4-OI). Mechanistically, inhibition of F3 by DMF and 4-OI involves suppression of Ifnb1 expression. Additionally, they block type I IFN- and caspase-11-mediated macrophage pyroptosis, and subsequent TF release. Thereby, DMF and 4-OI inhibit TF-dependent thrombin generation. In vivo, DMF and 4-OI suppress TF-dependent thrombin generation, pulmonary thromboinflammation, and lethality induced by LPS, E. coli, and S. aureus, with 4-OI additionally attenuating inflammation-associated coagulation in a model of SARS-CoV-2 infection. Our results identify the clinically approved drug DMF and the pre-clinical tool compound 4-OI as anticoagulants that inhibit TF-mediated coagulopathy via inhibition of the macrophage type I IFN-TF axis.

Topics: Anticoagulants; Caspases; COVID-19; Dimethyl Fumarate; Escherichia coli; Humans; Inflammation; Interferon Type I; Lipopolysaccharides; Macrophages; SARS-CoV-2; Staphylococcus aureus; Thrombin; Thromboplastin; Thrombosis

There has been increasing interest in the study of new pathogenic mechanisms in endometriosis (END), including the coagulation/fibrinolysis system and its link with inflammation and tissue remodeling. It has been suggested that END patients, especially with deep-infiltrating (DE) forms, could present a hypercoagulable state revealing higher levels of proinflammatory and procoagulant markers, such as total circulating microparticles (cMPs) and cMP-TF (tissue factor), released by cells in response to damage, activation, or apoptosis. However, no previous study has assessed the effect of END hormonal treatments on cMP and cMP-TF levels. Therefore, the aim of this study was to evaluate the impact of these treatments on cMP and cMP-TF levels in DE patients. Three groups were compared: DE patients receiving a continuous combined oral contraceptive regimen (CCOCR) (n = 41), DE patients without CCOCR (n = 45), and a control group (n = 43). cMP and cMP-TF levels were evaluated in platelet-free plasma. A significant decrease in the total cMP levels was found in the DE group with CCOCR versus the group without CCOCR, reflecting a higher chronic inflammatory status in DE patients that decreased with the treatment. cMP-TF levels were higher in DE patients receiving CCOCR versus those not receiving CCOCR, suggesting that treatments containing estrogens play a predominant role in suppressing the inhibitory pathway of TF.

Topics: Blood Coagulation; Cell-Derived Microparticles; Endometriosis; Ethinyl Estradiol; Female; Humans; Inflammation; Norpregnenes; Thromboplastin

Venous thromboembolism (VTE) is a leading cause of preventable deaths in hospitals, and its incidence is not decreasing despite extensive efforts in clinical and laboratory research. Venous thrombi are primarily formed in the valve pockets of deep veins, where activated monocytes play a crucial role in bridging innate immune activation and hemostatic pathways through the production of inflammatory cytokines, chemokines, and tissue factor (TF) - a principal initiator of coagulation. In the valve pocket inflammation and hypoxia (sustained/intermittent) coexist, however their combined effects on immunothrombotic processes are poorly understood. Inflammation is strongly associated with VTE, while the additional contribution of hypoxia remains largely unexplored. To investigate this, we modelled the intricate conditions of the venous valve pocket using a state-of-the-art hypoxia chamber with software-controlled oxygen cycling. We comprehensively studied the effects of sustained and intermittent hypoxia alone, and in combination with VTE-associated inflammatory stimuli on primary monocytes. TF expression and activity was measured in monocytes subjected to sustained and intermittent hypoxia alone, or in combination with IL-1β. Monocyte responses were further analyzed in detailed by RNA sequencing and validated by ELISA. Stimulation with IL-1β alone promoted both transcription and activity of TF. Interestingly, the stimulatory effect of IL-1β on TF was attenuated by sustained hypoxia, but not by intermittent hypoxia. Our transcriptome analysis further confirmed that sustained hypoxia limited the pro-inflammatory response induced by IL-1β, and triggered a metabolic shift in monocytes. Intermittent hypoxia alone had a modest effect on monocyte transcript. However, in combination with IL-1β intermittent hypoxia significantly altered the expression of 2207 genes and enhanced the IL-1β-stimulatory effects on several chemokine and interleukin genes (e.g., IL-19, IL-24, IL-32, MIF), as well as genes involved in coagulation (thrombomodulin) and fibrinolysis (VEGFA, MMP9, MMP14 and PAI-1). Increased production of CCL2, IL-6 and TNF following stimulation with intermittent hypoxia and IL-1β was confirmed by ELISA. Our findings provide valuable insights into how the different hypoxic profiles shape the immunothrombotic response of monocytes and shed new light on the early events in the pathogenesis of venous thrombosis.

Topics: Cytokines; Humans; Hypoxia; Inflammation; Monocytes; Thromboplastin; Venous Thromboembolism

Accumulating evidence into the pathogenesis of COVID-19 highlights a hypercoagulability state with high risk of life-threatening thromboembolic complications. However, the mechanisms of hypercoagulability and their link to hyperinflammation remain poorly understood. Here, we investigate functions and mechanisms of platelet activation and platelet-monocyte interactions in inflammatory amplification during SARS-CoV-2 infection. We used a combination of immunophenotyping, single-cell analysis, functional assays, and pharmacological approaches to gain insights on mechanisms. Critically ill patients with COVID-19 exhibited increased platelet-monocyte aggregates formation. We identified a subset of inflammatory monocytes presenting high CD16 and low HLA-DR expression as the subset mainly interacting with platelets during severe COVID-19. Single-cell RNA-sequencing analysis indicated enhanced fibrinogen receptor Mac-1 in monocytes from patients with severe COVID-19. Monocytes from patients with severe COVID-19 displayed increased platelet binding and hyperresponsiveness to P-selectin and fibrinogen with respect to tumor necrosis factor-α and interleukin-1β secretion. Platelets were able to orchestrate monocyte responses driving tissue factor (TF) expression, inflammatory activation, and inflammatory cytokines secretion in SARS-CoV-2 infection. Platelet-monocyte interactions ex vivo and in SARS-CoV-2 infection model in vitro reciprocally activated monocytes and platelets, inducing the heightened secretion of a wide panel of inflammatory mediators. We identified platelet adhesion as a primary signaling mechanism inducing mediator secretion and TF expression, whereas TF signaling played major roles in amplifying inflammation by inducing proinflammatory cytokines, especially tumor necrosis factor-α and interleukin-1β. Our data identify platelet-induced TF expression and activity at the crossroad of coagulation and inflammation in severe COVID-19.

Topics: Blood Platelets; COVID-19; Cytokines; Humans; Inflammation; Interleukin-1beta; Monocytes; SARS-CoV-2; Thromboinflammation; Thrombophilia; Thromboplastin; Thrombosis; Tumor Necrosis Factor-alpha

Ruscogenin (RUS) has anti-inflammatory and antithrombotic effects, while its potential effects on deep venous thrombosis (DVT) and pulmonary embolism (PE) remain unclear.. We aimed to elucidate the effects of RUS on DVT and PE induced by the inferior vena cava stenosis (IVCS) model and investigate the underlying mechanism.. Male C57/BL6 mice were used to explore whether IVCS model could be complicated with deep venous thrombosis and pulmonary embolism. Then, effects of RUS on DVT and PE related inflammatory factors and coagulation were examined using H&E staining, ELISA, and real-time PCR. Western blot analysis was used to examine the effects of RUS on MEK/ERK/Egr-1/TF signaling pathway in PE.. IVCS model induced DVT and complied with PE 48 h after surgery. Administration of RUS (0.01, 0.1, 1 mg/kg) inhibited DVT, decreased biomarker D-Dimer, cardiac troponin I, N-Terminal probrain natriuretic peptide in plasma to ameliorate PE induced by IVCS model. Meanwhile, RUS reduced tissue factor and fibrinogen content of lung tissue, inhibited P-selectin and C-reactive protein activity in plasma, and suppressed the expressions of interleukin-6 and interleukin-1β in mice. Furthermore, RUS suppressed the phosphorylation of ERK1/2 and MEK1/2, decreasing the expressions of Egr-1 and TF in the lung.. IVCS model contributed to the development of DVT and PE in mice and was associated with increased inflammation. RUS showed therapeutic effects by inhibiting inflammation as well as suppressing the activation of MEK/ERK/Egr-1/TF signaling pathway.

Topics: Animals; Constriction, Pathologic; Inflammation; Male; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase Kinases; Pulmonary Embolism; Signal Transduction; Spirostans; Thromboplastin; Vena Cava, Inferior; Venous Thrombosis

Deep vein thrombosis (DVT) is the third most common cause of cardiovascular mortality. Several studies suggest that DVT occurs at the intersection of dysregulated inflammation and coagulation upon activation of inflammasome and secretion of interleukin 1β (IL-1β) in restricted venous flow conditions. Our recent studies showed a signaling adapter protein, Gab2 (Grb2-associated binder 2), plays a crucial role in propagating inflammatory signaling triggered by IL-1β and other inflammatory mediators in endothelial cells. The present study shows that Gab2 facilitates the assembly of the CBM (CARMA3 [CARD recruited membrane-associated guanylate kinase protein 3]-BCL-10 [B-cell lymphoma 10]-MALT1 [mucosa-associated lymphoid tissue lymphoma translocation protein 1]) signalosome, which mediates the activation of Rho and NF-κB in endothelial cells. Gene silencing of Gab2 or MALT1, the effector signaling molecule in the CBM signalosome, or pharmacological inhibition of MALT1 with a specific inhibitor, mepazine, significantly reduced IL-1β-induced Rho-dependent exocytosis of P-selectin and von Willebrand factor (VWF) and the subsequent adhesion of neutrophils to endothelial cells. MALT1 inhibition also reduced IL-1β-induced NF-κB-dependent expression of tissue factor and vascular cell adhesion molecule 1. Consistent with the in vitro data, Gab2 deficiency or pharmacological inhibition of MALT1 suppressed the accumulation of monocytes and neutrophils at the injury site and attenuated venous thrombosis induced by the inferior vena cava ligation-induced stenosis or stasis in mice. Overall, our data reveal a previously unrecognized role of the Gab2-MALT1 axis in thromboinflammation. Targeting the Gab2-MALT1 axis with MALT1 inhibitors may become an effective strategy to treat DVT by suppressing thromboinflammation without inducing bleeding complications.

Topics: Adaptor Proteins, Signal Transducing; Animals; B-Cell CLL-Lymphoma 10 Protein; CARD Signaling Adaptor Proteins; Endothelial Cells; Guanylate Kinases; Inflammasomes; Inflammation; Inflammation Mediators; Interleukin-1beta; Mice; Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein; NF-kappa B; P-Selectin; Thromboinflammation; Thromboplastin; Thrombosis; Vascular Cell Adhesion Molecule-1; Venous Thrombosis; von Willebrand Factor

Topics: Acyclic Monoterpenes; Animals; Anti-Inflammatory Agents; Anticoagulants; Antioxidants; Chlorides; Diclofenac; Hemolysis; Humans; Inflammation; Interleukin-10; Interleukin-8; Lipopolysaccharides; Malondialdehyde; MAP Kinase Kinase Kinase 5; Molecular Docking Simulation; Nitric Oxide; Rats; Superoxide Dismutase; Thromboplastin; Tumor Necrosis Factor-alpha

Ischemic acute kidney injury is common, deadly, and accelerates the progression of chronic kidney disease, yet has no specific therapy. After ischemia, reperfusion is patchy with early and persistent impairment in regional renal blood flow and cellular injury. We tested the hypothesis that intrarenal coagulation results in sustained renal ischemia following reperfusion, using a well-characterized model. Markedly decreased, but heterogeneous, microvascular plasma flow with microthrombi was found postischemia by intravital microscopy. Widespread tissue factor expression and fibrin deposition were also apparent. Clotting was accompanied by complement activation and inflammation. Treatment with exosomes derived from renal tubular cells or with the fibrinolytic urokinase, given 24 h postischemia when renal failure was established, significantly improved microvascular flow, coagulation, serum creatinine, and histological evidence of injury. These data support the hypothesis that intrarenal clotting occurs early and the resultant sustained ischemia is a critical determinant of renal failure following ischemia; they demonstrate that the coagulation abnormalities are amenable to therapy and that therapy results in improvement in both function and postischemic inflammation.

Topics: Acute Kidney Injury; Animals; Creatinine; Disease Models, Animal; Fibrin; Inflammation; Ischemia; Kidney; Reperfusion; Reperfusion Injury; Thromboplastin; Urokinase-Type Plasminogen Activator

Protease-activated receptor 2 (PAR2) signaling controls skin barrier function and inflammation, but the roles of immune cells and PAR2-activating proteases in cutaneous diseases are poorly understood.. To dissect PAR2 signaling contributions to skin inflammation with new genetic and pharmacological tools.. Myeloid cells and the TF-FXa-PAR2 axis are key mediators and potential therapeutic targets in inflammatory skin diseases.

Topics: Animals; Factor Xa; Inflammation; Mice; Peptide Hydrolases; Receptor, PAR-2; Thromboplastin

Glomerular inflammation is a putative aggravation factor for type 2 diabetic nephropathy and urinary thrombin is a novel marker of glomerular inflammation. To clarify the relationship between glomerular inflammation and progression of the nephropathy, we measured urinary thrombin in 118 patients with type 2 diabetic nephropathy at different stages. To investigate the implications of urinary thrombin in the nephropathy, we compared urinary thrombin with expression of tissue factor, the trigger of blood coagulation activation, in glomeruli and with markers of renal injury (estimated glomerular filtration rate (eGFR) and proteinuria). Urinary thrombin was found in 4.9% (3/61), 0.0% (0/12), 29.6% (8/27) and 50.0% (9/18) of patient groups at stages 1, 2, 3 and 4, respectively. Thus, urinary thrombin was negligible in the patients at early stages (stages 1 and 2), but was present predominantly in the patients at advanced stages (stages 3 and 4). Tissue factor was expressed in accumulated macrophages in glomeruli, which indicates that thrombin may be generated in inflamed glomeruli presumably via inflammation-induced activation of the exudated coagulation factors into glomerular tissues and then be excreted in urine. Urinary thrombin was significantly associated with both decreased eGFR and increased proteinuria in type 2 diabetic nephropathy. Therefore, increased urinary thrombin in patients with advanced stages of type 2 diabetic nephropathy suggests that glomerular inflammation may injure the tissues, thereby impairing renal function. Monitoring an effect of anti-diabetic treatments on glomerular inflammation in the patients with type 2 diabetic nephropathy may be a possible application of urinary thrombin.

Topics: Antithrombin III; Biomarkers; C-Reactive Protein; Diabetes Mellitus, Type 2; Female; Glomerular Filtration Rate; Humans; Inflammation; Kidney Glomerulus; Male; Middle Aged; Peptide Hydrolases; Proteinuria; Thrombin; Thromboplastin

Patients with diabetes have an increased risk of heart failure (HF). Diabetes is highly prevalent in HF with preserved ejection fraction (HFpEF), which is on the rise worldwide. The role of diabetes in HF is less established, and available treatments for HF are not effective in patients with HFpEF. Tissue factor (TF), a transmembrane receptor, plays an important role in immune cell inflammation and atherothrombosis in diabetes. However, its role in diabetes-induced cardiac inflammation, hypertrophy, and HF has not been studied. In this study, we used wild-type (WT), heterozygous, and low-TF (with 1% human TF) mice to determine the role of TF in type 1 diabetes-induced HF. We found significant upregulation of cardiac TF mRNA and protein levels in diabetic WT hearts compared with nondiabetic controls. WT diabetic hearts also exhibited increased inflammation and cardiac hypertrophy versus controls. However, these changes in cardiac inflammation and hypertrophy were not found in low-TF mice with diabetes compared with their nondiabetic controls. TF deficiency was also associated with improved cardiac function parameters suggestive of HFpEF, which was evident in WT mice with diabetes. The TF regulation of inflammation and cardiac remodeling was further dependent on downstream ERK1/2 and STAT3 pathways. In summary, our study demonstrated an important role of TF in regulating diabetes-induced inflammation, hypertrophy, and remodeling of the heart leading to HFpEF.

Topics: Animals; Cardiomegaly; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Heart Failure; Inflammation; Male; Mice; Myocardium; Thromboplastin

Inflammation induces a procoagulant phenotype of endothelial cells (EC) with the exposure of tissue factor (TF), a potent initiator of the extrinsic coagulation cascade. Although systemic inflammation affects the whole vascular system, thrombotic lesions occur particularly in microcirculation. This raises the question of whether TF-procoagulant activity (TF-PCA) differs between EC from arterial, venous, and microvascular beds.. Functional coagulation tests, including TF-PCA, and inflammatory responses were investigated on arterial, venous and microvascular endothelial cells. Interleukin-6 (IL-6) and TF-levels were determined in cohort of 59 septic patients.. We found that tumor necrosis factor alpha (TNFα), lipopolysaccharide, and interleukin-1β induce a solid, dose-dependent increase in TF-PCA, which is highest in microvascular EC. A positive correlation of interleukin-6 (IL-6) with TF levels was observed in a cohort of 59 septic patients. In contrast, TF-PCA was independent of IL-6 concentrations in vitro. Re-analysis of publicly available gene expression data revealed that among the top 50 genes annotated to coagulation, TF is one of three regulated genes common to the three investigated EC subtypes. The response to inflammatory stimuli in terms of exposure of leukocyte-endothelial- and platelet-endothelial adhesion molecules (E-selectin and PECAM-1), remodeling of adherens junctions, co-exposure of negatively charged surfaces nor breakdown of the glycocalyx was comparable between the EC subtypes and did not explain the higher TF-PCA on microvascular cells. We found that the ratio of TF and TFPI exposure on the endothelial membrane significantly differs between the EC subtypes.. These findings indicate that the ratio of TF to its inhibitor TFPI is a determinant of endothelial TF-PCA, which is most pronounced on microvascular endothelial cells and might explain why the microvascular system is particularly susceptible to inflammation-induced thrombosis.

Topics: Arteries; Blood Coagulation; Endothelial Cells; Humans; Inflammation; Thromboplastin

Whitmania pigra Whitman (W pigra), a traditional Chinese medicine, has functions of breaking stagnant and eliminating blood stasis. The aim of this study was to investigate the underlying mechanism of W pigra against deep vein thrombosis (DVT).. A rat model of DVT induced by inferior vena cava stenosis was successfully established. Rats were administered vehicle (saline solution, p.o.), three doses of W pigra aqueous extract (34.7, 104.2, or 312.5 mg crude W pigra/kg, p.o.), heparin (200 U/kg, i.v.), or clopidogrel (25 mg/kg, p.o.) once daily for 2 d. Thrombus weight and histopathological changes were examined. Blood samples were collected to determine blood cell counts, blood viscosity, blood coagulation, blood fibrinolysis, serum levels of interleukin-1β, and tumor necrosis factor-α. Protein expressions of Sirtuin1 (SIRT1), acetylated p65 (Ace-p65), and phosphorylated p65 (p-p65) were determined by Western blot. Furthermore, SIRT1-specific inhibitor EX527 was applied to confirm the role of SIRT1 in the antithrombotic effect of W pigra.. W pigra significantly decreased thrombus weight. W pigra had no effects on blood cell counts, whole blood viscosity, blood coagulation, blood fibrinolysis. However, it reduced tissue factor protein expression in the vein wall and thrombus. Moreover, it sharply increased SIRT1 protein expression and decreased leukocytes recruitment in the thrombus and vein wall, serum levels of interleukin-1β and tumor necrosis factor-α, and protein expressions of Ace-p65 and p-p65. Furthermore, the antithrombotic effect of W pigra was significantly abolished by EX527.. Aqueous extract of W pigra effectively reduced DVT burden by inhibiting inflammation via SIRT1/nuclear factor-kappa B signaling pathway.

Topics: Animals; Biological Products; Carbazoles; Cytokines; Drug Evaluation, Preclinical; Female; Inflammation; Leeches; Male; Medicine, Chinese Traditional; NF-kappa B; Rats, Sprague-Dawley; Signal Transduction; Sirtuin 1; Thromboplastin; Venous Thrombosis

Diabetes mellitus is characterized by chronic vascular inflammation leading to pathological expression of the thrombogenic full length (fl) tissue factor (TF) and its isoform alternatively-spliced (as) TF. Blood-borne TF promotes factor (F) Xa generation resulting in a pro-thrombotic state and cardiovascular complications. MicroRNA (miR)s impact gene expression on the post-transcriptional level and contribute to vascular homeostasis. Their distinct role in the control of the diabetes-related procoagulant state remains poorly understood.. In a cohort of patients with poorly controlled type 2 diabetes (n = 46) plasma levels of miR-181b were correlated with TF pathway activity and markers for vascular inflammation. In vitro, human microvascular endothelial cells (HMEC)-1 and human monocytes (THP-1) were transfected with miR-181b or anti-miR-181b and exposed to tumor necrosis factor (TNF) α or lipopolysaccharides (LPS). Expression of TF isoforms, vascular adhesion molecule (VCAM) 1 and nuclear factor (NF) κB nuclear translocation was assessed. Moreover, aortas, spleen, plasma, and bone marrow-derived macrophage (BMDM)s of mice carrying a deletion of the first miR-181b locus were analyzed with respect to TF expression and activity.. In patients with type 2 diabetes, plasma miR-181b negatively correlated with the procoagulant state as evidenced by TF protein, TF activity, D-dimer levels as well as markers for vascular inflammation. In HMEC-1, miR-181b abrogated TNFα-induced expression of flTF, asTF, and VCAM1. These results were validated using the anti-miR-181b. Mechanistically, we confirmed a miR-181b-mediated inhibition of importin-α3 (KPNA4) leading to reduced nuclear translocation of the TF transcription factor NFκB. In THP-1, miR-181b reduced both TF isoforms and FXa generation in response to LPS due to targeting phosphatase and tensin homolog (PTEN), a principal inducer for TF in monocytes. Moreover, in miR-181-/- animals, we found that reduced levels of miR-181b were accompanied by increased TF, VCAM1, and KPNA4 expression in aortic tissue as well as increased TF and PTEN expression in spleen. Finally, BMDMs of miR-181-/- mice showed increased TF expression and FXa generation upon stimulation with LPS.. miR-181b epigenetically controls the procoagulant state in diabetes. Reduced miR-181b levels contribute to increased thrombogenicity and may help to identify individuals at particular risk for thrombosis.

Topics: Aged; alpha Karyopherins; Animals; Blood Coagulation; Diabetes Mellitus, Type 2; Down-Regulation; Endothelial Cells; Female; Fibrin Fibrinogen Degradation Products; Humans; Inflammation; Male; Mice, Knockout; MicroRNAs; Middle Aged; NF-kappa B; PTEN Phosphohydrolase; Signal Transduction; THP-1 Cells; Thromboplastin; Thrombosis; Vascular Cell Adhesion Molecule-1

Sepsis is a systemic inflammatory disease causing life-threatening multi-organ dysfunction. Accumulating evidences suggest that two forms of programmed necrosis, necroptosis and pyroptosis triggered by the pathogen component lipopolysaccharide (LPS) and inflammatory cytokines, play important roles in the development of bacterial sepsis-induced shock and tissue injury. Sepsis-induced shock and tissue injury required receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL) phosphorylation, caspase11 activation and gasdermin D (GSDMD) cleavage. However, the synergistic effect of necroptosis and pyroptosis in the pathological progress of sepsis remains elusive. In this study, we found that blockage of both necroptosis and pyroptosis (double deletion of Ripk3/Gsdmd or Mlkl/Gsdmd) resulted in accumulative protection against septic shock, systemic blood clotting and multi-organ injury in mice. Bone marrow transplantation confirmed that necroptosis and pyroptosis in both myeloid and nonmyeloid cells are indispensable in the progression of sepsis-induced multi-organ injury. Both RIPK3 and GSDMD signaling collaborated to amplify necroinflammation and tissue factor release in macrophages and endothelial cells, which led to tissue injury. Furthermore, cell death induced by inflammatory cytokines and high-mobility group box 1 could be prevented by double ablation of Ripk3/Gsdmd or Mlkl/Gsdmd, suggesting that a positive feedback loop interconnecting RIPK3/MLKL and GSDMD machinery and inflammation facilitated sepsis progression. Collectively, our findings demonstrated that RIPK3-mediated necroptosis and GSDMD-mediated pyroptosis collaborated to amply inflammatory signaling and enhance tissue injury in the process of sepsis, which may shed new light on two potential targets of combined therapeutic interventions for this highly lethal disorder.

Topics: Acute Lung Injury; Animals; Blood Coagulation; Cecum; Cell Movement; Endothelial Cells; Gene Deletion; Inflammation; Interleukin-1beta; Intestines; Intracellular Signaling Peptides and Proteins; Ligation; Mice, Inbred C57BL; Mice, Knockout; Monocytes; Myeloid Cells; Necroptosis; Neutrophils; Organ Specificity; Phosphate-Binding Proteins; Punctures; Pyroptosis; Receptor-Interacting Protein Serine-Threonine Kinases; Sepsis; Signal Transduction; Thromboplastin

In haemophilia, thrombin generation and fibrin deposition upon vascular injury critically depend on the tissue factor (TF)-driven coagulation pathway. TF expression by monocytes/macrophages and circulating microvesicles contributes to haemostasis, thrombosis and inflammation. Inflammation is a hallmark of blood-induced joint disease. The aim of this study is to correlate TF production by whole-blood monocytes with inflammatory markers and clinical parameters in patients with moderate-to-severe haemophilia A or B (n = 43) in comparison to healthy males (n = 23). Monocyte TF antigen and microvesicle-associated TF procoagulant activity (MV TF PCA) were measured immediately after blood draw (baseline) and following incubation of whole blood with buffer or lipopolysaccharide (LPS) using two-colour flow cytometry and chromogenic FXa generation assay, respectively. Patients with HIV or uncontrolled HBV/HCV infections were excluded. TF was hardly detectable and not different in baseline and buffer-treaded samples from both groups. Stimulation with LPS, however, induced monocyte TF production, with increased TF-specific mean fluorescence intensity (P = 0.08) and MV TF PCA (P < 0.05) in patients compared to controls. Patients also had elevated hs-CRP and IL-6 serum levels (P < 0.001), which correlated with LPS-induced TF parameters. Further exploratory analyses revealed that the presence of systemic (low-grade) inflammation and boosted LPS-induced monocyte TF production were mainly restricted to patients with clinically controlled HBV and/or HCV infection (n = 16), who were older and also had a significantly worse orthopaedic joint score than patients with no history of viral hepatitis (P < 0.01). Our study delineates a previously unrecognised link between systemic inflammation and inducible monocyte TF production in patients with haemophilia A or B.

Topics: Adult; Case-Control Studies; Female; Hemophilia A; Humans; Inflammation; Lipopolysaccharides; Male; Middle Aged; Monocytes; Secretory Pathway; Severity of Illness Index; Thromboplastin; Young Adult

Endothelial senescence is an emerging cause of vascular dysfunction. Because microparticles are effectors of endothelial inflammation and vascular injury after ischaemia-reperfusion, we examined leucocyte-derived microparticles of spleen origin as possible contributors. Microparticles were generated from primary rat splenocytes by either lipopolysaccharide or phorbol-myristate-acetate/calcium ionophore, under conditions mimicking innate and adaptive immune responses. Incubation of primary porcine coronary endothelial cells with either type of microparticles, but not with those from unstimulated splenocytes, leads to a similar threefold raise in senescence-associated β-galactosidase activity within 48 hours, indicating accelerated senescence, to endothelial oxidative stress, and a fivefold and threefold increase in p21 and p16 senescence markers after 24 hours. After 12-hour incubation, the endothelial-dependent relaxation of coronary artery rings was reduced by 50%, at distinct optimal microparticle concentration. In vitro, microparticles were pro-thrombotic by up-regulating the local angiotensin system, by prompting tissue factor activity and a secondary generation of pro-coagulant endothelial microparticles. They initiated an early pro-inflammatory response by inducing phosphorylation of NF-κB, MAP kinases and Akt after 1 hour, and up-regulated VCAM-1 and ICAM-1 at 24 hours. Accordingly, VCAM-1 and COX-2 were also up-regulated in the coronary artery endothelium and eNOS down-regulated. Lipopolysaccharide specifically favoured the shedding of neutrophil- and monocyte-derived microparticles. A 80% immuno-depletion of neutrophil microparticles reduced endothelial senescence by 55%, indicating a key role. Altogether, data suggest that microparticles from activated splenocytes prompt early pro-inflammatory, pro-coagulant and pro-senescent responses in endothelial cells through redox-sensitive pathways. The control of neutrophil shedding could preserve the endothelium at site of ischaemia-reperfusion-driven inflammation and delay its dysfunction.

Topics: Angiotensins; Animals; Apoptosis; Blood Coagulation; Cell Lineage; Cell-Derived Microparticles; Cellular Senescence; Cyclooxygenase 2; Endothelial Cells; Endothelium, Vascular; Inflammation; Lipopolysaccharides; Male; MAP Kinase Signaling System; Monocytes; Neutrophils; NF-kappa B; Oxidative Stress; Phosphatidylinositol 3-Kinases; Phosphorylation; Rats, Wistar; Reperfusion Injury; Spleen; Swine; Tetradecanoylphorbol Acetate; Thromboplastin; Vascular Cell Adhesion Molecule-1

Sepsis is an infection-induced aggressive and life-threatening organ dysfunction with high morbidity and mortality worldwide. Infection-associated inflammation and coagulation promote the progression of adverse outcomes in sepsis. Here, we report that phospho-Tyr705 of STAT3 (pY-STAT3), not total STAT3, contributes to systemic inflammation and coagulopathy in sepsis.. Cecal ligation and puncture (CLP)-induced septic mice were treated with BP-1-102, Napabucasin, or vehicle control respectively and then assessed for systemic inflammation, coagulation response, lung function and survival. Human pulmonary microvascular endothelial cells (HPMECs) and Raw264.7 cells were exposed to lipopolysaccharide (LPS) with pharmacological or genetic inhibition of pY-STAT3. Cells were assessed for inflammatory and coagulant factor expression, cell function and signaling.. Pharmacological inhibition of pY-STAT3 expression by BP-1-102 reduced the proinflammatory factors, suppressed coagulation activation, attenuated lung injury, alleviated vascular leakage and improved the survival rate in septic mice. Pharmacological or genetic inhibition of pY-STAT3 diminished LPS-induced cytokine production in macrophages and protected pulmonary endothelial cells via the IL-6/JAK2/STAT3, NF-κB and MAPK signaling pathways. Moreover, the increase in procoagulant indicators induced by sepsis such as tissue factor (TF), the thrombin-antithrombin complex (TAT) and D-Dimer were down-regulated by pY-STAT3 inhibition.. Our results revealed a therapeutic role of pY-STAT3 in modulating the inflammatory response and defective coagulation during sepsis. Video Abstract.

Topics: Aminosalicylic Acids; Animals; Benzofurans; Blood Coagulation; Cecum; Cell Membrane Permeability; Endothelial Cells; Humans; Inflammation; Inflammation Mediators; Ligation; Lipopolysaccharides; Macrophages; Male; Mice; Mice, Inbred C57BL; Models, Biological; Molecular Targeted Therapy; Naphthoquinones; Phosphotyrosine; Punctures; RAW 264.7 Cells; Sepsis; STAT3 Transcription Factor; Sulfonamides; Suppression, Genetic; Survival Analysis; Thromboplastin; Toll-Like Receptor 4

Heme is a critical danger molecule liberated from hemeproteins in various conditions, including from hemoglobin in hemolytic diseases. Heme may cause thromboinflammatory damage by activating inflammatory and hemostatic pathways, such as complement, the TLRs, coagulation, and platelets. In this study, we explored the effect of single and dual inhibition of complement component C5 and TLR coreceptor CD14 on heme-induced thromboinflammation in an ex vivo human whole blood model. Heme induced a dose-dependent activation of complement via the alternative pathway. Single inhibition of C5 by eculizumab attenuated the release of IL-6, IL-8, TNF, MCP-1, MIP-1α, IFN-γ, LTB-4, MMP-8 and -9, and IL-1Ra with more than 60% (

Topics: Adult; Anemia, Sickle Cell; Animals; Blood Coagulation; Complement Activation; Complement C5; Cytokines; Granulocytes; Heme; Hemolysis; Humans; Inflammation; Lipopolysaccharide Receptors; Male; Monocytes; Swine; Thromboplastin

Topics: Animals; Cell Line; Chemical and Drug Induced Liver Injury; Cytokines; Dose-Response Relationship, Drug; Fibrin; Gene Expression Regulation; Humans; Inflammation; Macrolides; Male; Mice; Mice, Inbred C57BL; Signal Transduction; Specific Pathogen-Free Organisms; Stilbenes; Thromboplastin

Peripheral venous (PV) catheters are often used for serial blood sampling, but studies suggest that PV catheters increase markers of coagulation activation and inflammation. Whether the increase is caused by irritation of the vessel wall or diurnal variation is unknown. We therefore compared the effects of a PV catheter and repeated venepunctures on markers of coagulation, inflammation, and endothelial function.A PV catheter was inserted at 07:45 in a hand vein in 10 healthy subjects, and blood samples were collected at 8:00, 10:00, 12:00, and 14:00. In the contralateral arm, blood was simultaneously obtained by venepunctures. Measures of coagulation, i.e., activated partial thromboplastin time (APTT), prothrombin time (PT), fibrinogen, prothrombin fragment 1 + 2 (F1 + 2) and thrombin-antithrombin (TAT), inflammation, i.e., interleukin 6 (IL-6) and C-reactive protein (CRP), and endothelial function, i.e., plasminogen activator inhibitor 1 (PAI-1), tissue plasminogen activator (tPA), von Willebrand factor (vWF), and tissue factor (TF) were measured in plasma.The concentrations of TAT and F1 + 2 were significantly increased (10:00;

Topics: Antithrombin III; Biomarkers; Blood Coagulation; Blood Specimen Collection; C-Reactive Protein; Catheterization, Peripheral; Catheters; Endothelial Cells; Fibrinogen; Humans; Inflammation; Interleukin-6; Partial Thromboplastin Time; Peptide Hydrolases; Phlebotomy; Plasminogen Activator Inhibitor 1; Thromboplastin; Tissue Plasminogen Activator; von Willebrand Factor

Topics: Adult; Aged; Biomarkers; Biopsy; Cholangitis, Sclerosing; Colitis, Ulcerative; Female; Flow Cytometry; Humans; Immunohistochemistry; Inflammation; Intestinal Mucosa; Leukocytes, Mononuclear; Male; Middle Aged; Thromboplastin; Young Adult

Tissue factor (TF)-dependent coagulation contributes to lung inflammation and the pathogenesis of acute lung injury (ALI). In this study, we explored the roles of targeted endothelial anticoagulation in ALI using two strains of transgenic mice expressing either a membrane-tethered human tissue factor pathway inhibitor (hTFPI) or hirudin fusion protein on CD31

Topics: Acute Lung Injury; Animals; Blood Coagulation; Chemokines; Chemotaxis, Leukocyte; Endothelial Cells; Hirudins; Humans; Inflammation; Leeches; Lipopolysaccharides; Lipoproteins; Lung; Mice, Inbred C57BL; Mice, Transgenic; Platelet Endothelial Cell Adhesion Molecule-1; Pseudomonas aeruginosa; Receptors, Proteinase-Activated; Recombinant Fusion Proteins; Thrombin; Thromboplastin

Platelet activation contributes to organs failure in inflammation and plays an important role in endotoxemia. Clopidogrel inhibits platelet aggregation and activation. However, the role of clopidogrel in modulating inflammatory progression of endotoxemia remains largely unexplored. Therefore, we investigated the role of clopidogrel on the activation of platelet and leukocytes in lipopolysaccharide (LPS)-induced inflammation in mice. Animals were treated with clopidogrel or vehicle before LPS induction. The expression of neutrophil-platelet aggregates and platelet activation and tissue factor was determined. Immunofluorescence was used to analyze platelet-leukocyte interactions and tissue factor (TF) expression on leukocytes. Clopidogrel pretreatment markedly decreased lung damage, inhibited platelet-neutrophil aggregates and TF expression. In addition, clopidogrel reduced thrombocytopenia and affected the number of circulating white blood cell in endotoxemia mice. Moreover, clopidogrel also reduced platelet shedding of CD40L and CD62P in endotoxemic mice. Taken together, clopidogrel played an important role through reducing platelet activation and inflammatory process in endotoxemia.

Topics: Animals; Blood Platelets; CD40 Ligand; Clopidogrel; Endotoxemia; Inflammation; Interleukin-1beta; Lipopolysaccharides; Mice, Inbred BALB C; Models, Animal; Neutrophils; P-Selectin; Platelet Aggregation Inhibitors; Pneumonia; Purinergic P2Y Receptor Antagonists; Thromboplastin; Tumor Necrosis Factor-alpha

The release of neutrophil extracellular traps (NETs) represents a novel neutrophil effector function in systemic lupus erythematosus (SLE) pathogenesis. However, the molecular mechanism underlying NET release and how NETs mediate end-organ injury in SLE remain elusive.. NET formation and NET-related proteins were assessed in the peripheral blood and biopsies from discoid lupus and proliferative nephritis, using immunofluorescence, immunoblotting, quantitative PCR and ELISA. Autophagy was assessed by immunofluorescence and immunoblotting. The functional effects of NETs in vitro were assessed in a primary fibroblast culture.. Neutrophils from patients with active SLE exhibited increased basal autophagy levels leading to enhanced NET release, which was inhibited in vitro by hydroxychloroquine. NETosis in SLE neutrophils correlated with increased expression of the stress-response protein REDD1. Endothelin-1 (ET-1) and hypoxia-inducible factor-1α (HIF-1α) were key mediators of REDD1-driven NETs as demonstrated by their inhibition with bosentan and L-ascorbic acid, respectively. SLE NETs were decorated with tissue factor (TF) and interleukin-17A (IL-17A), which promoted thrombin generation and the fibrotic potential of cultured skin fibroblasts. Notably, TF-bearing and IL-17A-bearing NETs were abundant in discoid skin lesions and in the glomerular and tubulointerstitial compartment of proliferative nephritis biopsy specimens.. Our data suggest the involvement of REDD1/autophagy/NET axis in end-organ injury and fibrosis in SLE, a likely candidate for repositioning of existing drugs for SLE therapy. Autophagy-mediated release of TF-bearing and IL-17A-bearing NETs provides a link between thromboinflammation and fibrosis in SLE and may account for the salutary effects of hydroxychloroquine.

Topics: Autophagy; Cell Culture Techniques; Extracellular Traps; Fibroblasts; Fibrosis; Humans; Inflammation; Interleukin-17; Lupus Erythematosus, Systemic; Signal Transduction; Thromboplastin; Thrombosis; Transcription Factors

Monocytes are active at the crossroads between inflammation and coagulation processes since they can secrete pro-inflammatory cytokines and express tissue factor (TF), a major initiator of coagulation. Cobalt-chrome (CoCr), a metal alloy, used as a biomaterial for vascular stents, has been shown to be potentially pro-thrombotic and pro-inflammatory. Research work with a polymer from a family of degradable-polar hydrophobic ionic polyurethanes (D-PHI), called HHHI, has been shown to exhibit anti-inflammatory responses from human monocytes. We have generated multifunctional polyurethane thin films (MPTF) based on the HHHI chemistry, as a thin coating for CoCr and have evaluated the reactivity of blood with MPTF-coated CoCr. The results showed that the coating of CoCr with MPTF derived from HHHI prevents thrombin generation, reduces coagulation activation, and suppresses fibrin formation in whole blood. Activation of monocytes was also suppressed at the surface of MPTF-coated CoCr and specifically the decrease in thrombin generation was accompanied by a significant decrease in TF and pro-inflammatory cytokine levels. Mass spectroscopy of the adsorbed proteins showed lower levels of fibrinogen, fibronectin and complement C3, C4, and C8 when compared to CoCr. We can conclude that MPTFs reduce the pro-thrombotic and pro-inflammatory phenotype of monocytes and macrophages on CoCr, and prevent clotting in whole blood.

Topics: Cell Shape; Chromium Alloys; Coated Materials, Biocompatible; Fibrin; Humans; Hydrophobic and Hydrophilic Interactions; Inflammation; Inflammation Mediators; Ions; Macrophages; Monocytes; Polyurethanes; Principal Component Analysis; Surface Properties; Thrombin; Thromboplastin; Thrombosis; Tumor Necrosis Factor-alpha

Monocytes are activated in inflammatory conditions via a variety of cytokine receptors as well as in a procoagulatory setting through thrombin, acting upon protease-activated receptors (PARs). This study investigated the expression pattern of PAR1 and PAR3 on human monocyte subsets. Furthermore, a possible regulation of the expression of PAR1 and PAR3 in these cells by inflammatory activation were studied. CD16

Topics: Adaptor Proteins, Signal Transducing; Cell Cycle Proteins; Cells, Cultured; Endotoxemia; Healthy Volunteers; Humans; Inflammation; Lactones; Lipopolysaccharides; Monocytes; Plasminogen Activator Inhibitor 1; Pyridines; Receptor, PAR-1; Receptors, IgG; Thromboplastin; Transcriptome; Up-Regulation

Overpressure blast-wave induced brain injury (OBI) and its long-term neurological outcome pose significant concerns for military personnel. Our aim is to investigate the mechanism of injury due to OBI.. Rats were divided into 3 groups: (1) Control, (2) OBI (exposed 30psi peak pressure, 2-2.5ms), (3) Repeated OBI (r-OBI) (three exposures over one-week period). Lung and brain (cortex and cerebellum) tissues were collected at 24h post injury.. The neurological examination score was worse in OBI and r-OBI (4.2±0.6 and 3.7±0.5, respectively) versus controls (0.7±0.2). A significant positive correlation between lung and brain edema was found. Malondialdehyde (index for lipid peroxidation), significantly increased in OBI and r-OBI groups in cortex (p<0.05) and cerebellum (p<0.01-0.001). The glutathione (endogenous antioxidant) level decreased in cortex (p<0.01) and cerebellum (p<0.05) of r-OBI group when compared with the controls. Myeloperoxidase activity indicating neutrophil infiltration, was significantly (p<0.01-0.05) elevated in r-OBI. Additionally, tissue thromboplastin activity, a coagulation marker, was elevated, indicating a tendency to bleed. NGF and NF-κB proteins along with Iba-1 and GFAP immunoreactivity significantly augmented in the frontal cortex demonstrating microglial activation. Serum biomarkers of injury, NSE, TNF-alpha and leptin, were also elevated.. OBI triggers both inflammation and oxidative injury in the brain. This data in conjunction with our previous observations suggests that OBI triggers a cascade of events beginning with impaired cerebral vascular function leading to ischemia and chronic neurological consequences.

Topics: Animals; Blast Injuries; Blood-Brain Barrier; Brain Edema; Cerebellum; Disease Models, Animal; Frontal Lobe; Gliosis; Glutathione; Inflammation; Leptin; Lung; Male; Malondialdehyde; Microglia; Oxidative Stress; Peroxidase; Rats, Sprague-Dawley; Thromboplastin

Antiphospholipid syndrome (APS) is a systemic autoimmune disorder of young adults associated with devastating pregnancy complications (recurrent miscarriages, preeclampsia and low birth weight) and vascular complications including thrombosis. The key components implicated in pathogenesis of APS are the complement cascade and tissue factor (TF) activity causing inflammation and coagulation. Purinergic signalling involving catabolism of ATP to adenosine by cell-surface enzymes CD39 and CD73 has anti-inflammatory and anti-thrombotic effects. We studied whether activities of CD39 and CD73 are important in preventing the development of miscarriages in APS.. We studied frequency of miscarriages and decidual pathology following passive transfer of human aPL-ab to pregnant wildtype mice, and mice deficient in CD39 and CD73, and also transgenic mice exhibiting 2-3X higher CD39 activity.. aPL-ab infusion in pregnant CD39-or CD73-knockout mice triggers an increase in miscarriages, associated with increased TF expression and complement deposition as well as elevated oxidative stress and pro-inflammatory TNF-α and IL-10 expression within the placental decidua. In contrast, aPL-ab induced miscarriages are prevented in mice over-expressing CD39, with reduced decidual TF expression and C3d deposition, diminished lipid peroxidation (4-hydroxynonenal or 4-HNE positive lipid adducts), and reduced TNF-α expression.. We demonstrate a protective role for CD39 in APS and provide rationale for both the development of endothelial cell-targeted soluble CD39 as a novel therapeutic for APS and analysis of perturbations in the purinergic pathway to explain human disease.

Topics: 5'-Nucleotidase; Abortion, Spontaneous; Adult; Animals; Antibodies, Antiphospholipid; Antigens, CD; Antiphospholipid Syndrome; Apyrase; Complement C3d; Disease Models, Animal; Female; Humans; Immunization, Passive; Inflammation; Inflammation Mediators; Lipid Peroxidation; Mice; Mice, Knockout; Mice, Transgenic; Pregnancy; Pregnancy Complications; Thromboplastin; Tumor Necrosis Factor-alpha

Oxidative stress and inflammation of the vessel wall contribute to prothrombotic states. The antioxidative protein paraoxonase-2 (PON2) shows reduced expression in human atherosclerotic plaques and endothelial cells in particular. Supporting a direct role for PON2 in cardiovascular diseases,

Topics: Animals; Aryldialkylphosphatase; Blood Coagulation; Cytokines; Endothelial Cells; Humans; Inflammation; Inflammation Mediators; Mice; Mice, Knockout; Models, Biological; Oxidation-Reduction; Oxidative Stress; Thromboplastin

Thrombosis is frequently diagnosed as a first symptom in tumor patients and the clinical management of hypercoagulability in cancer patients remains challenging due to concomitant changes in risk factors for severe bleeding. It therefore remains a priority to better understand interactions of the hemostatic system with cancer biology. Specifically, further research is needed to elucidate the details and effects of new anticoagulants on extravascular coagulation and the interplay between cancer progression and chronic inflammation. In addition, it will be important to identify subgroups of cancer patients benefiting from specific modulations of the coagulation system without increasing the bleeding risk. Here, we review recent findings on tissue factor (TF) regulation, its procoagulant activity and TF signaling in the various cell types of the tumor microenvironment.

Topics: Blood Coagulation; Chronic Disease; Humans; Inflammation; Neoplasms; Signal Transduction; Thromboplastin; Tumor Microenvironment

The crosstalk between immune and coagulation systems plays pivotal roles in host defense, which may involve monocyte-derived dendritic cells (moDCs). Our objectives were to elucidate the role of moDCs in coagulation under inflammatory conditions and the involvement of the complement system. We assessed the effects of lipopolysaccharide (LPS)-stimulated moDCs on coagulation using whole blood thromboelastometry in the presence of complement inhibitors. The sum of clotting time and clot formation time (CT plus CFT) in whole blood thromboelastometry was significantly more reduced in the presence of moDCs than in the absence of monocytes or moDCs and in the presence of monocytes, indicating a more potent coagulability of moDCs. The mRNA expression of coagulation-related proteins in moDCs was analyzed by quantitative PCR, which showed an increase only in the mRNA levels of tissue factor (TF). TF protein expression was assessed by western blot analysis and an activity assay, revealing higher TF expression in moDCs than that in monocytes. The in vitro moDC-associated hypercoagulable state was suppressed by a TF-neutralizing antibody, whereas LPS enhanced the in vitro hypercoagulation further. C1 inhibitor suppressed the in vitro LPS-enhanced whole blood hypercoagulability in the presence of moDCs and the increased TF expression in moDCs. These results suggest a significant role of moDCs and the complement system through TF expression in a hypercoagulable state under inflammatory conditions and demonstrate the suppressive effects of C1 inhibitor on moDC-associated hypercoagulation.

Topics: Blood Coagulation; Complement C1 Inhibitor Protein; Complement System Proteins; Dendritic Cells; Humans; Inflammation; Lipopolysaccharides; Monocytes; RNA, Messenger; Thrombelastography; Thrombophilia; Thromboplastin

Whether porcine cytokines are induced after pig-to-primate xenotransplantation and activate human cells remains unknown. First, we investigated the regulation of porcine IL-6, IFN-γ, IL-1β, and TNF-α in xenotransplantation using an in vitro model in which porcine aortic endothelial cells (PAECs) and porcine peripheral blood mononuclear cells (PBMCs) were stimulated with human serum. Downstream cytokines/chemokines were monitored. Pro-inflammatory cytokines (IL-6, IFN-γ, and IL-1β) and chemokines (IL-8, MCP-1, and CXCL2) were upregulated in the both cell types. TNF-α was induced 10-fold in PAECs, but not in PBMCs. Then, we assessed the role of porcine IL-6, IFN-γ, IL-1β, and TNF-α in xenotransplantation using western blotting and real-time PCR. Human umbilical vein endothelial cells (HUVECs) were selected as the target cells. Signaling pathways and downstream genes, such as those related to adhesion, inflammation, and coagulation, and chemokines were investigated. Porcine IL-1β and TNF-α significantly activated NF-κB and P38, and STAT3 was activated by porcine IL-6 in HUVECs. The adhesion genes (E-selectin, VCAM-1, and ICAM-1), inflammatory cytokines (IL-6, IL-1β, and TNF-α), chemokines (MCP-1 and IL-8), and the pro-coagulation gene (tissue factor) were upregulated by porcine IL-1β and TNF-α. Porcine IL-6 increased the expression of ICAM-1, IL-6, MCP-1, and tissue factor, but decreased IL-8 expression slightly. Surprisingly, porcine IFN-γ could not activate STAT1 or regulate the expression of any of the above genes in HUVECs. In conclusion, these findings suggest that porcine IL-6, IL-1β, and TNF-α activate HUVECs and regulate downstream genes expression, which may promote inflammation and coagulation response after xenotransplantation.

Topics: Animals; Chemokines; Cytokines; Gene Expression; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Interleukin-6; Leukocytes, Mononuclear; Swine; Thromboplastin; Transplantation, Heterologous; Tumor Necrosis Factor-alpha

Microvesicles (MVs) represent a subgroup of extracellular vesicles (EVs) emerging from various cells by blebbing of their outer membrane. Therefore, they share features such as membrane composition and antigenicity with their parental cells. Released by many immune and tumor cells, MVs act as intercellular messengers, account for horizontal gene transfer and can activate the coagulation system. With the aim to investigate their relevance for tumor cell biology, we characterized MVs released by human tumor cell lines of various origins in the absence or presence of TNF-α. After stimulation, we used the combination of low and high-speed centrifugation to enrich MVs from cell culture supernatants. We analyzed the presentation of phosphatidylserine (PS) and tissue factor (TF) activity on the cell surface and investigated their potency to induce tumor cell migration. In all tumor cell lines, TNF-α stimulation enhanced the release of MVs. While the expression of PS was universally increased, an elevated activity of procoagulant TF could be detected on MVs from lung, pancreatic, and colon carcinoma, but not from breast and ovarian cancer cell lines. Functionally, TNF-α stimulation significantly increased the potency of MVs to induce tumor cell migration. In conclusion, inflammatory conditions promote the release of MVs with increased procoagulant activity from tumor cell lines in vitro. PS-containing and TF-expressing MVs may account for systemic activation of the coagulation system as seen in cancer patients and, since they induce tumor cell migration, they may serve as biomarkers for tumor progression.

Topics: Biological Assay; Caspase 3; Cell Count; Cell Line, Tumor; Cell Movement; Extracellular Vesicles; Humans; Inflammation; Oligopeptides; Phosphatidylserines; Receptor, PAR-2; Signal Transduction; Thromboplastin; Tumor Necrosis Factor-alpha

Intestinal mesenchymal cells play essential roles in epithelial homeostasis, matrix remodeling, immunity, and inflammation. But the extent of heterogeneity within the colonic mesenchyme in these processes remains unknown. Using unbiased single-cell profiling of over 16,500 colonic mesenchymal cells, we reveal four subsets of fibroblasts expressing divergent transcriptional regulators and functional pathways, in addition to pericytes and myofibroblasts. We identified a niche population located in proximity to epithelial crypts expressing SOX6, F3 (CD142), and WNT genes essential for colonic epithelial stem cell function. In colitis, we observed dysregulation of this niche and emergence of an activated mesenchymal population. This subset expressed TNF superfamily member 14 (TNFSF14), fibroblastic reticular cell-associated genes, IL-33, and Lysyl oxidases. Further, it induced factors that impaired epithelial proliferation and maturation and contributed to oxidative stress and disease severity in vivo. Our work defines how the colonic mesenchyme remodels to fuel inflammation and barrier dysfunction in IBD.

Topics: Animals; Cell Proliferation; Colitis; Colon; Epithelial Cells; Fibroblasts; Genetic Heterogeneity; Homeostasis; Humans; Inflammation; Inflammatory Bowel Diseases; Intestinal Mucosa; Intestines; Mesenchymal Stem Cells; Mesoderm; Mice; Mice, Inbred C57BL; Myofibroblasts; Pericytes; RAW 264.7 Cells; Single-Cell Analysis; SOXD Transcription Factors; Thromboplastin; Tumor Necrosis Factor Ligand Superfamily Member 14; Wnt Signaling Pathway

Essentials Heparanase forms a complex with tissue factor and enhances the generation of factor Xa. The present study was aimed to identify the procoagulant domain of heparanase. Procoagulant peptides significantly shortened bleeding time and enhanced wound healing. Tissue factor pathway inhibitor (TFPI)-2 derived peptides inhibited the procoagulant peptides.. Background Heparanase, which is known to be involved in angiogenesis and metastasis, was shown to form a complex with tissue factor (TF) and to enhance the generation of activated factor X (FXa). Our study demonstrated that peptides derived from TF pathway inhibitor (TFPI)-2 impeded the procoagulant effect of heparanase, and attenuated inflammation, tumor growth, and vascularization. Aims To identify the procoagulant domain in the heparanase molecule, and to evaluate its effects in a model of wound healing that involves inflammation and angiogenesis. Methods Twenty-four potential peptides derived from heparanase were generated, and their effect was studied in an assay of FXa generation. Peptides 14 and 16, which showed the best procoagulant effect, were studied in a bleeding mouse model and in a wound-healing mouse model. Results Peptides 14 and 16 increased FXa levels by two-fold to three-fold, and, at high levels, caused consumption coagulopathy. The TFPI-2-derived peptides explored in our previous study were found to inhibit the procoagulant effect induced by peptides 14 and 16. In the bleeding model, time to clot formation was shortened by 50% when peptide 14 or peptide 16 was topically applied or injected subcutaneously. In the wound-healing model, the wound became more vascular, and its size was reduced to one-fifth as compared with controls, upon 1 week of exposure to peptide 14 or peptide 16 applied topically or injected subcutaneously. Conclusions The putative heparanase procoagulant domain was identified. Peptides derived from this domain significantly shortened bleeding time and enhanced wound healing.

Topics: Animals; Blood Coagulation; Coagulants; Factor Xa; Fibrin Fibrinogen Degradation Products; Fibrinogen; Glucuronidase; Glycoproteins; Hematologic Agents; Hemorrhage; Humans; Inflammation; Male; Mice; Neoplasm Metastasis; Neovascularization, Pathologic; Partial Thromboplastin Time; Peptides; Protein Domains; Prothrombin Time; Thrombelastography; Thromboplastin; Thrombosis; Wound Healing

Leukocyte adhesion to P-selectin on activated platelets and endothelial cells induces shedding of the P-selectin ectodomain into the circulation. Plasma soluble P-selectin (sP-selectin) is elevated threefold to fourfold in patients with cardiovascular disease. Circulating sP-selectin is thought to trigger signaling in leukocytes that directly contributes to inflammation and thrombosis. However, sP-selectin likely circulates as a monomer, and in vitro studies suggest that sP-selectin must dimerize to induce signaling in leukocytes. To address this discrepancy, we expressed the entire ectodomain of mouse P-selectin as a monomer (sP-selectin) or as a disulfide-linked dimer fused to the Fc portion of mouse immunoglobulin G (sP-selectin-Fc). Dimeric sP-selectin-Fc, but not monomeric sP-selectin, triggered integrin-dependent adhesion of mouse leukocytes in vitro. Antibody-induced oligomerization of sP-selectin or sP-selectin-Fc was required to trigger formation of neutrophil extracellular traps. Injecting sP-selectin-Fc, but not sP-selectin, into mice augmented integrin-dependent adhesion of neutrophils in venules, generated tissue factor-bearing microparticles, shortened plasma-clotting times, and increased thrombus frequency in the inferior vena cava. Furthermore, transgenic mice that overexpressed monomeric sP-selectin did not exhibit increased inflammation or thrombosis. We conclude that elevated plasma sP-selectin is a consequence rather than a cause of cardiovascular disease.

Topics: Animals; Antibodies; CD18 Antigens; Cell Adhesion; CHO Cells; Cricetulus; Disulfides; Extracellular Traps; Gene Expression Regulation; Immunoglobulin Fc Fragments; Inflammation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neutrophils; P-Selectin; Protein Domains; Protein Multimerization; Recombinant Fusion Proteins; Signal Transduction; Thromboplastin; Thrombosis; Vena Cava, Inferior

The pathomechanisms of morbidity due to blood transfusions are not yet entirely understood. Elevated levels of red blood cell-derived microparticles (RMPs) are found in coagulation-related pathologies and also in stored blood. Previous research has shown that RMPs mediate transfusion-related complications by the intrinsic pathway. We hypothesized that RMPs might play a role in post-transfusion thrombotic complications by enhancing procoagulant activity also through the extrinsic pathway of coagulation.. In this laboratory study, blood from 18 healthy volunteers was stimulated with microparticles from expired stored red blood cells. Various clotting parameters were recorded. Flow cytometry, enzyme-linked immunosorbent assays, and real-time polymerase chain reaction were used to investigate possible mediating mechanisms.. The addition of RMPs shortened the clotting time from 194 to 161 seconds (p < 0.001). After incubation with RMPs, there was increased expression of tissue factor (TF) on monocytes and in plasma. TF messenger RNA expression increased in a time-dependent and concentration-dependent manner. There was a significant induction of interleukin-1β and interleukin-6. After stimulation with RMPs, there was a significant increase in the number of activated platelets, an increased percentage of PAC-1/CD62P (procaspase activating compound-1/platelet surface P-selectin) double-positive platelets, and an increased number of platelet-neutrophil duplets and platelet-monocyte duplets, indicating enhanced interaction of platelets with neutrophils and monocytes. Levels of CXCL-8 (C-X-C motif chemokine ligand 1) and interleukin-6 were significantly higher after treatment with RMPs.. Our results suggest that RMPs trigger coagulation through TF signaling, induce the secretion of proinflammatory cytokines, and induce cell-cell interaction between platelets and neutrophils. Thus, under certain conditions, RMPs could play a role in post-transfusion complications through these mechanisms.

Topics: Blood Coagulation; Blood Preservation; Cell Communication; Cell-Derived Microparticles; Erythrocytes; Humans; Inflammation; Interleukin-6; Interleukin-8; Monocytes; Platelet Activation; RNA, Messenger; Thromboplastin; Thrombosis; Transfusion Reaction

In HIV infection, persistent inflammation despite effective antiretroviral therapy is linked to increased risk of noninfectious chronic complications such as cardiovascular and thromboembolic disease. A better understanding of inflammatory and coagulation pathways in HIV infection is needed to optimize clinical care. Markers of monocyte activation and coagulation independently predict morbidity and mortality associated with non-AIDS events. We identified a specific subset of monocytes that express tissue factor (TF), persist after virological suppression, and trigger the coagulation cascade by activating factor X. This subset of monocytes expressing TF had a distinct gene signature with up-regulated innate immune markers and evidence of robust production of multiple proinflammatory cytokines, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6, ex vivo and in vitro upon lipopolysaccharide stimulation. We validated our findings in a nonhuman primate model, showing that TF-expressing inflammatory monocytes were associated with simian immunodeficiency virus (SIV)-related coagulopathy in the progressive [pigtail macaques (PTMs)] but not in the nonpathogenic (African green monkeys) SIV infection model. Last, Ixolaris, an anticoagulant that inhibits the TF pathway, was tested and potently blocked functional TF activity in vitro in HIV and SIV infection without affecting monocyte responses to Toll-like receptor stimulation. Strikingly, in vivo treatment of SIV-infected PTMs with Ixolaris was associated with significant decreases in D-dimer and immune activation. These data suggest that TF-expressing monocytes are at the epicenter of inflammation and coagulation in chronic HIV and SIV infection and may represent a potential therapeutic target.

Topics: Animals; Anti-Retroviral Agents; Antibodies, Viral; Blood Coagulation; Blood Coagulation Disorders; Chlorocebus aethiops; Chronic Disease; Cytokines; HIV Infections; Humans; Inflammation; Inflammation Mediators; Lipopolysaccharide Receptors; Lipopolysaccharides; Monocytes; Receptor, PAR-1; Signal Transduction; Simian Acquired Immunodeficiency Syndrome; Simian Immunodeficiency Virus; Thromboplastin

Neisseria meningitidis (N. meningitidis) may cause sepsis and meningitis. N. meningitidis with a mutated lpxL1 gene has five, instead of six, acyl chains in the lipid A moiety. Compared with patients infected with the wild type (wt) meningococcus, patients infected with the lpxL1 mutant have a mild meningococcal disease with less systemic inflammation and less coagulopathy. Circulating tissue factor (TF), the main initiator of coagulation, has a central role in the development of coagulation disturbances during sepsis. To study how TF was influenced by the lpxL1 mutant, human primary monocytes and whole blood were incubated with the lpxL1 mutant or the wt meningococcus (H44/76). Monocyte and microvesicle (MV)-associated TF expression and TF-dependent thrombin generation were measured. In both purified monocytes and whole blood, our data show that the lpxL1 mutant is a weaker inducer of monocyte and MV-associated TF compared with the wt. Our data indicate that low levels of circulating TF may contribute to the reduced coagulopathy reported in patients infected with lpxL1 mutants.

Topics: Acyltransferases; Bacterial Proteins; Blood Coagulation; Cell-Derived Microparticles; Cells, Cultured; Disease Progression; Humans; Inflammation; Meningitis; Monocytes; Mutation; Neisseria meningitidis; Primary Cell Culture; Sepsis; Thromboplastin

Hypercoagulability is associated with chronic kidney disease (CKD). Tissue factor/factor VIIa complex and factor Xa in the coagulation cascade are known to activate protease-activated receptor 2 (PAR2), and to cause inflammation and tissue injury. Although PAR2 is highly expressed in the kidney, it is unclear whether PAR2 plays a pathogenic role in CKD. To test this, we fed the mice lacking Par2 (F2rl1

Topics: Adenine; Animals; Enzyme-Linked Immunosorbent Assay; Factor V; Factor Xa; Fibrin; Fibrosis; Gene Expression Regulation; Inflammation; Kidney; Kidney Diseases; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Oxidative Stress; Receptor, PAR-2; Renal Insufficiency, Chronic; Thromboplastin

Islet cell transplantation has limited effectiveness because of an instant blood-mediated inflammatory reaction (IBMIR) that occurs immediately after cell infusion and leads to dramatic β-cell death. In intraportal islet transplantation models using mouse and human islets, we demonstrated that α-1 antitrypsin (AAT; Prolastin-C), a serine protease inhibitor used for the treatment of AAT deficiency, inhibits IBMIR and cytokine-induced inflammation in islets. In mice, more diabetic recipients reached normoglycemia after intraportal islet transplantation when they were treated with AAT compared with mice treated with saline. AAT suppressed blood-mediated coagulation pathways by diminishing tissue factor production, reducing plasma thrombin-antithrombin complex levels and fibrinogen deposition on islet grafts, which correlated with less graft damage and apoptosis. AAT-treated mice showed reduced serum tumor necrosis factor-α levels, decreased lymphocytic infiltration, and decreased nuclear factor (NF)-κB activation compared with controls. The potent anti-inflammatory effect of AAT is possibly mediated by suppression of c-Jun N-terminal kinase (JNK) phosphorylation. Blocking JNK activation failed to further reduce cytokine-induced apoptosis in β-cells. Taken together, AAT significantly improves islet graft survival after intraportal islet transplantation by mitigation of coagulation in IBMIR and suppression of cytokine-induced JNK and NF-κB activation. AAT-based therapy has the potential to improve graft survival in human islet transplantation and other cellular therapies on the horizon.

Topics: alpha 1-Antitrypsin; Animals; Antithrombin III; Apoptosis; Blood Glucose; Cell Death; Cytokines; Diabetes Mellitus, Experimental; Fibrinogen; Graft Survival; Humans; Inflammation; Insulin-Secreting Cells; Islets of Langerhans; Islets of Langerhans Transplantation; JNK Mitogen-Activated Protein Kinases; Mice; NF-kappa B; Peptide Hydrolases; Phosphorylation; Serine Proteinase Inhibitors; Thromboplastin; Transplantation, Heterologous; Transplantation, Homologous; Tumor Necrosis Factor-alpha

During chronic kidney disease progression, kidney-specific risk factors for cardiovascular disease come into play. The present study investigated the impact of indoxyl sulfate, dietary tryptophan-derived uremic toxin, accumulated in the blood of patients with chronic kidney disease on hemostatic parameters, markers of inflammation, oxidative stress and monocyte to macrophage transition.. Fifty-one CKD patients not undergoing hemodialysis were enrolled in the study. Coagulation factors, fibrinolytic parameters, adhesion molecules, endothelial dysfunction markers, oxidative stress as well as inflammation markers were examined using immune-enzymatic method. Indoxyl sulfate levels were assessed using high-performance liquid chromatography. Biochemical parameters were determined by routine laboratory techniques using an automated analyzers. All assessed parameters were compared with controls and subjected to cross-sectional statistical analysis.. Elevated concentrations of indoxyl sulfate, the vast majority of parameters affecting hemostasis, and markers of renal insufficiency conditions were observed. Part of hemostatic factors, namely tissue factor, von Willebrand factor, thrombomodulin, soluble urokinase-type plasminogen activator receptor, soluble intercellular adhesion molecule-1, soluble vascular cell adhesion protein were correlated with the fraction of indoxyl sulfate. A significant quantity of assessed parameters showed strong correlations with superoxide-dismutase, renal insufficiency rate, C-reactive protein, and neopterin. Levels of indoxyl sulfate were independently associated with markers of impaired endothelial function (thrombomodulin, adhesion molecules), oxidative stress (superoxide-dismutase) and monocytes activation determinant (neopterin), which indicate unconventional links between these systems and the role of indoxyl sulfate. Furthermore, parameters that correlated with the levels of indoxyl sulfate (von Willebrand factor, soluble urokinase-type plasminogen activator receptor, soluble intercellular adhesion molecule-1) were positively associated with the prevalence of cardiovascular disease in a CKD patients.. The study demonstrated that in conditions of chronic exposure to uremic toxins, indoxyl sulfate seems to be one of the "missing links" between impaired renal function and prevalence of cardiovascular events, especially hemostatic disorders. The main functions of the action appear to be altered monocytes activation, intensified inflammatory process, and augmented oxidative stress by this uremic toxin.

Topics: Adult; Aged; C-Reactive Protein; Cardiovascular Diseases; Case-Control Studies; Female; Hemostasis; Humans; Hydrogen Peroxide; Indican; Inflammation; Intercellular Adhesion Molecule-1; Lipoproteins; Male; Middle Aged; Neopterin; Oxidative Stress; Peptide Fragments; Plasminogen Activator Inhibitor 1; Prevalence; Prothrombin; Renal Insufficiency, Chronic; Thrombin; Thromboplastin; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator; Vascular Cell Adhesion Molecule-1; von Willebrand Factor

Pro-inflammatory cytokines play important pathological effects in various diseases and allotransplantation; however, their pathological role in xenotransplantation remains elusive. In pig-to-human cell or organ transplantation, whether porcine cells or organs are activated by human cytokines or not as an important question needs to be investigated.. We investigated the effect of human IL-6, IFN-γ, IL-17, IL-1β, and TNF-α in xenotransplantation using several in vitro models and porcine aortic endothelial cells (PAECs) as target cells. The downstream signaling pathways activated by these cytokines were studied with Western blotting, the regulation of the pro-inflammatory related genes and pro-coagulation factor were assessed using real-time PCR or enzyme-linked immunosorbent assay, and swine leukocyte antigen (SLA) class I and SLA class II DR were analyzed by flow cytometry.. We found that NF-κB and mitogen-activated protein kinases (MAPKs) were activated by recombinant human IL-17 (rhIL-17), rhIL-1β, and rhTNF-α, while rhIL-6 activated signal transducer and activator of transcription 3 (STAT3) in PAECs. The adhesion molecules (E-selectin, VCAM-1, and ICAM-1), pro-inflammatory gene (IL-6), chemokines (IL-8 and MCP-1), and the pro-coagulation factor (tissue factor) were induced by rhIL-17, rhIL-1β, and rhTNF-α, while rhIL-6 only increased the expression of MCP-1 and tissue factor. Using flow cytometry analysis, SLA class I was upregulated in PAECs after exposure to rhIL-1β and rhTNF-α, but not rhIL-6 or rhIL-17, whereas SLA class II DR could not be induced by rhIL-6, rhIL-17, rhIL-1β, or rhTNF-α, although it could by recombinant porcine IFN-γ (rpIFN-γ). Although activation of PAECs by rhIL-17 alone was not strong, rhIL-17 combined with rhTNF-α amplified the expression of E-selectin, IL-6, and IL-8. Unexpectedly, we found that tocilizumab, a humanized anti-human IL-6 receptor antibody, could not block rhIL-6-mediated STAT3 activation in PAECs. Human IFN-γ could not activate STAT1 or induce the downstream gene expression in PAECs, which was consistent with a previous report.. In conclusion, our data suggest that human IL-6, IL-17, IL-1β, and TNF-α significantly activate PAECs and are likely to promote inflammation and coagulation reaction in response to xenograft.

Topics: Animals; Aorta; Cytokines; Endothelial Cells; Gene Expression; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Humans; Inflammation; Interleukin-17; Interleukin-1beta; Interleukin-6; Swine; Thromboplastin; Transplantation, Heterologous; Tumor Necrosis Factor-alpha

Tissue factor (TF) expression in human cancers has been associated with a procoagulant state and facilitation of metastasis. This study was conducted in order to evaluate if TF was expressed in canine mammary tumours. Forty epithelial mammary tumours from 28 dogs were included. TF expression of the tumours was evaluated by immunohistochemistry using a polyclonal antibody against recombinant canine TF. In addition, thromboelastography, haemostatic and inflammatory parameters were evaluated in the patients. TF was recognized in 44% of benign and 58% of malignant tumours. TF localized to the cytoplasmic membrane of neoplastic luminal epithelial cells and/or diffusely in the cytoplasm. No association was found between TF expression and stage or grade of disease. A significant association between TF expression and antithrombin and plasminogen was found, and extensive TF expression was seen in a lymph node metastasis classified as anaplastic mammary carcinoma from a dog with concomitant disseminated intravascular coagulation (DIC).

Topics: Adenoma; Animals; Antithrombins; Biomarkers, Tumor; Blood Coagulation; Carcinoma; Dog Diseases; Dogs; Female; Gene Expression Regulation, Neoplastic; Inflammation; Mammary Neoplasms, Animal; Neoplasm Grading; Neoplasm Staging; Plasminogen; Thromboplastin

Thrombin (coagulation factor IIa) is a serine protease encoded by the F2 gene. Pro-thrombin (coagulation factor II) is cut to generate thrombin in the coagulation cascade that results in a reduction of blood loss. Procoagulant states that lead to activation of thrombin are common in bone fracture sites. However, its physiological roles and relationship with osteoblasts in bone fractures are largely unknown. We herein report various effects of thrombin on mouse osteoblastic MC3T3-E1 cells. MC3T3-E1 cells expressed proteinase-activated receptor 1 (PAR1), also known as the coagulation factor II receptor. They also produced monocyte chemoattractant protein (MCP-1), tissue factor (TF), MCSF and IL-6 upon thrombin stimulation through the PI3K-Akt and MEK-Erk1/2 pathways. Furthermore, MCP-1 obtained from thrombin-stimulated MC3T3-E1 cells induced migration by macrophage RAW264 cells. All these effects of thrombin on MC3T3-E1 cells were abolished by the selective non-peptide thrombin receptor inhibitor SCH79797. We also found that thrombin, PAR-1, MCP-1, TF as well as phosphorylated AKT and p42/44 were significantly expressed at the fracture site of mouse femoral bone. Collectively, thrombin/PAR-1 interaction regulated MCP-1, TF, MCSF and IL-6 production by MC3T3-E1 cells. Furthermore, MCP-1 induced RAW264 cell migration. Thrombin may thus be a novel cytokine that regulates several aspects of osteoblast function and fracture healing.

Topics: Animals; Cell Movement; Chemokine CCL2; Disease Models, Animal; Fracture Healing; Humans; Inflammation; Interleukin-6; Male; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Models, Biological; Osteoblasts; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RAW 264.7 Cells; Receptor, PAR-1; Thrombin; Thromboplastin

Inflammation and hyperglycaemia are associated with a prothrombotic state. Cell-derived microparticles (MPs) are the conveyors of active procoagulant tissue factor (TF) and circulate at high concentration in diabetic patients. Liraglutide, a glucagon-like peptide (GLP)-1 analogue, is known to promote insulin secretion and β-cell preservation. In this in vitro study, we examined the link between insulin impairment, procoagulant activity and plasma membrane remodelling, under inflammatory conditions. Rin-m5f β-cell function, TF activity mediated by MPs and their modulation by 1 μM liraglutide were examined in a cell cross-talk model. Methyl-β-cyclodextrine (MCD), a cholesterol depletor, was used to evaluate the involvement of raft on TF activity, MP shedding and insulin secretion as well as Soluble N-éthylmaleimide-sensitive-factor Attachment protein Receptor (SNARE)-dependent exocytosis. Cytokines induced a two-fold increase in TF activity at MP surface that was counteracted by liraglutide. Microparticles prompted TF activity on the target cells and a two-fold decrease in insulin secretion via protein kinase A (PKA) and p38 signalling, that was also abolished by liraglutide. Large lipid raft clusters were formed in response to cytokines and liraglutide or MCD-treated cells showed similar patterns. Cells pre-treated by saturating concentration of the GLP-1r antagonist exendin (9-39), showed a partial abolishment of the liraglutide-driven insulin secretion and liraglutide-decreased TF activity. Measurement of caspase 3 cleavage and MP shedding confirmed the contribution of GLP-1r-dependent and -independent pathways. Our results confirm an integrative β-cell response to GLP-1 that targets receptor-mediated signalling and membrane remodelling pointing at the coupling of insulin secretion and inflammation-driven procoagulant events.

Topics: Animals; Caspase 3; Cell Membrane; Cell-Derived Microparticles; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Exocytosis; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Hyperglycemia; Inflammation; Insulin; Insulin-Secreting Cells; Liraglutide; MAP Kinase Signaling System; Peptide Fragments; Rats; SNARE Proteins; Thromboplastin

Clinical and epidemiologic studies reveal an association between vitamin D deficiency and increased risk of cardiovascular disease. Because vascular smooth muscle cell (VSMC)-derived tissue factor (TF) is suggested to be critical for arterial thrombosis, we investigated whether the vitamin D molecules calcitriol and paricalcitol could reduce the expression of TF induced by the proinflammatory cytokine TNF-α in human aortic VSMCs. We found that, compared with controls, incubation with TNF-α increased TF expression and procoagulant activity in a NF-κB-dependent manner, as deduced from the increased nuclear translocation of nuclear factor κ-light-chain-enhancer of activated B cells protein 65 (p65-NF-κB) and direct interaction of NF-κB to the TF promoter. This was accompanied by the up-regulation of TF signaling mediator protease-activated receptor 2 (PAR-2) expression and by the down-regulation of vitamin D receptor expression in a miR-346-dependent way. However, addition of calcitriol or paricalcitol blunted the TNF-α-induced TF expression and activity (2.01 ± 0.24 and 1.32 ± 0.14 vs. 3.02 ± 0.39 pmol/mg protein, P < 0.05), which was associated with down-regulation of NF-κB signaling and PAR-2 expression, as well as with restored levels of vitamin D receptor and enhanced expression of TF pathway inhibitor. Our data suggest that inflammation promotes a prothrombotic state through the up-regulation of TF function in VSMCs and that the beneficial cardiovascular effects of vitamin D may be partially due to decreases in TF expression and its activity in VSMCs.

Topics: Calcitriol; Cells, Cultured; Down-Regulation; Ergocalciferols; Humans; Inflammation; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NF-kappa B; Receptor, PAR-2; Receptors, Calcitriol; Signal Transduction; Thromboplastin; Tumor Necrosis Factor-alpha; Up-Regulation; Vitamin D

Topics: Anemia, Sickle Cell; Animals; Cardiomegaly; Disease Models, Animal; Inflammation; Mice; Thrombin; Thromboplastin

Our previous data demonstrated that nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) are involved in the process of anti-β2GPI/β2GPI-induced tissue factor (TF) expression in monocytes. However, the role of NF-κB and AP-1 in pathogenic mechanisms of antiphospholipid syndrome (APS) in vivo has been rarely studied. This study aimed to investigate whether NF-κB and c-Jun/AP-1 are involved in anti-β2GPI-induced expression of prothrombotic and proinflammatory molecules in mouse. IgG-APS or anti-β2GPI antibodies were injected into BALB/c mice in the presence or absence of PDTC (a specific inhibitor of NF-κB) and Curcumin (a potent inhibitor of AP-1) treatment. Our data showed that both IgG-APS and anti-β2GPI could induce the activation of NF-κB and c-Jun/AP-1 in mouse peritoneal macrophages. The anti-β2GPI-induced TF activity in homogenates of carotid arteries and peritoneal macrophages from mice could significantly decrease after PDTC and/or Curcumin treatment, in which PDTC showed the strongest inhibitory effect, but combination of two inhibitors had no synergistic effect. Furthermore, anti-β2GPI-induced expression of TF, VCAM-1, ICAM-1 and E-selectin in the aorta and expression of TF, IL-1β, IL-6 and TNF-α in peritoneal macrophages of mice were also significantly attenuated by PDTC and/or Curcumin treatment. These results indicate that both NF-κB and c-Jun/AP-1 are involved in regulating anti-β2GPI-induced expression of prothrombotic and proinflammatory molecules in vivo. Inhibition of NF-κB and c-Jun/AP-1 pathways may be beneficial for the prevention and treatment of thrombosis and inflammation in patients with APS.

Topics: Animals; Antibodies; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Curcumin; E-Selectin; Immunoglobulin G; Inflammation; Intercellular Adhesion Molecule-1; Interleukin-1beta; Interleukin-6; Macrophages, Peritoneal; Male; Mice, Inbred BALB C; NF-kappa B; Phosphorylation; Proline; Real-Time Polymerase Chain Reaction; Thiocarbamates; Thromboplastin; Thrombosis; Transcription Factor AP-1; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Topics: Blood Coagulation; Endothelium; Endothelium, Vascular; Humans; Inflammation; Thromboplastin

AMP activated protein kinase (AMPK) is a pivotal metabolic regulatory enzyme and novel target of controlling inflammation. Our previous studies had demonstrated that 5-amino-4-imidazolecarboxamide riboside (AICAR), an AMPK activator, attenuated lipopolysaccharide (LPS)/D-galactosamine (D-gal)-induced fulminant hepatitis via suppressing inflammatory response. Since inflammation usually activates the coagulation response and aggravates inflammation-induced tissue injury, the present study was to explore the effects of AICAR on inflammation-induced activation of coagulation. Male BALB/c mice received LPS/D-gal intraperitoneal injection were used as fulminant hepatitis model. Western blot was used to detect tissue factor (TF) and hypoxia-inducible factor 1α (HIF-1α) protein expressions in hepatic tissue, as well as nuclear factor kappa B (NF-κB) p65 translocation into the nucleus. Real-time quantitative PCR was used to analyze erythropoietin (EPO) mRNA expression level. Lactic acid (LA) level in hepatic tissue was detected by kit. The results showed that LPS/D-gal induced the enhanced expression of TF, elevation of NF-κB p65 nuclear translocation, up-regulation of HIF-1α and EPO expressions, and increased LA level. These above alterations could be suppressed by AICAR. These results suggest that AICAR may down-regulate LPS/D-gal-induced TF expression (coagulation activity), and relieve hepatic hypoxia and metabolic disorder via suppressing the activity of NF-κB, which may be a novel mechanism of the beneficial effect of AICAR on LPS/D-gal-induced fulminant hepatitis.

Topics: Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; Down-Regulation; Erythropoietin; Hepatitis; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation; Lipopolysaccharides; Male; Mice; NF-kappa B; Thromboplastin; Up-Regulation

Essentials H1N1 Influenza A virus (IAV) infection is a hemostatic challenge for the lung. Tissue factor (TF) on lung epithelial cells maintains lung hemostasis after IAV infection. Reduced TF-dependent activation of coagulation leads to alveolar hemorrhage. Anticoagulation might increase the risk for hemorrhages into the lung during severe IAV infection.. Background Influenza A virus (IAV) infection is a common respiratory tract infection that causes considerable morbidity and mortality worldwide. Objective To investigate the effect of genetic deficiency of tissue factor (TF) in a mouse model of IAV infection. Methods Wild-type mice, low-TF (LTF) mice and mice with the TF gene deleted in different cell types were infected with a mouse-adapted A/Puerto Rico/8/34 H1N1 strain of IAV. TF expression was measured in the lungs, and bronchoalveolar lavage fluid (BALF) was collected to measure extracellular vesicle TF, activation of coagulation, alveolar hemorrhage, and inflammation. Results IAV infection of wild-type mice increased lung TF expression, activation of coagulation and inflammation in BALF, but also led to alveolar hemorrhage. LTF mice and mice with selective deficiency of TF in lung epithelial cells had low basal levels of TF and failed to increase TF expression after infection; these two strains of mice had more alveolar hemorrhage and death than controls. In contrast, deletion of TF in either myeloid cells or endothelial cells and hematopoietic cells did not increase alveolar hemorrhage or death after IAV infection. These results indicate that TF expression in the lung, particularly in epithelial cells, is required to maintain alveolar hemostasis after IAV infection. Conclusion Our study indicates that TF-dependent activation of coagulation is required to limit alveolar hemorrhage and death after IAV infection.

Topics: Animals; Anticoagulants; Blood Coagulation; Bronchoalveolar Lavage Fluid; Epithelial Cells; Gene Deletion; Hemorrhage; Hemostasis; Inflammation; Influenza A Virus, H1N1 Subtype; Integrases; Lung; Male; Mice; Mice, Inbred C57BL; Orthomyxoviridae Infections; Pulmonary Alveoli; Thromboplastin

Thymoquinone (THQ) is a major component of black seeds. Given that both THQ and black seeds exhibit anti-cancer and anti-inflammatory activities, we hypothesized that THQ will affect cancer-associated thrombosis (CAT), which is primarily triggered by tissue factor (TF) and inflammation. The effect of both black seed-extracted and purchased ("pure") THQ on normal blood coagulation was tested with in vitro thromboelastography (TEG) and activated partial thromboplastin time (aPTT) coagulation assays. The effect of pure THQ on CAT was tested with aPTT assay using pancreatic cancer cell lines that are either positive or negative for TF, and with TEG assay using lipopolysaccharide as an inflammatory trigger. Additionally, the direct effect of THQ on the inactivation of factors IIa and Xa was assessed. Since TNF-α facilitates crosstalk between inflammation and thrombosis by triggering the NF-κB pathway, we tested THQ's ability to interfere with this communication with a luciferase assay. Both extracted and pure THQ had minimal effects on normal blood coagulation. Pure THQ reversed CAT initiated by both TF and inflammation to basal levels (p < 0.001). Mechanistically, while THQ had minimal to no effect on factor IIa and Xa inactivation, it strongly reduced the effects of TNF-α on NF-κB elements (p < 0.001). THQ has a minimal effect on basal coagulation and can reverse CAT in vitro, possibly by interfering with the crosstalk between inflammation and coagulation. This study suggests the utility of THQ as a preventative anticoagulant and/or as a supplement to existing chemotherapies and anticoagulant therapies.

Topics: Anticoagulants; Benzoquinones; Blood Coagulation; Cell Line, Tumor; Factor Xa; Humans; Inflammation; Lipopolysaccharides; NF-kappa B; Oils, Volatile; Partial Thromboplastin Time; Seeds; Thrombelastography; Thromboplastin; Thrombosis; Tumor Necrosis Factor-alpha

Tissue factor (TF) is the primary trigger of coagulation. Elevated levels of TF are found in atherosclerotic plaques, and TF leads to thrombus formation when released upon plaque rupture. Interleukin (IL)-33 was previously shown to induce angiogenesis and inflammatory activation of endothelial cells (ECs). Here, we investigated the impact of IL-33 on TF in human ECs, as a possible new link between inflammation and coagulation. IL-33 induced TF mRNA and protein in human umbilical vein ECs and coronary artery ECs. IL-33-induced TF expression was ST2- and NF-κB-dependent, but IL-1-independent. IL-33 also increased cell surface TF activity in ECs and TF activity in ECs-derived microparticles. IL-33-treated ECs reduced coagulation time of whole blood and plasma but not of factor VII-deficient plasma. In human carotid atherosclerotic plaques (n = 57), TF mRNA positively correlated with IL-33 mRNA expression (r = 0.691, p < 0.001). In this tissue, IL-33 and TF protein was detected in ECs and smooth muscle cells by immunofluorescence. Furthermore, IL-33 and TF protein co-localized at the site of clot formation within microvessels in plaques of patients with symptomatic carotid stenosis. Through induction of TF in ECs, IL-33 could enhance their thrombotic capacity and thereby might impact on thrombus formation in the setting of atherosclerosis.

Topics: Blood Coagulation; Cells, Cultured; Endothelial Cells; Humans; Inflammation; Interleukin-33; RNA, Messenger; Thromboplastin

Leptospirosis is a worldwide spread zoonotic and neglected infectious disease of human and veterinary concern that is caused by pathogenic Leptospira species. In severe infections, hemostatic impairments such as coagulation/fibrinolysis dysfunction are frequently observed. These complications often occur when the host response is controlled and/or modulated by the bacterial pathogen. In the present investigation, we aimed to analyze the modulation of the hemostatic and inflammatory host responses by the bacterial pathogen Leptospira. The effects of leptospires and their secreted products on stimulation of human intrinsic and extrinsic pathways of coagulation were investigated by means of altered clotting times, assembly and activation of contact system and induction of tissue factor. We show that both extrinsic and intrinsic coagulation cascades are modulated in response to Leptospira or leptospiral secreted proteins. We further find that the pro-inflammatory mediator bradykinin is released following contact activation at the bacterial surface and that pro-coagulant microvesicles are shed from monocytes in response to infection. Also, we show that human leptospirosis patients present higher levels of circulating pro-coagulant microvesicles than healthy individuals. Here we show that both pathways of the coagulation system are modulated by leptospires, possibly leading to altered hemostatic and inflammatory responses during the disease. Our results contribute to the understanding of the leptospirosis pathophysiological mechanisms and may open new routes for the discovery of novel treatments for the severe manifestations of the disease.

Topics: Bradykinin; Hemostasis; Humans; Inflammation; Kininogens; Leptospirosis; Thromboplastin

Atherosclerosis is characterized by endothelial dysfunction, mainly induced by inflammation and oxidative stress. Increased reactive oxygen species (ROS) production together with increased adhesion molecules and thrombogenic tissue factor (TF) expression on endothelial cells has a key role in proatherogenic mechanisms. Therefore downmodulation of these molecules could be useful for reducing the severity of inflammation and atherosclerosis progression. Dehydrozingerone (DHZ) is a nutraceutical compound with anti-inflammatory and antioxidant activities. In this study we evaluated the ability of DHZ and its symmetric dimer to modulate hydrogen peroxide- (H

Topics: Atherosclerosis; Cell Adhesion; Cell Survival; Dietary Supplements; Dimerization; Fluorometry; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Intercellular Adhesion Molecule-1; Intracellular Space; NF-kappa B; Oxidative Stress; Reactive Oxygen Species; Solubility; Styrenes; Thromboplastin; Vascular Cell Adhesion Molecule-1

Inhibition of the CD40-CD154 pathway controls inflammatory disorders. Unfortunately, administration of anti-CD154 monoclonal antibodies causes thromboembolism. Blockade of signalling downstream of CD40 may represent an approach to treat CD40-driven inflammatory disorders. Blocking tumour necrosis factor receptor-associated factor 6 (TRAF6) signalling downstream of CD40 in MHC II(+) cells diminishes inflammation. However, CD40-TRAF6 blockade may cause immunosuppression. We examined the role of CD40-TRAF2,3 and CD40-TRAF6 signalling in the development of pro-inflammatory responses in human non-haematopoietic and monocytic cells. Human aortic endothelial cells, aortic smooth muscle cells, renal proximal tubule epithelial cells, renal mesangial cells and monocytic cells were transduced with retroviral vectors that encode wild-type CD40, CD40 with a mutation that prevents TRAF2,3 recruitment (ΔT2,3), TRAF6 recruitment (ΔT6) or both TRAF2,3 plus TRAF6 recruitment (ΔT2,3,6). Non-haematopoietic cells that expressed CD40 ΔT2,3 exhibited marked inhibition in CD154-induced up-regulation of vascular cell adhesion molecule 1, intercellular adhesion molecule 1 (ICAM-1), monocyte chemotactic protein 1 (MCP-1), tissue factor and matrix metalloproteinase 9. Similar results were obtained with cells that expressed CD40 ΔT6. Although both mutations impaired ICAM-1 up-regulation in monocytic cells, only expression of CD40 ΔT6 reduced MCP-1 and tissue factor up-regulation in these cells. Treatment of endothelial and smooth muscle cells with cell-permeable peptides that block CD40-TRAF2,3 or CD40-TRAF6 signalling impaired pro-inflammatory responses. In contrast, while the CD40-TRAF2,3 blocking peptide did not reduce CD154-induced dendritic cell maturation, the CD40-TRAF6 blocking peptide impaired this response. Hence, preventing CD40-TRAF2,3 or CD40-TRAF6 interaction inhibits pro-inflammatory responses in human non-haematopoietic cells. In contrast to inhibition of CD40-TRAF6 signalling, inhibition of CD40-TRAF2,3 signalling did not impair dendritic cell maturation. Blocking CD40-TRAF2,3 signalling may control CD40-CD154-dependent inflammatory disorders.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; CD40 Antigens; CD40 Ligand; Cell Line; Chemokine CCL2; Dendritic Cells; Endothelial Cells; Humans; Inflammation; Intercellular Adhesion Molecule-1; Mesangial Cells; Mice; Myocytes, Smooth Muscle; Peptides; Peroxidases; Signal Transduction; Thromboplastin; TNF Receptor-Associated Factor 2; TNF Receptor-Associated Factor 3; TNF Receptor-Associated Factor 6; Up-Regulation

Culture of human pancreatic islets is now routinely carried out prior to clinical islet allotransplantation, using conditions that have been developed empirically. One of the major causes of early islet destruction after transplantation is the process termed instant blood-mediated inflammatory reaction (IBMIR). The aim of this study was to develop in vitro methods to investigate IBMIR and apply them to the culture conditions used routinely in our human islet isolation laboratory. Freshly isolated or precultured (24 h, 48 h) human islets were incubated in either ABO-compatible allogeneic human blood or Hank's buffered salt solution (HBSS) for 1 h at 37°C. Tissue factor (TF) expression and leukocyte migration were assessed by light microscopy. TF was also quantified by ELISA. To assess β-cell function, glucose-stimulated insulin secretion (GSIS) assay was carried out. The extent of islet β-cell damage was quantified using a proinsulin assay. Islets cultured for 24 h had higher GSIS when compared to freshly isolated or 48-h precultured islets. Freshly isolated islets had significantly higher TF content than 24-h and 48-h precultured islets. Incubation of freshly isolated human islets in allogeneic human blood released 6.5-fold higher level of proinsulin in comparison to freshly isolated human islets in HBSS. The high level of proinsulin released was significantly attenuated when precultured islets (24 h or 48 h) were exposed to fresh blood. Histological examination of fresh islets in blood clot showed that some islets were fragmented, showing signs of extraislet insulin leakage and extensive neutrophil infiltration and necrosis. These features were markedly reduced when the islets were cultured for 24 h. These results suggest that our standard 24-h islet culture is markedly beneficial in attenuating IBMIR, as evidenced by increased GSIS, lower content of TF, decrease islet fragmentation, and proinsulin release.

Topics: Adult; Cell Movement; Cells, Cultured; Female; Humans; Inflammation; Insulin-Secreting Cells; Islets of Langerhans Transplantation; Leukocytes; Male; Middle Aged; Necrosis; Neutrophil Infiltration; Neutrophils; Organ Culture Techniques; Proinsulin; Thromboplastin

For successful xenotransplantation, in addition to α1,3-galactosyltransferase gene-knockout and human complement regulatory protein (CD46, CD55, CD59) gene insertion, cloned pigs expressing human thrombomodulin (hTM) have been produced to solve the problem of molecular incompatibility in their coagulation system. Recombinant soluble hTM (S-hTM) which has been recently approved for treatment of disseminated intravascular coagulation might be potentially available. The purpose of this study is to examine the functional difference in endothelial cells between membrane-bound hTM (MB-hTM) and S-hTM and to elucidate effective strategy using both types of hTM.. The following factors regarding coagulation and inflammation were compared between hTM-expressing pig aortic endothelial cells (PAEC) derived from cloned pig and nontransgenic PAEC in the presence of S-hTM under tumor necrosis factor-α-activated conditions; (i) clotting time (ii) pig tissue factor (TF), (iii) pig E-selectin, (iv) direct prothrombinase activity, (v) activated protein C (APC), and (vi) prothrombinase activity.. The MB-hTM significantly suppressed the expression of pig TF and E-selectin and direct prothrombinase activity in tumor necrosis factor-α-activated PAEC, suggesting strong anti-inflammatory effect, compared to S-hTM. In contrast, S-hTM had more potent capacity to inhibit thrombin generation and to produce APC than MB-hTM, although MB-hTM had the same level of capacity as human endothelial cells.. It was speculated that S-hTM treatment would be of assistance during high-risk periods for excessive thrombin formation (e.g., ischemia reperfusion injury or severe infection/rejection). Considering the properties of MB-hTM exhibiting anti-inflammatory function as well as APC production, hTM-expressing cloned pigs might be indispensible to long-term stabilization of graft endothelial cells.

Topics: Animals; Animals, Genetically Modified; Anticoagulants; Blood Coagulation; Blood Coagulation Tests; Cell Membrane; Cells, Cultured; Dose-Response Relationship, Drug; E-Selectin; Endothelial Cells; Humans; Inflammation; Inflammation Mediators; Protein C; Solubility; Swine; Thrombomodulin; Thromboplastin; Time Factors; Tumor Necrosis Factor-alpha

Diabetes mellitus (DM) patients have an increased incidence of cardiovascular events. Blood tissue factor-procoagulant activity (TF-PCA), the initiating mechanism for blood coagulation, is elevated in DM. We have shown that hyperglycaemia (HG), hyperinsulinaemia (HI) and combined HG+HI (induced using 24-hour infusion clamps) increases TF-PCA in healthy and type 2 DM (T2DM) subjects, but not in type 1 DM (T1DM) subjects. The mechanisms for this are unknown. DM patients have elevated plasma lipopolysaccharide (LPS), a toll-like receptor (TLR) 4 ligand. We postulated that TLR4 plays a role in modulating TF levels. We studied the effect of HG+HI on TLR4 and TF-PCA in vivo during 24-hour HG+HI infusion clamps in healthy subjects, and T1DM and T2DM subjects, and in vitro in blood. In vivo, in healthy subjects, 24-hour HG + HI infusion increased TLR4 six-fold, which correlated with TF-PCA (r= 0.91, p<0.0001). T2DM patients showed smaller increases in both. In T1DM subjects, TLR4 declined (50%, p<0.05) and correlated with TF-PCA (r=0.55; p<0.05). In vitro, HG (200 mg/dl added glucose) and HI (1-100 nM added insulin) increased TF-PCA in healthy subjects (~2-fold, 2-4 hours). Insulin inhibited by ~30% LPS-induced increase in TF-PCA and high glucose reversed it. TLR4 levels paralleled TF-PCA (r=0.71, p<0.0001); HG and HI increased TLR4 and insulin inhibited LPS-induced TLR4 increase. This is first evidence that even in healthy subjects, HG of short duration increases TLR4 and TF-PCA, key players in inflammation and thrombosis. TLR4-TF interplay is strikingly different in non-diabetic, T1DM and T2DM subjects.

Topics: Adult; Biomarkers; Blood Coagulation; Blood Coagulation Tests; Blood Glucose; Case-Control Studies; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Female; Glucose Clamp Technique; Humans; Hyperglycemia; Hyperinsulinism; Inflammation; Insulin; Lipopolysaccharides; Male; Middle Aged; Thromboplastin; Thrombosis; Time Factors; Toll-Like Receptor 4

Carrageenan-induced paw edema has been described as a local and acute inflammatory process. In fact, little is known about the time course and systemic changes following a carrageenan injection. In this study, we examine the systemic changes that follow carrageenan injection in the paw.. Acute inflammation was produced by subplantar injection of carrageenan in a hind paw of Sprague-Dawley rats. Saline was used in control rats. Paw volume was measured with a plethysmometer. The hot plate latency test was used to quantify antinociception. C-reactive protein (CRP) levels were measured with a sandwich enzyme immunoassay. Fibrinogen concentration was measured using the gravimetric method. Lung morphometric analysis was performed using an image processing package. Lungs and paws were also examined for tissue factor (TF) and proinflammatory cytokines expression by immunohistochemistry.. We found diverse systemic changes including increased levels of acute phase proteins, such as CRP and fibrinogen, and a lung inflammatory process characterized by lung edema, fibrin deposition, and leukocyte infiltration. An elevated expression of TF, IL-6, IL-1β, and TNFα, was observed in paw and lung tissue sections by immunohistochemical methods.. This study provides new evidence that a local carrageenan injection induces a systemic response.

Topics: Animals; C-Reactive Protein; Carrageenan; Cytokines; Edema; Fibrinogen; Foot; Inflammation; Lung; Pain Measurement; Rats; Rats, Sprague-Dawley; Thromboplastin

To explore the functions of inflammatory cytokine, tissue factor (TF) and cancer procoagulant (CP) in non-tumor deep venous thrombosis (NT-DVT).. A total of 17 NT-DVT patients (5 males and 12 females) were selected for NT-DVT group while 20 voluntary (10 males and 10 females) blood donors for control group from May 2012 to March 2013. The levels of inflammatory cytokines interleukin 1β (IL-1 β), IL-18, tumor necrosis factor alpha (TNF-α), TF and CP were tested by enzyme-linked immunosorbent assay (ELISA) before and after treatment. Also the correlations of inflammatory cytokines and TF were determined.. The levels of inflammatory cytokines and TF were higher in NT-DVT than those in control group pre-treatment ((153.13 ± 2.30) vs (59.26 ± 1.57) ng/L, (364.27 ± 1.46) vs (67.46 ± 1.48) ng/L, (363.51 ± 1.85) vs (216.42 ± 1.55) µg/L, (66.90 ± 1.44) vs (14.55 ± 1.52) ng/L, all P < 0.05). And after anticoagulant therapy, the levels decreased (all P < 0.05). Also the levels of IL-1β,IL-18 and TNF-α were positively correlated with TF pre-treatment. And the correlation coefficients were 0.492 (P = 0.045), 0.652 (P = 0.005) and 0.511(P = 0.036) respectively. Compared with control group, the plasma level of CP had no obvious change before and after treatment (both P > 0.05).. A high level of inflammatory cytokines is an important risk factor for NT-DVT.

Topics: Cytokines; Enzyme-Linked Immunosorbent Assay; Female; Humans; Inflammation; Leg; Lower Extremity; Male; Neoplasms; Thromboplastin; Venous Thrombosis

Retinoic acid (RA) is the active vitamin A derivative and has diverse immunomodulatory actions. We hypothesized that RA reduces prothrombotic mediators such as tissue factor (TF) in endothelial cells during inflammatory conditions via an AMPK-dependent pathway, which attenuates cardiovascular complications.. RA significantly increased AMPK and Akt phosphorylation in a time- and concentration-dependent manner in endothelial cells (EC). RA downregulated TF expression at the transcriptional and translational levels in TNF-α activated ECs, which was reversed by the silencing of AMPK and transfection of DN-AMPK. Interestingly, the PI3-kinase inhibitor LY294002 reversed the RA effect on TF expression. Increased AMPK phosphorylation by RA was inhibited by LY294002. However, increased Akt phosphorylation was not reduced by compound C, indicating that PI3K/Akt signaling modulates AMPK activity. In addition, RA reduced HMGB1 release in TNF-α activated ECs, which was reversed by both LY294001 and siAMPK. Importantly, administration of RA (1 mg/kg) significantly reduced blood TF activity, circulating HMGB1 and PAI-1 levels and expression of hepatic TF mRNA as well as fibrin deposition in LPS (5 mg/kg)-injected mice.. Taken together, the activation of PI3K/Akt by RA modulates AMPK activity in ECs and plays a crucial role in the inhibition of coagulatory factors such as TF, PAI-1, and HMGB1 in inflammatory conditions.

Topics: AMP-Activated Protein Kinases; Animals; Chromones; Culture Media; Endothelial Cells; Fibrin; HMGB1 Protein; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Lipopolysaccharides; Liver; Male; Mice; Mice, Inbred BALB C; Morpholines; Phosphatidylinositol 3-Kinases; Phosphorylation; Plasminogen Activator Inhibitor 1; Serpin E2; Thromboplastin; Tretinoin; Tumor Necrosis Factor-alpha

We aimed to evaluate the mechanisms underlying the effects of red blood cells (RBCs) on the reactivity of monocytes to lipopolysaccharide (LPS) stimulation.. Measurements of tissue factor (TF) antigen and activity were performed on freshly isolated white blood cells (WBCs)/platelets resuspended in heparinized plasma, as well as cultured monocytic cells.. In a dose-dependent manner, RBCs significantly enhanced LPS-induced TF activity and antigen levels in blood monocytes; potentiation of TF activity by both human and murine RBCs did not require the presence of neutrophils and/or platelets. We also measured the levels of monocyte chemotactic protein-1 (MCP-1), the key proinflammatory chemokine that binds to duffy antigen receptor for chemokines (DARC) on RBC surface, in plasma and RBC lysates after the incubation of RBCs with WBC/platelets; at the concentrations corresponding to normal blood counts, RBCs exerted a significant influence on the free plasma levels of MCP-1, with about two-thirds of detectable MCP-1 post-LPS stimulation being associated with RBCs. Critically, DARC-deficient murine RBCs failed to enhance LPS-induced TF activity, confirming the mechanistic significance of RBC-DARC.. Our study reports a novel mechanism by which RBCs promote procoagulant and proinflammatory sequelae of WBC exposure to LPS, likely mediated by RBC-DARC in the microenvironment(s) that bring monocytes and RBCs in close proximity.

Topics: Adult; Animals; Blood Coagulation; Cell Line; Chemokine CCL2; Duffy Blood-Group System; Endotoxemia; Erythrocytes; Gene Expression Regulation; Humans; Inflammation; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Monocytes; Receptors, Cell Surface; RNA, Messenger; Thromboplastin

Metformin is a widely‑used antidiabetic drug with hypoglycemic activity and previously described anti‑inflammatory properties. Previous studies have demonstrated that metformin attenuates endotoxic hepatitis, however the mechanisms remain unclear. Inflammation and coagulation are closely associated pathological processes, therefore the potential effects of metformin on key steps in activation of the coagulation system were further investigated in endotoxic hepatitis induced by lipopolysaccharide/D‑galactosamine (LPS/D‑Gal). The current study demonstrated that treatment with metformin significantly suppressed the upregulation of tissue factor and plasminogen activator inhibitor‑1 in LPS/D‑Gal‑exposed mice. In addition, a reduction in the expression of interleukin 6 and inhibition of nuclear translocation of nuclear factor‑κB were observed. These data indicate that the LPS/D‑Gal‑induced elevation of the stable protein level of hypoxia inducible factor 1α, the mRNA level of erythropoietin, vascular endothelial growth factor and matrix metalloproteinase‑3, and the hepatic level of lactic acid were also suppressed by metformin. The current study indicates that the suppressive effects of metformin on inflammation‑induced coagulation may be an additional mechanism underlying the hepatoprotective effects of metformin in mice with LPS/D‑Gal‑induced fulminant hepatitis.

Topics: Animals; Anti-Inflammatory Agents; Blood Coagulation; Disease Models, Animal; Erythropoietin; Galactosamine; Hepatitis; Hypoglycemic Agents; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation; Interleukin-6; Lactic Acid; Lipopolysaccharides; Liver; Matrix Metalloproteinase 3; Metformin; Mice; NF-kappa B; Plasminogen Activator Inhibitor 1; RNA, Messenger; Thromboplastin; Up-Regulation; Vascular Endothelial Growth Factor A

Circulating microparticles play a pro-inflammatory and procoagulant detrimental role in the vascular dysfunction of septic shock. It was the objective of this study to investigate mechanisms by which a pharmacological modulation of microparticles could affect vascular dysfunction in a rat model of septic shock. Septic or sham rats were treated by activated protein C (aPC) and resuscitated during 4 hours. Their microparticles were harvested and inoculated to another set of healthy recipient rats. Haemodynamic parameters were monitored, circulating total procoagulant microparticles assessed by prothrombinase assay, and their cell origin characterised. Mesenteric resistance arteries, aorta and heart were harvested for western blotting analysis. We found that a) the amount and phenotype of circulating microparticles were altered in septic rats with an enhanced endothelial, leucocyte and platelet contribution; b) aPC treatment significantly reduced the generation of leucocyte microparticles and norepinephrine requirements to reach the mean arterial pressure target in septic rats; c) Microparticles from untreated septic rats, but not from aPC-treated ones, significantly reduced the healthy recipients' mean arterial pressure; d) Microparticle thromboxane content and aPC activity were significantly increased in aPC-treated septic rats. In inoculated naïve recipients, microparticles from aPC-treated septic rats prompted reduced NF-κB and cyclooxygenase-2 arterial activation, blunted the generation of pro-inflammatory iNOS and secondarily increased platelet and endothelial microparticles. In conclusion, in this septic shock model, increased circulating levels of procoagulant microparticles led to negative haemodynamic outcomes. Pharmacological treatment by aPC modified the cell origin and levels of circulating microparticles, thereby limiting vascular inflammation and favouring haemodynamic improvement.

Topics: Animals; Cell-Derived Microparticles; Coagulants; Cyclooxygenase 2; Hemodynamics; Humans; Inflammation; Male; NF-kappa B; Phenotype; Protein C; Rats; Rats, Wistar; Recombinant Proteins; Shock, Septic; Thromboplastin; Thromboxane A2; Time Factors; Treatment Outcome

Histamine is classified as an inflammatory mediator and has been reported to have anti- as well as pro-inflammatory properties. The aim of this study was to explore the role of histamine on the production of LPS-induced tissue factor (TF) activity and TNFα in monocytes of whole blood in the absence and presence of TNFα or PMA.. Human blood anticoagulated with Fragmin was subjected to stimulation by LPS in the presence and absence of TNFα or PMA and various concentrations of histamine. Tissue factor (TF) activity was measured in lyzed cells after isolation of mononuclear cells whereas TNFα was quantified in plasma after centrifugation of cells.. Histamine gave a dose dependent inhibitory effect on LPS-induced TF activity in monocytes of whole blood, with a 50% reduction at 0.033 μM. A similar effect was seen when the blood cells were stimulated with the combination of LPS and TNFα although TNFα enhanced LPS-induced TF activity almost two fold. In contrast, when blood was incubated with LPS and PMA in whole blood, histamine gave a significant rise in TF activity at 0.01 μM and 0.33 μM histamine. The effect of histamine was less at 0.1 μM or higher concentrations giving a biphasic profile. Contrary to the effect of histamine on LPS plus PMA induced TF activity, histamine caused a significant reduction in TNFα albeit less than in the absence of PMA. Intake of aspirin caused a significant rise in LPS-induced TF activity that was almost abolished by histamine at 0.033 μM.. Our study shows that histamine has an anti-inflammatory effect on LPS and LPS/TNFα stimulated monocytes of whole blood. In contrast when blood cells are activated by a combination of LPS and PMA whereby PKC is activated, histamine has a procoagulant/pro-inflammatory effect through enhancement of TF activity expression.

Topics: Dose-Response Relationship, Drug; Drug Synergism; Histamine; Humans; Inflammation; Lipopolysaccharides; Monocytes; Tetradecanoylphorbol Acetate; Thromboplastin; Tumor Necrosis Factor-alpha

We characterised a tissue factor (TF) and tissue factor pathway inhibitor (TFPI) expression in relation to severity of inflammatory infiltration of the gallbladder mucosa in a chronic cholecystitis. We prospectively studied the gallbladder specimens obtained from 54 patients who had undergone cholecystectomy due to chronic calculous cholecystitis and 16 calculosis-free gallbladder specimens obtained from patients who underwent cholecystectomy due to the polyp/polyps as well as in cases of gallbladder injury. To assess TF and TFPI immunoreactivity by immunohistochemistry, the monoclonal anti-human TF and TFPI antibodies were used. The inflammatory infiltration of the gallbladder mucosa was reflected by the number of CD3 and CD68 positive cells. The expression of TF and TFPI differed significantly between the cholecystitis and the control group. Most capillary endothelial cells of the cholecystitis group presented weak expression for TFPI. The mean number of CD3 positive lymphocytes in the cholecystitis group was 18.6 ± 12.2, but the mean number of CD68 positive cells was 29.7 ± 13.9. In the control sections, it was 3.1 ± 1.9 and 8.8 ± 3.9, respectively (P < 0.001). The results of the current study suggest that the tissue procoagulant state found may be engaged in the etiopathogenesis of the cholecystitis.

Topics: Antigens, CD; Antigens, Differentiation, Myelomonocytic; CD3 Complex; Female; Gallbladder; Humans; Immunohistochemistry; Inflammation; Lipoproteins; Male; Middle Aged; Mucous Membrane; Prospective Studies; Thromboplastin

Spreading of melanoma is associated with efficient extravasation of circulating tumor cells from the vascular system into distant target organs. This process is accompanied and supported by proinflammatory and procoagulatory conditions. In this study, we analysed the ability of human melanoma cell lines to activate endothelial cells (ECs) in vitro. Some melanoma cells, that is, MV3, were shown to trigger an prompt calcium-flux-dependent, procoagulatory endothelial response that was accompanied by luminal release of ultra-large von Willebrand factor (ULVWF) fibres that were immobilized to the endothelial surface layer. In contrast to MV3-derived supernatant, prolonged treatment of ECs with WM9-derived supernatant mediated a pronounced activation of nuclear factor kappa B (NFκB). NFκB activation in ECs was dependent on both IL-1α and IL-1β secreted from melanoma cells. Melanoma-derived IL-1 mediated an upregulation of proinflammatory cytokines IL-6 and IL-8, the intercellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule-1 (VCAM-1) and the procoagulatory tissue factor (TF) in ECs. Our data show that melanoma cells activate ECs either directly and within seconds or by an IL-1-mediated NFκB activation. Both pathways of EC activation convert the regular repressive function of ECs on inflammation and coagulation to a proinflammatory and procoagulatory surface that supports tumor progression.

Topics: Calcium Signaling; Capillary Permeability; Cell Line, Tumor; Cytokines; Disease Progression; Endothelium, Vascular; Humans; Inflammation; Intercellular Adhesion Molecule-1; Interleukin-1; Interleukin-6; Interleukin-8; Melanoma; Models, Biological; NF-kappa B; Phenotype; Thromboplastin; Up-Regulation; Vascular Cell Adhesion Molecule-1; von Willebrand Factor

Thrombin is essential for blood coagulation but functions also as a mediator of cellular signalling. Gene expression microarray experiments in human monocytes revealed thrombin-induced upregulation of a limited subset of genes, which are almost exclusively involved in inflammation and wound healing. Among these, the expression of F3 gene encoding for tissue factor (TF) was enhanced indicating that this physiological initiator of coagulation cascade may create a feed-forward loop to enhance blood coagulation. Activation of protease-activated receptor type 1 (PAR1) was shown to play a main role in promoting TF expression. Moreover, thrombin induced phosphorylation of ERK1/2, an event that is required for expression of thrombin-regulated genes. Thrombin also increased the expression of TF at the protein level in monocytes as evidenced by Western blot and immunostaining. Furthermore, FXa generation induced by thrombin-stimulated monocytes was abolished by a TF blocking antibody and therefore it is entirely attributable to the expression of tissue factor. This cellular activity of thrombin provides a new molecular link between coagulation, inflammation and wound healing.

Topics: Blood Coagulation; Cells, Cultured; Factor Xa; Feedback, Physiological; Gene Expression Regulation; Humans; Inflammation; MAP Kinase Signaling System; Monocytes; Oligonucleotide Array Sequence Analysis; Real-Time Polymerase Chain Reaction; Receptor, PAR-1; Thrombin; Thromboplastin; Transcription, Genetic; Up-Regulation; Wound Healing

Extracellular histones exert part of their prothrombotic activity through the stimulation of blood cells. Besides platelets, histones can bind to red blood cells (RBCs), which are important contributors to thrombogenesis, but little is known about the functional consequences of this interaction.. To evaluate the effect of histones on the procoagulant potential of human RBCs with particular regard to the expression of surface phosphatidylserine (PS).. PS exposure on human RBCs treated with a natural mixture of histones or recombinant individual histones was evaluated with fluorescein isothiocyanate-annexin-V binding and measured with flow cytometry. Calcium influx in RBCs loaded with the calcium-sensitive fluorophore Fluo-4 AM was assessed with flow cytometry. The procoagulant potential of histone-treated RBCs was evaluated with a purified prothrombinase assay and a one-stage plasma recalcification clotting test.. Natural histones induced PS exposure on RBCs in a dose-dependent manner, and neutralization or cleavage of histones by heparin or activated protein C, respectively, abolished PS externalization. H4 was mainly responsible for the stimulating activity of histones, whereas the other subtypes were almost ineffective. Similarly, natural histones and H4 induced influx of calcium into RBCs, whereas the other individual histones did not. Histone-induced exposure of PS on RBCs translated into increased prothrombinase complex-mediated prothrombin activation and accelerated fibrin formation in plasma.. Histones induce RBCs to express a procoagulant phenotype through the externalization of PS. This finding provides new insights into the prothrombotic activity of extracellular histones.

Topics: Aniline Compounds; Animals; Annexin A5; Blood Coagulation; Blood Platelets; Calcium; Cattle; Coagulants; Erythrocytes; Flow Cytometry; Fluorescein-5-isothiocyanate; Histones; Humans; Inflammation; Phenotype; Phosphatidylserines; Recombinant Proteins; Thromboplastin; Xanthenes

Immune responses to therapeutic factor VIII remain a major problem, affecting 30% of patients with severe hemophilia A. The primary factors that drive immune responses in these patients remain elusive. There have been conflicting reports on a role of coagulation (or thrombin) in anti-FVIII immune responses.. To assess the importance of coagulation-associated processes for the onset of the anti-FVIII immune response.. Using FVIII-deficient mice, we compared the immunogenicity of recombinant FVIII or the inactive FVIII(V) (634M) mutant. In parallel, the involvement of tissue factor (TF) activity in the anti-FVIII immune response was investigated upon injection of a neutralizing anti-TF antibody or by the use of chimeric mice that lack TF expression in myeloid cells. The development of the anti-FVIII immune response was also monitored after treatment with warfarin.. The kinetics of the development of antibody responses to FVIII(V) (634M) were indistinguishable from those of wild-type FVIII. Inhibition of TF activity did not modulate immune responses to exogenous FVIII. Additionally, global inhibition of coagulation with warfarin failed to reduce the anti-FVIII immune response.. Thrombin generation or coagulation-associated processes do not modulate the anti-FVIII antibody response in mouse model of severe hemophilia A.

Topics: Animals; Antibodies, Neutralizing; Blood Coagulation; Disease Models, Animal; Factor VIII; Hemophilia A; Immunity, Humoral; Inflammation; Mice; Mutation; Plasmids; Protein Structure, Tertiary; Recombinant Proteins; Thrombin; Thromboplastin; Warfarin

Tuberculosis (TB) is a chronic lung infectious disease characterized by severe inflammation and lung granulomatous lesion formation. Clinical manifestations of TB include hypercoagulable states and thrombotic complications. We previously showed that Mycobacterium tuberculosis (M.tb) infection induces tissue factor (TF) expression in macrophages in vitro. TF plays a key role in coagulation and inflammation. In the present study, we investigated the role of TF in M.tb-induced inflammatory responses, mycobacterial growth in the lung and dissemination to other organs. Wild-type C57BL/6 and transgenic mice expressing human TF, either very low levels (low TF) or near to the level of wild-type (HTF), in place of murine TF were infected with M.tb via aerosol exposure. Levels of TF expression, proinflammatory cytokines and thrombin-antithrombin complexes were measured post M.tb infection and mycobacterial burden in the tissue homogenates were evaluated. Our results showed that M.tb infection did not increase the overall TF expression in lungs. However, macrophages in the granulomatous lung lesions in all M.tb-infected mice, including low TF mice, showed increased levels of TF expression. Conspicuous fibrin deposition in the granuloma was detected in wild-type and HTF mice but not in low TF mice. M.tb infection significantly increased expression levels of cytokines IFN-γ, TNF-α, IL-6 and IL-1ß in lung tissues. However, no significant differences were found in proinflammatory cytokines among the three experimental groups. Mycobacterial burden in lungs and dissemination into spleen and liver were essentially similar in all three genotypes. Our data indicate, in contrast to that observed in acute bacterial infections, that TF-mediated coagulation and/or signaling does not appear to contribute to the host-defense in experimental tuberculosis.

Topics: Animals; Bronchoalveolar Lavage Fluid; Inflammation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mycobacterium tuberculosis; Thromboplastin; Tuberculosis

Atherosclerosis is a chronic inflammatory process, in which vascular endothelial cells (ECs) become dysfunctional owing to the effects of chemical substances, such as inflammatory factor and growth factors. Tissue factor (TF) expression is induced by the above chemical substances in activated ECs. TF initiates thrombosis on disrupted atherosclerotic plaques which plays an essential role during the onset of acute coronary syndromes (ACS). Increasing evidences suggest the important role of microRNAs as epigenetic regulators of atherosclerotic disease. The aim of our study is to identify if microRNA-223 (miR-223) targets TF in ECs.. Bioinformatic analysis showed that TF is a target candidate of miR-223. Western blotting analysis revealed that tumor necrosis factor α (TNF-α) increased TF expression in aorta of C57BL/6J mice and cultured ECs (EA.hy926 cells and HUVEC) after 4 h treatment. In TNF-α treated ECs, TF mRNA was also increased measured by real-time PCR. Real-time PCR results showed that miR-223 levels were downregulated in TNF-α-treated aorta of C57BL/6J mice and cultured ECs. Transfection of ECs with miR-223 mimic or miR-223 inhibitor modified TF expression both in mRNA and protein levels. Luciferase assays confirmed that miR-223 suppressed TF expression by binding to the sequence of TF 3'-untranslated regions (3'UTR). TF procoagulant activity was inhibited by overexpressing miR-223 with or without TNF-α stimulation.. MiR-223-mediated suppression of TF expression provides a novel molecular mechanism for the regulation of coagulation cascade, and suggests a clue against thrombogenesis during the process of atherosclerotic plaque rupture.

Topics: Animals; Aorta; Base Sequence; Binding Sites; Blood Coagulation; Cells, Cultured; Endothelial Cells; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Male; Mice; Mice, Inbred C57BL; MicroRNAs; Molecular Sequence Data; Plaque, Atherosclerotic; Thromboplastin; Tumor Necrosis Factor-alpha

Activation of coagulation pathways may contribute to risk for non-AIDS-related conditions among HIV-positive patients. Tissue factor (TF)-dependent procoagulant activity on circulating microparticles (MP-TF) in the plasma of 163 HIV-positive participants, both untreated and treated, with viral suppression was measured. MP-TF activity was 39% lower among treated versus untreated participants (P < 0.001), which persisted in adjusted models (-36%, P = 0.03). Among treated participants, MP-TF activity correlated modestly with D-dimer (r = 0.24, P = 0.01), von Willebrand factor (r = 0.36, P < 0.001), and interleukin-6 (r = 0.20, P = 0.04) levels. Future research should focus on mechanisms driving residual functional TF activity and whether these alterations have clinical consequences for non-AIDS-defining complications.

Topics: Adult; Anti-HIV Agents; Blood Coagulation; Blood Coagulation Tests; C-Reactive Protein; Cell-Derived Microparticles; Female; HIV Infections; Humans; Inflammation; Interleukin-6; Male; Middle Aged; Thromboplastin; von Willebrand Factor

S100A12 is elevated in the circulation in patients with chronic inflammatory diseases and recent studies indicate pleiotropic functions. Serum amyloid A induces monocyte cytokines and tissue factor. S100A12 did not stimulate IL-6, IL-8, IL-1β or TNF-α production by human peripheral blood mononuclear cells but low amounts consistently reduced cytokine mRNA and protein levels induced by serum amyloid A, by ∼49% and ∼46%, respectively. However, S100A12 did not affect serum amyloid A-induced monocyte tissue factor. In marked contrast, LPS-induced cytokines or tissue factor were not suppressed by S100A12. S100A12 did not alter cytokine mRNA stability or the cytokine secretory pathway. S100A12 and serum amyloid A did not appear to form complexes and although they may have common receptors, suppression was unlikely via receptor competition. Serum amyloid A induces cytokines via activation of NF-κB and the MAPK pathways. S100A12 reduced serum amyloid A-, but not LPS-induced ERK1/2 phosphorylation to baseline. It did not affect JNK or p38 phosphorylation or the NF-κB pathway. Reduction in ERK1/2 phosphorylation by S100A12 was unlikely due to changes in intracellular reactive oxygen species, Ca(2+) flux or to recruitment of phosphatases. We suggest that S100A12 may modulate sterile inflammation by blunting pro-inflammatory properties of lipid-poor serum amyloid A deposited in chronic lesions where both proteins are elevated as a consequence of macrophage activation.

Topics: Calcium; Cytokines; Dose-Response Relationship, Drug; Gene Expression Regulation; Humans; Inflammation; Inflammation Mediators; JNK Mitogen-Activated Protein Kinases; Leukocytes, Mononuclear; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Reactive Oxygen Species; S100 Proteins; S100A12 Protein; Serum Amyloid A Protein; Thromboplastin

Hyperlipidaemia is a causal factor in the ethiopathogenesis of atherosclerosis. Statins are the cornerstone drug therapy for LDL-cholesterol (LDL-c) lowering, that exert beneficial effects beyond lipid lowering. Circulating microparticles (cMPs), microvesicles released by activated cells into the bloodstream, are markers of vascular and inflammatory cell activation with tentative role in disease progression. However, the role of statins on cMPs seems controversial. We aimed at the evaluation of the effects of lipid-lowering treatment (LLT) on cMP generation in patients in primary prevention of atherosclerosis. A case-control study was conducted in hypercholesterolaemic patients receiving LLT with statins and normocholesterolaemic controls (LLT+ and LLT-, respectively, n=37/group), matched by age, gender and LDL-c levels. cMPs were characterised by flow cytometry using annexin-V and cell-specific antibodies. In LLT+-patients overall numbers of cMPs (p<0.005) were lower than in controls. Levels of cMPs carrying parental cell markers from vascular and circulating cell origin (platelet, endothelial cell, pan-leukocyte and specific-leukocyte subsets) were significantly lower in blood of LLT+ compared to LLT--patients. Moreover, MPs from LLT+-patients had reduced markers of activated platelets (αIIbβ3-integrin), activated inflammatory cells (αM-integrin) and tissue factor. The effect of LLT on cMP shedding was found to be accumulative in years. cMP shedding associated to cardiovascular risk in LLT+-patients. In summary, at similar plasma cholesterol levels patients on statin treatment had a significant lower number of cMPs carrying markers of activated cells. These findings indicate that statins protect against vascular cell activation.

Topics: Adult; Annexin A5; Atherosclerosis; Blood Platelets; Case-Control Studies; Cell-Derived Microparticles; Cholesterol, LDL; Endothelial Cells; Female; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hyperlipidemias; Hypolipidemic Agents; Inflammation; Leukocytes; Male; Middle Aged; Primary Prevention; Thromboplastin

In the present study, we tested whether serum amyloid A (SAA) protein, an established biomarker of inflammation, also plays a role in stimulating neovascularization. To evaluate this possibility, human carotid artery endothelial (HCtAE) cells were cultured and cellular migration and the proinflammatory and/or thrombotic activity of SAA (0, 1 or 10 μg/mL) on vascular endothelial cells was verified by determining gene regulation relative to control (in the absence of SAA). Exposure of HCtAE cells to SAA increased expression of the transcription factor nuclear factor-κB (NFKB), tumour necrosis factor (TNF) and pro-coagulative tissue factor (F3), and stimulated phosphorylation of the P65 subunit of the NFKB complex. Enhanced production of TNF and NFKB was paralleled by increased vascular endothelial growth factor (VEGF) mRNA and protein expression, as demonstrated by quantitative polymerase chain reaction, western blotting and ELISA. Administration of 10 μg/mL SAA enhanced endothelial cell migration (1.6-fold vs control), stimulated regrowth of HCtAE cells after mechanical injury (~1.2-fold vs control) and increased endothelial tube formation relative to control after 6 h. The SAA-mediated enhancement of endothelial cell migration, proliferation and tube formation were markedly inhibited by pretreatment of HCtAE cells with the multi-angiokinase receptor inhibitor BIBF1120 (100 nmol/L), although SAA-stimulated gene responses for F3 and NFKB were unaffected by 100 nmol/L BIBF1120 pretreatment. Overall, BIBF1120 inhibited the pro-angiogenic activity of SAA on vascular endothelial cells in this experimental model of inflammation.

Topics: Carotid Arteries; Carrier Proteins; Cell Movement; Cell Proliferation; Cells, Cultured; Endothelial Cells; Endothelium, Vascular; Gene Expression Regulation; Humans; Indoles; Inflammation; Intracellular Signaling Peptides and Proteins; Neoplasm Proteins; Neovascularization, Pathologic; NF-kappa B; Phosphorylation; Serum Amyloid A Protein; Thromboplastin; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

Xiang-Qi-Tang (XQT) is a Chinese herbal formula containing Cyperus rotundus, Astragalus membranaceus and Andrographis paniculata. Alpha-Cyperone (CYP), astragaloside IV (AS-IV) and andrographolide (AND) are the three major active components in this formula. XQT may modulate the inflammatory or coagulant responses. We therefore assessed the effects of XQT on lipopolysaccharide (LPS)-induced inflammatory model of rat cardiac microvascular endothelial cells (RCMECs). XQT, CYP, AS-IV and AND inhibited the production of tumor necrosis factor alpha (TNF-α), intercellular cell adhesion molecule-1 (ICAM-1) and plasminogen activator inhibitor-1 (PAI-1), and up-regulated the mRNA expression of Kruppel-like factor 2 (KLF2). XQT and CYP inhibited the secretion of tissue factor (TF). To further explore the mechanism, we found that XQT, or its active components CYP, AS-IV and AND significantly inhibited extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK) and p38 phosphorylation protein expression as well as decreased the phosphorylation levels of nuclear factor κB (NF-κB) p65 proteins in LPS-stimulated RCMECs. These results suggested that XQT and its active components inhibited the expression of inflammatory and coagulant mediators via mitogen-activated protein kinase (MAPKs) and NF-κB signaling pathways. These findings may contribute to future research on the action mechanisms of this formula, as well as therapy for inflammation- or coagulation-related diseases.

Topics: Animals; Anti-Inflammatory Agents; Anticoagulants; Drugs, Chinese Herbal; Endothelial Cells; Endothelium, Vascular; Extracellular Signal-Regulated MAP Kinases; Heart; Inflammation; Intercellular Adhesion Molecule-1; JNK Mitogen-Activated Protein Kinases; Kruppel-Like Transcription Factors; Lipopolysaccharides; Microvessels; Mitogen-Activated Protein Kinases; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Plasminogen Activator Inhibitor 1; Rats; Rats, Wistar; Signal Transduction; Thromboplastin; Tumor Necrosis Factor-alpha

Previous studies have shown that the innate immune stimulant LPS augments mechanical ventilation-induced pulmonary coagulation and inflammation. Whether these effects are mediated by alveolar epithelial cells is unclear. The alveolar epithelium is a key regulator of the innate immune reaction to pathogens and can modulate both intra-alveolar inflammation and coagulation through up-regulation of proinflammatory cytokines and tissue factor (TF), the principal initiator of the extrinsic coagulation pathway. We hypothesized that cyclic mechanical stretch (MS) potentiates LPS-mediated alveolar epithelial cell (MLE-12) expression of the chemokine keratinocyte-derived cytokine (KC) and TF. Contrary to our hypothesis, MS significantly decreased LPS-induced KC and TF mRNA and protein expression. Investigation into potential mechanisms showed that stretch significantly reduced LPS-induced surface expression of TLR4 that was not a result of increased degradation. Decreased cell surface TLR4 expression was concomitant with reduced LPS-mediated NF-κB activation. Immunofluorescence staining showed that cyclic MS markedly altered LPS-induced organization of actin filaments. In contrast to expression, MS significantly increased LPS-induced cell surface TF activity independent of calcium signaling. These findings suggest that cyclic MS of lung epithelial cells down-regulates LPS-mediated inflammatory and procoagulant expression by modulating actin organization and reducing cell surface TLR4 expression and signaling. However, because LPS-induced surface TF activity was enhanced by stretch, these data demonstrate differential pathways regulating TF expression and activity. Ultimately, loss of LPS responsiveness in the epithelium induced by MS could result in increased susceptibility of the lung to bacterial infections in the setting of mechanical ventilation.

Topics: Animals; Cell Line; Cell Survival; Chemokines; Coagulants; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Inflammation; Keratinocytes; Lipopolysaccharides; Lung; Mice; NF-kappa B; Signal Transduction; Thromboplastin; Toll-Like Receptor 4; Toll-Like Receptors

Tissue factor (TF) expression on islets has been shown to trigger instant blood-mediated inflammatory reaction (IBMIR), leading to rapid islet loss in portal vein islet transplantation. This study investigated whether antisense RNA-mediated TF gene knockdown in islets could suppress IBMIR as a strategy to overcome IBMIR. Neonatal porcine islet cell clusters (NICCs) were transfected with or without TF-specific antisense RNA or a nonspecific RNA by a lipid-based method. Expression of both TF gene and protein in NICCs was analyzed after transfection by real-time PCR, Western blot, and FACS, respectively. The impact of antisense RNA transfection on NICC viability and in vitro function was examined by FACS and insulin release test, respectively. The effect of TF knockdown in NICCs on IBMIR was assessed with an in vitro tubing loop assay using human blood. A significant reduction in TF gene and protein expression was achieved in TF antisense RNA but not control RNA transfected NICCs, which did not affect NICCs' viability or their insulin secreting capacity. Incubation of TF antisense RNA transfected with human blood resulted in a considerable reduction in blood clot formation, platelet consumption, and complement and coagulation activation compared to that observed in the loops containing human blood and untreated or control RNA transfected NICCs. Consistent with these findings, infiltrating neutrophils in the blood clots with entrapped TF antisense RNA transfected NICCs was also reduced substantially compared to that seen in the clots containing untreated or control RNA transfected NICCs. This study presents a nontoxic TF antisense RNA-mediated TF knockdown in porcine islets that leads to an effective suppression of IBMIR, suggesting a potentially new strategy to improve islet transplantation outcomes.

Topics: Animals; Blood Coagulation; Blood Platelets; Cells, Cultured; Complement Activation; Female; Gene Silencing; Humans; Inflammation; Insulin; Insulin Secretion; Islets of Langerhans; Islets of Langerhans Transplantation; Male; Neutrophils; RNA, Antisense; Swine; Thromboplastin; Transfection; Transplantation, Heterologous

Infective endocarditis (IE) remains a serious complication after heart valve replacement. Autologous valves constructed by matrix-based tissue engineering are under investigation to increase biocompatibility. The impact of the underlying matrix on the risk to develop IE is not known. The IE is characterized by bacterial adhesion and subsequent interactions of disseminating bacteria with endothelial cells (ECs) and monocytes, evoking endothelial proinflammatory and procoagulant activity, leading to heart valve destruction. In the current study, we, therefore, have seeded human ECs on a fibrin versus collagen gel matrix and, at confluence, infected them with Staphylococcus aureus, Streptococcus sanguis, and Staphylococcus epidermidis. Especially Sta. aureus infected ECs grown on fibrin (4.2% of the inoculum) and collagen (3.7%) matrices, more than on ECs grown on noncoated plates (1.2%; p<0.01). This was associated with higher monocyte adhesion (61%; p<0.01 on fibrin and 43%; p<0.05 on collagen) versus control cultures (30%), even at comparable EC surface expression of intercellular adhesion molecule-1 and vascular adhesion molecule-1. The collagen matrix attenuated the Sta. aureus-induced monocyte chemoattractant protein 1 expression 2.0-fold, compared with the noncoated control ECs. This reduction coincided with a 4.2-5.0-fold reduction of the procoagulant activity, triggered in ECs grown on noncoated wells, as a consequence of tissue factor (TF) expression by ECs, further stimulated by EC-bound monocytes. Overall, moderate responses were seen on infection with Str. sanguis and Sta. epidermidis for both gel matrices. Thus, even when fibrin and collagen gel matrices equally increase bacterial adhesion, and subsequent monocyte adhesion to infected ECs, these matrices modulate EC responses to these stimuli, thus resulting in attenuated cytokine production and attenuated adherent monocyte-dependent TF production by the ECs. Further investigations will need to confirm whether also in vivo, EC-matrix interactions can attenuate EC responses to bacteria and inflammatory cells to reduce IE at infected endovascular sites.

Topics: Bacterial Adhesion; Blood Coagulation; Cell Adhesion; Cells, Cultured; Collagen; Cytokines; Endothelial Cells; Fibrin; Flow Cytometry; Humans; Inflammation; Monocytes; Staphylococcus aureus; Thromboplastin; Tissue Scaffolds

Diabetes predisposes to aortic stenosis (AS). We aimed to investigate if diabetes affects the expression of selected coagulation proteins and inflammatory markers in AS valves. Twenty patients with severe AS and concomitant type 2 diabetes mellitus (DM) and 40 well-matched patients without DM scheduled for valve replacement were recruited. Valvular tissue factor (TF), TF pathway inhibitor (TFPI), prothrombin, C-reactive protein (CRP) expression were evaluated by immunostaining and TF, prothrombin, and CRP transcripts were analyzed by real-time PCR. DM patients had elevated plasma CRP (9.2 [0.74-51.9] mg/l vs. 4.7 [0.59-23.14] mg/l, p = 0.009) and TF (293.06 [192.32-386.12] pg/ml vs. 140 [104.17-177.76] pg/ml, p = 0.003) compared to non-DM patients. In DM group, TF-, TFPI-, and prothrombin expression within valves was not related to demographics, body mass index, and concomitant diseases, whereas increased expression related to DM was found for CRP on both protein (2.87 [0.5-9]% vs. 0.94 [0-4]%, p = 0.01) and transcript levels (1.3 ± 0.61 vs. 0.22 ± 0.43, p = 0.009). CRP-positive areas were positively correlated with mRNA TF (r = 0.84, p = 0.036). Diabetes mellitus is associated with enhanced inflammation within AS valves, measured by CRP expression, which may contribute to faster AS progression.

Topics: Aged; Aortic Valve; Aortic Valve Stenosis; C-Reactive Protein; Diabetes Complications; Diabetes Mellitus; Disease Progression; Female; Humans; Inflammation; Lipoproteins; Male; Middle Aged; Prothrombin; RNA, Messenger; Thromboplastin

Excessive activation of coagulation, which can culminate in overt disseminated intravascular coagulation, is a prominent feature of heat stroke. However, neither the mechanism that initiates the coagulation activation nor its pathogenic role is known. We examined whether the tissue factor/factor VIIa complex initiates the coagulation activation in heat stroke and, if so, whether upstream inhibition of coagulation activation through its neutralization may minimize cellular injury and organ dysfunction. We also examined whether coagulation inhibition influences heat stroke-induced fibrinolytic and inflammatory responses.. Randomized controlled study.. Comparative Medicine Department, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.. Baboons (Papio Hamadryas).. Twelve anesthetized baboons assigned randomly to recombinant nematode anticoagulant protein c2, a powerful inhibitor of tissue factor/factor VIIa-dependent coagulation (n = 6), or a control group (n = 6) were heat-stressed in a prewarmed neonatal incubator at 44-47°C until systolic blood pressure fell <90 mm Hg, signaling the onset of severe heat stroke. Recombinant nematode anticoagulant protein c2 was administered as a single intravenous dose of 30 μg/kg body weight at onset of heat stroke. The control group received an equivalent volume of sterile saline intravenously.. Heat stroke was associated with coagulation activation and fibrin formation as evidenced by the increased plasma thrombin-antithrombin complexes, endogenous thrombin potential, and D-dimer levels. Recombinant nematode anticoagulant protein c2 induced significant inhibition of thrombin generation and fibrin formation. Inhibition of coagulation in recombinant nematode anticoagulant protein c2-treated animals did not influence either fibrinolysis (assessed by tissue plasminogen activator, plasmin-α2-antiplasmin complexes, and plasminogen activator inhibitor) or the release of pro- and anti-inflammatory cytokines. No difference in markers of cell injury and organ dysfunction was observed between recombinant nematode anticoagulant protein c2-treated and control groups.. Tissue factor/factor VIIa-dependent pathway initiates coagulation activation in induced-heat stroke in the baboon without an effect on fibrinolysis and inflammation. The findings suggest also that coagulation activation is not a prerequisite of cell injury and organ dysfunction.

Topics: Animals; Blood Coagulation; Blood Gas Analysis; Factor VIIa; Heat Stroke; Helminth Proteins; Inflammation; Papio hamadryas; Partial Thromboplastin Time; Prothrombin Time; Recombinant Proteins; Thrombomodulin; Thromboplastin

In preterm infants, inflammation and intra-alveolar fibrin formation characterize respiratory distress syndrome (RDS). Tissue factor (TF) is a link between inflammation and coagulation pathways. We investigated the relationship between TF and cytokines in preterm infants to gain information of the role of TF in the inflammatory response.. We measured TF in plasma and in tracheal aspirates and analysed TF on monocytes by flow cytometry and 13 cytokines from plasma, in 56 preterm infants (birthweight 600-1500 g) during their first week.. Plasma TF increased and peaked on day 3 and correlated with both RDS and inversely with paO2/FIO2. On day 1, TF in tracheal aspirates was 10-fold higher than in plasma and correlated with plasma TF (4888 vs. 506 pg/mL, R = 0.692, p = 0.013, n = 12). Of main pro-inflammatory cytokines, plasma TF correlated post-natally with IL-8 and IL-6 but not with IL-1 or TNF-α.. Respiratory morbidity associates with high TF in lungs and plasma. In sick newborn infants, upregulation of TF may be mediated by IL-6 and IL-8. High TF and pro-inflammatory cytokines may together participate in the pathogenesis of pulmonary and extrapulmonary injury in preterm infants through pro-inflammatory mechanisms.

Topics: Bronchopulmonary Dysplasia; Cytokines; Female; Humans; Infant, Newborn; Infant, Premature; Inflammation; Lung; Male; Morbidity; Plasma; Respiratory Distress Syndrome, Newborn; Thromboplastin

The implantation of synthetic medical devices is known to generate an immediate and complex material-related inflammatory response. Consequently, 15 candidate materials for a new microfabricated sensor were investigated. A human whole blood model that permits the interaction of all the putative inflammatory systems was used. The experiments were performed by administering 500 μL of lepirudin-anticoagulated blood in each well of a 24-well polystyrene microtiter plate preloaded with the respective materials. The degree of inflammation was evaluated by assessing four complement activation markers, six proinflammatory cytokines, and chemokines, the expression of monocyte tissue factor (TF), as well as platelet activation. The complement system was inhibited with the C5-inhibitor eculizumab. Three of the materials distinctly activated complement through the alternative pathway, whereas the rest of the materials were virtually inert. Notably, the same three materials induced a marked and selective expression of TF as well as the release of five of the six cytokines. All these increases were statistically significant (p < 0.05). Inhibition of complement by the C5-inhibitor virtually abolished TF expression and markedly reduced several of the cytokines, suggesting that complement is a particularly useful tool to reveal the immediate inflammatory-inducing properties of these biomaterials.

Topics: Biocompatible Materials; Biosensing Techniques; Chemokines; Complement Activation; Glucose; Humans; Inflammation; Microtechnology; Platelet Activation; Prostheses and Implants; Thromboplastin

Sickle cell disease (SCD) is associated with a complex vascular pathophysiology that includes activation of coagulation and inflammation. However, the crosstalk between these 2 systems in SCD has not been investigated. Here, we examined the role of tissue factor (TF) in the activation of coagulation and inflammation in 2 different mouse models of SCD (BERK and Townes). Leukocytes isolated from BERK mice expressed TF protein and had increased TF activity compared with control mice. We found that an inhibitory anti-TF antibody abrogated the activation of coagulation but had no effect on hemolysis or anemia. Importantly, inhibition of TF also attenuated inflammation and endothelial cell injury as demonstrated by reduced plasma levels of IL-6, serum amyloid P, and soluble vascular cell adhesion molecule-1. In addition, we found decreased levels of the chemokines MCP-1 and KC, as well as myeloperoxidase in the lungs of sickle cell mice treated with the anti-TF antibody. Finally, we found that endothelial cell-specific deletion of TF had no effect on coagulation but selectively attenuated plasma levels of IL-6. Our data indicate that different cellular sources of TF contribute to activation of coagulation, vascular inflammation, and endothelial cell injury. Furthermore, it appears that TF contributes to these processes without affecting intravascular hemolysis.

Topics: Anemia, Sickle Cell; Animals; Blood Coagulation; Chemokine CCL2; Chemokine CXCL1; Disease Models, Animal; Endothelial Cells; Erythrocytes; Female; Hemolysis; Inflammation; Interleukin-6; Leukocytes; Male; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Transgenic; Neutrophils; Serum Amyloid P-Component; Thromboplastin; Vascular Cell Adhesion Molecule-1

Blood-sucking arthropods' salivary glands contain a remarkable diversity of antihemostatics. The aim of the present study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades.. Several L. longipalpis salivary proteins were expressed in human embryonic kidney 293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, noncompetitive, and reversible inhibitor of factor Xa (FXa). Notably, Lufaxin's primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, Dansyl-Glu-Gly-Arg-FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both prothrombin time and activated partial thromboplastin time. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with an equilibrium constant ≈3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents protease-activated receptor 2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl(3)-induced carotid artery thrombus formation and prolongs activated partial thromboplastin time ex vivo, implying that it works as an anticoagulant in vivo. Finally, salivary gland of sand flies was found to inhibit FXa and to interact with the enzyme.. Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and anti-inflammatory activities. It is a useful tool to understand FXa structural features and its role in prohemostatic and proinflammatory events.

Topics: Amino Acid Sequence; Animals; Anti-Inflammatory Agents; Blood Coagulation; Calorimetry; Cell Line, Tumor; Chlorides; Cloning, Molecular; Disease Models, Animal; Dose-Response Relationship, Drug; Factor Xa; Factor Xa Inhibitors; Female; Ferric Compounds; Fibrinolytic Agents; HEK293 Cells; Humans; Inflammation; Insect Proteins; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Molecular Weight; Partial Thromboplastin Time; Protein Binding; Prothrombin Time; Psychodidae; Rats; Receptor, PAR-2; Recombinant Proteins; Salivary Glands; Surface Plasmon Resonance; Thromboplastin; Thrombosis; Time Factors

The link between platelet activation and vascular injury in Systemic Sclerosis (SSc) is poorly characterized. Here we report that platelet activation results in i) the translocation from the cytoplasm to the surface of HMGB1, a prototypical DAMP signal associated with tissue regeneration and ii) the release of platelet derived microparticles (PDμP) expressing HMGB1. Decreased HMGB1 content (334.6 ± 21.2 vs 587.1 ± 11.1 AUF, P < 0.001) and HMGB1 translocation to the outer leaflet of the plasma membrane (17.8 ± 3.5 vs 4.5 ± 0.5%, P < 0.001) characterize circulating platelets of SSc patients (n = 29) when compared with age-matched healthy controls (HC, n = 20). Conversely, a significantly higher fraction of PDμP in the blood of SSc patients, but not of HC, consistently expose (HMGB1 (MFI 62.8 ± 3.95 vs 4.3 ± 0.7). Platelet HMGB1 depletion is significantly associated in SSc patients with degranulation and with expression of P-selectin and of tissue factor as well as with fibrinogen binding to their plasma membrane. These findings indicate that platelets represent a source of HMGB1, an ancestral signal of necrosis, in the vasculature of SSc patients, possible contributing to persistent microvascular injury and endothelial cell activation.

Topics: Blood Platelets; Cell Membrane; Cell-Derived Microparticles; Fibrinogen; HMGB1 Protein; Humans; Inflammation; P-Selectin; Platelet Activation; Protein Transport; Scleroderma, Systemic; Thromboplastin

Systemic blockade of tissue factor (TF) attenuates acute lung injury (ALI) in animal models of sepsis but the effects of global TF deficiency are unknown. We used mice with complete knockout of mouse TF and low levels (∼1%) of human TF (LTF mice) to test the hypothesis that global TF deficiency attenuates lung inflammation in direct lung injury.. LTF mice were treated with 10 μg of lipopolysaccharide (LPS) or vehicle administered by direct intratracheal injection and studied at 24 h.. Contrary to our hypothesis, LTF mice had increased lung inflammation and injury as measured by bronchoalveolar lavage cell count (3.4×10(5) wild-type (WT) LPS vs 3.3×10(5) LTF LPS, p=0.947) and protein (493 μg/ml WT LPS vs 1014 μg/ml LTF LPS, p=0.006), proinflammatory cytokines (TNF-α, IL-10, IL-12, p<0.035 WT LPS vs LTF LPS) and histology compared with WT mice. LTF mice also had increased haemorrhage and free haemoglobin in the airspace accompanied by increased oxidant stress as measured by lipid peroxidation products (F(2) isoprostanes and isofurans).. These findings indicate that global TF deficiency does not confer protection in a direct lung injury model. Rather, TF deficiency causes increased intra-alveolar haemorrhage following LPS leading to increased lipid peroxidation. Strategies to globally inhibit TF may be deleterious in patients with ALI.

Topics: Acute Lung Injury; Analysis of Variance; Animals; Blotting, Western; Bronchoalveolar Lavage; Cytokines; Electrophoresis, Polyacrylamide Gel; Furans; Hemoglobins; Hemorrhage; Inflammation; Isoprostanes; Lipopolysaccharides; Mice; Mice, Knockout; Oxidative Stress; Pulmonary Alveoli; Real-Time Polymerase Chain Reaction; Statistics, Nonparametric; Thromboplastin

Although angiogenesis is viewed as a fundamental component of inflammatory bowel disease (IBD) pathogenesis, we presently lack a thorough knowledge of the cell type(s) involved in its induction and maintenance in the inflamed intestinal mucosa. This study aimed to determine whether platelet (PLT) adhesion to inflamed intestinal endothelial cells of human origin may favour angiogenesis. Unstimulated or thrombin-activated human PLT were overlaid on resting or tumour necrosis factor (TNF)-α-treated human intestinal microvascular endothelial cells (HIMEC), in the presence or absence of blocking antibodies to either vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, integrin α(v)β(3) , tissue factor (TF) or fractalkine (FKN). PLT adhesion to HIMEC was evaluated by fluorescence microscopy, and release of angiogenic factors (VEGF and soluble CD40L) was measured by ELISA. A matrigel tubule formation assay was used to estimate PLT capacity to induce angiogenesis after co-culturing with HIMEC. TNF-α up-regulated ICAM-1, α(v)β(3) and FKN expression on HIMEC. When thrombin-activated PLT were co-cultured with unstimulated HIMEC, PLT adhesion increased significantly, and this response was further enhanced by HIMEC activation with TNF-α. PLT adhesion to HIMEC was VCAM-1 and TF independent but ICAM-1, FKN and integrin α(v)β(3) dependent. VEGF and sCD40L were undetectable in HIMEC cultures either before or after TNF-α stimulation. By contrast, VEGF and sCD40L release significantly increased when resting or activated PLT were co-cultured with TNF-α-pre-treated HIMEC. These effects were much more pronounced when PLT were derived from IBD patients. Importantly, thrombin-activated PLT promoted tubule formation in HIMEC, a functional estimate of their angiogenic potential. In conclusion, PLT adhesion to TNF-α-pre-treated HIMEC is mediated by ICAM-1, FKN and α(v)β(3) , and is associated with VEGF and sCD40L release. These findings suggest that inflamed HIMEC may recruit PLT which, upon release of pro-angiogenic factors, actively contribute to inflammation-induced angiogenesis.

Topics: Blood Platelets; CD40 Ligand; Cells, Cultured; Chemokine CX3CL1; Coculture Techniques; Endothelial Cells; Enzyme-Linked Immunosorbent Assay; Female; Humans; Inflammation; Inflammatory Bowel Diseases; Integrin alphaVbeta3; Intercellular Adhesion Molecule-1; Intestines; Male; Microscopy, Fluorescence; Microvessels; Neovascularization, Pathologic; Platelet Adhesiveness; Thromboplastin; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1; Vascular Endothelial Growth Factor A

Increased cardiovascular mortality and risk of venous thromboembolism are serious extra-pulmonary complications of chronic obstructive pulmonary disease (COPD). Previously, circulating active tissue factor (TF) and factor XIa (FXIa) have been reported to be associated with acute coronary syndromes.. To measure plasma FXIa and active TF, prothrombin fragment 1.2 (F1.2), and markers of systemic inflammation (C-reactive protein [CRP], interleukin-6 [IL-6], tumor necrosis factor α [TNFα] and matrix metalloproteinase 9 [MMP-9]) in 60 patients with documented stable COPD free of previous thromboembolic events.. In-house clotting assays using inhibitory monoclonal antibodies against FXIa and TF.. FXIa was detected in 9 (15%) and TF activity in 7 (11.7%) COPD patients. Subjects positive for FXIa and/or TF (n=10; 16.7%) had higher F1.2 (median [interquartile range], 398 [216] vs 192 [42] pM, p<0.000001), fibrinogen (5.58 [2.01] vs 3.97 [2.47] g/L, p=0.0007), CRP (14.75 [1.20] vs 1.88 [2.95] mg/L, p<0.000001), IL-6 (8.14 [4.74] vs 2.45 [2.24] pg/mL, p=0.00002), and right ventricular systolic pressure (47 [15] vs 38 [12] mmHg, p=0.023), and lower vital capacity (66 [15] vs 80 [17] % predicted, p=0.04) than COPD patients without detectable FXIa and TF. COPD severity was not associated with the presence of circulating FXIa and active TF.. This is the first study to show that active FXIa and TF are present in stable COPD patients, who exhibit enhanced systemic inflammation and thrombin generation. Our findings suggest a new prothrombotic mechanism which might contribute to elevated risk of thromboembolic complications in COPD.

Topics: Aged; Factor XIa; Female; Humans; Inflammation; Male; Middle Aged; Pulmonary Disease, Chronic Obstructive; Thrombin; Thromboplastin

Bad pregnancy outcomes have been associated with increased activation of the coagulation cascade and inflammation, in particular the activation of the complement cascade. Recent studies have suggested that inflammatory processes modulate thrombogenic pathways and vice versa. We studied the cross-talk between the coagulation and the complement cascade in the pathogenesis of recurrent miscarriages and preeclampsia in mice. We identified tissue factor (TF) as a crucial mediator of fetal and placental damage in mouse models of recurrent miscarriages and preeclampsia. Increased TF expression increases the release of reactive oxygen species and antiangiogenic molecules from inflammatory cells inducing trophoblast damage and bad pregnancy outcomes. We also demonstrated that pravastatin, by downregulating TF expression, prevents miscarriages and the onset of preeclampsia in mice.

Topics: Abortion, Habitual; Animals; Anticholesteremic Agents; Blood Coagulation; Complement Activation; Complement System Proteins; Female; Humans; Inflammation; Mice; Pravastatin; Pre-Eclampsia; Pregnancy; Thromboplastin

Hepatocyte transplantation (HcTx) has proven to be a safe procedure, although the functional results have been unsatisfactory, probably due to insufficient engraftment or a loss of transplanted mass or function. In this study, we investigate whether hepatocytes in contact with blood induce an inflammatory reaction leading to, similar to what happens in clinical islet transplantation, an instant blood-mediated inflammatory reaction (IBMIR) resulting in an early loss of transplanted cells.. By using an experimental model that mimics the portal vein blood flow, we could study different parameters reflecting the effects on the innate immunity elicited by hepatocytes in contact with ABO-matched human blood.. We report that all aspects of the IBMIR such as platelet and granulocyte consumption, coagulation, and complement activation were demonstrated. Addition of various specific inhibitors of coagulation allowed us to clearly delineate the various stages of the hepatocyte-triggered IBMIR and show that the reaction was triggered by tissue factor. Analysis of a case of clinical HcTx showed that hepatocyte-induced IBMIR also occurs in vivo. Both the inflammatory and the coagulation aspects were controlled by low-molecular-weight dextran sulfate.. Isolated hepatocytes in contact with blood induce the IBMIR in vitro, and there are indications that these events are also relevant in vivo. According to these findings, HcTx would benefit from controlling a wider range of signals from the innate immune system.

Topics: ABO Blood-Group System; Blood Coagulation; Cells, Cultured; Dextran Sulfate; Hepatocytes; Humans; Immunohistochemistry; Inflammation; Postoperative Complications; Thromboplastin

Nutritional compounds that potentially limit inflammation and tissue factor expression may decrease the progression of chronic kidney disease (CKD) and associated cardiovascular disease. This project aimed to determine the effect of curcumin, bovine colostrum, and fish oil on inflammatory cytokine and tissue factor procoagulant activity of peripheral blood mononuclear cells (PBMCs) from patients with CKD before dialysis.. Peripheral blood mononuclear cells from patients with CKD before dialysis (n = 13) and age- and sex-matched healthy controls (n = 12) were cultured alone and with low and high doses of the nutritional compounds for 24 h. Cells were cultured with and without lipopolysaccharide. Supernatants were analyzed for tumor necrosis factor-α, interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, IL-1β, C-reactive protein, and tissue factor procoagulant activity.. The production of C-reactive protein, monocyte chemoattractant protein-1, IL-6, and IL-1β by PBMCs was inhibited by low- and high-dose fish oil in the CKD group (P < 0.05). Curcumin decreased secretion of IL-6 (P = 0.015) and IL-1 β (P = 0.016). Curcumin was more effective than colostrum at decreasing the procoagulant activity of PBMCs in the CKD and control groups (P < 0.019).. Fish oil decreased inflammatory cytokine secretion from CKD PBMCs. In addition, the beneficial effects of curcumin were demonstrated in decreasing inflammation in vitro, often to a similar magnitude as fish oil.

Topics: Adult; Animals; Anti-Inflammatory Agents; C-Reactive Protein; Case-Control Studies; Cattle; Chemokine CCL2; Colostrum; Curcuma; Curcumin; Female; Fish Oils; Humans; Inflammation; Inflammation Mediators; Interleukin-1beta; Interleukin-6; Kidney Failure, Chronic; Leukocytes, Mononuclear; Lipopolysaccharides; Male; Middle Aged; Phytotherapy; Thromboplastin

Our objectives were to characterize sex differences during venous thrombosis, using the electrolytic inferior vena cava model of the disease.. Male and female C57BL/6 mice (6-8 weeks) underwent inferior vena cava thrombosis. Time points included 6 hours, day 2, day 6, and day 14 post surgery, along with surgically naïve true controls and surgical shams. Analyses included thrombus weight, vein wall morphometrics, vein wall protein and gene expression for P-selectin, interleukin-1β, and tumor necrosis factor-α; hematology, soluble P-selectin, and plasma microparticle tissue factor activity assays.. Male venous thrombi were significantly larger than females at days 2 (13.1 ± 1.0 vs. 6.8 ± 0.5 × 10(-3) grams, p < 0.01), 6 (10.4 ± 0.8 vs. 5.4 ± 0.5 × 10(-3) grams, p < 0.01) and 14 (6.3 ± 0.5 vs. 4.1 ± 0.3 × 10(-3) grams, p < 0.01). Both male and female mice exhibited significantly increased vein wall P-selectin at 6 hours, vs. true controls (p < 0.05). Males had increased vein wall interleukin-1β, versus females, at 6 hours (180.926 ± 24.596 vs. 60.417 ± 10.478 pg/mL, p < 0.05) and day 6 (76.966 ± 13.081 vs. 33.834 ± 4.198 pg/mL, p < 0.01). Males showed decreased tumor necrosis factor-α expression (-66 %) at 6 hours. Females had increased tumor necrosis factor-α expression at 6 hours (+541%) and day 6 (+539%). Both sexes demonstrated decreased peripheral platelets at 6 hours (p < 0.05), coinciding with thrombogenesis. Plasma P-selectin increased in both sexes, versus controls, through day 6 (p < 0.05).. Males had significantly larger venous thrombi than females. Sex differences in vascular anatomy and response to inflammation may influence thrombus formation in our mouse thrombosis model.

Topics: Animals; Disease Models, Animal; Female; Gene Expression; Inflammation; Interleukin-1beta; Male; Mice; Mice, Inbred C57BL; P-Selectin; Sex Factors; Thromboplastin; Thrombosis; Tumor Necrosis Factor-alpha; Veins; Venous Thrombosis

In islet transplantation, tissue factor (TF) has been reported to be involved in triggering the instant blood-mediated inflammatory reaction (IBMIR), which causes early massive loss of islets transplanted intraportally. TF is synthesized and secreted by several cell sources including islets and inflammatory cells such as neutrophils, monocytes, and platelets. In this study, we investigated whether xenografts-mediated IBMIR could be inhibited by selectively inhibiting TF production by islets using small interfering RNA (siRNA)-mediated TF gene knockdown.. Porcine neonatal islet cell clusters (NICC) were transfected with siRNA specific for TF or a nonspecific siRNA. TF gene and protein expression were analyzed by real-time polymerase chain reaction and fluorescence-activated cell sorting, respectively. The effect of TF knockdown on IBMIR was evaluated using an in vitro tubing loop model of human blood-NICC interactions.. TF siRNA transfection of NICC resulted in reduced TF gene and protein expression. TF siRNA transfected NICC showed a significant reduction in the formation of blood clots, platelet activation, thrombin generation, and complement activation after exposure to human ABO compatible blood in vitro. In addition, there was reduced neutrophil infiltration within blood clots containing TF siRNA transfected NICC.. TF expression on porcine NICC is an important initiator of IBMIR in islet xenotransplantation. This study identifies porcine TF as a potential target for inhibiting this response.

Topics: Animals; Animals, Newborn; Blood Coagulation; Cell Separation; Cell Survival; Flow Cytometry; Humans; Inflammation; Islets of Langerhans; Islets of Langerhans Transplantation; Neutrophil Infiltration; Polymerase Chain Reaction; RNA Interference; Swine; Thromboplastin; Time Factors; Tissue Culture Techniques; Transfection; Transplantation, Heterologous

Instant blood-mediated inflammatory reaction (IBMIR) causes rapid islet loss in islet transplantation. Endothelial colony-forming cells (ECFCs) display unique abilities to promote angiogenesis and repair vascular injury compared to those of endothelial cells (ECs), which inhibits the allogeneic and xenogeneic IBMIR. We investigated the coating of pig islets with ex vivo-expanded ECFCs as a strategy to overcome xenogeneic IBMIR. Porcine islets were cocultured with human ECFCs in a specially modified culture medium for 2 days to obtain 70-90% coverage. The coating of pig islets with human ECFCs did not affect the glucose-stimulated insulin secretion capacity or diabetes reversal rate after the transplantation of a marginal islet mass under the kidney capsules of diabetic nude mice compared to that of untreated islets. Uncoated islets, PBS control without islets, and the ECFC-coated islets were examined with an in vitro tubing loop assay using human blood. After 60 min of incubation in human blood, the ECFC-coated islets showed platelet consumption inhibition and low C3a and TAT assay results compared to those of the uncoated islets. Furthermore, there was very little macroscopic or microscopic clotting in the human ECFC-coated pig islets. The protective effect was more prominent compared to that of human EC coating of pig islets in our previous study. We investigated the changes in human-specific MCP-1, IL-8, and tissue factor (TF) levels after the coating of pig islets with human ECFCs or human ECs. The IL-8 levels after coating pig islets with ECFCs were significantly lower than those after coating pig islets with ECs, but there were no significant differences in the MCP-1 or TF levels between the ECFCs and ECs. In conclusion, the coating of pig islets with ECFCs completely prevented all components of xenogeneic IBMIR. ECFCs may be a better source of protection against xenogeneic IBMIR than are mature ECs.

Topics: Animals; Antithrombin III; Chemokine CCL2; Coculture Techniques; Complement C3a; Endothelial Cells; Fetal Blood; Humans; Inflammation; Insulin; Interleukin-8; Islets of Langerhans; Islets of Langerhans Transplantation; Mice; Mice, Inbred BALB C; Mice, Nude; Peptide Hydrolases; Sus scrofa; Thromboplastin; Transplantation, Heterologous

Bullous pemphigoid (BP) is a skin disease caused by autoantibodies to hemidesmosomal proteins BP180 and BP230, with eosinophils participating in blister formation. Tissue factor (TF), the initiator of coagulation, is embodied within the eosinophil granules and exposed upon activation. We evaluated the coagulation activation in patients with BP (63), chronic urticaria (CU; 20), atopic dermatitis (AD; 14), cutaneous drug reactions (CDRs; six), psoriasis (20), dermatitis herpetiformis (DH; four) and primary cutaneous T cell lymphoma (CTCL; five), and in 40 healthy controls. Prothrombin fragment F1+2 and d-dimer (coagulation markers) were measured by enzyme-linked immunosorbent assay (ELISA) in all plasma samples and BP blister fluid. Skin TF expression was evaluated immunohistochemically in the patients and 20 controls. F1+2 and d-dimer levels were higher in BP plasma than in control plasma (P = 0·0001 for both), and dramatically high in blister fluid; both correlated positively with disease severity, esinophil counts and anti-BP180 antibodies (P = 0·006-0·0001). Plasma F1+2 and d-dimer levels were higher in the CU, AD and CDR patients than in controls (P = 0·0001 for all), but normal in the psoriasis, DH and CTCL patients. Skin TF was expressed in the BP (P = 0·0001), CU (P = 0·0001), AD (P = 0·001) and CDR patients (P = 0·01), but not in the psoriasis, DH or CTCL patients. Co-localization confocal microscopy studies confirmed eosinophils as the source of TF in 10 BP patients. The coagulation cascade is activated in BP and other eosinophil-mediated skin disorders, but not in non-eosinophil driven conditions. This hypercoagulability may contribute to inflammation, tissue damage and, possibly, thrombotic risk.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Autoantibodies; Autoantigens; Blood Coagulation; Carrier Proteins; Cell Count; Collagen Type XVII; Cytoskeletal Proteins; Disease Progression; Dystonin; Eosinophils; Female; Fibrin Fibrinogen Degradation Products; Humans; Inflammation; Lymphoma, T-Cell, Cutaneous; Male; Membrane Glycoproteins; Middle Aged; Nerve Tissue Proteins; Non-Fibrillar Collagens; Pemphigoid, Bullous; Peptide Fragments; Prothrombin; Skin Neoplasms; Thromboplastin

Thrombosis is initiated by tissue factor (TF), a coagulation cofactor/receptor expressed in the vessel wall, on myeloid cells, and on microparticles (MPs) with variable procoagulant activity. However, the molecular pathways that generate prothrombotic TF in vivo are poorly defined. The oxidoreductase protein disulfide isomerase (PDI) is thought to be involved in the activation of TF. Here, we found that in mouse myeloid cells, ATP-triggered signaling through purinergic receptor P2X, ligand-gated ion channel, 7 (P2X7 receptor; encoded by P2rx7) induced activation (decryption) of TF procoagulant activity and promoted release of TF+ MPs from macrophages and SMCs. The generation of prothrombotic MPs required P2X7 receptor-dependent production of ROS leading to increased availability of solvent-accessible extracellular thiols. An antibody to PDI with antithrombotic activity in vivo attenuated the release of procoagulant MPs. In addition, P2rx7-/- mice were protected from TF-dependent FeCl3-induced carotid artery thrombosis. BM chimeras revealed that P2X7 receptor prothrombotic function was present in both hematopoietic and vessel wall compartments. In contrast, an alternative anti-PDI antibody showed activities consistent with cellular activation typically induced by P2X7 receptor signaling. This anti-PDI antibody restored TF-dependent thrombosis in P2rx7-/- mice. These data suggest that PDI regulates a critical P2X7 receptor-dependent signaling pathway that generates prothrombotic TF, defining a link between inflammation and thrombosis with potential implications for antithrombotic therapy.

Topics: Adenosine Triphosphate; Animals; Bone Marrow Transplantation; Cell-Derived Microparticles; Enzyme Inhibitors; Inflammation; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Myeloid Cells; NADPH Oxidases; Protein Disulfide-Isomerases; Receptors, Purinergic P2X7; Signal Transduction; Thromboplastin; Thrombosis

The acute-phase protein serum amyloid A (SAA) is elevated during inflammation and may be deposited in atheroma where it promotes atherosclerosis. We investigated the proatherogenic effects of SAA on the vascular endothelium and their regulation by high-density lipoprotein (HDL). Exposure of human aortic endothelial cells (HAEC) to SAA (0.25-25μg/ml) decreased nitric oxide ((•)NO) synthesis/bioavailability, although the endothelial NO synthase monomer-to-dimer ratio was unaffected. SAA (10μg/ml) stimulated a Ca(2+) influx linked to apocynin-sensitive superoxide radical anion (O(2)(•-)) production. Gene expression for arginase-1, nuclear factor κB (NF-κB), interleukin-8, and tissue factor (TF) increased within 4h of SAA stimulation. Enzymatically active Arg-1/2 was detected in HAEC cultured with SAA for 24h. Therefore, in addition to modulating (•)NO bioavailability by stimulating O(2)(•-) production in the endothelium, SAA modulated vascular l-Arg bioavailability. SAA also diminished relaxation of preconstricted aortic rings induced by acetylcholine, and added superoxide dismutase restored the vascular response. Preincubation of HAEC with HDL (100 or 200, but not 50, μg/ml) before (not after) SAA treatment ameliorated the Ca(2+) influx and O(2)(•-) production; decreased TF, NF-κB, and Arg-1 gene expression; and preserved overall vascular function. Thus, SAA may promote endothelial dysfunction by modulating (•)NO and l-Arg bioavailability, and HDL pretreatment may be protective. The relative HDL to SAA concentrations may regulate the proatherogenic properties of SAA on the vascular endothelium.

Topics: Animals; Aorta; Arginase; Atherosclerosis; Blotting, Western; Calcium; Endothelial Cells; Endothelium, Vascular; Gene Expression; Humans; Inflammation; Lipoproteins, HDL; Male; NF-kappa B; Nitric Oxide; Rats; Rats, Wistar; Serum Amyloid A Protein; Signal Transduction; Superoxide Dismutase; Superoxides; Thromboplastin

A high mortality occurs in dogs with idiopathic immune-mediated haemolytic anaemia (IMHA) during the first 2 weeks after the diagnosis. The aim of this study was to investigate the inflammatory response and coagulation abnormalities in dogs with IMHA in relation to the prognosis and to establish the contribution of whole blood tissue factor (TF) and IL-8 gene expressions. Gene expressions in dogs with IMHA were compared to healthy dogs, dogs with DIC, dogs with sepsis, and in two groups of dogs that underwent intensive care treatment but had no evidence for either DIC or sepsis. The whole blood TF and IL-8 expressions were up regulated in all non-IMHA groups. Similarly, the TF expression in IMHA dogs was high, but the intravascular IL-8 expression was not increased. The dogs with IMHA had a pronounced inflammatory response that included a high WBC, left shift and monocytosis in comparison to the other disease groups. Coagulation factor activities in IMHA dogs were decreased fitting consumptive coagulopathy and the acute phase proteins FVIII and fibrinogen were increased. The platelet parameters suggested platelet activation and high platelet turnover in IMHA dogs. The model that best explained mortality contained monocytosis, increased activated partial thromboplastin time and elevated creatinine. Whole blood TF gene expression is up regulated and may contribute to consumptive coagulopathy in dogs with IMHA. Increased TF expression by activated platelets is an alternative explanation and should be investigated.

Topics: Anemia, Hemolytic, Autoimmune; Animals; Blood Coagulation; Disseminated Intravascular Coagulation; Dog Diseases; Dogs; Female; Inflammation; Interleukin-8; Male; Prognosis; Real-Time Polymerase Chain Reaction; Sepsis; Thromboplastin

Tissue factor, the initiator of the coagulation cascade, mediates coagulation factor VIIa-dependent activation of protease-activated receptor 2 (PAR2). Here we delineate a role for this signaling pathway in obesity and its complications. Mice lacking PAR2 (F2rl1) or the cytoplasmic domain of tissue factor were protected from weight gain and insulin resistance induced by a high-fat diet. In hematopoietic cells, genetic ablation of tissue factor-PAR2 signaling reduced adipose tissue macrophage inflammation, and specific pharmacological inhibition of macrophage tissue factor signaling rapidly ameliorated insulin resistance. In contrast, nonhematopoietic cell tissue factor-VIIa-PAR2 signaling specifically promoted obesity. Mechanistically, adipocyte tissue factor cytoplasmic domain-dependent VIIa signaling suppressed Akt phosphorylation with concordant adverse transcriptional changes of key regulators of obesity and metabolism. Pharmacological blockade of adipocyte tissue factor in vivo reversed these effects of tissue factor-VIIa signaling and rapidly increased energy expenditure. Thus, inhibition of tissue factor signaling is a potential therapeutic avenue for improving impaired metabolism and insulin resistance in obesity.

Topics: Adipose Tissue; Animals; Bone Marrow Transplantation; Diet, High-Fat; Factor VIIa; Glucose; Inflammation; Insulin Resistance; Lipid Metabolism; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Obesity; Receptor, PAR-2; Signal Transduction; Thromboplastin; Transplantation Chimera

Topics: Adipose Tissue; Animals; Diet, High-Fat; Inflammation; Male; Obesity; Receptor, PAR-2; Signal Transduction; Thromboplastin

Altered hepatic lipid homeostasis, hepatocellular injury, and inflammation are features of nonalcoholic steatohepatitis, which contributes significantly to liver-related morbidity and mortality in the Western population. A collection of inflammatory mediators have been implicated in the pathogenesis of steatohepatitis in mouse models. However, the pathways essential for coordination and amplification of hepatic inflammation and injury caused by steatosis are not completely understood. We tested the hypothesis that tissue factor (TF)-dependent thrombin generation and the thrombin receptor protease activated receptor-1 (PAR-1) contribute to liver inflammation induced by steatosis in mice. Wild-type C57Bl/6J mice fed a diet deficient in methionine and choline for 2 weeks manifested steatohepatitis characterized by increased serum alanine aminotransferase activity, macrovesicular hepatic steatosis, hepatic inflammatory gene expression, and lobular inflammation. Steatohepatitis progression was associated with thrombin generation and hepatic fibrin deposition. Coagulation cascade activation was significantly reduced in low TF mice, which express 1% of normal TF levels. Hepatic triglyceride accumulation was not affected in low TF mice or PAR-1-deficient mice. In contrast, biomarkers of hepatocellular injury, inflammatory gene induction, and hepatic accumulation of macrophages and neutrophils were greatly reduced by TF-deficiency and PAR-1-deficiency. The results suggest that TF-dependent thrombin generation and activation of PAR-1 amplify hepatic inflammation and injury during the pathogenesis of steatohepatitis.

Topics: Alanine Transaminase; Animals; Blood Coagulation; Blood Coagulation Factors; Chemokine CCL2; Choline; Diet; Fatty Liver; Gene Expression Regulation; Inflammation; Liver; Methionine; Mice; Mice, Inbred C57BL; Models, Biological; Receptor, PAR-1; Thrombin; Thromboplastin; Triglycerides

To overcome the limitations of balloon expandible metal stent-induced neointimal smooth muscle cell proliferation, drug-coated stent devices have been developed. Drug eluting stents release high concentrations of antiproliferative agents, such as paclitaxel, to reduce neointimal hyperplasia. The proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), is known to cause severe endothelial dysfunction and accelerate atherosclerotic lesion progression. The interaction of TNF-alpha and paclitaxel on the release of prothrombotic molecules was examined in endothelial cells. Treatment of endothelial cells with paclitaxel had no direct effect on tissue factor (TF) expression, but TNF-alpha increased TF. Cotreatment of paclitaxel with TNF-alpha markedly augmented the release of TF. TNF-alpha induced release of plasminogen activator inhibitor but no synergism occurred with paclitaxel. Treatment of endothelial cells with paclitaxel and TNF-alpha reduced expression of thrombomodulin and protein C receptor. Tissue factor pathway inhibitor expression was reduced by prolonged treatment with either paclitaxel or TNF-alpha. The adhesion molecule, CD62 E, was induced by TNF-alpha; however, CD31, CD62 P, and CD106 were not affected by paclitaxel and TNF-alpha. Apoptosis was not observed with cotreatment of endothelial cells with paclitaxel and TNF-alpha. CD59-positive microparticles were released in response to TNF-alpha, but the release was not augmented by paclitaxel. Paclitaxel and TNF-alpha increased the nitrotyrosination of proteins. These findings indicate that paclitaxel enhances TNF-alpha-induced release of TF, and downregulated thrombomodulin, increased protein nitration, which may subsequently favor prothrombotic intimal surface.

Topics: Cells, Cultured; Cytokines; Down-Regulation; Endothelial Cells; Endothelium, Vascular; Gene Expression Regulation; Humans; Inflammation; Nitrates; Paclitaxel; Plasminogen Inactivators; Proteins; Thrombomodulin; Thromboplastin; Tubulin Modulators; Tumor Necrosis Factor-alpha

The metabolic syndrome (MetS) is a cluster of cardiovascular risk factors closely linked to inflammation and insulin resistance (IR). Tissue factor (TF) is an initiator of the extrinsic coagulation cascade and is expressed on peripheral blood monocytes and macrophages in atherosclerotic plaques. Monocytes are the principle cells capable of TF synthesis. Therefore, TF plays an important role in both thrombosis and atherosclerosis. Elevated levels of lipopolysaccharide (LPS), a strong stimulator of TF, have been observed in patients with MetS. No study has investigated the relationship between monocyte TF activity and inflammation, and IR in MetS.. Peripheral blood mononuclear cells (PBMCs) were collected from 40 normal subjects and 77 patients with MetS. Mononuclear cell TF procoagulant activity (MPCA) was measured with and without 100 pg/ml LPS stimulation using a 1-stage clotting assay and expressed as the mean +/- SD (mU TF/10(6) PBMCs). MPCA in MetS was significantly greater than in normal subjects (without LPS: 88.0+/-74.8 vs 52.6+/-9.8 mU TF/10(6) PBMCs, P<0.001; with LPS: 269.6+/-165.6 vs 158.6+/-42.8 mU TF/10(6) PBMCs, P<0.001). The LPS-stimulated log MPCA in MetS patients was significantly associated with homeostasis model assessment of IR (r=0.256, P=0.024) and log high-sensitivity C-reactive protein (r=0.332, P=0.003).. Upregulation of monocyte TF is significantly associated with low-grade inflammation and IR in MetS.

Topics: Aged; C-Reactive Protein; Chronic Disease; Disease Progression; Female; Homeostasis; Humans; In Vitro Techniques; Inflammation; Insulin Resistance; Lipopolysaccharides; Male; Metabolic Syndrome; Middle Aged; Monocytes; Risk Factors; Thromboplastin; Up-Regulation

In clinical islet transplantation, inflammatory responses initiated by the transplanted islets and by the host immune system cause acute and chronic graft loss. The resolution of acute inflammation is an active process mediated by specific signals and mediators such as resolvin E1 (RvE1). We investigated the effect of RvE1 on i) the inflammatory status of human pancreatic islets, ii) islet viability and apoptosis, and iii) the instant blood-mediated inflammatory reaction (IBMIR) IN VITRO.. Pro-inflammatory cytokines and tissue factor (TF) in isolated human islets were determined by real-time RT-qPCR (mRNA levels), CBA and Gyrolab bioaffy (protein levels) after lipopolysaccaride (LPS) stimulation. Islet viability was measured using insulin secretion in a dynamic model, ADP/ATP ratio and total ATP content. Apoptosis was measured using commercial kits after stimulation with proinflammatory cytokines. To assess effect on IBMIR, human islets were mixed with non-anticoagulated, RvE1 or vehicle pretreated ABO-compatible blood in heparin-coated tubing loops.. Treatment of human islets with RvE1 (500 nM) for 24 h reduced LPS-induced increase in mRNA and protein levels of selected pro-inflammatory markers (IL-8, MCP-1, and TF). RvE1 lowered the ADP/ATP ratio, but had no effect on insulin secretion. RvE1 reduced the apoptotic effect of proinflammatory cytokines. Additionally, RvE1 reduced platelet consumption and TAT complex formation during the first 5 min after islet-blood contact.. RvE1 suppresses proinflammatory markers and lowers the ADP/ATP ratio in human islets IN VITRO. RvE1 demonstrates anti-apoptotic effects in a proinflammatory milieu. Additionally, RvE1 has modest dampening effects on IBMIR. We conclude that RvE1 may have potential in clinical islet transplantation.

Topics: Analysis of Variance; Apoptosis; Biomarkers; Cell Survival; Cytokines; Eicosapentaenoic Acid; Humans; Immunoenzyme Techniques; Inflammation; Inflammation Mediators; Insulin; Insulin Secretion; Islets of Langerhans; Organ Culture Techniques; Receptors, Leukotriene B4; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thromboplastin

Sickle cell anemia is accompanied by the activation of coagulation and thrombosis. We have studied the abnormal expression of tissue factor (TF) by the pulmonary vein endothelium of the mild-phenotype NY1DD sickle transgenic. As detected by immunofluorescence microscopy, this occurs only after the NY1DD mouse is exposed to hypoxia/reoxygenation (H/R), which actually causes ischemia/reperfusion in the sickle cell disease-but not the normal-mouse model. We tested the hypothesis that the nuclear factor-kappa B (NFkappaB)-activating inflammation that develops in post-H/R NY1DD mice is responsible for this phenotype switch. Various NFkappaB inhibitors (including p50-specific andrographolide) demonstrated that endothelial TF positivity is NFkappaB dependent. Several systemic inflammatory stimulators (tumor necrosis factor [TNFalpha], lipopolysaccharide, thioglycollate, and carageenan) given to control mice showed that the inflammatory promotion of TF expression by only pulmonary vein endothelium is not specific to the sickle cell disease model. We bred the NFkappaB(p50)-/- state into the NY1DD mouse. Combined with marrow transplantation, this allowed the creation of NY1DD mice that were NFkappaB(p50)-/- only in peripheral blood cells (and marrow) versus only in vessel walls (and tissues). This process revealed that endothelial TF expression in the NY1DD mouse is highly dependent on NFkappaB(p50) in peripheral blood mononuclear cells-but not in the vessel wall. In confirmation, the infusion of post-H/R sickle mouse blood mononuclear cells into naïve NY1DD mice stimulated endothelial TF expression; the infusion of such cells from unstimulated sickle cell disease mice at ambient air did not stimulate TF expression. We conclude that peripheral blood mononuclear cells indirectly promote endothelial TF expression via a NFkappaB(p50)-dependent mechanism. This approach may be relevant to the role of coagulopathy in clinical sickle cell disease.

Topics: Anemia, Sickle Cell; Animals; Blood Coagulation; Endothelium, Vascular; Gene Knockout Techniques; Humans; Inflammation; Leukocytes, Mononuclear; Mice; Mice, Transgenic; NF-kappa B p50 Subunit; Thromboplastin

Topics: Animals; Blood Coagulation; Blood Platelets; Bradykinin; Calcium; Cytoplasmic Granules; Enzyme Activation; Factor XII; Factor XII Deficiency; Hermanski-Pudlak Syndrome; Humans; Inflammation; Models, Biological; Polyphosphates; Prokaryotic Cells; Thrombophilia; Thromboplastin; Thrombosis

Atrial fibrillation (AF) is a common complication of coronary artery bypass grafting (CABG). We sought to determine the diagnostic validity of plasma biomarkers of i) inflammation (marked by interleukin-6 [IL-6] and high-sensitivity C-reactive protein [hs-CRP]), ii) extracellular matrix remodelling (matrix metalloproteinase [MMP-9], tissue inhibitor of matrix metalloproteinase [TIMP-1]) and iii) the prothrombotic state (tissue factor and von Willebrand factor [vWF]) in the risk prediction of post-operative AF. Samples were obtained preoperatively from peripheral/femoral vein and from intracardiac chambers (right atrium [RA], the right atrial appendage [RAA], the left atrium [LA] and the left atrial appendage [LAA]) amongst 100 consecutive patients free of AF and inflammatory disease undergoing elective CABG. Biomarker concentrations were related to incident AF (30 days). At 30 days post CABG, 30 patients were proven to have had AF. Concentrations of tissue factor (TF) and vWF were unrelated to postoperative AF. Peripheral (p=0.018), and intracardiac levels (RAA (p=0.029) and LA (p=0.026)) of hs-CRP were associated with the presence of AF after CABG. Intracardiac levels of IL-6 in samples from the RAA (p=0.031), LA (p=0.042) and LAA (p=0.006), and MMP-9 in the LAA sample were also associated with AF (p=0.007). Our data suggest that an intra-cardiac inflammatory environment that is manifest peri-operatively may predispose to the development of post-operative AF. This intracardiac inflammatory state was reflected by increased peripheral hs-CRP levels. These differences may indicate local substrate abnormalities contributing to the development of AF post-operatively.

Topics: Aged; Atrial Fibrillation; Biomarkers; C-Reactive Protein; Coronary Artery Bypass; Coronary Artery Disease; Female; Humans; Inflammation; Interleukin-6; Male; Matrix Metalloproteinase 9; Middle Aged; Postoperative Complications; Predictive Value of Tests; Prognosis; Reproducibility of Results; Thromboplastin

Endothelial progenitor cells (EPC) are good candidates for cell-based therapy in cardiovascular diseases. However, concerns have been raised about the potential risks of EPC-based cell therapy, in terms of thrombogenicity particularly in inflammatory conditions, currently observed in such patients. Tissue factor (TF) can trigger coagulation and may support thrombogenicity. TF is also a key receptor in angiogenesis.. The present study was designed to (i) evaluate the capacity of resting and tumour necrosis factor-alpha (TNF)-α-stimulated late-outgrowth endothelial colony-forming cells (ECFCs) to express TF and (ii) investigate the effect of TF/FVII(a) interaction on procoagulant and non-procoagulant activities of ECFCs in vitro.. ECFCs from cord blood (cb) and adult peripheral blood (ab) were analyzed for TF expression and activity using reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry, Western blot and a thrombin generation assay. Non-procoagulant properties of TF-expressing ECFCs were investigated in vitro using wound-healing, cell proliferation, tube formation and spheroid-based assays.. ECFCs expressed TF in response to TNF-α. The up-regulation of TF conferred to ECFCs a FVII(a)-dependent thrombin generation activity. Compared with cb-ECFC, ab-ECFCs can display a higher level of constitutive TF expression and activity, with a notable heterogeneity among donors. TF/FVIIa interaction did not modify non-procoagulant properties of TNF-α stimulated cb-ECFCs in vitro.. Proinflammatory conditions up-regulate TF expression in ECFCs. This expression confers to ECFCs a strong thrombin generation capacity without influencing their non-coagulant properties. Our results suggest that EPC-based cell therapy may be associated with prothrombotic risk which could be limited by inhibiting TF without affecting the proangiogenic capacity of the cells.

Topics: Blotting, Western; Cell Proliferation; Coagulants; Endothelium, Vascular; Factor VII; Fetal Blood; Flow Cytometry; Humans; Inflammation; Risk; Thrombin; Thromboplastin; Thrombosis; Tumor Necrosis Factor-alpha; Up-Regulation; Wound Healing

Platelets have been implicated in the pathogenesis of liver damage after orthotopic liver transplantation (OLT). Early graft dysfunction is frequently caused by reperfusion injury subsequent to cold ischemia (IRI). Therefore, we investigated activation of the pivotal haemostatic cells, platelets and monocytes, from patients with elevated markers of IRI and from patients with uneventful course (control-group), respectively during the first week after OLT. Flow cytometry analysis of citrate anticoagulated blood samples revealed that platelets from IRI patients became significantly activated within 48 h after OLT in vivo, with increased surface presentation of P-selectin, CD40L, thrombospondin-1 and tissue-factor. Platelet activation in IRI patients on post-transplant day 2 was accompanied by significantly enhanced tissue-factor expression on peripheral blood monocytes, significant elevated levels of C-reactive protein and hepatocellular damage. Towards post-transplant day 4, levels of platelet-derived microparticles rose significantly in IRI patients if contrasted to control patients. Thus, activated cellular haemostasis is involved in the early inflammatory response of hepatocellular damage subsequent to reperfusion of the transplanted liver. Targeting distinct activation patterns of platelets and monocytes in an early phase of hepatic grafting may counteract the extent of IRI via inhibition of micro-thrombus formation and inflammation without exacerbating the existing bleeding risk.

Topics: Adult; Blood Platelets; Case-Control Studies; Female; Humans; Inflammation; Liver; Liver Diseases; Liver Transplantation; Male; Middle Aged; Monocytes; Platelet Activation; Prospective Studies; Reperfusion Injury; Thromboplastin

Shear stress has an established effect on mature endothelial cells, but less is known about how shear stress regulates endothelial progenitor cells (EPCs). In vitro expanded EPCs isolated from adult human blood represent a novel tool in regenerative vessel therapy. However, in vitro culturing may generate cells with unfavourable properties. The aim of the present study was therefore to assess whether shear stress may influence the inflammatory and thrombotic phenotype of in vitro expanded EPCs. In late outgrowth EPCs, 6 hours of shear stress (6.0 dynes/cm2) significantly reduced the mRNA levels of IL-8, COX2, and tissue factor (TF) compared to static controls. This was associated with a reduced TF activity. In contrast, mRNA expression of NOS3 was significantly increased following 6 and 24 hours of shear stress. In accordance with this, NOS3 protein expression was increased following 24 hours of shear stress. Overall stimulation with the proinflammatory mediator, TNFalpha, for the final 2 hours increased the mRNA expression of IL-6, IL-8, MCP-1, ICAM1, and TF. However exposure to 6 hours of shear stress significantly suppressed the inductory potential of TNFalpha to increase the mRNA levels of IL-6, IL-8, COX2, and TF. Additionally, TNFalpha increased TF activity approximately 10 times, an effect that was also significantly reduced by exposure to 6 and 24 hours of shear stress. The effect of shear on the gene levels of TF and NOS3 were not blocked by the NOS inhibitor L-NAME. These observations suggest that EPCs are capable of functionally responding to shear stress.

Topics: Cells, Cultured; Chemokine CCL2; Cyclooxygenase 2; Endothelial Cells; Enzyme Inhibitors; Gene Expression Regulation; Humans; Inflammation; Inflammation Mediators; Intercellular Adhesion Molecule-1; Interleukin-6; Interleukin-8; Mechanotransduction, Cellular; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type III; RNA, Messenger; Stem Cells; Stress, Mechanical; Thromboplastin; Thrombosis; Time Factors; Tumor Necrosis Factor-alpha

Within atherosclerotic lesions Tissue Factor (TF)-Factor VIIa (FVIIa) not only contributes to thrombotic events but also alters vascular remodeling through enhancement of migration. Moreover, the TF-FVIIa-FXa complex activates protease-activated receptors (PAR). TF/FVIIa/PAR-2 signaling has also been shown to promote proliferation and metastasis of tumor cells. Since coagulation factors promote inflammation which plays a major role during atherosclerosis as well as tumor metastasis this study sought to investigate the effects of FVIIa on the inflammatory response in vascular cells.. FVIIa induces interleukin-8 (IL-8) and IL-6 in primary smooth muscle cells (SMC), which was correlated to the expression of TF and PAR-2 as shown by immunoassay and qRT-PCR. The effect was dose-dependent and required TF, the proteolytic activity of FVIIa and PAR-2. Secondary effects of downstream coagulation factors were excluded. No proinflammatory FVIIa effect was observed in endothelial cells (EC) and mononuclear cells (MNC), expressing either TF or PAR-2. In atherosclerotic lesions mRNA expression of PAR-1, PAR-2 and IL-8 was elevated compared to healthy vessels indicating a role for PAR-1 and PAR-2 signaling in atherosclerosis.. In addition to the procoagulant and promigratory role of the TF-FVIIa complex we identify a proinflammatory role of FVIIa in human SMC dependent on expression of TF and PAR-2 that provides yet another link between coagulation and inflammation.

Topics: Blood Coagulation; Cell Membrane; Coronary Vessels; Cytokines; Factor VIIa; Gene Expression Regulation; Humans; Inflammation; Interleukin-6; Interleukin-8; Myocytes, Smooth Muscle; Receptor, PAR-1; Receptor, PAR-2; RNA Interference; Thromboplastin

Topics: Animals; Cell Movement; Factor VIIa; Genetic Predisposition to Disease; Hemophilia A; Hemorrhage; Inflammation; Leukocytes; Mice; Mice, Inbred C57BL; Recombinant Proteins; Thrombocytopenia; Thromboplastin; Wound Healing

Complement 5a factor (C5a) elicits a broad range of proinflammatory effects, including chemotaxis of inflammatory cells and cytokine release. C5a is also linked to the coagulant activity in autoimmune diseases. Therefore, C5a most likely plays a crucial role in the instant blood-mediated inflammatory reaction.. Intraportal transplantation of 2.5 islet equivalents/g of syngeneic rat islet grafts was performed in two groups of streptozotocin-induced diabetic rats: controls and C5a inhibitory peptide (C5aIP)-treated group.. The thrombin-antithrombin complex was significantly suppressed in the C5aIP group (P=0.003), and both the curative rate and the glucose tolerance were significantly improved in the C5aIP group (P<0.05 and P<0.005, respectively). Expression of tissue factor on granulocytes in recipient livers was up-regulated 1 h after islet infusion (P<0.0001), which was significantly suppressed by C5aIP (P<0.005). However, C5aIP was unable to regulate tissue factor expression on isolated islets. Furthermore, no differences were detected between the groups, regarding infiltration of CD11b-positive cells and deposition of C5b-9 on the islet grafts.. These data suggest that C5aIP attenuates cross-talk between the complement and coagulation cascades through suppressing up-regulation of tissue factor expression on leukocytes in recipient livers but not on islet grafts, a process leading to improvement in islet engraftment. Therefore, C5aIP in combination with conventional anticoagulants could be a strong candidate strategy to control the instant blood-mediated inflammatory reaction induced in clinical islet transplantation.

Topics: Animals; Anticoagulants; Antithrombins; Blood Coagulation; Complement C5a; Complement System Proteins; Diabetes Mellitus, Experimental; Granulocytes; Inflammation; Islets of Langerhans Transplantation; Liver; Portal System; Rats; Rats, Inbred Lew; Thrombin; Thromboplastin; Transplantation, Isogeneic; Treatment Outcome; Up-Regulation

C-reactive protein (CRP), the prototypic marker of inflammation, is a cardiovascular risk marker and recent in vitro studies suggest that it may promote atherogenesis. CRP promotes oxidative stress in vitro and induces tissue factor (TF) release. However, there is a paucity of data examining the effects of CRP on oxidative stress and tissue factor procoagulant activity (PCA) in vivo. Thus, we tested the effects of CRP administration on superoxide anion release and tissue factor activity and examined mechanistic pathways using a rat sterile air pouch model. Intraperitoneal administration of CRP (20mg/kg body weight) compared to human serum albumin (HuSA) increased superoxide anion release and tissue factor activity from peritoneal macrophages in vivo (p<0.01). This was confirmed using intrapouch administration of CRP (25mug/mL) compared to HuSA. Pretreatment with reactive oxygen species (ROS) scavengers or protein kinase C (PKC) inhibitor significantly abrogated CRP-induced superoxide anion release and tissue factor activity. Pretreatment with extracellular signal-regulated kinase (ERK) and Jun N-terminal kinase (JNK) inhibitors, but not p38 mitogen-activated protein kinase (p38MAPK) significantly decreased CRP-induced superoxide anion release from macrophages in vivo. CRP-induced tissue factor activity in vivo was abrogated by pretreatment with inhibitors to p38MAPK, JNK and NFkappab (nuclear factor-kappab), but not ERK. Antibodies to Fc gamma receptors, CD32 and CD64 resulted in significant reduction in CRP-induced superoxide and tissue factor activity in vivo. Thus, CRP appears to induce oxidative stress in vivo by stimulating NADPH oxidase via PKC, ERK and JNK phosphorylation, and induces tissue factor PCA in vivo via upregulation of PKC, p38MAPK, JNK, ROS and NFkappab. CRP-induced ROS appears to precede tissue factor release. These effects are abrogated by blocking Fc gamma receptors, CD32 and CD64. This in vivo demonstration provides further evidence for a role for CRP in atherothrombosis.

Topics: Animals; C-Reactive Protein; Coagulants; Inflammation; Male; Models, Biological; Oxidative Stress; Rats; Rats, Wistar; Reactive Oxygen Species; Receptors, IgG; Superoxides; Thromboplastin; Thrombosis

Prompt coronary thrombus resolution, reducing time of ischemia, improves cardiac recovery. The factors triggered by ischemia that contribute to the clinical outcome are not fully known. We hypothesize that unabated inflammation due to cardiac ischemia may be a contributing factor.. As a proof-of-concept, we evaluated the effect of short-term myocardial ischemia on the local and systemic inflammatory response.. Pigs underwent either 90-min mid-left anterior descending (LAD) coronary artery balloon occlusion (infarct size 25% +/- 1% left ventricle; 29% heart function deterioration) or a sham-operation procedure. Peri-infarcted and non-ischemic cardiac tissue was obtained for histopathologic, molecular and immunohistochemical analysis of inflammatory markers [interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), modified C-reactive protein (mCRP), and human alveolar macrophage-56 (HAM-56)]. Blood (femoral vein) was withdrawn prior to myocardial infarction (MI) induction (t = 0) and at 30 and 90 min to evaluate: (i) systemic cytokine levels (IL-6, TNF-alpha, CRP); (ii) proinflammatory gene and protein expression in peripheral blood mononuclear cells (PBMCs) of tissue factor (TF), cyclo-oxygenase-2 (Cox-2), monocyte chemoattractant protein-1 (MCP-1), and CRP; and (iii) platelet activation (assessed by perfusion studies and RhoA activation).. Short-term ischemia triggered cardiac IL-6 and TNF-alpha expression, recruitment of inflammatory cells, and mCRP expression in infiltrated macrophages (P < 0.05 vs. t = 0 and sham). PBMC mRNA and protein expression of MCP-1, Cox-2 and TF was significantly increased by ischemia, whereas no differences were detected in CRP. Ischemia increased cardiac troponin-I, IL-6 and TNF-alpha systemic levels, and was associated with higher platelet deposition and RhoA activation (P < 0.001 vs. t = 0 and sham).. Short-term myocardial ischemia, even without atherosclerosis, induces an inflammatory phenotype by inducing local recruitment of macrophages and systemic activation of mononuclear cells, and renders platelets more susceptible to activation.

Topics: Animals; Biomarkers; C-Reactive Protein; Chemokine CCL2; Cyclooxygenase 2; Cytokines; Immune System; Immunologic Factors; Inflammation; Myocardial Ischemia; Platelet Activation; Swine; Thromboplastin; Up-Regulation

Topics: Animals; Blood Coagulation; Humans; Inflammation; Myocardial Reperfusion Injury; Receptor Cross-Talk; Signal Transduction; Thromboplastin

Myocardial inflammation is associated with an increase in circulating microparticles (MPs) and procoagulability.. We determined whether acute inflammation was associated with altered full-length tissue factor (flTF) expression and increased procoagulability in cardiomyocytic cells.. This study examined the transcriptional regulation of flTF expression in murine cardiomyocytic (HL-1) cells. Also, the generation of MPs by HL-1 cells and their ability to diffuse through an artificial endothelium was evaluated.. Constitutive and tumor necrosis factor-alpha (TNF-alpha)-induced flTF expression of HL-1 was reduced when c-Jun N-terminal kinase (JNK) was inhibited. Tissue factor (TF)-positive procoagulant MPs were released from HL-1 cells in response to TNF-alpha. JNK inhibition potentiated the release of MPs from HL-1 cells without affecting MP-associated TF activity. MP generation was dependent on RhoA activation and associated with a reorganization of the actin cytoskeleton. Increased diffusion of HL-1-derived MPs through an endothelial monolayer was found after TNF-alpha treatment. The increased diffusion was dependent not only on TNF-alpha but also on HL-1-released mediators.. Full-length TF expression in HL-1 cells was regulated through JNK. The TNF-alpha-induced increase in procoagulability was mediated through RhoA-dependent release of flTF-bearing MPs. These MPs were able to diffuse through an endothelial barrier adjacent to HL-1 cells and increased the procoagulability of the extracellular endothelial space. Cardiomyocytes seem to be a likely source of flTF-bearing procoagulant MPs.

Topics: Actins; Animals; Cell Line; Coculture Techniques; Inflammation; Mice; Myocardium; Polymerase Chain Reaction; RNA, Messenger; Thromboplastin; Transcription, Genetic; Tumor Necrosis Factor-alpha

Eosinophil counts in the bronchoalveolar lavage fluid of wild-type (WT) mice increased after ovalbumin (OVA) challenge, a response that was diminished in comparably challenged low-expressing coagulation factor VII (FVII(tTA/tTA)) mice. Levels of T helper type 2 (Th2) cytokines, IL-4, IL-5, and IL-13, and eosinophil-attracting chemokines, eotaxin and RANTES, were also lower in the OVA-challenged FVII(tTA/tTA) mice. Eosinophils purified from low-FVII mice underwent apoptosis at a faster rate compared with WT eosinophils, and eosinophil migration in response to eotaxin was reduced in eosinophils obtained from FVII(tTA/tTA) mice. Airway hyperresponsiveness and mucous layer thickness were reduced in OVA-treated FVII(tTA/tTA) mice, and addition of exogenous coagulation factor X (FX) enhanced mucin production in human epithelial NCI-H292 cells. Correspondingly, incubation of FX with NCI-H292 cells resulted in activated (a) FX production, suggesting that the components required for FX activation were present on NCI-H292 cells. These results demonstrate that FVIIa functions in the asthmatic response to an allergen by stimulating lung eosinophilia, airway hyperresponsiveness, and mucin production, this latter effect through its ability to activate FX in conjunction with tissue factor.

Topics: Animals; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Cell Count; Cell Line; Cell Movement; Cell Survival; Cytokines; Eosinophils; Epithelial Cells; Factor VIIa; Female; Gene Expression Regulation; Inflammation; Inflammation Mediators; Lung; Mice; Mice, Inbred C57BL; Mucins; Ovalbumin; RNA, Messenger; Thromboplastin

Complement component C5 is crucial for experimental animal inflammatory tissue damage; however, its involvement in human inflammation is incompletely understood. The responses to gram-negative bacteria were here studied taking advantage of human genetic complement-deficiencies--nature's own knockouts--including a previously undescribed C5 defect. Such deficiencies provide a unique tool for investigating the biological role of proteins. The experimental conditions allowed cross-talk between the different inflammatory pathways using a whole blood model based on the anticoagulant lepirudin, which does not interfere with the complement system. Expression of tissue factor, cell adhesion molecules, and oxidative burst depended highly on C5, mediated through the activation product C5a, whereas granulocyte enzyme release relied mainly on C3 and was C5a-independent. Release of cytokines and chemokines was mediated to varying degrees by complement and CD14; for example, interleukin (IL)-1beta and IL-8 were more dependent on complement than IFN-gamma and IL-6, which were highly dependent on CD14. IL-1 receptor antagonist (IL-1ra) and IFN-gamma inducible protein 10 (IP-10) were fully dependent on CD14 and inversely regulated by complement, that is, complement deficiency and complement inhibition enhanced their release. Granulocyte responses were mainly complement-dependent, whereas monocyte responses were more dependent on CD14. Notably, all responses were abolished by combined neutralization of complement and CD14. The present study provides important insight into the comprehensive role of complement in human inflammatory responses to gram-negative bacteria.

Topics: Adolescent; Adult; Case-Control Studies; Cell Adhesion; Complement Activation; Complement C2; Complement C5; Complement System Proteins; Escherichia coli; Female; Gram-Negative Bacteria; Humans; Immunity, Innate; In Vitro Techniques; Inflammation; Lipopolysaccharide Receptors; Male; Models, Immunological; Monocytes; Neisseria meningitidis; Phagocytosis; Respiratory Burst; Thromboplastin

Ischemia is a leading causes of morbidity in giant cell arteritis (GCA). We studied circulating platelets and leukocytes in patients with GCA and with polymyalgia rheumatica. Normal healthy donors (>60 a) served as controls. Patients had a significantly greater fraction of platelets expressing P-selectin, of platelet-Nph and platelet-Mo aggregates, and of Nph and Mo expressing tissue factor. These differences were correlated with the percentage of platelets expressing P-selectin and were not influenced by clinical features or by systemic inflammation. Activated circulating leukocytes and platelets could contribute to indolent vessel inflammation and possibly to thromboembolic events in patients with systemic large vessel vasculitis.

Topics: Aged; Blood Platelets; Giant Cell Arteritis; Humans; Inflammation; Leukocytes; Middle Aged; P-Selectin; Platelet Activation; Polymyalgia Rheumatica; Risk Factors; Thromboembolism; Thromboplastin

The effects of anti-inflammatory plant extracts, such as black tea extract (BTE) and resveratrol (RSV) could modulate cell activation leading to atherosclerosis, however there is little comparative information about how different endothelial cell types are affected by these compounds. In order to compare human endothelial cells derived from different origins (umbilical vein or HUVEC, coronary artery or HCAEC, microvascular or HMVEC) and their interleukin-1beta (IL-1beta) responsiveness, IL-6 ELISA, RT-PCR, tissue factor assay, and prostacyclin responses using 6-keto PGF1alpha ELISA were determined. The IL-1beta-induced IL-6 levels were dose-dependent with highest responses seen in HCAEC. Significant inhibition of IL-1beta responses was achieved with BTE and RSV, with the largest decrease of IL-6 and TF seen in HCAEC. Prostacyclin levels were highest in HUVEC and were inhibited by RSV in all cell types. The differences between the endothelial cell types could account for greater susceptibility of coronary arteries to inflammation and atherogenesis.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Antioxidants; Blood Coagulation; Cells, Cultured; Coronary Vessels; Dose-Response Relationship, Drug; Endothelial Cells; Humans; Inflammation; Interleukin-1beta; Interleukin-6; Plant Extracts; Resveratrol; Stilbenes; Tea; Thromboplastin

Topics: Disseminated Intravascular Coagulation; Heat Stroke; Humans; Inflammation; Thromboplastin

MIAMI was a prospective multicenter clinical study designed to investigate the relationship between changes in carotid intima-media thickness (C-IMT) and those in the levels of circulating markers of inflammation, thrombosis and endothelial dysfunction. The study was performed in a group of stable coronary patients treated for two years with a moderate dosage of atorvastatin (20mg/day). In this paper the cross-sectional relationship between C-IMT and the same circulating markers of inflammation, thrombosis and endothelial dysfunction measured at baseline was investigated.. Eighty-five subjects that had not used statins for at least two months were enrolled in the study. At time of enrollment, the levels of vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), E-selectin, interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-alpha, high-sensitivity C-reactive protein (hs-CRP), tissue factor (TF), tissue factor pathway inhibitor (TFPI), von Willebrand factor (vWF), fibrinogen, total cholesterol (TC), high-density lipoprotein (HDL) and low-density lipoprotein (LDL), and triglycerides were measured, in parallel with C-IMT assessment.. In cross-sectional analyses, markers of endothelial perturbation (i.e. E-selectin) and TFPI were more strongly correlated with arherosclerotic burden than markers of inflammation. The baseline picture in this study indicates that E-selectin and TFPI are linked with atherosclerotic burden.

Topics: Atorvastatin; Biomarkers; C-Reactive Protein; Carotid Artery Diseases; Cholesterol; E-Selectin; Endothelium, Vascular; Female; Fibrinogen; Heptanoic Acids; Humans; Inflammation; Intercellular Adhesion Molecule-1; Interleukin-6; Interleukin-8; Lipoproteins; Male; Middle Aged; Prospective Studies; Pyrroles; Thromboplastin; Triglycerides; Tumor Necrosis Factor-alpha; Tunica Intima; Vascular Cell Adhesion Molecule-1; von Willebrand Factor

Procoagulant microparticles constitute valuable hallmarks of cell damage. Microparticles also behave as cellular effectors.. We hypothesized that the extent of the vascular cell damage measured by circulating microparticles could be related to the severity of pulmonary arterial hypertension (PAH).. Circulating biomarkers of vascular damage and cell activation were measured in blood samples from 20 patients with PAH. Samples were withdrawn from occluded pulmonary artery and jugular vein. Peripheral venous blood samples were obtained in 23 control subjects. The microparticle procoagulant abilities were quantified by functional prothrombinase and tissue factor assays and their cellular origin was determined.. Soluble vascular cellular adhesion molecule-1 and proinflammatory markers, such as monocyte chemoattractant protein-1 and highly specific C-reactive protein, were elevated in patients with PAH compared with control subjects. Microparticles bearing active tissue factor and CD105 (endoglin) were also elevated in patients with PAH compared with control subjects (29 +/- 13 vs. 16 +/- 6 fmol/L, P < 0.001, and 1.10 +/- 0.46 vs. 0.49 +/- 0.33 nmol/L phosphatidylserine equivalent, P < 0.001, respectively). A further increase in endothelium-derived CD105 microparticles was observed in pulmonary arterial blood compared with venous blood in patients with PAH (1.73 +/- 0.77, P = 0.038). Microparticles bearing active tissue factor were at a higher level in patients in functional class III and IV and who were walking fewer than 380 m with the six-minute-walk test.. Circulating markers of endothelium damage, proinflammatory markers, and cell stimulation estimated with circulating microparticles appear to be valuable tools in determining the severity of PAH.

Topics: Adult; Aged; Antigens, CD; Blood Coagulation; Blood Gas Analysis; C-Reactive Protein; Chemokine CCL2; Chemokine CCL5; Endoglin; Endothelium, Vascular; Female; Humans; Hypertension, Pulmonary; Inflammation; Lung; Male; Microcirculation; Middle Aged; Particle Size; Receptors, Cell Surface; Severity of Illness Index; Thromboplastin; Vascular Cell Adhesion Molecule-1

It has been established that inflammation and enhanced pro-coagulant activity are associated with the pathogenesis of atherosclerotic vascular disease. We evaluated and compared the contributions of the factor (F)XIa and tissue factor (TF) activity in plasma of patients with coronary artery disease (CAD). Citrate plasma was obtained prior to therapy from 53 patients with stable angina (29 with a history of previous myocardial infarction; CAD-MI) and 30 with acute coronary syndrome (ACS) within 12 hours from pain onset. Four ACS patients treated with heparin were excluded. FXIa and TF activity were determined in clotting assays based upon the prolongation of clotting time by inhibitory monoclonal antibodies. Twenty-five of 26ACS patients (96%) and 22 of 29 CAD-MI patients (76%) had quantifiable FXIa (50 +/- 33 and 42 +/- 45pM, respectively). Ten of 26 (38%) ACS patients and only three of 53 (6%) stable CAD patients showed TF activity (<0.4pM). No FXIa or TF activity was observed in age-matched healthy controls (n = 12). For both CAD-MI and ACS patients, there were correlations (p < 0.05) between FXIa and interleukin-6 (R(2) = 0.59 and 0.39, respectively) and between FXIa and TAT (R(2) = 0.64 and 0.63, respectively). In conclusion, the majority of ACS and CAD-MI patients have circulating FXIa that correlates with markers of coagulation and inflammation.

Topics: Acute Coronary Syndrome; Aged; Angina Pectoris; Antithrombin III; Biomarkers; Blood Coagulation; Blood Coagulation Tests; Case-Control Studies; Coronary Artery Disease; Factor Xa; Humans; Inflammation; Interleukin-6; Male; Middle Aged; Myocardial Infarction; Peptide Hydrolases; Thromboplastin

P-selectin inhibition has been evaluated as a therapeutic for prevention and treatment of venous thrombosis. In this study, a novel oral small-molecule inhibitor of P-selectin, PSI-421, was evaluated in a baboon model of stasis induced deep vein thrombosis (DVT). Experimental groups included i) primates receiving a single oral dose of 1 mg/kg PSI-421 two days prior and continued six days after thrombosis (n = 3); ii) primates receiving a single daily subcutaneous dose of 0.57 mg/kg enoxaparin sodium two days prior and continued six days post thrombosis (n = 3); and iii) primates receiving no treatment (n = 3). PSI-421 treated primates had greater percent vein reopening and less vein wall inflammation than the enoxaparin and controls at day 6. Microparticle tissue factor activity (MPTFA) was significantly lower in the animals receiving PSI-421 immediately after thrombosis (T+6 hours day 0) suggesting lower potential for thrombogenesis in these animals. PSI-421 also reduced soluble P-selectin levels versus controls at T+6 hours day 0, day 2 and 6. Experimental animals in any group showed no adverse effects on coagulation. This study is the first to demonstrate a reduction in MPTFA associated with vein reopening and reduced vein inflammation due to oral P-selectin inhibition in a baboon model of DVT.

Topics: Administration, Oral; Animals; Anticoagulants; Blood Coagulation; Blood Platelets; Disease Models, Animal; Enoxaparin; Factor Xa Inhibitors; Fibrinolytic Agents; Hydroxyquinolines; Iliac Vein; Inflammation; Injections, Subcutaneous; Male; P-Selectin; Papio anubis; Phlebography; Thromboplastin; Time Factors; Ultrasonography, Doppler, Color; Vascular Patency; Venous Thrombosis

Leptin is an adipocyte-derived cytokine primarily involved in the regulation of body weight and energy balance. In vivo studies suggest that leptin promotes platelet aggregation and thrombosis. Neutrophils are involved in the crosstalk between inflammation and thrombosis in clinical disorders. Leptin is also involved in the regulation of inflammation.. We examined the in vitro effects of leptin on the expression of tissue factor (TF), the primary initiator of coagulation, in healthy neutrophils.. The effects on TF expression were assayed functionally using a modified prothrombin time (mPT), as well as at mRNA and protein levels. The same experiments were performed in parallel with PBMC. Leptin induced functional TF and increased TF mRNA and protein expression in both cell types, as determined by mPT, real-time RT-PCR, western blot, flow cytometry, immunocytochemistry. Inhibition studies revealed that the effect of leptin on TF expression is mediated, at least in part, by JAK2 and PI3K. Our findings, after neutralising TNFalpha in supernatants of leptin-treated cells, also suggest the involvement of TNFalpha in the leptin-induced TF expression in leukocytes.. This study indicates a novel link between inflammation, obesity and thrombosis by showing that leptin is able to trigger the extrinsic coagulation cascade. This work suggests a possible mechanism of the thrombotic effects of hyperleptinemic-associated clinical disorders.

Topics: Blood Coagulation; Humans; Inflammation; Janus Kinase 2; Leptin; Leukocytes, Mononuclear; Neutrophils; Obesity; Phosphatidylinositol 3-Kinases; Platelet Aggregation; Prothrombin Time; RNA, Messenger; Thromboplastin; Thrombosis; Tumor Necrosis Factor-alpha

Severe heatstroke is a leading cause of morbidity and mortality during heat waves. The pathogenesis of tissue injury, organ failure, and death in heatstroke is not well understood.. We investigated the pathways of heatstroke-induced tissue injury and cell death in anesthetized baboons (Papio hamadyras) subjected to environmental heat stress until core temperature attained 42.5 degrees C (moderate heatstroke; n = 3) or onset of severe heatstroke (n = 4) signaled by a fall in systolic blood pressure to < 90 mm Hg and rise in core temperature to 43.1+/-0.1 degrees C. Three sham-heated animals served as controls. Light and electron microscopy revealed widespread hemorrhage and thrombosis, transmural migration of leukocytes, and microvascular endothelium injury in severe heatstroke. Immunohistology and ultrastructural analysis demonstrated increased staining of endothelial von Willebrand factor (vWF), tissue factor (TF), and endothelial leukocyte-platelet interaction. Extensive apoptosis was noted in spleen, gut, and lung, and in hematopoeitic cells populating these organs. Double-labeling studies colocalized active caspase-3 and TF with apoptotic cells. Findings in sham-heated animals were unremarkable.. These data suggested that microvascular injury, thrombosis, inflammation, and apoptosis may play an important role in the pathogenesis of heatstroke injury.

Topics: Animals; Apoptosis; Blood Pressure; Body Temperature; Disease Models, Animal; Disseminated Intravascular Coagulation; Endothelium, Vascular; Heat Stress Disorders; Heat Stroke; Inflammation; Papio hamadryas; Thromboplastin; Thrombosis; von Willebrand Factor

Tissue factor (TF) activation of the coagulation proteases enhances inflammation in animal models of arthritis and endotoxemia, but the mechanism of this effect is not yet fully understood - in particular, whether this is primarily due to fibrin formation or through activation of protease activated receptors (PARs).. We induced extravascular inflammation by injection of recombinant soluble murine TF (sTF1-219) in the hind paw. The effects of thrombin inhibition, fibrinogen and platelet depletion were evaluated, as well as the effects of PAR deficiency using knockout mice deficient for each of the PARs.. Injection of soluble TF provoked a rapid onset of paw swelling. Inflammation was confirmed histologically and by increased serum IL-6 levels. Inflammation was significantly reduced by depletion of fibrinogen (P < 0.05) or platelets (P = 0.015), and by treatment with hirudin (P = 0.04) or an inhibitor of activated factor VII (P < 0.001) compared with controls. PAR-4-deficient mice exhibited significantly reduced paw swelling (P = 0.003). In contrast, a deficiency in either PAR-1, PAR-2 or PAR-3 did not affect the inflammatory response to soluble TF injection.. Our results show that soluble TF induces acute inflammation through a thrombin-dependent pathway and both fibrin deposition and platelet activation are essential steps in this process. The activation of PAR-4 on platelets is crucial and the other PARs do not play a major role in soluble TF-induced inflammation.

Topics: Animals; Blood Coagulation; Blotting, Western; Factor VIIa; Fibrin; Immunohistochemistry; Inflammation; Mice; Mice, Inbred C57BL; Mice, Knockout; Platelet Activation; Receptors, Proteinase-Activated; Thromboplastin

Dengue fever is the most prevalent viral disease transmitted by vectors (Aedes aegypti, Aedes albopictus) in worldwide. More than 100 million cases occur annually with a mortality rate of 5% and no safe vaccine is available. The pathogenesis of Dengue, where host and viral factors participate in the establishment of Dengue haemorrhagic fever (DHF) and Dengue shock syndrome (DSS) remains unresolved. Clinical observations have revealed significant abnormalities in coagulation and inflammation systems, with increased levels of tissue factor (TF) and the chemokine IL-8, correlating with the severity of the disease and implicating damage to endothelial vascular cells (EVC). Here we present novel insights concerning the crosstalk between the regulatory signaling pathways of the coagulation-inflammation processes, during Dengue virus (DV) infection of EVC. We found that DV up-regulates Protease Activated receptor type-1 (inflammation) and TF (coagulation) receptors, via the phosphorylation of p38 and ERK1/2 MAPKs, which favor the activation of NF-kappaB transcription factor. This induces pro-inflammatory (IL-8) or pro-adhesive (VCAM-1) gene expression which may lead to EVC activation. The elucidation of the basic principles that signal these processes has important implications for the design of new therapeutic strategies for DHF/DSS.

Topics: Blood Coagulation; Cells, Cultured; Dengue Virus; Endothelial Cells; Humans; Inflammation; Inflammation Mediators; Interleukin-8; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Prothrombin; Receptor, PAR-1; Severe Dengue; Signal Transduction; Thrombin; Thromboplastin; Time Factors; Vascular Cell Adhesion Molecule-1; Virulence

Sepsis is a life-threatening disorder resulting from systemic inflammatory and coagulatory responses to infection. High-mobility group box 1 protein (HMGB1), an abundant intranuclear protein, was recently identified as a potent lethal mediator of sepsis. However, the precise mechanisms by which HMGB1 exerts its lethal effects in sepsis have yet to be confirmed. We recently reported that plasma HMGB1 levels correlated with disseminated intravascular coagulation (DIC) score, indicating that HMGB1 might play an important role in the pathogenesis of DIC.. To investigate the mechanisms responsible for the lethal effects of HMGB1, and more specifically, to explore the effects of HMGB1 on the coagulation system.. Rats were exposed to thrombin with or without HMGB1, and a survival analysis, pathologic analyses and blood tests were conducted. The effects of HMGB1 on the coagulation cascade, anticoagulant pathways and surface expression of procoagulant or anticoagulant molecules were examined in vitro.. Compared to thrombin alone, combined administration of thrombin and HMGB1 resulted in excessive fibrin deposition in glomeruli, prolonged plasma clotting times, and increased mortality. In vitro, HMGB1 did not affect clotting times, but inhibited the anticoagulant protein C pathway mediated by the thrombin-thrombomodulin complex, and stimulated tissue factor expression on monocytes.. These findings demonstrate the procoagulant role of HMGB1 in vivo and in vitro. During sepsis, massive accumulation of HMGB1 in the systemic circulation would promote the development of DIC.

Topics: Animals; Blood Coagulation; Blood Coagulation Tests; Cells, Cultured; Coagulants; Cytokines; Disease Models, Animal; Disseminated Intravascular Coagulation; Enzyme Activation; Fibrin; Hemolysis; High Mobility Group Proteins; HMGB1 Protein; Humans; Inflammation; Kidney; Lung; Male; Monocytes; Protein C; Rats; Rats, Sprague-Dawley; Repressor Proteins; Thrombin; Thromboplastin; Thrombosis

In patients with chronic hypercholesterolemia, the CD40-CD40L dyad is upregulated, contributing to the initiation and progression of atherosclerosis. Our aim was to describe the role of postprandial lipemia and inflammatory stimulation on platelet and monocyte activation and CD40-ligand (CD40L) levels.. Before and 2 h after consumption of a defined fatty meal, whole blood samples of 31 healthy subjects were incubated with endotoxin (LPS). CD40-ligand and CD62P expression on platelets, tissue-factor expression on monocytes and platelet-monocyte aggregates were measured with flow cytometry. Soluble CD40-ligand plasma levels were measured with an ELISA. After the meal, serum triglyceride levels increased from 137.6+/-60.5 mg/dl to 201.5+/-75.0 mg/dl. Expression of CD40L and CD62P on platelets and plasma levels of soluble CD40L were significantly decreased. No significant changes after the meal were observed concerning tissue factor expression on monocytes and platelet-monocyte aggregates. Addition of LPS showed no significant effect concerning CD40L or CD62P expression on platelets, whereas the amount of platelet-monocyte aggregates significantly increased under LPS stimulation after the fatty meal.. Acute alimenatry lipemia leads to a decreased expression of CD40L on platelets and a reduced plasma level of sCD40L, suggesting an increased turnover in the CD40L system.. Before and after a fatty meal, blood samples of 31 healthy subjects were incubated with LPS. After the meal, expression of CD40L and CD62P on platelets and plasma levels of soluble CD40L were significantly decreased. Addition of LPS showed no effect concerning CD40L or CD62P expression, whereas the amount of platelet-monocyte aggregates significantly increased under LPS stimulation after the fatty meal.

Topics: Adult; Aged; Blood Platelets; CD40 Antigens; CD40 Ligand; Female; Gene Expression Regulation; Humans; Hypercholesterolemia; Hyperlipidemias; Inflammation; L-Selectin; Lipopolysaccharides; Male; Middle Aged; Postprandial Period; Thromboplastin

There is growing evidence for an interplay between inflammatory and coagulation pathways in acute and chronic inflammatory diseases. However, it remains unclear whether components of the coagulation pathway, such as tissue factor (TF), contribute to intestinal inflammation, and whether targeting TF will blunt the inflammatory cell recruitment, tissue injury, and enhanced thrombus formation that occur in experimental colitis. Mice were fed 3% dextran sodium sulfate (DSS) to induce colonic inflammation, with some mice receiving a mouse TF-blocking antibody (muTF-Ab). The adhesion of leukocytes and platelets in colonic venules, light/dye-induced thrombus formation in cremaster muscle microvessels, as well as disease activity index, thrombin-antithrombin (TAT) complexes in plasma, and histopathologic changes in the colonic mucosa were monitored in untreated and muTF-Ab-treated colitic mice. In untreated mice, DSS elicited the recruitment of adherent leukocytes and platelets in colonic venules, caused gross and histologic injury, increased plasma TAT complexes, and enhanced thrombus formation in muscle arterioles. muTF-Ab prevented elevation in TAT complexes, reduced blood cell recruitment and tissue injury, and blunted thrombus formation in DSS colitic mice. These findings implicate TF in intestinal inflammation and support an interaction between inflammation and coagulation in experimental colitis.

Topics: Animals; Antithrombins; Blood Coagulation; Blood Platelets; Colitis; Colon; Disease Models, Animal; Inflammation; Leukocytes; Mice; Microcirculation; Thromboplastin; Thrombosis

Tissue factor (TF) is the primary initiator of the coagulation cascade. Ca(2+) signaling is involved in TF gene expression. Monocyte chemoattractant protein-1 (MCP-1) and its receptor (CCR2) play a pivotal role in the inflammation of atherosclerosis. Although nitric oxide (NO) impairment appears to promote thrombogenicity in monocyte adhesion to endothelial cells (ECs), little is known about its mechanism. N(omega)-nitro-L-arginine methyl ester (L-NAME) promoted MCP-1 expression in EC culture. In response to monocyte adhesion, increased TF expression accompanied by NF-kappaB p65 activation was observed in L-NAME-treated ECs compared with non-treated ECs. This increased TF expression was prevented by BAPTA-AM, an intracellular Ca(2+) chelator. Monocyte attachment to L-NAME- treated ECs increased Ca(2+) influx compared with non-treated ECs, which was prevented by the blockade of MCP1/CCR2. These findings suggest that increased production of MCP-1 caused by L-NAME contributes to the enhancement of Ca(2+) influx only when monocytes adhered to ECs and that this may accelerate TF expression in ECs triggered by monocyte adhesion. We demonstrate the role of Ca(2+) influx via MCP-1/CCR2 under NO impairment in TF expression in monocyte-EC interaction.

Topics: Calcium; Calcium Signaling; Cell Adhesion; Cells, Cultured; Chemokine CCL2; Endothelium, Vascular; Humans; Inflammation; Monocytes; NG-Nitroarginine Methyl Ester; Nitric Oxide; Thromboplastin

Ionizing radiation (IR) is associated with thrombotic vascular occlusion predicting a poor clinical outcome. Our study examined whether IR induced tissue factor (TF) expression and procoagulability. We further investigated coordinated gene alterations associated with TF upregulation in the myelomonocytic leukemia THP-1 cells.. TF expression was determined by quantitative Reverse Transcriptase (TaqMan) PCR, TF ELISA and TF activity by a two stage chromogenic assay in the time course of days 1, 3, 7, 10, and 17 post IR. To detect IR-induced alterations in gene expression, Affymetrix HG U133 Plus 2.0 microarrays were used. RESULTS IR induced a significant increase in TF/GAPDH mRNA ratios and cellular TF protein on days 3 and 7 post IR (20 Gy [p>or=0.01] and 40 Gy [p or=0.001] vs. control respectively), suggesting IR immediately alters the cellular thrombogenicity. TF upregulation post IR was confirmed in PBMNCs. Gene expression profiling showed IR increased the expression of inflammatory and apoptosis-related pathways known to be involved in the regulation of TF expression.. TF upregulation together with inflammation and apoptosis may increase the thrombogenicity of tissues. The demonstrated upregulation of TF might play a pivotal role in radiation associated thrombosis.

Topics: Apoptosis; Blood Coagulation Factors; Cell Line, Tumor; Enzyme-Linked Immunosorbent Assay; Factor Xa; Gene Expression Profiling; Gene Expression Regulation, Leukemic; Humans; Inflammation; Leukemia, Myelomonocytic, Acute; Neoplasm Proteins; NF-kappa B; Nitriles; Oligonucleotide Array Sequence Analysis; Particle Accelerators; Radiation, Ionizing; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Sulfones; Thrombophilia; Thromboplastin

Recent findings indicate that atherosclerosis, a chronic inflammatory process, might start during childhood. Nevertheless, the expression of inflammation-related molecules of endothelial cell isolated from healthy neonates with a strong family history of myocardial infarction (SFHMI) has been rarely analyzed.. Human umbilical vein endothelial cells (HUVECs) from children with SFHMI were assessed for the expression of CD40 and CD40L, in the presence of TNF-alpha and oxLDL. The intracellular content of CD80, CXCL8 and tissue factor by HUVECs stimulated with a CD40 agonist monoclonal antibody as well as monocytes/lymphocyte adhesion to TNF-alpha-stimulated HUVECs was also evaluated.. The basal expression of CD40 and CD40L was higher in SFHMI-positive HUVECs in comparison to controls. TNF-alpha and oxLDL upregulated the expression of CD40 and CD40L in SFHMI versus control HUVECs (p<0.001). The intracellular expression of CXCL8, tissue factor and CD80 was also higher than in controls, and the adhesion of lymphocyte- and monocyte-like cells augmented upon TNF-alpha stimulation.. It is possible that the modifications observed in the SFHMI-positive HUVECs, all of them relevant to the atherosclerosis process, may lead to early inflammatory reactions, thus contributing to the premature initiation of atherosclerotic lesions in these children.

Topics: CD40 Antigens; CD40 Ligand; Cell Adhesion; Endothelial Cells; Humans; Infant, Newborn; Inflammation; Interleukin-8; Lipoproteins, LDL; Lymphocytes; Monocytes; Myocardial Infarction; Thromboplastin; Tumor Necrosis Factor-alpha; Umbilical Veins

Thrombotic meningoencephalitis (TME) is a neurological condition in cattle characterized by fibrinopurulent meningitis with hemorrhage, abscess formation and thrombotic vasculitis throughout the central nervous system. The etiologic agent of TME is Haemophilus somnus, a gram-negative pleomorphic coccobacillus. Although the pathogenesis of TME is not well understood, the propensity of H. somnus to cause vasculitis and intravascular thrombosis suggests a critical role for the interactions between the bacteria and endothelial cells in inciting the disease. The goal of this study was to determine if H. somnus elicits an inflammatory and procoagulative response in bovine brain microvascular endothelial cells (BBEC) in vitro. We demonstrate that BBEC exposed to H. somnus secrete significant levels of the proinflammatory and procoagulative cytokines TNF-alpha and IL-1beta. BBEC treated with H. somnus also display increased levels of IL-6 mRNA, another cytokine associated with coagulopathy in vivo. H. somnus-treated BBEC exhibited increased procoagulant activity and tissue factor expression and activity, along with a decreased ability to activate protein C and decreased expression of thrombomodulin mRNA. These changes would be expected to promote thrombus formation in vessels of the CNS, and potentially contribute to the pathogenesis of TME.

Topics: Animals; Brain; Cattle; Central Nervous System Infections; Coagulants; Cytokines; Endothelial Cells; Fibrin; Haemophilus somnus; Inflammation; Models, Biological; Protein C; Thromboplastin; Thrombosis

Topics: Cell Line, Tumor; Chemokine CXCL1; Coagulants; Dose-Response Relationship, Drug; Factor VIIa; Factor X; Gene Expression Regulation; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inflammation; Interleukin-8; Models, Biological; RNA; Thromboplastin; Transcription, Genetic

The NF-kappaBeta transcription factors modulate the expression of tissue factor (TF), E-selectin (CD62E) and vascular cell adhesion molecule-1 (VCAM-1), which are essential for thrombosis and inflammation. We have previously shown that andrographolide (Andro) covalently modifies the reduced cysteine(62) of p50 - a major subunit of NF-kappaBeta transcription factors, thus blocking the binding of NF-kappaBeta transcription factors to the promoters of their target genes, preventing NF-kappaBeta activation and inhibiting inflammation in vitro and in vivo. Here we report that Andro, but not its inactive structural analog 4H-Andro, significantly suppressed the proliferation of arterial neointima ( approximately 60% reduction) in a murine model of arterial restenosis. Consistently, p50(-/-) mice manifested attenuated neointimal hyperplasia upon arterial ligation. Notably, the same dosage of Andro did not further reduce neointimal formation in p50(-/-) mice, which implicates the specificity of Andro on p50 for treating experimental arterial restenosis. The upregulation of NF-kappaBeta target genes, including TF, E-selectin and VCAM-1, and the increased deposition of leukocytes (mainly CD68+ macrophages) were clearly detected within the injured arterial walls, all of which were significantly abolished by treatment with Andro or genetic deletion of p50. The expression of TF, E-selectin and VCAM-1 was also markedly upregulated in the patient sample of thrombotic vasculitis, indicating the clinical relevance of NF-kappaBeta activation in the pathogeneses of occlusive arterial diseases. Our data thus indicate that, by the downregulation of the NF-kappaBeta target genes that are critical in thrombosis and inflammation, specific inhibitors of p50, such as Andro, may be therapeutically valuable for preventing and treating thrombotic arterial diseases, including neointimal hyperplasia in arterial restenosis.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Coronary Restenosis; Coronary Vessels; Cysteine; Disease Models, Animal; Diterpenes; E-Selectin; Gene Deletion; Humans; Hyperplasia; Inflammation; Macrophages; Mice; Mice, Knockout; NF-kappa B p50 Subunit; Thromboplastin; Tunica Intima; Up-Regulation; Vascular Cell Adhesion Molecule-1

Tissue factor (TF) is one of the proteins that participate in hemostatic and inflammatory processes. Activated monocytes present in the liver increase expression of TF, and while accumulating in the organ they can intensify inflammation. The aim of the present study was to evaluate the expression of TF on monocytes in advanced liver cirrhosis with regard to other activation markers. The flow cytometric analysis of TF (CD142), CD14, adhesive molecules CD11b and CD11c, costimulatory molecules CD40, CD80 and CD86, and HLA-DR on monocytes was carried out in 45 patients with postalcoholic liver cirrhosis (Child Pugh B, 20 patients; Child Pugh C, 25 patients) and in 25 healthy persons. The positive correlation between monocytic TF expression and monocyte [soluble CD14 (sCD14), CD11b, monocyte aggregates] activation, the expression of costimulatory molecules on monocytes (CD40, CD80), blood platelet (soluble P-selectin, microplatelets) activation, the level of tumor necrosis factor-alpha, biochemical parameters of liver damage (alanine aminotransferase, aspartate aminotransferase, alkaline phosphate, gamma-glutamyltransferase, and bilirubin) as well as coagulation disorders were observed in the study. In conclusion, the study revealed that the activation of monocytes and blood platelets is accompanied by the elevation of monocytic TF expression in advanced liver cirrhosis. The monocytic TF is a significant link connecting clotting processes and inflammatory and immunological phenomena in liver cirrhosis.

Topics: Adult; Case-Control Studies; Female; Humans; Inflammation; Liver Cirrhosis, Alcoholic; Male; Middle Aged; Monocytes; Platelet Activation; Thromboplastin

Increased circulating endothelial cells (CECs, reflecting endothelial damage) in acute coronary syndromes (ACS) has been reported. However, the inter-relationships of indices of endothelial damage/injury with development of vascular (dys)function, plasma levels of tissue factor (TF, an index of coagulation) and interleukin-6 (IL-6, a pro-inflammatory cytokine) have not been investigated in ACS. We hypothesized that increased CECs can be related to impaired flow-mediated vasodilatation (FMD, an index of endothelial dysfunction) and elevated plasma von Willebrand factor (vWf, also marking endothelial damage/dysfunction), TF and IL-6 in patients with ACS.. We studied 120 patients with ACS (80 acute myocardial infarction and 40 unstable angina; 86 male, age 65+/-12 years) and 40 matched patients with stable CAD and 40 healthy controls (HC) in a cross-sectional analysis. Plasma vWf, TF and IL-6 levels were measured by ELISA. CECs were quantified using epifluorescence microscope after immunomagnetic separation with CD146. Brachial artery FMD was assessed in a subset of 39 ACS patients.. ACS patients had significantly higher CECs, vWf, TF and IL-6 levels, but lower FMD, when compared to stable CAD and HC (all p<0.001) and all were inter-correlated significantly. In ACS, CECs was strongly correlated with FMD (r=-0.64, p<0.001) and TF (r=0.7, p<0.001). In stable CAD, significant correlations were again found between many indices, but on multivariate analysis, IL-6 and vWf were both independently related to FMD.. Increased CECs in ACS patients are closely associated with endothelial damage/dysfunction (vWf and FMD), coagulation (TF) and inflammation (IL-6). These inter-relationships support the concept of a central role of endothelial damage/injury in the activation of vascular and coagulation abnormalities in ACS.

Topics: Aged; Biomarkers; Blood Flow Velocity; Chest Pain; Coronary Disease; Endothelium, Vascular; Humans; Inflammation; Interleukin-6; Male; Middle Aged; Multivariate Analysis; Thromboplastin; Vasodilation; von Willebrand Factor

Tissue factor (TF) is a transmembrane glycoprotein that binds its zymogen cofactor, Factor VIIa (FVIIa) on the cell surface. Together (TF/FVIIa) they activate Factor X (FX) and Factor IX (FIX) and start the extrinsic pathway of blood coagulation. As such, the TF/FVIIa complex plays an important role in normal physiology as well as in thrombotic diseases such as unstable angina (UA), disseminated intravascular coagulation (DIC), and deep vein thrombosis (DVT). In addition to its function as an initiator of coagulation, TF/FVIIa plays an important role in inflammation. Expression of TF on the cell surface and its appearance as a soluble molecule are characteristic features of acute and chronic inflammation in conditions such as sepsis and atherosclerosis. Here we demonstrate that BCX-3607, a small molecule potent inhibitor of TF/FVIIa, reduces thrombus weight in an animal model of DVT. BCX-3607 also decreases the level of interleukin-6 (IL-6) in a LPS-stimulated mouse model of endotoxemia. Additionally, in vitro studies indicate that BCX-3607 blocks the generation of TF/FVIIa-induced IL-8 mRNA in human keratinocytes and reduces the TF/FVIIa-mediated generation of IL-6 and IL-8 in human umbilical vein endothelial cells (HUVEC). Therefore, BCX-3607 might block the TF/FVIIa-mediated coagulation and inflammation associated with pathological conditions.

Topics: Animals; Anti-Inflammatory Agents; Atherosclerosis; Blotting, Northern; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelium, Vascular; Endotoxemia; Factor VIIa; Fibrinolytic Agents; Humans; Inflammation; Interleukin-6; Interleukin-8; Keratinocytes; Lipopolysaccharides; Male; Mice; Models, Biological; Models, Chemical; Prothrombin Time; Pyridines; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sepsis; Thromboplastin; Time Factors

Detailed histochemical analysis of coronary thrombi obtained freshly from acute phase of myocardial infarction patients may provide information necessary to understand the mechanism of coronary occlusive thrombus formation.. Coronary thrombi causing myocardial infarction were obtained from 10 consecutive patients of myocardial infarction in the acute phase, using a newly developed aspiration catheter. All the fixed specimens of coronary thrombi, by hematoxylin and eosin staining, were found to contain three major constituents, namely, platelets, densely packed fibrin and inflammatory cells, including polymorphonuclear and mononuclear cells, although their distribution in each specimen is totally heterogeneous. Immunohistochemical staining revealed the prominent presence of von Willebrand factor (VWF) at the sites of platelet accumulation, presence of tissue factor and platelets at the sites of deposition of fibrin fibrils. It also revealed the presence of CD16-, CD45- and CD34-positive cells, yet the functional roles of these cells have still to be elucidated. There are weak positive correlation between the number of inflammatory cells involved in the unit area of coronary thrombi specimen and the time of collection of the specimens after the onset of chest pain.. In spite of various limitations, our results contain information suggesting the possible role of VWF in platelet-thrombus formation, possible important role played by tissue factor and activated platelets in the formation of fibrin fibrils, and the positive relationship between inflammatory cells migration and the formation of occlusive thrombi in human coronary arteries.

Topics: Adult; Aged; Aged, 80 and over; Biopsy, Needle; Blood Platelets; Coronary Thrombosis; Female; Fibrin; Humans; Immunohistochemistry; Inflammation; Male; Middle Aged; Myocardial Infarction; Thromboplastin; von Willebrand Factor

Tissue factor (TF) is a transmembrane protein, which is essential for initiation of the coagulation cascade. TF has been reported to play an important role in the progression of endotoxin (lipopolysaccharide, LPS)-mediated endotoxemia, being induced in numerous tissues, such as kidney, spleen and lung. We developed and validated a rabbit anti-murine TF peptide antiserum to localize TF protein in a murine sepsis model. TF protein distribution was compared to localization of TF mRNA and fibrin deposits, the ultimate resultant of procoagulant TF activity. Evident LPS mediated TF mRNA induction was observed in the tubular area at the cortico-medullar junction in the kidney, and TF activity was increased after 6 hours of endotoxemia. In the spleen, however, TF mRNA was induced in the interfollicular region upon LPS injection, corresponding to increased TF protein in the same area. The clusters of TF-protein positive cells in the spleen are predominantly granulocytes, but no TF mRNA expression was observed within these cells. Based on these observations and the presence of TF-protein positive granulocytes after splenectomy, we hypothesize that granulocytes take-up TF for transport to other locations in order to initiate fibrin formation or to induce pro-inflammatory gene expression upon interaction with factor VIIa.

Topics: Animals; Endotoxemia; Female; Fibrin; Gene Expression Regulation; Granulocytes; Immune Sera; Inflammation; Kidney; Lipopolysaccharides; Mice; Protein Transport; Rabbits; RNA, Messenger; Sepsis; Spleen; Thromboplastin; Tissue Distribution

The association between brain damage and respiratory dysfunction has been recognized although mechanistic link between the two is still poorly defined. Intracerebral hemorrhage is accompanied by brain injury, stroke, and parenchymal hematoma formation with surrounding inflammation. Increase intracranial pressure as a result of intracerebral hemorrhage may promote localized activation of cytokines and coagulation system including tissue factor release. However, whether intracerebral hemorrhage triggers inflammation in noncerebral organs has not been elucidated. The aim of the present study was to examine the impact of intracerebral hemorrhage on lung inflammatory response. Intracerebral hemorrhage was induced by stereotaxic intrastriatal administration of bacterial collagenase. Expression of intracellular adhesion molecule-1 (ICAM-1), IKB-alpha, tissue factor, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) was evaluated by Western blot analysis. Our results revealed that intracerebral hemorrhage upregulated expression of ICAM-1 and tissue factor in both brain and lung, whereas it enhanced TNF-alpha and IL-1beta mainly in brain within 6 and 24 h of the brain injury. Levels of IKB-alpha remained unchanged in brain and lung tissues. Appearance of inflammatory markers in the lung was accompanied by morphological pulmonary damage. These data suggest that intracerebral hemorrhage may trigger acute inflammatory response in both brain and lung.

Topics: Animals; Brain; Cerebral Hemorrhage; Female; I-kappa B Proteins; Inflammation; Intercellular Adhesion Molecule-1; Interleukin-1; Lung; NF-KappaB Inhibitor alpha; Rats; Rats, Sprague-Dawley; Thromboplastin; Time Factors; Tumor Necrosis Factor-alpha

After activation, platelets expose CD40 ligand (CD40L) on their surface, then subsequently release the inflammatory mediator as a soluble fragment (sCD40L). Because sickle cell anemia (SCA) is noted for both platelet activation and chronic inflammation, we asked whether platelet-released CD40L potentially plays a role in SCA.. ELISAs demonstrate that SCA patient plasma contains 30-fold more sCD40L than control plasma. Correspondingly, platelets from these patients contain less than half the CD40L found in control platelets. Platelets from patients in painful crises are further depleted of CD40L, with even higher plasma levels, suggesting a correlation to the patient's clinical state. In addition, elevated sCD40L correlates with increased tissue factor in SCA plasma. Blockage of the CD40L receptor CD40 reduces SCA plasma-induced production of tissue factor and endothelial intercellular adhesion molecule-1 (ICAM-1). Finally, sCD40L activity in SCA plasma is confirmed by its induction of B-cell proliferation.. Platelet-derived sCD40L is elevated in SCA, further elevated in crises, and biologically active. The participation of sCD40L in SCA plasma-induced production of B cells, tissue factor, and ICAM-1 suggests that CD40L may contribute to the chronic inflammation and increased thrombotic activity known to occur in SCA.

Topics: Adult; Anemia, Sickle Cell; B-Lymphocytes; Blood Platelets; Case-Control Studies; CD40 Antigens; CD40 Ligand; Cell Proliferation; Hemoglobin, Sickle; Humans; Inflammation; Intercellular Adhesion Molecule-1; Middle Aged; Monocytes; Thromboplastin

Topics: Animals; Endotoxins; Gene Expression Regulation; Genetic Variation; Humans; Inflammation; Lipoproteins; Polymorphism, Genetic; Thromboplastin

Topics: Alternative Splicing; Animals; Arteries; Blood Coagulation; Inflammation; Lung; Mice; Protein Structure, Tertiary; Streptococcus pneumoniae; Thromboplastin; Thrombosis

The aim of the present study was to assess the effects of two 30-min consecutive exercise bouts on a treadmill at 80% VO2max separated by a 4-h rest interval, on blood cell counts and the production of tissue factor, cytokines, and eicosanoids in lipopolysaccharide (LPS)-stimulated blood.. Blood samples were taken from eight endurance athletes (mean+/-SD: age, 23.4+/-1.6 yr; VO2max, 66.0+/-6.4 L.min.kg), both immediately before and after each exercise bout. Cell counts were performed, and the heparinized blood was subjected to LPS-stimulation for 2 h.. There was a significant rise in white blood cell counts after the first exercise bout (81%, P<0.001), increasing to 123% (P<0.001) after the second bout. After the first and second runs, the tissue factor activity in LPS-stimulated monocytes was enhanced by 70% (not significant) and almost 200% (P=0.012), respectively, compared with baseline values. The high monocyte responsiveness after the second bout remained undiminished 2 h later. Similarly, the interleukin (IL)-8 production had risen 70% (P=0.022) after the first run and 100% (P=0.005) after the second run, relative to baseline values. IL-6 or leukotriene B4 levels did not change significantly. The rise in LPS-induced thromboxane B2 was 80% (P=0.024) after the first run and 63% after the second run (P=0.071, not significant). VO2max correlated negatively with the concentration of granulocytes immediately after the second exercise bout (R=0.864, P=0.006).. The results of this study are evidence that two physical exercise bouts separated by a 4-h rest are associated with an enhanced propensity of the blood cells to generate tissue factor activity and some proinflammatory products compared with one exercise bout.

Topics: Cytokines; Eicosanoids; Exercise; Exercise Test; Humans; Inflammation; Leukocyte Count; Leukocytes; Norway; Physical Exertion; Thromboplastin

Clinically, the vascular pathobiology of human sickle cell disease includes an abnormal state of chronic inflammation and activation of the coagulation system. Since these biologies likely underlie development of vascular disease in sickle subjects, they offer attractive targets for novel therapeutics. Similar findings characterize the sickle transgenic mouse, which therefore provides a clinically relevant inflammation model.. The authors tested two polyhydroxyphenyl hydroxamic acid derivatives, didox and trimidox, in sickle transgenic mice. Animals were examined by intravital microscopy (cremaster muscle and dorsal skin fold preparations) and by histochemistry before and after transient exposure to hypoxia, with versus without preadministration of study drug. Previous studies have validated the application of hypoxia/reoxygenation to sickle transgenic mice as a disease-relevant model.. Animals pretreated with these agents exhibited marked improvements in leukocyte/ endothelial interaction, hemodynamics and vascular stasis, and endothelial tissue factor expression. Thus, these drugs unexpectedly exert powerful inhibition on both the inflammation and coagulation systems.. Each of these changes is expected to be therapeutically beneficial in systemic inflammatory disease in general, and in sickle disease in particular. Thus, these novel compounds offer the advantage of having multiple therapeutic benefits in a single agent.

Topics: Anemia, Sickle Cell; Animals; Benzamidines; Blood Coagulation; Cell Communication; Chronic Disease; Disease Models, Animal; Drug Evaluation, Preclinical; Endothelial Cells; Enzyme Inhibitors; Humans; Hydroxamic Acids; Inflammation; Leukocytes; Mice; Mice, Transgenic; Thromboplastin

Triggering of tissue factor (TF)-mediated blood coagulation leads to the development of disseminated intravascular coagulation during rewarming from hypothermia. We studied post-rewarming TF levels, activity, and surface redistribution, along with the regulation of TF gene transcription in mononuclear cells (MNCs) obtained from an in vivo rat model.. Rewarming after a 5-hour episode of 15 degrees C hypothermia caused an increase in TF activity, protein levels, and externalization of TF antigen in rat MNCs. This was accompanied by a dramatic elevation of c-Jun and JNK phosphorylation, and the absence of EGR-1 and NF-kappaB activation. To search for a stimulus to counteract c-Jun-mediated induction of TF activity in MNCs from rewarmed rats, we applied heat shock pretreatment one day before the hypothermia/rewarming experiment. This restored post-rewarming TF activity, protein levels, and surface-to-total TF ratio in rat MNCs to normothermic levels. Furthermore, in heat shock-pretreated animals, rewarming failed to increase phosphorylated c-Jun and JNK levels. We attribute this to the profound overexpression of heat shock protein 70 and inhibition of JNK.. MNCs respond to rewarming from hypothermia by an induction of active TF antigen. This effect is dependent on c-Jun activation and is abolished by heat shock pretreatment.

Topics: Animals; Enzyme Activation; Hot Temperature; Hyperthermia, Induced; Hypothermia; Inflammation; JNK Mitogen-Activated Protein Kinases; Male; Monocytes; Phosphorylation; Rats; Rats, Wistar; Thromboplastin; Transcription, Genetic

Tissue factor (TF) triggers upstream coagulation signaling via the activation of protease-activated receptors (PARs) of relevance for inflammation and angiogenesis. TF pathway inhibitor 1 (TFPI-1) is the physiologic inhibitor of TF-initiated coagulation, but its role in regulating TF signaling is poorly understood. Here, we demonstrate that endogenous, endothelial cell-expressed TFPI-1 controls TF-mediated signaling through PARs. In endothelial cells transduced with TF to mimic exacerbated TF expression in vascular cells, TF-VIIa-Xa ternary complex-dependent activation of PAR1 remained intact when TF-mediated Xa generation was blocked with 2.5 to 5 nM recombinant TFPI-1 (rTFPI-1). Concordantly, inhibition of signaling in PAR1-expressing Chinese hamster ovary (CHO) cells required about 30-fold higher rTFPI-1 concentrations than necessary for anticoagulation. Studies with proteoglycan-deficient CHO cells document a crucial role of accessory receptors in supporting the anticoagulant and antisignaling activities of rTFPI-1. Coexpression of PAR2 with TF enhanced rTFPI-mediated inhibition of TF-VIIa-Xa-mediated PAR1 signaling, suggesting an unexpected role of PAR2 in the inhibitory control of TF signaling. These experiments are of potential significance for the limited therapeutic benefit of rTFPI-1 in systemic inflammation and recommend caution in using anticoagulant potency as a measure to predict how efficacious TF-directed inhibitors block cell signaling during initiation of coagulation.

Topics: Animals; Blood Coagulation; CHO Cells; Cricetinae; Cricetulus; Factor VIIa; Factor Xa; Humans; Inflammation; Lipoproteins; Multienzyme Complexes; Proteoglycans; Receptor, PAR-1; Receptor, PAR-2; Signal Transduction; Thrombin; Thromboplastin

The presence of antiphospholipid antibodies (APLA) is associated with an increased risk of recurrent thrombosis and pregnancy loss. APLA are able to activate endothelial cells (EC) and induce an increase in the expression of inflammatory marker proteins, such as leukocyte adhesion molecules, tissue factor or the monocyte chemoattractant protein-1 (MCP-1). Our objective was to investigate the effect of statins on EC activation induced by APLA in vitro. IgG was purified from the plasma of six patients with APLA and from healthy controls. EC were incubated with patient IgG or with control IgG, in the presence or absence of 5microM of fluvastatin, and expression of the leukocyte adhesion molecules, VCAM-1 and E-selectin, analyzed by flow cytometry and by quantitative reverse transcriptase-PCR (QRT-PCR). The expression of tissue factor and the chemokine MCP-1 was analyzed by QRT-PCR alone. Incubation of EC with patient IgG increased the expression of VCAM-1, E-selectin, tissue factor and MCP-1. Prior treatment of the cells with fluvastatin further increased the expression of these proteins. The fluvastatin effect was reversed by co-incubation with mevalonate or geranylgeranylpyrophosphate and mimicked by the geranylgeranyl transferase inhibitor GGTI-286. Our results show that in cultured human EC, statins increase the extent of inflammatory activation induced by APLA. This effect appears to be mediated by an inhibitory effect of statins on one or more geranylgeranylated protein(s).

Topics: Adolescent; Adult; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Cell Adhesion Molecules; Cells, Cultured; Chemokine CCL2; Endothelium, Vascular; Fatty Acids, Monounsaturated; Female; Fluvastatin; Gene Expression Regulation; Humans; Immunoglobulin G; Indoles; Inflammation; Male; Middle Aged; Thromboplastin; Umbilical Cord

Topics: Atrial Fibrillation; C-Reactive Protein; Echocardiography, Transesophageal; Heart Atria; Humans; Inflammation; Interleukin-6; Intracranial Embolism; Risk Factors; Thrombophilia; Thromboplastin

Microvascular occlusion, the pathophysiological hallmark of sickle cell disease (SCD), is a complex multifactorial process with alterations in coagulation, endothelial function and inflammation. However, relationships between these process in the two most common genotypes, HbSS and HbSC, are unknown. We hypothesized differences in the hypercoagulable state [as assessed by tissue factor (TF), fibrinogen and D-dimer], endothelial function [markers soluble E-selectin (sE-sel) and von Willebrand factor (vWf)], and inflammation [markers interleukin-6 (IL-6) and high-sensitivity C-reactive protein (hsCRP)] in these two SCD genotypes. Citrated plasma TF, sE-sel, vWf, fibrinogen and fibrin D-dimer, and serum IL-6 and hsCRP (enzyme-linked immunosorbent assay/Clauss) were measured in 64 patients with SCD (27 with HbSS disease) and 42 AA subjects matched for age and ethnic origin. TF (P = 0.0014), sE-sel (P = 0.001) and, as expected, vWf, D-dimer, and hsCRP (all P < or = 0.01), but not fibrinogen or IL-6, were raised in the SCD patients compared with the AA subjects. However, only vWf and, as expected, D-dimer (all P < or = 0.01) were higher in HbSS disease than in HbSC disease. Raised plasma TF and sE-sel in SCD compared with HbAA subjects may contribute to the increased risk of thrombotic disease in this group. Raised vWf in HbSS compared with HbSC may be important in determining pathophysiology in these two genotypes. Positive correlations between IL-6 and TF in both HbSC and HbSS disease leads us to speculate that inflammation may be important in coagulation activation in these patients, or vice versa. However, lack of correlation of sE-sel with inflammatory markers implies that other mechanisms are responsible for increased levels of this marker of endothelial activation.

Topics: Adult; Anemia, Sickle Cell; C-Reactive Protein; Cross-Sectional Studies; E-Selectin; Female; Fibrin Fibrinogen Degradation Products; Genotype; Humans; Inflammation; Interleukin-6; Male; Thromboplastin; von Willebrand Factor

Tissue remodeling by matrix metalloproteinases (MMPs) and plasminogen activators such as tissue factor (TF) is postulated to be involved in the pathogenesis of atherosclerosis. The in situ expression of MMP9 and TF in unstable atherosclerotic plaques has not been examined in detail. Moreover, interference of tissue remodeling by vascular inflammation, apoptosis, and Chlamydia pneumoniae inside plaque subregions is unclear. A total of 40 autopsy carotid arteries (controls) and 20 atherosclerotic carotid endarterectomy specimens (with type VI lesions, according to the American Heart Association classification) from stroke patients were analyzed for expression of MMP9 and TF using in situ techniques. The data on tissue remodeling were correlated with the presence of inflammatory cells (T cells, B-cells, macrophages), apoptosis, and the presence of C. pneumoniae using immunohistochemistry and Western blot analyses. We found a significant overexpression of MMP9 and TF in progressive atherosclerotic carotid arteries, especially in the shoulder and cap subregions (both p < 0.05). Expression of MMP9 and TF correlated significantly with T-cell and macrophage infiltrates as well as with apoptosis (p < 0.05). C. pneumoniae infection was significantly associated with elevated TF expression (p < 0.01) but not with MMP9. MMP9 and TF are thus significantly overexpressed in progressive atherosclerotic plaques, and their relevant subregions (shoulder and cap) are involved in plaque instability. This process is associated with local inflammatory cell infiltrates and apoptosis, which might be influenced by infectious agents such as C. pneumoniae.

Topics: Aged; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Apoptosis; Carotid Artery Diseases; Chlamydophila Infections; Chlamydophila pneumoniae; Female; Humans; Immunohistochemistry; Inflammation; Macrophages; Male; Matrix Metalloproteinase 9; Middle Aged; Prospective Studies; Thromboplastin; Up-Regulation

Topics: Animals; Apoptosis; Disease Models, Animal; Humans; Inflammation; Membrane Microdomains; Neutrophils; Secretory Vesicles; Thromboplastin

During malperfusion and inflammation leukocyte adhesion is common. The purpose of this study was to examine the effects of reduced shear stress on leukocyte-endothelial interactions and subsequent inflammatory reactions such as up-regulation of tissue factor.. Isolated neutrophils and monocytes were co-incubated with human umbilical venous endothelium at 0-3 dynes/cm(2) in a flow chamber. Adhesion and tissue factor expression on adherent leukocytes were examined at various flow conditions.. At 2-3 dynes/cm(2) adhesion occurred only on TNFalpha-activated endothelium. Below 1 dyne/cm(2) similarly increased adhesion was also observed on non-activated endothelium. As was observed for leukocyte adhesion, these shear stress-dependent cell interactions also resulted in an up-regulation of tissue factor on adherent monocytes from non-activated co-cultures.. Apart from additional activators of inflammation, reduced shear forces may directly contribute to inflammation.

Topics: Cell Adhesion; Cell Adhesion Molecules; Cells, Cultured; Coculture Techniques; Endothelial Cells; Flow Cytometry; Humans; Inflammation; Leukocytes; Monocytes; Stress, Mechanical; Thromboplastin; Tumor Necrosis Factor-alpha; Up-Regulation

Topics: Apoptosis; Blood Coagulation Disorders; Blood Platelets; Cell Membrane; Endothelium, Vascular; Hemostasis; Humans; Inflammation; Ischemia; Microspheres; Neovascularization, Physiologic; Platelet Activation; Thrombin; Thromboplastin

There are strong indications that only a small fraction of grafts successfully engraft in clinical islet transplantation. One explanation may be the instant blood-mediated inflammatory reaction (IBMIR) elicited by tissue factor, which is produced by the endocrine cells. In the present study, we show that islets intended for islet transplantation produce tissue factor in both the transmembrane and the alternatively spliced form and that the membrane-bound form is released as microparticles often associated with both insulin and glucagon granules. A low-molecular mass factor VIIa (FVIIa) inhibitor that indirectly blocks both forms of tissue factor was shown in vitro to be a promising drug to eliminate the IBMIR. Thrombin-antithrombin complex (TAT) and FVIIa-antithrombin complex (FVIIa-AT) were measured in nine patients who together received 20 infusions of isolated human islets. Both the TAT and FVIIa-AT complexes increased rapidly within 15-60 min after infusion. When the initial TAT and FVIIa-AT levels were plotted against the increase in C-peptide concentration after 7 days, patients with an initially strong IBMIR showed no significant increase in insulin synthesis after 7 days. In conclusion, tissue factor present in both the islets and the culture medium and elicits IBMIR, which affects the function of the transplanted islets.

Topics: Adult; Alternative Splicing; Antithrombin III; Factor VIIa; Female; Humans; Immunohistochemistry; Inflammation; Islets of Langerhans; Islets of Langerhans Transplantation; Male; Middle Aged; Peptide Hydrolases; Thromboplastin; Time Factors

Inflammatory cytokines alter the hemostatic balance of endothelial cells (ECs). Alternatively spliced human tissue factor (asHTF), a soluble isoform of tissue factor (TF), has recently been detected in ECs, possibly contributing to procoagulability. Agonists regulating asHTF expression and release are yet unknown. This study examines the effect of TNF-alpha and IL-6 on the endothelial expression of both TF variants and delineates the impact of asHTF on the procoagulability of extracellular fluids. asHTF and TF mRNA were assessed by real-time PCR, and asHTF, TF, and tissue factor pathway inhibitor (TFPI) proteins by Western blot and fluorescence microscopy before and after stimulation with TNF-alpha (10 ng/mL) or IL-6 (10 ng/L). The procoagulability of cell supernatant was analyzed by a chromogenic assay with or without phospholipid vesicles. We found asHTF mRNA to be maximally increased 10 minutes after TNF-alpha and 40 minutes after IL-6 treatment (asHTF/GAPDH ratio 0.0223+/-0.0069 versus 0.0012+/-0.0006 for control, P<0.001 and 0.0022+/-0.0004 versus 0.0012+/-0.0007, P<0.05, respectively). Not only was asHTF increased, but also TFPI decreased after cytokine treatment. asHTF was found in the supernatant as early as 5 hours after TNF-alpha stimulation, supporting factor Xa generation after relipidation (6.55+/-1.13 U versus 2.99+/-0.59 U in control supernatant, P<0.00001). Removal of asHTF from supernatants by immunoprecipitation diminished its procoagulability to baseline. The soluble TF isoform expressed and released from ECs in response to inflammatory cytokines becomes procoagulant in the presence of phospholipids. Thus, asHTF released from ECs is a marker for and a contributor to imbalanced hemostasis.

Topics: Blood Coagulation; Cells, Cultured; Endothelial Cells; Humans; Inflammation; Interleukin-6; Lipoproteins; p38 Mitogen-Activated Protein Kinases; Phospholipids; RNA, Messenger; Thromboplastin; Tumor Necrosis Factor-alpha

Topics: Blood Coagulation; Humans; Inflammation; Interleukin-6; RNA, Messenger; Thromboplastin; Tumor Necrosis Factor-alpha

The initial phase of wound repair involves inflammation, induction of tissue factor (TF), formation of a fibrin matrix, and growth of new smooth muscle actin (alpha-SMA)-positive vessels. In diabetes, TF induction in response to cutaneous wounding, which ordinarily precedes increased expression of vascular endothelial growth factor (VEGF) and alpha-SMA transcription, is diminished, though not to a degree causing excessive local bleeding. Enhanced TF expression in wounds of diabetic mice caused by somatic TF gene transfer increased VEGF transcription and translation and, subsequently, enhanced formation of new blood vessels and elevated blood flow. Furthermore, increased levels of TF in wounds of diabetic mice enhanced wound healing; the time to achieve 50% wound closure was reduced from 5.5 days in untreated diabetic mice to 4.1 days in animals undergoing TF gene transfer (this was not statistically different from wound closure in nondiabetic mice). Thus, cutaneous wounds in diabetic mice display a relative deficiency of TF compared with nondiabetic controls, and this contributes to delayed wound repair. These data establish TF expression as an important link between the early inflammatory response to cutaneous wounding and reparative processes.

Topics: Animals; Diabetes Mellitus, Type 1; DNA Primers; Inflammation; Mice; Mice, Inbred NOD; Polymerase Chain Reaction; Skin; Thromboplastin; Vascular Endothelial Growth Factor A; Wound Healing; Wounds and Injuries

Bacterial compounds signal the presence of foreign pathogens in the innate immune system. These microbial components are key players in infectious diseases and implicate toll-like receptors in the activation of inflammation and coagulation. Nevertheless, the existence of a synergistic relationship between peptidoglycan and bacterial DNA on these two physiological responses has not been investigated. The present study reports new findings on the regulation of tumor necrosis factor alpha and tissue factor in peripheral blood mononuclear cells by peptidoglycan and bacterial DNA. These were found to induce tumor necrosis factor alpha and tissue factor simultaneously and in a synergistic manner. These findings provide a new proinflammatory and procoagulant mechanism likely to play a role in sepsis pathogenesis.

Topics: Biomarkers; Blood Coagulation; DNA, Bacterial; Dose-Response Relationship, Drug; Drug Synergism; Humans; Inflammation; Leukocytes, Mononuclear; Monocytes; Peptidoglycan; Thromboplastin; Tumor Necrosis Factor-alpha

Inflammation is a key pathogenic component of atherosclerosis; it also promotes thrombosis, a process underlying acute coronary events and stroke. Cells present in atherosclerotic plaque show abnormal tissue factor (TF) expression. Macrolides, in addition to their antimicrobial properties, have antiinflammatory effects that might help prevent atherothrombosis. The aim of this study was to determine the effect of an immunosuppressant macrolide, rapamycin (Sirolimus), on the expression of TF and its inhibitor (TFPI) by monocytic cells (human blood mononuclear and THP-1 cells) and human aortic smooth muscle cells, in comparison with FK-506 and azithromycin. In monocytic cells, rapamycin and FK-506 inhibited LPS-induced TF activity, antigen and mRNA expression through a transcriptional mechanism involving NF-kappaB. In smooth muscle cells, rapamycin and azithromycin had no effect on serum-induced TF expression, while FK-506 increased serum-induced TF protein and mRNA expression. TFPI levels in the culture supernatants of serum-stimulated smooth muscle cells were not modified by any of the three macrolides. Rapamycin slightly inhibits TFPI induction by LPS in monocytic cells. In addition to its recently established efficacy in the prevention of stent restenosis, the inhibitory effect of rapamycin on the TF pathway might have interesting therapeutic implications.

Topics: Anti-Bacterial Agents; Anti-Inflammatory Agents; Aorta; Azithromycin; Cell Nucleus; Cell Survival; Cells, Cultured; Coagulants; Dose-Response Relationship, Drug; Humans; Immunosuppressive Agents; Inflammation; Leukocytes, Mononuclear; Lipopolysaccharides; Monocytes; Muscle, Smooth; Myocytes, Smooth Muscle; NF-kappa B; Plasmids; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sirolimus; Tacrolimus; Thromboplastin; Transcription, Genetic

Inhibition of blood coagulation appears to be an important therapeutic strategy to improve the outcome in sepsis. However, the beneficial effect of anticoagulant treatment in sepsis is solely based on experimental data using inhibitors of the extrinsic coagulant pathway. The role of the intrinsic pathway of coagulation in the pathogenesis of sepsis has not been explored yet.. In the current study, we contribute to determine the role of factor (F)VIII, the key player of the intrinsic coagulant pathway, on host defense against peritonitis.. Hemizygous FVIII-deficient mice and their wild-type littermates were challenged with 1 x 10(4) bacteria in a septic peritonitis model.. The intraperitoneal injection of Escherichia coli led to growth and dissemination of bacteria and provoked an inflammatory response as evident from elevated cytokine levels, increased cell influx into tissues, liver necrosis, and endothelialitis resulting in mortality. The FVIII-deficient genotype slightly reduced bacterial outgrowth but had no effect on markers of inflammation and/or survival. In addition, FVIII-deficient mice showed profound activation of coagulation, thereby improving the hemophilic phenotype of FVIII-deficient mice.. FVIII deficiency slightly modifies host defense in septic peritonitis in mice, but does not influence the final outcome of peritonitis. Therefore, we question the importance of the intrinsic coagulant pathway during sepsis.

Topics: Animals; Blood Coagulation; Colony Count, Microbial; Escherichia coli; Factor VIII; Hemophilia A; Immunity; Inflammation; Lipoproteins; Mice; Peritonitis; RNA, Messenger; Sepsis; Survival Rate; Thromboplastin

Activation of inflammatory and procoagulant mechanisms is thought to contribute significantly to the initiation of restenosis, a common complication after balloon angioplasty of obstructed arteries. During this process, expression of tissue factor (TF) represents one of the major physiologic triggers of coagulation that results in thrombus formation and the generation of additional signals leading to vascular smooth muscle cell (VSMC) proliferation and migration. In this study, we have investigated the mechanisms by which inhibition of coagulation at an early stage through overexpression of tissue factor pathway inhibitor (TFPI), an endogenous inhibitor of TF, might reduce restenosis. In a rabbit femoral artery model, percutaneous delivery of TFPI using a recombinant adenoviral vector resulted in a significant reduction of the intimamedia ratio 21 days after injury. Investigating several markers of inflammation and coagulation, we found reduced neointimal expression of monocyte chemoattractant protein-1 (MCP-1), lesional monocyte infiltration, and expression of vascular TF, matrix metalloproteinase-2 (MMP-2), and MMP-9. Moreover, overexpression of TFPI suppressed the autocrine release of platelet-derived growth factor BB (PDGF-BB), MCP-1, and MMP-2 in response to factors VIIa and Xa from VSMCs in vitro and inhibited monocyte TF activity. These results suggest that TFPI exerts its action in vivo through not only thrombotic, but also nonthrombotic mechanisms.

Topics: Adenoviridae; Angioplasty; Animals; Becaplermin; Cell Division; Cell Movement; Chemokine CCL2; Cloning, Molecular; Constriction, Pathologic; DNA, Complementary; Factor VIIa; Factor Xa; Graft Occlusion, Vascular; Humans; Immunohistochemistry; In Vitro Techniques; Inflammation; Lipoproteins; Matrix Metalloproteinase 2; Microscopy, Confocal; Monocytes; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Platelet-Derived Growth Factor; Precipitin Tests; Proto-Oncogene Proteins c-sis; Rabbits; Reverse Transcriptase Polymerase Chain Reaction; Thromboplastin; Time Factors; Transfection; Transgenes; U937 Cells

In addition to its central role in hemostasis, thrombin may play a role in inflammation and remodeling. To investigate the contribution of thrombin to allergic airway inflammation in asthma, we used an enzymatic assay to determine thrombin activity in bronchoalveolar lavage fluid obtained from 19 subjects with atopic asthma before (Day 0) and 48 hours after (Day 2) segmental bronchoprovocation with antigen. Thrombin activity increased from 0 (0, 2.9) on Day 1 to 41.1 (0.3, 75.6) U x 10(-3)/ml on Day 2 (p = 0.002) and correlated with total protein levels in lavage fluid on Day 2 (r = 0.885, p < 0.001). After antigen challenge, thrombin activity also showed significant correlations with interleukin-5 (r = 0.66, p = 0.002), transforming growth factor beta1 (r = 0.70, p < 0.001), fibronectin (r = 0.85, p < 0.001) and tissue factor (r = 0.55, p = 0.03) levels in lavage fluid. Furthermore, Day 2, but not Day 0 lavage fluid, induced proliferation of human airway fibroblasts. This mitogenic effect was significantly reduced with hirudin, a specific thrombin inhibitor. Taken together, our findings suggest that allergen-driven airway inflammation in asthma is associated with enhanced potential for fibroblast proliferation that is related, at least in part, to increased thrombin activity. We propose that enhanced thrombin activity provides a potential link between allergic inflammation and initiation of airway remodeling.

Topics: Adult; Allergens; Asthma; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Cohort Studies; Enzyme-Linked Immunosorbent Assay; Female; Fibronectins; Humans; Inflammation; Inflammation Mediators; Interleukin-5; Male; Middle Aged; Probability; Prospective Studies; Sensitivity and Specificity; Statistics, Nonparametric; Thrombin; Thromboplastin; Transforming Growth Factor beta

Nuclear factor kappa B (NF-kappa B) activation has been observed in human atherosclerotic plaques and is enhanced in unstable coronary plaques, but whether such activation has a protective or pathophysiological role remains to be determined. We addressed this question by developing a short-term culture system of cells isolated from human atherosclerotic tissue, allowing efficient gene transfer to directly investigate signaling pathways in human atherosclerosis. We found that NF-kappa B is activated in these cells and that this activity involves p65, p50, and c-Rel but not p52 or RelB. This NF-kappa B activation can be blocked by overexpression of I kappa B alpha or dominant-negative I kappa B kinase (IKK)-2 but not dominant-negative IKK-1 or NF-kappa B-inducing kinase, resulting in selective inhibition of inflammatory cytokines (tumor necrosis factor alpha, IL-6, and IL-8), tissue factor, and matrix metalloproteinases without affecting the antiinflammatory cytokine IL-10 or tissue inhibitor of matrix metalloproteinases. Our results demonstrate that the canonical pathway of NF-kappa B activation that involves p65, p50, c-Rel, and IKK-2 is activated in human atherosclerosis and results in selective up-regulation of major proinflammatory and prothrombotic mediators of the disease.

Topics: Actins; Adenoviridae; Arteriosclerosis; CD3 Complex; CD40 Ligand; Cells, Cultured; Gene Transfer, Horizontal; Humans; I-kappa B Kinase; Inflammation; Interleukin-6; Interleukin-8; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Myocytes, Smooth Muscle; NF-kappa B; NF-kappa B p50 Subunit; NF-kappaB-Inducing Kinase; Protein Serine-Threonine Kinases; Thromboplastin; Thrombosis; Tissue Inhibitor of Metalloproteinase-1; Transcription Factor RelA

Macrophages associated with arterial wall lipid deposition contribute to inflammatory processes. Tissue factor (TF) has been implicated in the thrombogenicity of atherosclerotic plaques. Intimal cells undergoing apoptosis have been postulated as a source for TF. However, there is only limited knowledge of cell type, plaque component, and conditions associated with TF expression and apoptosis. We examined the hypothesis that macrophages exposed to conditions of lipid-rich plaque undergo apoptosis and express TF.. In human carotid (n=15) and coronary (n=6) atherosclerotic plaques, TF and caspase-3 mRNA and protein expression (evaluated by in situ hybridization and immunohistochemistry) were increased significantly in lipid-rich compared with fibrous plaque components (P<0.01) and correlated with high macrophage content (P<0.05). Double-labeling studies demonstrated colocalization of TF and active caspase-3. In hyperlipidemic mice, expression of TF and active caspase-3 was observed simultaneously and colocalized in neointimal macrophages after arterial injury. In neointima of normolipidemic animals, TF and active caspase-3 were absent after arterial injury. In monocytes cultured in the presence of oxidized LDL, strong induction and colocalization of TF and active caspase-3 were found compared with baseline (P<0.05). Both antigens were significantly decreased after cotreatment with a caspase inhibitor (P<0.05) and were absent in untreated control cells.. The expression of TF as the primary cell-associated activator of the coagulation pathway proves to be closely related to macrophages undergoing apoptosis in conditions of lipid-rich plaque, pointing to a key role of lipid content and inflammatory cell viability in determining plaque thrombogenicity.

Topics: Animals; Apoptosis; Arteriosclerosis; Caspase 3; Caspases; Foam Cells; Gene Expression; Humans; Inflammation; Male; Mice; Mice, Inbred C57BL; Monocytes; Thromboplastin; Thrombosis

High levels of the soluble fragment of CD40 ligand (sCD40L) have previously been associated with adverse cardiovascular outcomes. CD40L-CD40 interaction has been linked to the pathogenesis of atherothrombotic complications in cardiovascular disease (CVD). We sought to determine whether a "package of care" of intensified multifactorial cardiovascular risk intervention could reduce indices of platelet activation, inflammation, and coagulation in diabetes and whether patients with overt CVD would derive similar benefit compared with those without.. We measured plasma sCD40L, soluble P-selectin (sP-sel, an index of platelet activation), interleukin-6 (IL-6, a proinflammatory cytokine), and tissue factor (TF, an initiator of coagulation) in 97 patients with diabetes mellitus (41 with and 56 without overt CVD) and 39 comparable healthy control subjects. Thirty-six patients with and 32 without overt CVD then participated in a package of care of cardiovascular risk intervention over a period of 1 year. Plasma levels of sCD40L (P<0.001), sP-sel (P<0.001), IL-6 (P=0.001), and TF (P<0.001) were higher in patients with diabetes than in control subjects, with TF levels highest in patients with overt CVD. Multifactorial intervention was associated with significant reductions in sCD40L in both patient groups (both P<0.001), but reductions in sP-sel and TF were seen only in patients without overt CVD. There was no significant change in IL-6 levels in both patient groups.. Intensive multifactorial risk management can reduce high levels of sCD40L but can only partially correct abnormal platelet activation, inflammation, and coagulation in diabetes, particularly in patients with overt CVD.

Topics: Aged; Aspirin; Biomarkers; Blood Coagulation; Cardiovascular Diseases; CD40 Ligand; Cohort Studies; Diabetes Mellitus; Female; Follow-Up Studies; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypercholesterolemia; Hypoglycemic Agents; Inflammation; Interleukin-6; Male; Middle Aged; P-Selectin; Platelet Activation; Platelet Aggregation Inhibitors; Risk Factors; Risk Management; Thromboplastin

Tissue factor (TF) is the main initiator of coagulation in vivo. Recently, however, a role for TF as a cell receptor involved in signal transduction has been suggested. The aim of the present study was to assess whether activated factor VII (FVIIa) binding to TF could induce smooth muscle cell (SMC) proliferation and to clarify the possible intracellular mechanism(s) responsible for this proliferation.. Cell proliferation was induced by FVIIa in a dose-dependent manner, as assessed by [3H]thymidine incorporation and direct cell counting, whereas no response was observed with active site-inhibited FVIIa (FVIIai), which is identical to FVIIa but is devoid of enzymatic activity. Similarly, no proliferation was observed when binding of FVIIa to TF was prevented by the monoclonal anti-TF antibody AP-1. Activation of the p44/42 mitogen-activated protein (MAP) kinase (extracellular signal-regulated kinases 1 and 2 [ERK 1/2]) pathway on binding of FVIIa to TF was demonstrated by transient ERK phosphorylation in Western blots and by suppression of proliferation with the specific MEK (MAP kinase/ERK kinase) inhibitor UO126. ERK phosphorylation was not observed with FVIIai or when cells were pretreated with AP-1.. These data indicate a specific effect by which binding of FVIIa to TF on the surface of SMCs induces proliferation via a coagulation-independent mechanism and possibly indicate a new link between coagulation, inflammation, and atherosclerosis.

Topics: Animals; Antibodies, Monoclonal; Aorta, Thoracic; Arteriosclerosis; Binding Sites; Blood Coagulation; Butadienes; Cell Division; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Factor VIIa; Humans; Inflammation; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nitriles; Phosphorylation; Protein Binding; Protein Processing, Post-Translational; Rabbits; Recombinant Fusion Proteins; Signal Transduction; Thromboplastin

Anticoagulants have gained increasing attention for the treatment of sepsis. Inhibition of the tissue factor (TF)/factor (F) VIIa pathway has been shown to attenuate the activation of coagulation and to prevent death in a primate model of sepsis caused by gram-negative bacteria.. To determine the role of the TF/FVIIa complex in the host response to peritonitis, mice received an intraperitoneal injection of live Escherichia coli with or without concurrent treatment with recombinant nematode anticoagulant protein c2 (rNAPc2), a selective inhibitor of the TF/FVIIa pathway.. Peritonitis was associated with an increase in the expression of TF at the tissue level and activation of coagulation, as reflected by elevated levels of thrombin-antithrombin complexes and by increased fibrin(ogen) deposition in the liver and lungs. rNAPc2 strongly attenuated this procoagulant response but did not influence the inflammatory response (histopathology, leukocyte recruitment to the peritoneal cavity, and cytokine and chemokine levels). Moreover, rNAPc2 did not alter bacterial outgrowth locally or dissemination of the infection, and survival was not different between rNAPc2-treated mice and control mice.. These data suggest that TF/FVIIa activity contributes to the activation of coagulation during E. coli peritonitis but does not play a role in the inflammatory response or antibacterial host defense.

Topics: Animals; Blood Coagulation; Disease Models, Animal; Escherichia coli; Escherichia coli Infections; Factor VIIa; Helminth Proteins; Inflammation; Male; Mice; Mice, Inbred C57BL; Peritonitis; Thromboplastin

Pentraxin-3 (PTX3), an acute-phase protein that belongs to the family of the PTXs, is found elevated in septic shock and increased in patients with acute myocardial infarction. As tissue factor (TF) plays a key role in thrombosis and inflammation associated with atherosclerosis and as we have recently reported that PTX3 increases TF synthesis in endothelial cells, we tested whether PTX3 could modulate TF expression in monocytes. Monocytes from peripheral blood of healthy donors were incubated with highly purified PTX3 with or without lipopolysaccharide (LPS). Cells were then disrupted, and procoagulant activity was assessed by a one-stage clotting time. PTX3 enhanced TF activity and antigen from LPS-stimulated monocytes in a dose-dependent way. The effect was specific, as other PTXs, such as C-reactive protein and serum amyloid P component, were ineffective. Moreover, the increase in activity was specific for LPS, as in the presence of other TF-inducing agents such as interleukin-1beta and tumor necrosis factor alpha, PTX3 was not effective. The increase in TF activity requires mRNA synthesis, as assessed by polymerase chain reaction. The mechanism by which PTX3 modulates TF synthesis resides in an enhanced IkappaB, alpha phosphorylation and degradation and increased migration of the transacting factor c-Rel/p65 into the nucleus, as determined by Western blot and electro-mobility shift assay. These results show that PTX3 is an enhancer of the expression of TF by mononuclear cells. In the area of vascular injury, during the inflammatory response, cell-mediated fibrin deposition takes place. PTX3 increases TF expression, thus potentially playing a role in thrombogenesis and wound healing.

Topics: Blood Coagulation; Blotting, Western; C-Reactive Protein; Dose-Response Relationship, Drug; Electrophoretic Mobility Shift Assay; Humans; I-kappa B Proteins; Inflammation; Lipopolysaccharides; Monocytes; Phosphorylation; Polymerase Chain Reaction; Proto-Oncogene Proteins c-rel; RNA, Messenger; Serum Amyloid P-Component; Thromboplastin; Up-Regulation

Atrial fibrillation (AF) has been shown to confer a prothrombotic or hypercoagulable state, which could be related to inflammation. Factor XIII (FXIII) catalyses the cross-linking of fibrin monomers, increasing clot resistance; specifically, a common polymorphism, Val34Leu, in the FXIII-A subunit gene has been associated with more rapid FXIII activation. We hypothesised a role for this polymorphism in the prothrombotic state and inflammation in AF, and tested this hypothesis by measurement of indices of coagulation (tissue factor (TF) and fibrinogen), inflammation (interleukin-6 (IL6)) and platelet activation (soluble P selectin (sPsel)).. We studied 90 stable outpatients (73 +/- 8 years) with persistent AF. The FXIII Val34Leu polymorphism was determined by polymerase chain reaction-allelic specific restriction assay (PCR-ASRA). Prevalence of Val34Leu polymorphism of patients was compared to 585 unrelated subjects from the same geographical area. Plasma fibrinogen (Clauss), TF, IL6 and sPsel (all ELISA) were quantified in patient group. Research indices were compared to 74 controls in sinus rhythm with similar clinical characteristics.. There were no statistical differences in FXIII polymorphism prevalence between AF patients and controls. Patients carrying the Leu34 allele had higher plasma levels of TF, IL6 and sPsel (all P < 0.05) compared to controls. Plasma IL6 and TF levels were significantly correlated (Spearman coefficient, r = 0.33, P < 0.01). On multivariate analysis, the Leu34 allele was independently associated with IL6 levels (P < 0.01), whereas TF levels were only associated with IL6 concentrations. However, sPsel and fibrinogen levels were not related to Leu34 allele.. FXIII Val34Leu polymorphism was independently associated with IL6 levels in AF. The Leu34 allele may potentially influence the prothrombotic state in these patients by modulating the inflammatory state.

Topics: Aged; Aged, 80 and over; Alleles; Amino Acid Substitution; Atrial Fibrillation; Factor XIII; Female; Fibrinogen; Genetic Predisposition to Disease; Humans; Inflammation; Interleukin-6; Male; P-Selectin; Polymorphism, Genetic; Thromboplastin

Intervention studies blocking tissue factor (TF) driven coagulation show beneficial effects on survival in endotoxemia models by reducing cytokine production. It is unknown, however, if moderately reduced TF levels influence endotoxemia. The objective was to investigate whether TF haploinsufficiency reduces endotoxin-induced cytokine production in murine cells or in mice. We analyzed the intrinsic capacity of heterozygous TF deficient (TF+/-) leukocytes to produce cytokines. In addition, we determined the consequences of TF haploinsufficiency on endotoxin-induced inflammation during murine endotoxemia. Endotoxin induced the production of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and keratinocyte-derived chemokine (KC) in both whole blood and macrophages. Heterozygous TF deficiency reduced endotoxin induced IL-6 and KC levels about two-fold, while TNF-alpha levels were indistinguishable between TF+/- and wild-type cells. In vivo, endotoxin induced a biphasic coagulant response and significant increases in cytokine levels. Surprisingly, both the inflammatory and the coagulant responses were indistinguishable between wild-type and TF+/- mice. At baseline tissues of TF+/- mice showed a 50% reduction in TF activity compared to wild type. Upon endotoxin administration, TF activity increased and the difference between TF+/- and wild-type mice disappeared after 4 h. After 12 h the baseline difference in TF activity was re-established. TF deficiency reduces cytokine production in vitro, but an enhanced induction of TF during murine endotoxemia eliminates this effect in vivo.

Topics: Animals; Antithrombins; Blood Coagulation; Cell Line; Chemokines; Cytokines; Endotoxemia; Endotoxins; Heterozygote; Immunohistochemistry; Inflammation; Interleukin-6; Keratinocytes; Leukocytes; Lipopolysaccharides; Macrophages; Mice; Mice, Transgenic; Thrombin; Thromboplastin; Time Factors; Tumor Necrosis Factor-alpha

Streptococcal toxic shock syndrome (StrepTSS) is an invasive infection characterized by marked coagulopathy, multiple organ failure, and rapid tissue destruction and is strongly associated with M type 1 and 3 group A streptococci (GAS). Initiation of the coagulation cascade with formation of microvascular thrombi contributes to multiple organ failure in human cases of gram-negative bacteremia; however, little is known regarding the mechanism of coagulopathy in StrepTSS. Thus, we investigated the abilities of several strains of M type 1 and 3 GAS isolated from human cases of StrepTSS to stimulate production of tissue factor (TF), the principal initiator of coagulation in vivo. Washed, killed M type 1 and 3 GAS, but not M type 6 GAS, elicited high-level TF-mediated procoagulant activity from both isolated human monocytes and cultured human umbilical vein endothelial cells. M type 1 GAS consistently elicited higher levels of TF from monocytes than did M type 3 GAS. GAS-induced TF synthesis in monocytes did not correlate with production of tumor necrosis factor alpha or interleukin-8. Conversely, M type 3 GAS were consistently more potent than M type 1 GAS in stimulating endothelial cell TF synthesis. These results demonstrate that (i) M type 1 and 3 strains of GAS are potent inducers of TF synthesis, (ii) GAS-induced TF synthesis is not simply an epiphenomenon of cytokine generation, and (iii) induction of TF in endothelial cells and monocytes may be M type specific. In total, these findings suggest that a novel interaction between GAS and host cells contributes to the observed coagulopathy in StrepTSS.

Topics: Bacterial Typing Techniques; Blood Coagulation; Cells, Cultured; Cytokines; Endothelium, Vascular; Humans; Inflammation; Monocytes; Shock, Septic; Streptococcal Infections; Streptococcus pyogenes; Thromboplastin; Umbilical Veins

Blockade of tissue factor before lethal sepsis prevents acute lung injury and renal failure in baboons, indicating that activation of coagulation by tissue factor is an early event in the pathogenesis of acute lung injury and organ dysfunction. We hypothesized that blockade of tissue factor would also attenuate these injuries in established sepsis by prevention of further fibrin deposition and inflammation. Twelve male baboons received heat-killed Escherichia coli intravenously followed 12 hours later by live E. coli infusion. Six animals were treated 2 hours after the live bacteria with site-inactivated Factor VIIa, a competitive tissue factor inhibitor, and six animals were vehicle-treated sepsis control subjects. Animals were ventilated and monitored for 48 hours. Physiologic and hematologic parameters were measured every 6 hours, and pathologic evaluation was performed after 48 hours. Animals treated with site inactivated Factor VIIa had less severe lung injury, with preserved gas exchange, better lung compliance and histology scores, and decreased lung wet/dry weight. In treated animals, urine output was higher, metabolic acidosis was attenuated, and renal tubular architecture was protected. Coagulopathy was attenuated, and plasma interleukin-6, interleukin-8, and soluble tumor necrosis factor receptor-1 levels were significantly lower in the treated animals. These results show that blockade of coagulation attenuates acute lung and renal injury in established Gram-negative sepsis accompanied by antiinflammatory effects of therapy.

Topics: Acute Kidney Injury; Animals; Antigens, CD; Cytokines; Disease Models, Animal; Drug Monitoring; Escherichia coli Infections; Factor VIIa; Hemodynamics; Inflammation; Interleukin-6; Interleukin-8; Lung Compliance; Male; Papio; Pulmonary Gas Exchange; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Respiratory Distress Syndrome; Sepsis; Severity of Illness Index; Thromboplastin

Expression of membrane-bound CX3CL1, a CX(3)C chemokine, can be strongly induced by inflammatory cytokines in primary endothelial cells, mediating capture and tight adhesion of cells, such as monocytes, that carry the CX(3)CR1 receptor. Here, we measured CX3CL1 mRNA and protein induction by human aortic smooth muscle cells (SMCs), another major component of vessel walls, in response to inflammatory stimuli, and analyzed the effect of membrane-bound CX3CL1 on monocyte adhesion, tissue factor (TF) expression, and tumor necrosis factor-alpha (TNF-alpha) released. In human vascular SMCs, CX3CL1 transcripts were induced after 4h of stimulation with a combination of TNF-alpha and interferon-gamma. Cell-associated and shedded CX3CL1 were measured with a specific ELISA, showing that only 30% of the protein was cleaved from the membrane. Expression of CX3CL1 by SMC increased adhesion of monocytic cells, an effect, which was blocked by soluble CX3CL1. Interestingly, monocyte adhesion to CX3CL1-coated plates partially inhibited lipopolysaccharide-induced TF expression and TNF-alpha release. Thus, CX3CL1, in addition to its adhesive/chemotactic functions, directly promotes monocyte antiinflammatory and antiprocoagulant responses. This could have important implications in clinical settings such as atherosclerosis, in which SMCs and monocytic cells are in close proximity.

Topics: Aorta; Blood Coagulation Factors; Cell Adhesion; Cell Communication; Cells, Cultured; Chemokine CX3CL1; Chemokines, CX3C; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Humans; Inflammation; Interferon-gamma; Lipopolysaccharides; Membrane Proteins; Monocytes; Muscle, Smooth, Vascular; RNA, Messenger; Thromboplastin; Tumor Necrosis Factor-alpha

The immune system contributes to the outcome of pregnancy by complex immunological interactions. Cytokines especially influence the immune milieu pro or contra pregnancy. T helper 1 (Th1) cytokines [tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)] cause inflammation and together are thought to threaten the maintenance of pregnancy. It has been proposed that increased levels of these Th1 cytokines activate coagulation via up-regulating the novel prothrombinase, fgl2. This study further investigates the Th1 cytokine up-regulation of fgl2 expression in a pathophysiological, stress induced abortion model, and an inflammatory, interleukin-12 (IL-12) triggered abortion model.. The DBA/2J-mated CBA/J female mice were exposed to sonic sound stress or were injected with IL-12 during early gestation. On day 13.5 of pregnancy the uteri were removed and the resorption rate was calculated. We evaluated TNF-alpha, IFN-gamma, fgl2 as well as IL-12 messenger RNA (mRNA) expression in decidual samples of all mice by quantitative, real-time polymerase chain reaction (PCR).. A similar resorption rate of 24% was detected in stressed mice, as well as in IL-12 injected mice compared with approximately 11% in non-stressed, non-injected control mice. In stressed mice compared with controls, we observed on day 13.5 up-regulated TNF-alpha, unchanged IFN-gamma down-regulated fgl2, and a slightly increased levels of IL-12. In the IL-12 triggered abortion model, we observed up-regulated levels of TNF-alpha, IFN-gamma and fgl2.. These novel data suggest two distinct cytokine patterns leading to similar abortion rates. A physiological cascade associated with up-regulation of TNF-alpha, and an IL-12-triggered cascade characterized by persistent up-regulation of TNF-alpha and IFN-gamma as well as a persistent increase in fgl2.

Topics: Abortion, Spontaneous; Adjuvants, Immunologic; Animals; Female; Inflammation; Interferon-gamma; Interleukin-12; Mice; Models, Animal; Pregnancy; Reverse Transcriptase Polymerase Chain Reaction; Stress, Psychological; Thromboplastin; Tumor Necrosis Factor-alpha; Up-Regulation

Tissue factor (TF) is the main initiator of blood coagulation in vivo. Its increased expression on activated monocytes is associated with thrombotic complications and mortality in conditions such as sepsis, disseminated intravascular coagulation and coronary artery disease.. The effect of the vitamin B derivative nicotinamide on endotoxin-induced monocyte TF and CD11b expression, soluble interleukin(IL)-6, and clotting onset time (COT) was studied.. Experiments were conducted in human peripheral blood leukocyte suspensions and in whole blood from eight healthy volunteers. Free oscillating rheometry (measuring COT) and flow cytometry were applied to evaluate the effect of endotoxin on TF, CD11b, IL-6 and the overall coagulation response of plasma supplemented with activated autologous leukocytes.. In response to endotoxin, there was an increase in IL-6, TF and CD11b expression and a procoagulant shift of COT. At 4 mmol L-1 nicotinamide, inhibition of TF expression and IL-6 and a normalization of COT were seen. At 16 mmol L-1 nicotinamide, CD11b decreased also. The level of monocyte TF expression correlated with the COT readings, and the endotoxin-induced procoagulant shift of COT could be totally inhibited by blocking TF with an inhibitory antibody.. These results demonstrate the ability of nicotinamide to inhibit the activation of coagulation associated with endotoxemia. We have previously shown that nicotinamide exerts strong anti-inflammatory effects. Evidence is accumulating for nicotinamide to have a therapeutic potential in modulating disease states in which there is a profound activation of coagulation and inflammation, such as in sepsis and disseminated intravascular coagulation.

Topics: Adult; Blood Coagulation; Blood Coagulation Tests; CD11b Antigen; Drug Antagonism; Endotoxins; Humans; Inflammation; Interleukin-6; Leukocytes; Middle Aged; Monocytes; Niacinamide; Thromboplastin

Tissue factor plays a key role in the extrinsic coagulation pathway and is induced by inflammatory cytokines. Atrial myocarditis has been detected recently in some patients with lone atrial fibrillation. Virchow's triad of low blood flow, hypercoagulability, and endothelial dysfunction, enhances thrombus formation. The present study was designed to elucidate the role of endothelial dysfunction in thrombogenesis associated with nonvalvular atrial fibrillation.. We investigated tissue factor expression in the endothelia of left atrial appendages obtained from seven patients with nonvalvular atrial fibrillation and cardiogenic thromboembolism. Tissues were divided into 7-13 sections and compared with control specimens from four patients who died of noncardiac events. Expression of tissue factor, von Willebrand factor and tissue factor pathway inhibitor was evaluated by immunohistochemistry.. Histopathologically, inflammatory cells infiltrated the endocardium and all seven patients showed features of persistent myocarditis. Activated T cells [15.3+/-9.4 cells/high power field (HPF, mean+/-S.D.) vs. control 2.2+/-4.4/HPF (P=0.0294)] and a few macrophages [5.1+/-8.4 cells/HPF vs. control 2.4+/-3.5 cells/HPF (P=NS)] infiltrated the endocardium. Tissue factor was overexpressed in the endothelia particularly in tissues containing inflammatory cells and denuded matrix of the endocardium, compared with the control group. Von Willebrand factor, but not tissue factor pathway inhibitor, was also overexpressed in these tissues.. Tissue factor expression induced by local inflammation is involved in the pathogenesis of thrombosis in patients with nonvalvular atrial fibrillation.

Topics: Aged; Atrial Fibrillation; Endocardium; Endothelium, Vascular; Female; Heart Atria; Humans; Immunohistochemistry; Inflammation; Male; Middle Aged; Myocarditis; Thromboplastin; von Willebrand Factor

Topics: Anticoagulants; Antithrombin III; Blood Coagulation; Heparin; Humans; Inflammation; Multiple Organ Failure; Protein C; Thromboplastin

Inflammation is a major contributing factor to atherosclerotic plaque development and ischemic heart disease. PTX3 is a long pentraxin that was recently found to be increased in patients with acute myocardial infarction. Because tissue factor (TF), the in vivo trigger of blood coagulation, plays a dominant role in thrombus formation after plaque rupture, we tested the possibility that PTX3 could modulate TF expression. Human umbilical vein endothelial cells, incubated with endotoxin (lipopolysaccharide) or the inflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha, expressed TF. The presence of PTX3 increased TF activity and antigen severalfold in a dose-dependent fashion. PTX3 exerted its effect at the transcription level, inasmuch as the increased levels of TF mRNA, mediated by the stimuli, were enhanced in its presence. The increase in mRNA determined by PTX3 originated from an enhanced nuclear binding activity of the transacting factor c-Rel/p65, which was mediated by the agonists and measured by electrophoretic mobility shift assay. The mechanism underlying the increased c-Rel/p65 activity resided in an enhanced degradation of the c-Rel/p65 inhibitory protein IkappaBalpha. In the area of vascular injury, during the inflammatory response, cell-mediated fibrin deposition takes place. Our results suggest that PTX3, by increasing TF expression, potentially plays a role in thrombogenesis and ischemic vascular disease.

Topics: Acute-Phase Proteins; Antigens; Blood Coagulation; C-Reactive Protein; Cell Separation; Cells, Cultured; Dimerization; DNA-Binding Proteins; Endothelium, Vascular; Humans; I-kappa B Proteins; Inflammation; Interleukin-1; Lipopolysaccharides; NF-kappa B; NF-KappaB Inhibitor alpha; Proto-Oncogene Proteins c-rel; RNA, Messenger; Serum Amyloid P-Component; Thromboplastin; Transcription Factor RelA; Tumor Necrosis Factor-alpha; Umbilical Veins; Up-Regulation

Tissue factor (TF) is a transmembrane glycoprotein and the main triggering element of blood coagulation. TF expression on monocytes and endothelial cells is induced by exposure to endotoxin, tumor necrosis factor, and IL-1 and is considered to appear in consequence of inflammation. In order to assess the proinflammatory capacity of TF itself, the recombinant extracellular domain of TF was injected intra-articularly into healthy mice. To characterize the role of immune cells in the TF-induced arthritis, mice deprived of lymphocytes, neutrophils and monocytes were used. Histomorphological analysis of the joints with respect to inflammatory cell infiltration, pannus formation and erosion formation revealed development of arthritis in 80% of animals injected with TF. In most of the cases synovial proliferation was accompanied by pannus formation and cartilage destruction. Inflammatory cell infiltrate consisted of CD4-Mac1+ macrophages. Depletion of monocytes was, however, not enough to abolish inflammation. Indeed, combined deficiency of monocytes and lymphocytes was required to prevent inflammation following the injection of TF. We observed that TF induced chemokine production (MIP-1alpha and RANTES), but did not induce a proliferative response nor cytokine release by mouse spleen cells. TF has strong inflammatogenic properties mediated predominantly by monocytes and their release of chemokines. Our study shows that TF can simultaneously trigger the immune and coagulation systems.

Topics: Animals; Arthritis, Experimental; CD4 Antigens; Chemokine CCL3; Chemokine CCL4; Chemokine CCL5; Female; Immunocompromised Host; Immunoenzyme Techniques; Inflammation; Knee Joint; Lymphocyte Activation; Lymphocytes; Macrophage Inflammatory Proteins; Macrophages; Mice; Mice, Inbred BALB C; Mice, SCID; Monocytes; Nuclear Proteins; Recombinant Proteins; Synovial Membrane; Thromboplastin; Transcription Factors

Monocytes and macrophages express cytokines and procoagulant molecules in various inflammatory diseases. In sepsis, lipopolysaccharide (LPS) from Gram-negative bacteria induces tumor necrosis factor-alpha (TNF-alpha) and tissue factor (TF) in monocytic cells via the activation of the transcription factors Egr-1, AP-1, and nuclear factor-kappa B. However, the signaling pathways that negatively regulate LPS-induced TNF-alpha and TF expression in monocytic cells are currently unknown. We report that inhibition of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway enhances LPS-induced activation of the mitogen-activated protein kinase pathways (ERK1/2, p38, and JNK) and the downstream targets AP-1 and Egr-1. In addition, inhibition of PI3K-Akt enhanced LPS-induced nuclear translocation of nuclear factor-kappa B and prevented Akt-dependent inactivation of glycogen synthase kinase-beta, which increased the transactivational activity of p65. We propose that the activation of the PI3K-Akt pathway in human monocytes limits the LPS induction of TNF-alpha and TF expression. Our study provides new insight into the inhibitory mechanism by which the PI3K-Akt pathway ensures transient expression of these potent inflammatory mediators.

Topics: Cell Line; DNA-Binding Proteins; Early Growth Response Protein 1; Escherichia coli; Gene Expression Regulation; Humans; Immediate-Early Proteins; Inflammation; Lipopolysaccharides; Monocytes; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Thromboplastin; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Tumor Necrosis Factor-alpha; Zinc Fingers

The expression of tissue factor (TF) by monocytes that have transmigrated across the endothelium to sites of extravascular inflammation acts both to focus and amplify the inflammatory response. Because clustering of the integrins responsible for endothelial adhesion and transmigration induces tyrosine phosphorylation and activation of the mitogen-activated protein (MAP) kinases, we postulated that transmigration might lead to monocyte activation and TF production. Monocytes were migrated across TNFalpha-primed ECV304 cells grown on fibronectin-coated Transwell chambers in response to FMLP (10(-8) M). After transmigration, monocytes showed a time-dependent increase in surface TF expression and biological procoagulant activity. TF expression was dependent on monocyte adhesion to ECV304 cells. Specifically, TF was not induced by FMLP treatment of suspended monocytes, by migration across fibronectin alone, or by soluble factors induced during migration, whereas monocyte-ECV304 adhesion was sufficient to stimulate TF. Antibodies against CD29 (beta1 integrin), but not against CD18 (beta2 integrin) or CD31 (PECAM-1), inhibited TF expression. Monocyte adhesion to ECV304 cells induced tyrosine phosphorylation of cellular proteins and specifically of the ERK and p38 MAP kinases. Tyrosine kinase inhibition with genistein (10 microg/mL) blocked transmigration, whereas selective ERK inhibition with PD98059 (50 microM) or p38 inhibition with SB203580 (20 microM) did not. However, both ERK and p38 inhibition dose dependently abolished TF expression. These studies suggest that an extravascular focus of infection or inflammation can promote both intravascular thrombosis and extravascular fibrin deposition during the process of adhesion and transmigration across the endothelial barrier. The selective inhibition of the mitogen-activated protein kinases may offer a novel therapeutic means of modulating this inflammatory sequence.

Topics: Antibodies; CD18 Antigens; Cell Adhesion; Cell Movement; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Fibronectins; Flavonoids; Humans; Imidazoles; Inflammation; Integrin beta1; Mitogen-Activated Protein Kinases; Monocytes; N-Formylmethionine Leucyl-Phenylalanine; Phosphorylation; Platelet Endothelial Cell Adhesion Molecule-1; Pyridines; Thromboplastin; Tumor Necrosis Factor-alpha

The relationship between platelet and leukocyte activation, coagulation, and neointima development was investigated in noninjured murine blood vessels subjected to blood stasis. The left common carotid artery of C57BL/6J mice was ligated proximal to the bifurcation. Tissue-factor expression in luminal leukocytes progressively increased over 2 weeks. On day 3 after ligation, in addition to infiltrated granulocytes, platelet microthrombi and platelet-covered leukocytes as well as tissue-factor-positive fibrin deposits lined the endothelium. Maximal neointima formation in carotid artery cross sections of control mice equaled 28+/-3.7% (n=11) and 42+/-5.1% (n=8) of the internal elastic lamina cross-sectional area 1 and 2 weeks after ligation. In FVIII(-/-) mice, stenosis was significantly lower 1 (11+/-3.6%, n=8) and 2 (21+/-4.7%, n=7) weeks after ligation (both P:<0.01 versus background-matched controls). In u-PA(-/-) mice, luminal stenosis was significantly higher 1 (38+/-7.0%, n=7) and 2 (77+/-5.6%, n=6) weeks after ligation (P:<0.05 and P:<0.01, respectively, versus matched controls). In alpha(2)-AP(-/-) mice, stenosis was lower at 1 week (14+/-2.6%, n=7, P:<0.01) but not at 2 weeks. Responses in tissue-type plasminogen activator or plasminogen activator inhibitor-1 gene-deficient mice equaled that in controls. Reducing plasma fibrinogen levels in controls with ancrod or inducing partial thrombocytopenia with busulfan resulted in significantly less neointima, but inflammation was inhibited only in busulfan-treated mice. We conclude that stasis induces platelet activation, leading to microthrombosis and platelet-leukocyte conjugate formation, triggering inflammation and tissue-factor accumulation on the carotid artery endothelium. Delayed coagulation then results in formation of a fibrin matrix, which is used by smooth muscle cells to migrate into the lumen.

Topics: Afibrinogenemia; Animals; Blood Coagulation; Blood Platelets; Carotid Arteries; Cell Division; Disease Models, Animal; Endothelium, Vascular; Fibrin; Hemostatic Disorders; Inflammation; Leukocytes; Ligation; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Smooth, Vascular; Platelet Activation; Thrombocytopenia; Thromboplastin; Thrombosis; Tunica Intima

When blood is subjected to contact with foreign surfaces, as during cardiopulmonary bypass (CPB), the whole body inflammatory response is initiated, resulting in the expression of procoagulant molecules on the vascular endothelium and white blood cells. These surface bound procoagulants participate in the extrinsic coagulation pathway. It appears that the primary source of thrombin generation during CPB is due to extrinsic pathway activation. Thrombin not only converts fibrinogen to fibrin, it also acts as a proinflammatory agent resulting in a positive feedback loop or the inflammo-coagulatory response. Extrinsic pathway thrombin generation occurs as a membrane bound event. Membrane bound factors are resistant to heparin/ATIII inhibition. Therefore, the anticoagulant effect of heparin/ATIII is due to thrombin inhibition, not the inhibition of thrombin generation. Interpretation of the activated clotting time (ACT) must take into account the thrombin concentration [T]; this results in the coagulatory ratio, ACT is proportional to ([Hep -ATIII]/[T]). Considering this proportionality, it can be seen that the ACT cannot be used to quantitate heparin concentration. Changes in the ACT can reflect changes in [Hep - ATIII], changes in [T], or changes in both concentrations. Anti-inflammatory agents which suppress or inhibit the extrinsic pathway, such as aprotinin, result in decreased thrombin generation. As thrombin generation decreases, the ACT-heparin dose response curve is warped, resulting in a dose response curve resembling a PTT-heparin dose response curve. We can no longer assume that the disproportionate rise in the ACT relative to the [HEP - ATIII] when aprotinin is used as indicative of failure of the ACT to provide a credible indication of anticoagulation.

Topics: Animals; Anticoagulants; Antithrombin III; Blood Coagulation Factors; Cardiopulmonary Bypass; Cell Membrane; Dose-Response Relationship, Drug; Endothelium, Vascular; Heparin; Humans; Inflammation; Models, Biological; Thrombin; Thrombophilia; Thromboplastin; Whole Blood Coagulation Time

To determine the precise relationship between tissue factor and tissue factor pathway inhibitor (TFPI) after trauma, as well as to test the hypothesis that low TFPI levels are not sufficient to prevent tissue factor-dependent intravascular coagulation, leading to multiple organ dysfunction syndrome (MODS).. Prospective, observational cohort study.. Emergency room and intensive care unit in a university hospital.. Thirty-three trauma patients, 18 with disseminated intravascular coagulation (DIC) and 15 without DIC were studied. Ten normal, healthy volunteers served as control subjects.. None.. Antigen concentration of tissue factor and TFPI, and global parameters of coagulation and fibrinolysis were measured on the day of admission, and on days 1-4 after admission. The number of systemic inflammatory response syndrome (SIRS) criteria that patients met and the DIC score were determined, simultaneously. The results of these measurements, incidence of MODS, and outcome were compared between the DIC patients and those without DIC. In the DIC patients, significantly higher tissue factor levels (p =.0049) and lower platelet counts (p =.0016) were found compared with the non-DIC patients and control subjects. However, the TFPI values remained at normal levels during the study period. No correlation was found between the peak levels of tissue factor and TFPI. The mean duration of SIRS and the maximum number of the SIRS criteria being met by the patients in the DIC group were statistically longer and higher than those in the non-DIC patients. The incidence of MODS and the number of the dysfunctioning organs were higher in the DIC patients compared with those in the non-DIC patients, and the DIC patients had a poor outcome.. We systematically elucidated the relationship between tissue factor and TFPI in post-trauma patients. Highly activated tissue factor-dependent coagulation pathway is not sufficiently prevented by the normal TFPI levels in patients with DIC. The DIC associated with thrombotic and inflammatory responses causes MODS, and leads to poor outcome in post-trauma patients.

Topics: Analysis of Variance; APACHE; Case-Control Studies; Disseminated Intravascular Coagulation; Female; Humans; Incidence; Inflammation; Lipoproteins; Male; Middle Aged; Multiple Organ Failure; Multiple Trauma; Platelet Count; Prognosis; Prospective Studies; Systemic Inflammatory Response Syndrome; Thromboplastin; Time Factors; Treatment Outcome

It was previously shown that plasmin activates human peripheral monocytes in terms of lipid mediator release and chemotactic migration. Here it is demonstrated that plasmin induces proinflammatory cytokine release and tissue factor (TF) expression by monocytes. Plasmin 0.043 to 1.43 CTA U/mL, but not active site-blocked plasmin, triggered concentration-dependent expression of mRNA for interleukin-1alpha (IL-1alpha), IL-1beta, tumor necrosis factor-alpha (TNF-alpha), and TF with maximum responses after 4 hours. Plasmin-mediated mRNA expression was inhibited in a concentration-dependent manner by the lysine analogue trans-4-(aminomethyl)cyclohexane-1-carboxylic acid (t-AMCA). Increases in mRNA levels were followed by concentration- and time-dependent release of IL-1alpha, IL-1beta and TNF-alpha and by TF expression on monocyte surfaces. Neither cytokines nor TF could be detected when monocytes were preincubated with actinomycin D or cycloheximide. Electrophoretic mobility shift assays indicated plasmin-induced activation of NF-kappaB; DNA-binding complexes were composed of p50, p65, and c-Rel, as shown by supershift experiments. Nuclear translocation of NF-kappaB/Rel proteins coincided with IkappaBalpha degradation. At variance with endotoxic lipopolysaccharide, plasmin elicited the rapid degradation of another cytoplasmic NF-kappaB inhibitor, p105. Proteolysis of NF-kappaB inhibitors was apparently due to transient activation of IkappaB kinase (IKK) beta that reached maximum activity at 1 hour after plasmin stimulation. In addition, AP-1 binding was increased in plasmin-treated monocytes, with most complexes composed of JunD, c-Fos, and FosB. These findings further substantiate the role of plasmin as a proinflammatory activator of human monocytes and reveal an important new link between the plasminogen-plasmin system and inflammation. (Blood. 2001;97:3941-3950)

Topics: Active Transport, Cell Nucleus; Cytokines; Fibrinolysin; Humans; I-kappa B Kinase; Inflammation; Kinetics; Monocytes; NF-kappa B; Protein Serine-Threonine Kinases; RNA, Messenger; Signal Transduction; Thromboplastin; Transcription Factor AP-1; Up-Regulation

Topics: Animals; Arteriosclerosis; Humans; Inflammation; Mice; Models, Cardiovascular; Thromboplastin; Thrombosis

Hypoxic induction of the early growth response-1 (Egr-1) transcription factor initiates proinflammatory and procoagulant gene expression. Orthotopic/isogeneic rat lung transplantation triggers Egr-1 expression and nuclear DNA binding activity corresponding to Egr-1, which leads to increased expression of downstream target genes such as interleukin-1b, tissue factor, and plasminogen activator inhibitor-1. The devastating functional consequences of Egr-1 up-regulation in this setting are prevented by treating donor lungs with a phosphorothioate antisense oligodeoxyribonucleotide directed against the Egr-1 translation initiation site, which blocks expression of Egr-1 and its gene targets. Post-transplant graft leukostasis, inflammation, and thrombosis are consequently diminished, with marked improvement in graft function and recipient survival. Blocking expression of a proximal transcription factor, which activates deleterious inflammatory and coagulant effector mechanisms, is an effective molecular strategy to improve organ preservation.

Topics: Animals; Blotting, Northern; Blotting, Western; DNA-Binding Proteins; DNA, Antisense; Early Growth Response Protein 1; Fibrin; Gene Expression; Gene Expression Regulation; Graft Survival; Immediate-Early Proteins; Inflammation; Interleukin-1; Lung Transplantation; Plasminogen Activator Inhibitor 1; Rats; RNA, Messenger; Signal Transduction; Thromboplastin; Thrombosis; Transcription Factors

Sepsis-induced tissue factor (TF) expression activates coagulation in the lung and leads to a procoagulant environment, which results in fibrin deposition and potentiates inflammation. We hypothesized that preventing initiation of coagulation at TF-Factor VIIa (FVIIa) complex would block fibrin deposition and control inflammation in sepsis, thereby limiting acute lung injury (ALI) and other organ damage in baboons. A model of ALI was used in which adult baboons were primed with killed Escherichia coli (1 x 10(9) CFU/kg), and bacteremic sepsis was induced 12 h later by infusion of live E. coli at 1 x 10(10) CFU/kg. Animals in the treatment group were given a competitive inhibitor of TF, site-inactivated FVIIa (FVIIai), intravenously at the time of the infusion of live bacteria and monitored physiologically for another 36 h. FVIIai dramatically protected gas exchange and lung compliance, prevented lung edema and pulmonary hypertension, and preserved renal function relative to vehicle (all p < 0.05). Treatment attenuated sepsis-induced fibrinogen depletion (p < 0.01) and decreased systemic proinflammatory cytokine responses, for example, interleukin 6 (p < 0.01). The protective effects of TF blockade in sepsis-induced ALI were confirmed by using tissue factor pathway inhibitor. The results show that TF-FVIIa complex contributes to organ injury in septic primates in part through selective stimulation of proinflammatory cytokine release and fibrin deposition.

Topics: Acute Kidney Injury; Animals; Bacteremia; Blood Coagulation; Disease Models, Animal; Drug Evaluation, Preclinical; Escherichia coli Infections; Factor VIIIa; Fibrinogen; Hemodynamics; Inflammation; Interleukin-6; Kidney Function Tests; Lung Compliance; Male; Papio; Pulmonary Edema; Pulmonary Gas Exchange; Random Allocation; Respiratory Distress Syndrome; Thromboplastin; Tumor Necrosis Factor-alpha

CC chemokine receptors are important modulators of inflammation. Although CC chemokine receptors have been found predominantly on leukocytes, recent studies have suggested that vascular smooth muscle cells respond to CC chemokines. We now report that human smooth muscle cells express CCR5, a co-receptor for human immunodeficiency virus. CCR5 mRNA was detectable by RNA blot hybridization in human aortic and coronary artery smooth muscle cells. The cDNA generated by reverse transcription-polymerase chain reaction from aortic smooth muscle cells had 100% identity throughout the entire coding region with the CCR5 cloned from THP-1 cells. By immunohistochemistry, CCR5 and the CCR5 ligand, macrophage inflammatory protein-1beta (MIP-1beta), were detected in smooth muscle cells and macrophages of the atherosclerotic plaque. In smooth muscle cell culture, MIP-1beta induced a significant increase in intracellular calcium concentrations, which was blocked by an antibody to CCR5. In addition, MIP-1beta caused a calcium-dependent increase in tissue factor activity. Tissue factor is the initiator of coagulation and is thought to play a key role in arterial thrombosis. These data suggest that human arterial smooth muscle cells express functional CCR5 receptors and MIP-1beta is an agonist for these cells.

Topics: Aorta; Arteriosclerosis; Calcium; Chelating Agents; Chemokine CCL4; Coronary Vessels; Dose-Response Relationship, Drug; Egtazic Acid; Endothelium, Vascular; Humans; Immunohistochemistry; Inflammation; Macrophage Inflammatory Proteins; Muscle, Smooth, Vascular; Receptors, CCR5; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thromboplastin; Thrombosis; Time Factors; Umbilical Cord

Adhesive interactions between monocytes and vascular endothelial cells increase the expression of the inflammatory genes, tissue factor (TF) and E-selectin, thus contributing to the inflammatory process. In this study, we have shown that these responses could be regulated by the immunomodulatory cytokine interleukin 10 (IL-10). IL-10 reduced TF generation in monocyte/endothelium co-cultures (64. 3 +/- 3.3% reduction, P < 0.01, n = 4) by acting directly on monocytes, whereas IL-4 inhibited TF expression in both monocytes and endothelium. Similarly, IL-10 reduced the induction of endothelial E-selectin by monocytes (100% reduction at 21 h), but had no effect on cytokine-induced E-selectin expression. IL-10 itself was not able to induce E-selectin protein or mRNA in endothelial cells. IL-10 mRNA was detected in monocytes after 6 h co-culture with endothelial cells, and was sustained for up to 30 h. Finally, IL-10 significantly reduced the adhesion of monocytes to endothelium (45% reduction), which may account in part for the inhibitory actions of IL-10. We conclude that IL-10 has an anti-inflammatory effect on monocyte/endothelium interactions, and may itself be produced as a result of such interactions.

Topics: Cell Adhesion; Cells, Cultured; Coculture Techniques; E-Selectin; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique, Indirect; Humans; Inflammation; Interleukin-10; Interleukin-4; Monocytes; NF-kappa B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thromboplastin

Elevated plasma levels of C-reactive protein (CRP) in population studies and in patients with unstable coronary syndromes are predictive of future adverse events, including cardiac death and myocardial infarction, implicating inflammation in pathogenesis. Although CRP is considered a marker of inflammation, it induces monocyte tissue factor (TF) and may play a prothrombotic role in atherosclerosis and its complications.. Peripheral blood mononuclear cells (PBMCs) from 79 healthy men and women aged 26 to 83 years and 21 healthy postmenopausal women taking hormone replacement therapy (HRT) were stimulated with CRP, lipopolysaccharide (LPS), interferon-gamma (IFN), or their combination. Levels of CRP in the normal range (1 to 5 microg/mL) increased basal monocyte TF 4- to 6-fold and 40-fold at higher concentrations (25 microg/mL). Coincubation of LPS with CRP produced a greater-than-additive response. IFN did not induce TF but synergized with CRP to approximately double activity. There was a striking positive correlation between age and monocyte TF induction, with a dramatic rise on monocytes from postmenopausal women that was not apparent on cells from women taking HRT.. Synergy between CRP and inflammatory mediators may play a direct prothrombotic role in the pathogenesis of coronary atherosclerosis and its acute complications by increasing monocyte/macrophage TF. This may contribute to age and sex differences in coronary events and to the protective effects of HRT.

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Analysis of Variance; C-Reactive Protein; Coronary Artery Disease; Drug Synergism; Female; Hormone Replacement Therapy; Humans; Inflammation; Interferon-gamma; Lipopolysaccharides; Male; Middle Aged; Monocytes; Postmenopause; Sex Factors; Thromboplastin

The capacity of inflammatory cell-derived matrix metalloproteinases (MMPs) to cleave tissue factor pathway inhibitor (TFPI) and alter its activity was investigated. MMP-7 (matrilysin) rapidly cleaved TFPI to a major 35-kDa product. In contrast, MMP-1 (collagenase-1), MMP-9 (gelatinase B), and MMP-12 (macrophage elastase) cleaved TFPI into several fragments including the 35-kDa band. However, rates of cleavage were most rapid for MMP-7 and MMP-9. NH(2)-terminal amino acid sequencing revealed that MMP-12 cleaved TFPI at Lys(20)-Leu(21)(close to Kunitz I domain and producing a 35-kDa band), Arg(83)-Ile(84) (between Kunitz I and II domains), and Ser(174)-Thr(175) (between Kunitz II and III domains). MMP-7 and MMP-9 cleaved TFPI at Lys(20)-Leu(21) with additional COOH-terminal processing. These MMPs did not cleave tissue factor (TF), factor VII, and factor Xa. Proteolytic cleavage by MMP-1, MMP-7, MMP-9, and MMP-12 resulted in considerable loss of TFPI activity. These observations indicate specific cleavage of TFPI by MMPs, which broadens their substrate profile. Co-localization of MMPs, TF, and TFPI in atherosclerotic tissues suggests that release of MMPs from inflammatory cell leukocytes may effect TF-mediated coagulation.

Topics: Base Sequence; Blood Coagulation; DNA Primers; Factor VII; Factor Xa; Humans; Hydrolysis; Inflammation; Lipoproteins; Matrix Metalloproteinases; Recombinant Proteins; Thromboplastin

Tissue factor (TF), a main initiator of clotting, is up-regulated in vasculopathy. We tested the hypothesis that chronic in vivo angiotensin (ANG) II receptor AT(1) receptor blockade inhibits TF expression in a model of ANG II-induced cardiac vasculopathy. Furthermore, we explored the mechanisms by examining transcription factor activation and analyzing the TF promoter. Untreated transgenic rats overexpressing the human renin and angiotensinogen genes (dTGR) feature hypertension and severe left ventricular hypertrophy with focal areas of necrosis, and die at age 7 weeks. Plasma and cardiac ANG II was three- to fivefold increased compared to Sprague-Dawley rats. Chronic treatment with valsartan normalized blood pressure and coronary resistance completely, and ameliorated cardiac hypertrophy (P < 0.001). Valsartan prevented monocyte/macrophage infiltration, nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) activation, and c-fos expression in dTGR hearts. NF-kappaB subunit p65 and TF expression was increased in the endothelium and media of cardiac vessels and markedly reduced by valsartan treatment. To analyze the mechanism of TF transcription, we then transfected human coronary artery smooth muscle cells and Chinese hamster ovary cells overexpressing the AT(1) receptor with plasmids containing the human TF promoter and the luciferase reporter gene. ANG II induced the full-length TF promoter in both transfected cell lines. TF transcription was abolished by AT(1) receptor blockade. Deletion of both AP-1 and NF-kappaB sites reduced ANG II-induced TF gene transcription completely, whereas the deletion of AP-1 sites reduced transcription. Thus, the present study clearly shows an aberrant TF expression in the endothelium and media in rats with ANG II-induced vasculopathy. The beneficial effects of AT(1) receptor blockade in this model are mediated via the inhibition of NF-kappaB and AP-1 activation, thereby preventing TF expression, cardiac vasculopathy, and microinfarctions.

Topics: Angiotensin II; Angiotensin Receptor Antagonists; Animals; Animals, Genetically Modified; Antihypertensive Agents; Blood Coagulation Factors; Blood Pressure; Cell Line; CHO Cells; Coronary Disease; Coronary Vessels; Cricetinae; Extracellular Matrix Proteins; Heart Ventricles; Humans; Inflammation; Integrin alpha4beta1; Integrins; NF-kappa B; Promoter Regions, Genetic; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Receptors, Lymphocyte Homing; RNA, Messenger; Tetrazoles; Thromboplastin; Transcription Factor AP-1; Valine; Valsartan; Vascular Resistance

Reports on the effectiveness of endothelin receptor blockers in angiotensin (Ang) II-induced end-organ damage are conflicting, and the mechanisms involved are uncertain. We tested the hypothesis that endothelin (ET)(A/B) receptor blockade with bosentan (100 mg/kg by gavage after age 4 weeks) ameliorates cardiac and renal damage by decreasing inflammation in rats harboring both human renin and angiotensinogen genes (dTGR). Furthermore, we elucidated the effect of bosentan on tissue factor (TF), which is a key regulator of the extrinsic coagulation cascade. We compared bosentan with hydralazine (80 mg/L in the drinking water for 3 weeks) as a blood pressure control. Untreated dTGR featured hypertension, focal necrosis in heart and kidney, and a 45% mortality rate (9 of 20) at age 7 weeks. Compared with Sprague-Dawley controls, both systolic blood pressure and 24-hour albuminuria were increased in untreated dTGR (203+/-8 versus 111+/-2 mm Hg and 67.1+/-8.6 versus 0.3+/-0.06 mg/d at week 7, respectively). Bosentan and hydralazine both reduced blood pressure and cardiac hypertrophy. Mortality rate was markedly reduced by bosentan (1/15) and partially by hydralazine (4/15). However, only bosentan decreased albuminuria and renal injury. Untreated and hydralazine-treated dTGR showed increased nuclear factor (NF)-kappaB and AP-1 expression in the kidney and heart; the p65 NF-kappaB subunit was increased in the endothelium, vascular smooth muscles cells, infiltrating cells, glomeruli, and tubules. In the heart and kidney, ET(A/B) receptor blockade inhibited NF-kappaB and AP-1 activation compared with hydralazine treatment. Macrophage infiltration, ICAM-1 expression, and the integrin expression on infiltrating cells were markedly reduced. Renal vasculopathy was accompanied by increased tissue factor expression on macrophages and vessels of untreated and hydralazine-treated dTGR, which was markedly reduced by bosentan. Thus, ET(A/B) receptor blockade inhibits NF-kappaB and AP-1 activation and the NF-kappaB- and/or AP-1-regulated genes ICAM-1, VCAM-1, and TF, independent of blood pressure-related effects. We conclude that Ang II-induced NF-kappaB and AP-1 activation and subsequent inflammation and coagulation involve at least in part the ET(A/B) receptors.

Topics: Albuminuria; Angiotensin II; Angiotensinogen; Animals; Animals, Genetically Modified; Antihypertensive Agents; Blood Pressure; Bosentan; Cardiomegaly; Fibronectins; Heart; Humans; Hydralazine; Immunohistochemistry; Inflammation; Intercellular Adhesion Molecule-1; Kidney; Macrophages; Male; NF-kappa B; Rats; Rats, Sprague-Dawley; Renin; Sulfonamides; Thromboplastin; Transcription Factor AP-1; Vascular Cell Adhesion Molecule-1

Topics: Blood Coagulation; Blood Coagulation Disorders; Cell Communication; Cytokines; Humans; Inflammation; Sepsis; Thromboplastin

The association between cancer and thromboembolic disease has been known for over a century. Increased tissue factor expression by endothelial cells, monocytes or macrophages is implicated. Thus, monocyte tissue factor measurements may reflect disease presence or progression.. Using a 2 stage kinetic chromogenic assay, monocyte tissue factor levels were assessed in normal controls (n=60), patient controls (hernia or cholecystectomy, n=60) and in patients with benign and malignant disease of the bladder (n=73), prostate (n=81), breast (n=83) and colorectum (n=62). This was performed as baseline (resting cells) and after 6 hours incubation with (stimulated) and without (unstimulated) lipopolysaccharide. Each benign disease group was sub-divided into inflammatory and non-inflammatory categories.. The relative operating characteristic curve for the lipopolysaccharide-stimulated monocyte tissue factor assay showed sensitivity and specificity for cancer, the area under the curve being 0.71. The control groups and the benign non-inflammatory groups gave similar results and were pooled for further analysis. Each malignant group showed higher monocyte tissue factor levels than the control groups for baseline (P< 0.05) and lipopolysaccharide-stimulated cells (P< 0.05). All benign inflammatory groups apart from breast, showed increased monocyte tissue factor levels over controls for baseline (P< 0.05) and lipopolysaccharide-stimulated cells (P< 0.05). In all cases there was no significant difference between the malignant and the benign inflammatory groups. Monocyte tissue factor levels were related to tumor grade or stage, patients' survival time, serum prostate specific antigen and static bone scan images. Levels were also higher in patients with bladder cancer recurrence and in those who subsequently died.. Lipopolysaccharide-stimulated monocyte tissue factor assay showed sensitivity and specificity for cancer compared to controls. Monocyte tissue factor levels are raised in malignant groups compared to controls and non-inflammatory diseases but not when compared with inflammatory conditions. Stimulated cells give better discrimination between the groups and may be useful in identifying high risk individuals. Monocyte tissue factor levels were related to tumor progression.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Diseases; Breast Neoplasms; Case-Control Studies; Colonic Diseases; Colorectal Neoplasms; Discriminant Analysis; Disease Progression; Female; Humans; Inflammation; Male; Middle Aged; Monocytes; Prostatic Diseases; Prostatic Neoplasms; Risk Factors; Sensitivity and Specificity; Thromboembolism; Thromboplastin; Urinary Bladder Diseases; Urinary Bladder Neoplasms

Vascular endothelial cell growth factor (VEGF) is a major regulator of angiogenesis. We report here that treatment of endothelial cells with VEGF leads to upregulation of tissue factor mRNA and protein expression on the cell surface. Reporter gene studies show that transcriptional activation of the tissue factor gene by VEGF is mediated by a GC-rich promoter element containing overlapping binding sites for Sp1 and EGR-1. As shown by immunofluorescence and electrophoretic mobility shift assays, upon VEGF treatment EGR-1 rapidly accumulates in the nucleus and binds to its respective recognition site in the tissue factor promoter. Sp1 occupies this element in unstimulated cells and seems to be partially displaced by increasing amounts of EGR-1. Transfection of endothelial cells with an EGR-1 expression plasmid mimics the upregulation of tissue factor transcription observed after VEGF treatment. In contrast, NFkappaB, the major transcription factor involved in tissue factor upregulation by inflammatory stimuli, is not activated by VEGF. These data show that VEGF induces a response in endothelial cells largely distinct from inflammatory stimuli, and suggest that EGR-1 is a major mediator of the activation of the tissue factor and possibly other VEGF-responsive genes.

Topics: Cells, Cultured; DNA-Binding Proteins; Early Growth Response Protein 1; Endothelial Growth Factors; Endothelium, Vascular; Gene Expression Regulation; Humans; Immediate-Early Proteins; Inflammation; Lymphokines; Signal Transduction; Thromboplastin; Transcription Factors; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The aim of this study was to see if a short-term period of exposure to cold in young healthy subjects causes changes in hematological factors known to be associated with the promotion of thrombogenesis. Over a period of 48 hours, changes in the distribution of erythrocytes, granulocytes, and blood platelets, as well as several coagulation, inflammatory, and fibrinolytic parameters, were monitored in 11 young healthy male subjects following a short period (1 hour) of cold exposure (CE) (ambient temperature, 11 degrees C) or exposure to thermoneutral conditions (ambient temperature, 26 degrees C) in winter (November). The major findings were: (1) a CE-induced hemoconcentration as indicated by an increase in erythrocyte count (3.2% increase); (2) after appropriate adjustments for changes in hemoconcentration, a cold-induced mobilization of granulocytes (14.5% increase) and a cold-induced decrease in lymphocytes (7% decrease); (3) thromboxane B2 release following endotoxin stimulation of whole blood was increased by 27.4% in the CE experiments; (4) diurnal rhythms were observed in granulocytes, blood platelets, middle plate volume, tissue plasminogen activator, and plasma activator inhibitor; and (5) CE caused no significant changes in lipopolysaccharide-induced tissue factor, nor in the blood coagulation factor VII or cytokines, interleukin-6, and tumor necrosis factor. It is concluded that short-term cold exposure in young healthy subjects initiates a mild inflammatory reaction and a tendency for an increased state of hypercoagulability.

Topics: Adult; Blood Cell Count; Blood Coagulation; Cardiovascular Diseases; Cell Differentiation; Cold Temperature; Cytokines; Environmental Exposure; Fibrinogen; Fibrinolytic Agents; Hematocrit; Humans; Inflammation; Male; Norway; Pain Measurement; Plasminogen Activator Inhibitor 1; Risk Factors; Self-Assessment; Serine Proteinase Inhibitors; Thromboplastin; Thromboxane B2; Tissue Plasminogen Activator

A role of membrane microparticles (MP) released by vascular cells in endothelial cell (EC) activation was investigated. Flow cytofluorimetric analysis of blood samples from normal volunteers revealed the presence of an heterogeneous MP population, which increased by approximately 2-fold after inflammatory stimulation with the chemotactic peptide, N-formyl-Met-Leu-Phe (2,799 +/- 360 versus 5241 +/- 640, p < 0.001). Blood-derived MP stimulated release of EC cytokines interleukin (IL)-6 (377 +/- 68 pg/ml) and MCP-1 (1, 282 +/- 79) and up-regulated de novo expression of tissue factor on the EC surface. This was associated with generation of a factor Xa-dependent procoagulant response (2.28 +/- 0.56 nM factor Xa/min/10(4) cells), in a reaction inhibited by a monoclonal antibody to tissue factor. Fluorescent labeling with antibodies to platelet GPIbalpha or leukocyte lactoferrin demonstrated that circulating MP originated from both platelets and leukocytes. However, depletion of platelet MP with an antibody to GPIbalpha did not reduce EC IL-6 release, and, similarly, MP from thrombin-stimulated platelets did not induce IL-6 release from endothelium. EC stimulation with leukocyte MP did not result in activation of the transcription factor NF-kappaB and was not associated with tyrosine phosphorylation of extracellular signal-regulated protein kinase, ERK1. In contrast, leukocyte MP stimulated a sustained, time-dependent increased tyrosine phosphorylation of approximately 46-kDa c-Jun NH(2)-terminal kinase (JNK1) in EC. These findings demonstrate that circulating leukocyte MP are up-regulated by inflammatory stimulation in vivo and activate a stress signaling pathway in EC, leading to increased procoagulant and proinflammatory activity. This may provide an alternative mechanism of EC activation, potentially contributing to dysregulation of endothelial functions during vascular injury.

Topics: Base Sequence; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Chemokine CCL2; Coagulants; DNA Primers; Endothelium, Vascular; Humans; Inflammation; Interleukin-6; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinases; Neutrophils; Signal Transduction; Thromboplastin

In the present study, the involvement of both procoagulants and anticoagulants from adherent peritoneal cells in an inflammatory model was investigated.. Mice were injected with thioglycollate broth or lipopolysaccharide (LPS).. Cells were harvested by peritoneal lavage. Adherent peritoneal cells were cultured further +/- LPS. Subsequently, their ability to activate either Factor X or prothrombin, and their ability to inactivate thrombin through a thrombin-degrading mast cell chymase, was assayed.. Inflammatory cells expressed reduced amounts of thrombin-inactivating activity as compared with control resident peritoneal cells. Both resident and inflammatory cells expressed potent prothrombinase activities. Further stimulation of the various cellular populations with LPS in vitro had very little effect on the prothrombinase and thrombin-inactivating activities, bud had a strong stimulatory effect on the Factor X-activating activities.

Topics: Animals; Cells, Cultured; Chymases; Disease Models, Animal; Factor X; Female; Inflammation; Lipopolysaccharides; Macrophages, Peritoneal; Mice; Mice, Inbred Strains; Serine Endopeptidases; Thioglycolates; Thrombin; Thromboplastin

The potential for tissue factor (TF) to enhance inflammation by factor VIIa-dependent induction of proinflammatory changes in macrophages was explored. Purified recombinant human factor VIIa enhanced reactive oxygen species production by human monocyte-derived macrophages expressing TF in vitro. This effect was dose- and time-dependent, ligand- and receptor-specific, and independent of other coagulation proteins. This receptor/ligand binding induced phospholipase C-dependent intracellular calcium fluxes. Transfection studies using a human monocyte-derived cell line (U937) demonstrated that an intact intracytoplasmic domain of TF is required for factor VIIa-induced intracellular calcium fluxes. The capacity of TF to enhance proinflammatory functions of rabbit peritoneal-elicited macrophages (production of reactive oxygen species and expression of major histocompatibility complex class II and cell adhesion molecules) was demonstrated in vivo by treatment with an anti-TF antibody. These data demonstrate that, in addition to its role in activation of coagulation, TF can directly augment macrophage activation. These effects are initiated by binding factor VIIa and are independent of other coagulation proteins. These studies provide the first demonstration of a direct proinflammatory role for TF acting as a cell-signaling receptor.

Topics: Antibodies; Biological Transport; Calcium; Cell Differentiation; Cytoplasm; Dose-Response Relationship, Drug; Factor VIIa; Humans; Inflammation; Inflammation Mediators; Ligands; Macrophage Activation; Macrophages; Macrophages, Peritoneal; Monocytes; Reactive Oxygen Species; Signal Transduction; Thrombin; Thromboplastin; Time Factors; U937 Cells; Up-Regulation

Monocyte and urinary tissue factors (mTF and uTF) are both elevated in a number of pathologic conditions, including cancer. This study validates the best available uTF and mTF assays as diagnostic tools for cancer and examines if uTF levels reflect monocyte activation. Using kinetic chromogenic assays for uTF and mTF (measured on fresh resting cells [baseline], unstimulated cells, and lipopolysaccharide [LPS]-stimulated cells), we assessed TF levels in normal individuals, surgical controls, and patients with benign and malignant diseases. Each benign disease group was stratified as inflammatory or noninflammatory. Controls and benign noninflammatory results were indistinguishable. The malignant and inflammatory groups showed raised uTF levels over controls (p < 0.001). mTF levels differ similarly. For mTF and uTF assays, there was no significant difference between the malignant and inflammatory groups. The relative operating characteristic (ROC) curve plots sensitivity against false positive rate (1-specificity) for all possible cutoff values of a diagnostic test. Assay performance is assessed as the area under the curve (AUC). The ROC curve for the uTF assay displayed both sensitivity and specificity for cancer, the AUC being 0.83. Of the three mTF levels, LPS-stimulated cells gave the optimum curve (AUC = 0.71). uTF showed a weak to moderate association with mTF levels but correlated best and was statistically significant when compared with levels in the LPS-stimulated cells. uTF represents an intrinsic, kidney-derived, physiologic concentration rather than that of preactivated or postactivated monocytes. In conclusion, both uTF and LPS-stimulated mTF levels showed sensitivity and specificity in detecting cancer and inflammatory diseases. However, the two forms of TF appear to be independently derived.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Area Under Curve; Biomarkers, Tumor; Breast Neoplasms; Child; Child, Preschool; Cholelithiasis; Colorectal Neoplasms; Diagnosis, Differential; False Positive Reactions; Female; Hernia, Inguinal; Humans; Inflammation; Kidney Calculi; Lipopolysaccharides; Male; Middle Aged; Monocytes; Neoplasms; Prostatic Neoplasms; ROC Curve; Sensitivity and Specificity; Thromboplastin; Urinary Bladder Neoplasms

The levels of circulating monocyte chemoattractant protein-1 (MCP-1) and tissue factor (TF) were examined on admission in 46 consecutive patients with acute coronary syndromes (ACS) and 30 patients with stable exertional angina (SEA). The plasma levels of both MCP-1 and TF were higher in the ACS patients than in the SEA patients (MCP-1: p<0.001; TF: p<0.001). Only the circulating TF level related to the number of diseased vessels. A positive correlation between plasma MCP-1 and TF levels was found (r=0.476, p<0.001). These results suggest that circulating MCP-1 plays an important role in the pathogenesis and/or development of ACS.

Topics: Acute Disease; Aged; Angina Pectoris; Angina, Unstable; Chemokine CCL2; Female; Humans; Inflammation; Male; Middle Aged; Monocytes; Myocardial Infarction; Thromboplastin

Tissue factor (TF), a principal initiator of the vertebrate coagulation cascade, is expressed in organ tissues, cells and blood. TF is known to be induced in endothelial cells, monocytes and macrophages by inflammatory stimuli and in many pathologic conditions. By using the modified method for in vivo TF activity assay, we found that turpentine oil injection as an inflammatory stimulus also induced the TF activity in lung and brain tissues of rats. And the age-related increase in TF activity was observed in healthy rat brain tissue.

Topics: Aging; Animals; Body Weight; Brain Chemistry; Calibration; Inflammation; Irritants; Lung; Male; Rats; Rats, Sprague-Dawley; Thromboplastin; Turpentine

Tissue factor (TF) is an integral membrane glycoprotein that serves as a cofactor for blood coagulation factor VIIa. The induction of TF on the surface of endothelial cells is initiated by various stimuli including lipopolysaccharide, interleukin-1 beta, and tumor necrosis factor alpha. We have demonstrated that recombinant human C5a induces TF activity in a dose-dependent fashion in human umbilical vein endothelial cells (HUVEC). Peak activity (4.9-fold increase) was obtained 3-6 h after treatment with 10 microM C5a. TF mRNA as assessed by RT-PCR method was also significantly increased (3.75-fold) after 3 h incubation with C5a, suggesting that C5a induces TF activity on HUVEC, at least in part, by enhancing the level of TF mRNA. The increase in TF activity by C5a was inhibited by methylprednisolone. The induction of TF on endothelial cells by C5a may represent one of many potential interrelationships between the inflammatory and coagulation schemes.

Topics: Blood Coagulation; Cells, Cultured; Complement C5a; Dose-Response Relationship, Drug; Endothelium, Vascular; Gene Expression Regulation; Humans; Inflammation; Methylprednisolone; Recombinant Proteins; RNA, Messenger; Thromboplastin; Umbilical Veins

Endothelial-monocyte-activating polypeptide II (EMAP II) is a novel mediator isolated from conditioned medium of methylcholanthrene A-induced tumor cells which modulates properties of endothelial cells, mononuclear phagocytes (MPs), and polymorphonuclear leukocytes (PMNs) in vitro and induces an acute inflammatory response in vivo. A synthetic peptide comprising 15 residues from the N-terminal region (residues 6-20) was shown to induce directional migration of MPs and PMNs, with half-maximal effect at approximately 200-250 pM, whereas a peptide from the C terminus of EMAP II, as well as other irrelevant peptides, were without effect. Modulation of cellular phenotype by EMAP II-derived peptide was suggested by peptide-induced elevation of cytosolic free calcium concentration in fura-2-loaded MPs and PMNs and by stimulation of peroxidase release in PMNs. Consistent with these in vitro data, EMAP II-derived N-terminal peptide-albumin conjugates injected into the mouse footpad elicited inflammatory cell tissue infiltration, whereas albumin alone or EMAP II-derived C-terminal peptide conjugated to albumin incited little response. Binding of 125I-labeled EMAP II-derived peptide (residues 12-20) to MPs was saturable (Kd approximately 200 pM) and was blocked in a dose-dependent manner by the addition of intact EMAP II and unlabeled EMAP II-derived peptides (residues 6-20 and 12-20), whereas interleukin 1, tumor necrosis factor, formyl-methionyl-leucinyl-phenylalanine, or irrelevant peptides were without effect. Cross-linking of 125I-EMAP II-derived peptide (residues 12-20) by disuccinimidyl suberate to human MPs demonstrated a band, approximately 73 kDa, on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 125I-EMAP II-derived peptide also demonstrated specific binding to human PMNs and murine RAW cells. These data indicate that the N-terminal region of EMAP II defines a biologically active locus of the molecule which interacts with target cells via a potentially novel cellular receptor.

Topics: Amino Acid Sequence; Animals; Calcium; Cell Line, Transformed; Cell Membrane; Chemotaxis, Leukocyte; Cytokines; Humans; Inflammation; Kinetics; Leukocytes, Mononuclear; Macrophages; Mice; Molecular Sequence Data; Neoplasm Proteins; Neutrophils; Peptide Fragments; Peroxidase; Phagocytes; Protein Binding; RNA-Binding Proteins; Structure-Activity Relationship; Thromboplastin

Reciprocal interactions between elements of the acute inflammatory response and the coagulation system play important roles in host defense homeostasis during Gram-negative bacterial sepsis. However, derangements in the regulation of the inflammatory-coagulant axis in this setting may result in progressive tissue damage and disseminated intravascular coagulation. In this article, the integrated responses in the baboon model of Escherichia coli sepsis are analyzed as a basis of understanding these response interactions in the critically ill. In particular, three topics will be reviewed. First, the role of tissue factor in mediating the coagulant response to inflammation and the role of tumor necrosis factor (TNF) in initiating and amplifying this coagulant response into a full-blown consumptive coagulopathy are defined. A second and parallel topic concerns the role played by tissue factor pathway inhibitor and other anticoagulant systems in not only regulating this coagulant response, but also in attenuating the initial inflammatory response. The third topic concerns the use of assays of enzyme inhibitor complexes composed of components of these regulatory anticoagulant systems to help define the hypercoagulable state and possibly to make an early, specific diagnosis of sepsis prior to overt failure of the hemostatic system.

Topics: Animals; Blood Coagulation; Blood Coagulation Factors; Disseminated Intravascular Coagulation; Endothelium, Vascular; Escherichia coli Infections; Fibrin; Inflammation; Lipoproteins; Papio; Sepsis; Thromboplastin; Tumor Necrosis Factor-alpha

We recently proposed that endothelium may represent the primary physiologic site of synthesis of the tissue factor pathway inhibitor (TFPI). In support of this conclusion, we have now found that the poly(A)+ RNAs obtained from rabbit and bovine lung tissues contain abundant amounts of TFPI messenger RNAs (mRNAs), whereas the poly(A)+ RNAs obtained from the liver of these animals contain less than 5% of that found in the lung tissues. Because inflammatory mediators are known to upregulate tissue factor (TF) expression by the endothelium, we have examined the effect of these agents on the TFPI expression by the cultured endothelial cells. When cultured human umbilical vein endothelial cells were stimulated (in 10% fetal bovine serum) with phorbol myristate acetate (PMA), endotoxin, interleukin-1, or tumor necrosis factor-alpha, the TF mRNA increased approximately 7- to 10-fold within 2 to 4 hours. Unstimulated cells constitutively expressed TFPI mRNA and its levels either did not change or increased slightly (up to 1.5-fold) upon stimulation with these inflammatory agents. TF mRNA abruptly declined to a negligible level and the TFPI mRNA returned essentially to the basal level at approximately 24 hours. The membrane-bound TF clotting activity of induced cells peaked between 4 and 8 hours, and finally declined. The cumulative TFPI activity secreted into the media was either unchanged or slightly higher in the induced cell cultures as compared with that present in the noninduced cultures. Endothelial cells were also cultured in 10% heat-inactivated human serum derived from plasma or whole blood. TFPI secreted into the media containing whole blood serum was consistently higher (approximately 1.5-fold at 8 hours) than that secreted into the media supplemented with serum obtained from plasma lacking the formed elements; these cells also expressed similarly increased levels of TFPI mRNA. Moreover, PMA-stimulated cells cultured in whole blood serum expressed modestly increased levels of TFPI mRNA (approximately 1.5-fold); supernatants from these cells also contained similarly increased TFPI activity. Cumulatively, our data indicate that, unlike thrombomodulin and fibrinolytic enzymes synthesized by the endothelial cells, TFPI synthesis is not downregulated and may be slightly upregulated during an inflammatory response. Inspection of the 5' flanking region of the TFPI gene showed a conserved GATA-binding motif located approximately 400 bp upstream of the proposed

Topics: Animals; Base Sequence; Blood; Blotting, Northern; Cattle; Cell Line; Cells, Cultured; Endothelium, Vascular; Endotoxins; Factor VII; Gene Expression; Humans; Inflammation; Interleukin-1; Lipoproteins; Liver; Lung; Molecular Sequence Data; Rabbits; RNA, Messenger; Tetradecanoylphorbol Acetate; Thromboplastin; Tumor Necrosis Factor-alpha; Umbilical Veins

We have previously reported the presence in normal human placentae of coagulation, macrophages and helper T lymphocytes in inflammatory foci known as villitis of unestablished etiology. In order to investigate the link between coagulation and immunity, we have studied fetal stem vessel endothelium for tissue factor, which is made available by cytokines and activates coagulation via the extrinsic pathway. We found that fetal stem vessel endothelial cells of normal chorionic villi did not react with antibody to tissue factor. Normal placentae contain small numbers of villitis areas and endothelium in these areas was reactive with antibody to tissue factor. Endothelial tissue factor reactivity was more prominent in placentae from secondary recurrent spontaneous aborters and these placentae have greatly increased numbers of villitis areas. The tissue factor availability on fetal stem vessel endothelium may result from immunologically mediated cytokine release. The net effect of these reactions is the presence of lymphocytes, macrophages, coagulation, necrosis and vasculitis in villitis.

Topics: Abortion, Habitual; Chorionic Villi; Endothelium, Vascular; Female; Fetus; Humans; Inflammation; Placenta Diseases; Pregnancy; Pregnancy Trimester, Third; Thromboplastin

Monocytes can be induced to synthesize and express tissue factor procoagulant activity. They can also be stimulated to release a broad spectrum of inflammatory agents including superoxide anion (O2-) that are thought to contribute to the pathogenesis of inflammatory diseases. Dipyridamole, an inhibitor of platelet aggregation blocks the lipopolysaccharide (LPS)-induced increase in monocyte-associated tissue factor activity and phorbol myristate acetate (PMA) stimulated O2- release from monocytes and polymorphonuclear leukocytes (PMN). Dipyridamole inhibition of O2- release can be reversed by increased glucose in the culture media, whereas dipyridamole inhibition of tissue factor can not be reversed by increased glucose in the culture media. These results reveal that dipyridamole influences monocytes by at least two distinct mechanisms. Further, it may serve as an anti-thrombotic agent by virtue of its effect on both platelet aggregation and monocyte tissue factor activity.

Topics: Biological Transport; Cells, Cultured; Depression, Chemical; Dipyridamole; Endotoxins; Fibrinolytic Agents; Glucose; Humans; Inflammation; Leukocytes, Mononuclear; Lipopolysaccharides; Neutrophils; Superoxides; Tetradecanoylphorbol Acetate; Thromboplastin

Extravascular coagulation and fibrinolysis is an integral part of inflammatory reactions. Disordered expression of procoagulant and profibrinolytic factors by mononuclear phagocytes of the lung (i.e. lung alveolar macrophages (LAM) and interstitial macrophages) may have important bearings on inflammatory lung tissue destruction and repair. Based on this hypothesis we have measured the presence of trigger molecules and activation products of the coagulation and fibrinolytic system in cell-free bronchoalveolar lavage fluid and in bronchoalveolar cells. Patient groups with chronic obstructive disease (COLD) (n = 76), idiopathic pulmonary fibrosis (IPF) (n = 29), sarcoidosis (n = 22), lung cancer (n = 36), pneumonia (n = 39), acquired immunodeficiency syndrome (AIDS) (n = 17) and a control group (n = 60) were studied by bronchoalveolar lavage (BAL). In all patient groups tissue thromboplastin (TPL) and fibrinopeptide A (FPA) were significantly increased compared to controls. Plasminogen activator (PA) activity was significantly lower in patients than in normals, and usually associated with high levels of antifibrinolytic activity. The level of PA inhibitor (PAI-2) was not significantly higher in any patient group compared to controls. The sensitivity of the method for fibrin degradation products (FDP) analysis was not high enough to detect FDP in BAL fluid of control individuals, whereas such products could be demonstrated in 25-53% of patients in various categories. We conclude that disordered expression of procoagulant and plasminogen activator activities in bronchoalveolar lavage fluid may reflect a milieu that favours accumulation of fibrin in inflammatory lung tissue and form the basis for the development of pulmonary fibrosis.

Topics: Acquired Immunodeficiency Syndrome; Blood Coagulation; Bronchoalveolar Lavage Fluid; Fibrin Fibrinogen Degradation Products; Fibrinolysis; Fibrinopeptide A; Humans; Inflammation; Lung Diseases; Plasminogen Inactivators; Pulmonary Fibrosis; Thromboplastin

Topics: Animals; Anticoagulants; Fibrin; Glomerulonephritis; Humans; Inflammation; Macrophages; Microcirculation; Monocytes; Rabbits; Rats; Thromboplastin

We used a murine mAb, H4/18, raised by immunization with IL-1-treated human umbilical vein endothelial cell cultures, to localize an endothelial activation antigen in induced human delayed hypersensitivity reactions (DHR) and in pathological tissues. We used streptococcus varidase to elicit DHR in human skin and we examined sequential skin biopsies with the immunoperoxidase technique. There was no staining for H4/18 binding antigen in normal endothelium of skin and other tissues; strong positive staining, localized to vascular endothelium, was seen at 16 and 23 h but disappeared by 6 d, when the DHR had faded. H4/18 binding antigen, also confined to endothelium, was detected in lymph nodes, skin, and other tissues exhibiting immune/inflammatory reactions. The studies indicate that H4/18 is a useful marker for activated endothelium in vivo and they support the relevance of in vitro studies on inducible endothelial cell functions.

Topics: Animals; Antibodies, Monoclonal; Antigens; Antigens, Surface; Blood Coagulation Factors; Endothelium; Humans; Hypersensitivity, Delayed; Immunoblastic Lymphadenopathy; Inflammation; Mice; Mice, Inbred BALB C; Skin; Thromboplastin

Fibrin deposition is prominent in the histopathologic features of chronic interstitial lung disease. Human alveolar macrophages can potentially modulate this process because normal macrophages synthesize and express the initial enzymes of both coagulation and fibrinolytic pathways. In the present study, we examined the cell-associated procoagulant activity of macrophages lavaged from patients with sarcoidosis (n = 14) or idiopathic pulmonary fibrosis (n = 13) and compared the enzyme activities with that of a group of normal volunteers (n = 16). Cells from sarcoid patients had a mean (+/- 1 SD) tissue factor activity of 1,491 +/- 2,160 units/5 X 10(5) cells, as compared with a mean of 480 units (range, 140 to 1,000 units) for normal control subjects. The same cells had a mean plasma Factor VII equivalent of 4.7 ng/10(6) cells, as compared with 0.81 ng/10(6) cells (range, 0.2 to 2.0 ng) for the normal control subjects. The enhanced activity correlated with disease activity as judged by radiographic stage: only patients with Stage II or Stage III disease had consistently elevated procoagulant activity. There was no correlation of procoagulant activity with the percentage of lymphocytes in the alveolar fluid. Cells from patients with idiopathic pulmonary fibrosis also had increased tissue factor (mean, 2,980 +/- 2,619 units) but less consistently elevated Factor VII. There was considerable variation in both procoagulant activity and cell differentials between lavage sites in 10 patients in whom 2 separate lobes were studied concurrently. In addition, we examined the plasminogen activator (PA) activities of lavaged cells and concentrated alveolar fluids.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Aged; Factor VII; Female; Fibrin; Fibrinolysis; Humans; Inflammation; Macrophages; Male; Middle Aged; Plasminogen Activators; Pulmonary Alveoli; Pulmonary Fibrosis; Sarcoidosis; Thromboplastin

Interleukin-1 (IL-1), an inflammatory/immune mediator, acts directly and selectively on cultured human vascular endothelial cells to alter two important functional properties. First, IL-1 induces endothelial cell biosynthesis and surface expression of a tissue factor-like procoagulant activity. Second, IL-1 dramatically increases the adhesiveness of the endothelial cell surface for human peripheral blood polymorphonuclear leukocytes (6-42-fold increase) and monocytes (2-5-fold increase), as well as the related leukocyte cell lines HL-60 and U937. These IL-1 effects are concentration-dependent (maximum, 5-10 U/ml), time-dependent (peak 4-6 hours), and reversible. Cycloheximide and actinomycin D block these IL-1 actions on endothelium, which suggests the requirement for de novo protein synthesis. Human-monocyte-derived IL-1, cell-line--derived IL-1, and recombinant IL-1 exhibited comparable biologic activities in our assays, whereas two other mediators, IL-2 and immune interferon, were without effect. IL-1 stimulated procoagulant activity and leukocyte adhesion in human endothelial cells cultured from both umbilical veins and adult saphenous veins but not in other cultured cell types, including SV-40-transformed human endothelial cells and human dermal fibroblasts. Similar actions of IL-1 on vascular endothelium in vivo may contribute to the development of intravascular coagulation and enhanced leukocyte--vessel wall adhesion at sites of inflammation.

Topics: Adhesiveness; Blood Coagulation; Cells, Cultured; Endothelium; Humans; Inflammation; Interleukin-1; Monocytes; Neutrophils; Thromboplastin; Umbilical Veins

Several inflammatory particles (zymosan, two forms of asbestos, carrageenan) and the soluble inflammatory agent dextran sulphate induced a dose- and time-dependent increase in thromboplastin activity of cultured human monocytes. Two weakly inflammatory particles (latex and anatase) had little effect. The increase largely depended on protein synthesis; was slow, reaching a peak at about 18 h; and was not due to endotoxin. This procoagulant may contribute to the fibrin deposition frequently seen in inflammatory lesions.

Topics: Asbestos; Carrageenan; Cells, Cultured; Dextran Sulfate; Dextrans; Endotoxins; Humans; Inflammation; Latex; Monocytes; Polymyxin B; Thromboplastin; Zymosan

The release of procoagulant material by connective tissue cells as a sequel to cell injury can initiate blood coagulation and may thus play a role in he pathogenesis of inflammatory lesions. Human foreskin fibroblasts were shown to synthesize high levels of the procoagulant TF in vitro. Generation of TF by fibroblasts was inhibited by addition of supernatants of PHA-stimulated human mononuclear cells to fibroblast cultures. The inhibition was independent of supernatant effects on cellular proliferation and was accompanied by up to a 20-fold increases in PGE2 synthesis in the fibroblast cultures. The inhibition of TF generation by MC-SNs was reversed by adding indomethacin to the fibroblast cultures, suggesting that mononuclear cells suppress fibroblast TF generation by stimulation of endogenous fibroblast synthesis of prostaglandin. Regulation of fibroblast PCA by products of immune cells may be important in the pathogenesis of inflammatory lesions.

Topics: Cells, Cultured; Dinoprostone; Fibroblasts; Humans; Indomethacin; Inflammation; Lymphocyte Activation; Male; Monocytes; Phytohemagglutinins; Prostaglandins E; Thromboplastin

Forty-eight virgin guinea pigs were subjected to bilateral sectioning of the uterine horn and cuff type salpingostomy. Avitene, Topical Thrombin, or Gelfoam was used as the sole hemostatic agent in each of three study groups. Bipolar cauterization was used in a control series. These agents were compared with regard to ease of application, degree of adhesion formation, net surface area, and microscopic evaluation of degree of fibrosis and inflammatory reaction. The results revealed no statistically significant differences among the various methods of hemostasis. Data were evaluated by uni- and multivariate analyses. Several trends in the data were noted: the greatest amount of fibrosis was associated with Topical Thrombin and Avitene, and the greatest inflammatory response was associated with Avitene. These agents show no superiority to currently used cautery methods of obtaining hemostasis.

Topics: Animals; Cautery; Collagen; Fallopian Tubes; Female; Gelatin; Guinea Pigs; Hemostasis, Surgical; Inflammation; Prothrombin; Thromboplastin; Tissue Adhesions

Intravascular coagulation, thrombosis, and fibrin deposition often produce tissue damage in allogeneic inflammatory reactions such as allograft rejection. The mechanisms which initiate blood clotting in these reactions are poorly understood. We find that allogeneic stimulation of human leukocytes in vitro increases production and expression of tissue thromboplastin-like activity. In our experiments mixed leukocyte cultures (MLC) of cells from allogeneic (unrelated) donors produced and expressed more procoagulant activity than control cultures of cells from each donor alone. After 7 days, allogeneic MLC had 5- to 50-fold more total procoagulant activity than controls, as shown by assaying lysed whole cultures. Additionally, allogeneic MLC had 8- to 240-fold more procoagulant activity expressed on leukocyte surfaces and in culture supernates than controls after 7 days, as shown by assaying intact whole cultures and cell-free supernates. These increases were largely accounted for by gains in the amounts of procoagulant activity produced and expressed per cell in MLC as compared to controls. Controls and MLC produced and expressed considerable amounts of procoagulant activity during the 1st day of culture, and there were no differential effects of allogeneic stimulation on day 1. However, after day 1, the total amount of procoagulant activity produced and the amount expressed declined steadily in controls, nearly reaching preculture levels by day 7. In contrast, the total amount of procoagulant activity in allogeneic MLC remained high, and the amount of activity expressed on cell surfaces and in supernates increased severalfold by day 7. MLC of syngeneic (identical twin) cells produced and expressed the same amount of activity as controls over a 7-day period, whereas MLC of cells from each twin and an allogeneic donor produced and expressed more activity than controls (at least 9- and 35-fold more, respectively). Thus, increases of procoagulant activity production and expression were found only in MLC of genetically dissimilar cells. Therefore, these increases must have resulted from allogeneic stimulation.

Topics: Blood Coagulation; Cell Membrane; Female; Humans; Immunity; In Vitro Techniques; Inflammation; Leukocytes; Lymphocyte Culture Test, Mixed; Pregnancy; Thromboplastin; Transplantation Immunology; Twins, Monozygotic

Topics: Animals; Aspirin; Blood Coagulation Tests; Capillary Permeability; Factor XII; Guinea Pigs; Inflammation; Oxyphenbutazone; Thromboplastin

Topics: Angiography; Blood Platelets; Blood Vessels; Dextrans; Humans; Inflammation; Ischemia; Platelet Adhesiveness; Thrombin; Thromboplastin; Thrombosis; Veins

Topics: Arthritis; Blood Coagulation Factors; Factor XI; Factor XII; Gout; Humans; In Vitro Techniques; Inflammation; Synovial Fluid; Thromboplastin

The relation of intravascular fibrin to the leucocytic sticking reaction in ear chambers of rabbits injured by heat was investigated in two ways. First, attempts were made to destroy the thin layer of fibrin believed to coat the surfaces of cells involved in the sticking reaction. Second, white cell sticking was studied after fibrinogen had been removed from the blood stream. The results of these experiments were as follows:- 1. Activation of fibrinolysin in vivo by streptokinase did not impair sticking of white blood cells. 2. Administration of streptokinase parenterally did not lower fibrinogen blood levels appreciably even when the amount used was large. 3. Thromboplastin infusions alone reduced circulating fibrinogen to low levels but leucocytic sticking was not prevented. Furthermore, frequent death of animals due to pulmonary embolism made such experiments prohibitive. 4. Addition of streptokinase to thromboplastin infusions protected against embolic deaths but did not influence sticking even though the fibrinogen levels achieved were quite low. 5. Finally, when thrombin was added to infusions of thromboplastin and streptokinase, no circulating fibrinogen could be detected. Under such circumstances leucocytic sticking following heat injury occurred without reduction. These findings were interpreted as evidence against a primary role of the blood clotting mechanism in causing the sticking of white blood cells to injured endothelium. Alternative explanations were discussed.

Topics: Animals; Endothelium; Fibrin; Fibrinogen; Hot Temperature; Inflammation; Leukocyte Count; Leukocytes; Rabbits; Thrombin; Thromboplastin