thromboplastin and Thrombophilia

Tissue factor (TF) is the primary initiator of the coagulation cascade, though its effects extend well beyond hemostasis. When TF binds to Factor VII, the resulting TF:FVIIa complex can proteolytically cleave transmembrane G protein-coupled protease-activated receptors (PARs). In addition to activating PARs, TF:FVIIa complex can also activate receptor tyrosine kinases (RTKs) and integrins. These signaling pathways are utilized by tumors to increase cell proliferation, angiogenesis, metastasis, and cancer stem-like cell maintenance. Herein, we review in detail the regulation of TF expression, mechanisms of TF signaling, their pathological consequences, and how it is being targeted in experimental cancer therapeutics.

Topics: Amino Acid Sequence; Cell Hypoxia; Factor VIIa; Gene Expression Regulation, Neoplastic; Humans; Immunotherapy, Adoptive; Integrins; Molecular Sequence Data; Molecular Targeted Therapy; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; Neoplastic Stem Cells; Neovascularization, Pathologic; Protein Conformation; Protein Domains; Protein Isoforms; Receptor Protein-Tyrosine Kinases; Receptors, Proteinase-Activated; Signal Transduction; Thrombophilia; Thromboplastin

Topics: Anticoagulants; Betacoronavirus; Coronavirus Infections; COVID-19; Cytokine Release Syndrome; Endothelium, Vascular; Extracellular Traps; Extracellular Vesicles; Fibrinolytic Agents; Hemostasis; Humans; Intermittent Pneumatic Compression Devices; Neoplasms; Pandemics; Pneumonia, Viral; Risk; SARS-CoV-2; Thrombophilia; Thromboplastin; Thrombosis; Venous Thromboembolism

It has long been recognised that pancreatic cancer induces a hypercoagulable state that may lead to clinically apparent thrombosis. Although the relationship between pancreatic cancer and hypercoagulability is well described, the underlying pathological mechanism(s) and the interplay between these pathways remain a matter of intensive study. This review summarises existing data on epidemiology and pathogenesis of thrombotic complications in pancreatic cancer with a particular emphasis on novel pathophysiological pathways. Pancreatic cancer is characterised by high tumoural expression of tissue factor, activation of leukocytes with the release of neutrophil extracellular traps, the dissemination of tumour-derived microvesicles that promote hypercoagulability and increased platelet activation. Furthermore, other coagulation pathways probably contribute to these processes, such as those that involve heparanase, podoplanin and hypofibrinolysis. In the era in which heparin and its derivatives-the currently recommended therapy for cancer-associated thrombosis-might be superseded by direct oral anticoagulants, novel data from mouse models of cancer-associated thrombosis suggest the possibility of future personalised therapeutic approaches. In this dynamic era for cancer-associated thrombosis, the discovery of novel prothrombotic and proinflammatory mechanisms will potentially uncover pharmacological targets to prevent and treat thrombosis without adversely affecting haemostasis.

Topics: Animals; Anticoagulants; Antithrombin III; Cell-Derived Microparticles; Disease Models, Animal; Extracellular Traps; Factor VIII; Factor Xa Inhibitors; Fibrin Fibrinogen Degradation Products; Fibrinogen; Fibrinolysis; Glucuronidase; Heparin; Humans; Leukocytes; Membrane Glycoproteins; Mice; Molecular Targeted Therapy; Pancreatic Neoplasms; Protein C; Thrombophilia; Thromboplastin; Thrombosis

Factor V (FV) is a regulator of both pro- and anticoagulant pathways. It circulates as a single-chain procofactor, which is activated by thrombin or FXa to FVa that serves as cofactor for FXa in prothrombin activation. The cofactor function of FVa is regulated by activated protein C (APC) and protein S. FV can also function as an anticoagulant APC cofactor in the inhibition of FVIIIa in the membrane-bound tenase complex (FIXa/FVIIIa). In recent years, it has become clear that FV also functions in multiple ways in the tissue factor pathway inhibitor (TFPI) anticoagulant pathway. Of particular importance is a FV splice variant (FV-Short) that serves as a carrier and cofactor to TFPIα in the inhibition of FXa. FV-Short is generated through alternative splicing of exon 13 that encodes the large activation B domain. A highly negatively charged binding site for TFPIα is exposed in the C-terminus of the FV-Short B domain, which binds the positively charged C-terminus of TFPIα, thus keeping TFPIα in circulation. The binding of TFPIα to FV-Short is also instrumental in localizing the inhibitor to the surface of negatively charged phospholipids, where TFPIα inhibits FXa in process that is stimulated by protein S. Plasma FV activation intermediates and partially proteolyzed platelet FV similarly bind TFPIα with high affinity and regulate formation of prothrombinase. The novel insights gained into the interaction between FV isoforms, TFPIα, and protein S have opened a new avenue for research about the mechanisms of coagulation regulation and also for future development of therapeutics aimed at modulating coagulation.

Topics: Alternative Splicing; Anticoagulants; Binding Sites; Blood Coagulation; Blood Coagulation Tests; Exons; Factor V; Factor VIIIa; Humans; Lipoproteins; Point Mutation; Protein Binding; Protein C; Protein Domains; Protein Isoforms; Protein S; Thrombin; Thrombophilia; Thromboplastin

It is widely recognized that a strong correlation exists between cancer and aberrant hemostasis. Patients with various types of cancers often develop thrombosis, a phenomenon commonly referred to as Trousseau syndrome. Tissue factor (TF) is expressed by tumor cells and contributes to a variety of pathologic processes, such as thrombosis, tumor growth, tumor angiogenesis, and metastasis. Tissue factor is expressed in two naturally occurring protein isoforms: membrane-bound full-length TF (flTF) and soluble alternatively spliced TF (asTF). Tissue factor is the primary initiator of blood coagulation, and it triggers intracellular signaling through protease-activated receptors (PARs). PARs are activated either by TF/FVIIa complexes or by thrombin generated following coagulation activation. Furthermore, the noncoagulant asTF retains an integrin-binding site and stimulates angiogenesis by ligating endothelial integrins αvβ3 and α6β1. Lastly, the increased TF expression in tumors is associated with the release in blood of TF-positive procoagulant microparticles that favor thromboembolic complications. Therefore, the interruption of asTF and flTF signaling represents a potential antiangiogenic strategy. In this review, we summarize the current knowledge on the role of TF in cancer, and we explore therapeutic perspectives based on TF targeting.

Topics: Animals; Biomarkers, Tumor; Forecasting; Humans; Neoplasms; Neovascularization, Pathologic; Thrombophilia; Thromboplastin; Tumor Burden; Vascular Neoplasms

Forming an interface with virtually every other organ, endothelium has a strategic role in modulating vascular homeostasis. While its miscellany of functions includes regulation of vasomotor tone, promotion, and prevention of vascular growth, and modulation of inflammatory and hemostatic processes, it functions critically in maintaining the balance of hemostasis in health. Whereas endothelium has been recognized to influence all stages of hemostasis, new evidence suggests it to have a primary role for thrombin generation. Endothelial dysfunction is being increasingly appreciated in several pathological states and particularly, by virtue of its critical role in hemostasis, in causing thrombosis in a multitude of diseases.

Topics: Animals; Antigens, CD; Endothelial Cells; Endothelial Protein C Receptor; Endothelium, Vascular; Hemostasis; Homeostasis; Hormones; Humans; Lipoproteins; Paracrine Communication; Receptors, Cell Surface; Thrombin; Thrombomodulin; Thrombophilia; Thromboplastin; Thrombosis; von Willebrand Factor

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

Thrombosis is a major cause of morbidity and mortality in cancer patients. Many clinical factors contribute to the high thrombotic risk of this condition, including the type of malignancy, its disease stage, anticancer therapies, and comorbidities. However, the cancer cell-specific prothrombotic properties together with the host cell inflammatory response are important players in the pathogenesis of the cancer-associated hypercoagulability. Tissue factor (TF) is the most important procoagulant protein expressed by cancer cells, and with other cancer tissue procoagulant properties highly contributes to the procoagulant phenotype of malignant cells. Recent discoveries indicate that oncogenes determine the procoagulant protein expression, including TF, in cancer tissues. In addition, in malignancy, TF is also overexpressed by host normal blood cells triggered by cancer-derived inflammatory stimuli. As a consequence, a subclinical activation of blood coagulation is typically present in cancer patients, as demonstrated by abnormalities of circulating thrombotic biomarkers. The relevance of measuring these biomarkers to determine the patient thrombotic risk level is under active investigation. The goal is to identify the high-risk subgroups to establish more accurate and targeted anticoagulation strategies to prevent thrombosis in cancer patients. Ultimately, the clarification of specific molecular mechanisms triggering blood coagulation in specific cancer types may also indicate alternative ways to inhibit clotting activation in these conditions.

Topics: Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Neoplasms; Thrombophilia; Thromboplastin; Thrombosis

Coagulopathy is a unique component of the pathology of acute promyelocytic leukaemia (APL). Though many causative factors have been elucidated, therapies to rectify the coagulopathy are far from being realised. Thrombotic and bleeding complications remain the major causes of early deaths. In this chapter, the known causes of abnormalities in haemostatic function, namely the coagulopathy and changes in the fibrinolytic system, will be reviewed. Major risk factors for these complications are identified. Current available measures for correction of the coagulopathy and their effectiveness are critically examined. Unless the coagulopathy can be effectively controlled, bleeding complications will remain an obstacle to achieving a cure for this disease. The issues that need to be addressed in next phase of investigations are also discussed.

Topics: Annexin A2; Anticoagulants; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Blood Coagulation Disorders; Blood Coagulation Tests; Carboxypeptidase B2; Disseminated Intravascular Coagulation; Fibrinolysis; Forecasting; Granulocyte Precursor Cells; Hemorrhagic Disorders; Humans; Leukemia, Promyelocytic, Acute; Oxides; Recombinant Proteins; Risk Factors; S100 Proteins; Thrombomodulin; Thrombophilia; Thromboplastin; Tretinoin; Urokinase-Type Plasminogen Activator

Anticoagulants are effective at preventing and treating thrombosis, but can cause bleeding. For decades, vitamin K antagonists (VKAs) have been the only available oral anticoagulants. The development of non-VKA oral anticoagulants (NOACs), which inhibit either factor Xa or thrombin stoichiometrically, has provided alternatives to VKAs for several indications. The results of recent large-scale randomised controlled trials comparing NOACs with VKAs for the prevention of stroke in patients with non-valvular atrial fibrillation (AF) have produced some unexpected results. As a group, NOACs showed similar efficacy as warfarin, but a reduced risk of major bleeding. The reduction in bleeding with NOACs was greatest with intracranial hemorrhage. In contrast, the relative risk of gastro-intestinal bleeding was increased with some NOACs. In this review, we explore the potential mechanisms as well as the implications of these organ-specific bleeding patterns.

Topics: Anticoagulants; Atrial Fibrillation; Blood Coagulation Factors; Endothelium, Vascular; Factor Xa Inhibitors; Gastrointestinal Hemorrhage; Hemorrhage; Humans; International Normalized Ratio; Intestinal Absorption; Intracranial Hemorrhages; Organ Specificity; Randomized Controlled Trials as Topic; Stroke; Thrombophilia; Thromboplastin; Vitamin K; Warfarin

Inhibition of coagulation greatly limits cancer metastasis in many experimental models. Cancer cells trigger coagulation, through expression of tissue factor or P-selectin ligands that have correlated with worse prognosis in human clinical studies. Cancer cells also affect coagulation through expression of thrombin and release of microparticles that augment coagulation. In the cancer-bearing host, coagulation facilitates tumour progression through release of platelet granule contents, inhibition of Natural Killer cells and recruitment of macrophages. We are revisiting this literature in the light of recent studies in which treatment of clinical cohorts with anticoagulant drugs led to diminished metastasis.

Topics: Animals; Anticoagulants; Blood Coagulation; Blood Platelets; Cysteine Endopeptidases; Cytoplasmic Granules; Hirudins; Humans; Killer Cells, Natural; Lung Neoplasms; Macrophages; Mice; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms, Experimental; Neoplastic Cells, Circulating; Neuraminidase; P-Selectin; Platelet Activation; Platelet Aggregation Inhibitors; Rats; Thrombin; Thrombophilia; 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

It is known that peripheral blood eosinophilia (PBE) is a normal hematopoietic response to several parasitic diseases, but it is less known that PBE promotes a hypercoagulable state that may favor thrombosis. Scope of this article is to explore which parasitic infestations are most likely to be complicated by thrombosis and to highlight the pathogenetic contribution of PBE to vascular occlusions in this setting. A review of the world literature revealed 18 cases in which PBE was associated with vascular occlusion though no specific surveys were dedicated to this topic. The eosinophil exerts its thrombogenic potential by inhibition of the natural anticoagulant pathways and release of tissue factor with enhanced coagulation activation leading to vascular occlusion. It is hoped that this review contributes to the awareness of the link between PBE and thrombosis in parasitic disorders to foster research in this area.

Topics: Adult; Aged; Blood Coagulation; Eosinophilia; Eosinophils; Female; Humans; Male; Middle Aged; Parasitic Diseases; Thrombophilia; Thromboplastin; Thrombosis

Topics: Blood Coagulation Disorders; Cell-Derived Microparticles; Endothelium, Vascular; Hemostasis; Humans; Models, Biological; Neoplasm Proteins; Neoplasms; Thrombophilia; Thromboplastin

Topics: Animals; Blood Coagulation Factors; Cell Line, Tumor; Enzyme Activation; Humans; Mice; Neoplasm Proteins; Neoplasms; Thromboembolism; Thrombophilia; Thromboplastin

Topics: Angiogenesis Inhibitors; Blood Coagulation; Blood Coagulation Factors; Blood Platelets; Clinical Trials, Phase II as Topic; Humans; Neoplasm Proteins; Neoplasms; Neovascularization, Pathologic; Neovascularization, Physiologic; Thromboembolism; Thrombophilia; Thromboplastin

Topics: Anticoagulants; Antineoplastic Agents; Blood Coagulation Factors; Clinical Trials as Topic; Disease Progression; Glucuronidase; Heparin; Heparin, Low-Molecular-Weight; Humans; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; Selectins; Thrombin; Thromboembolism; Thrombophilia; Thromboplastin

Preeclampsia is a life-threatening hypertensive disease of pregnancy. The condition is characterized by the presence of autoantibodies that activate the major angiotensin receptor, AT(1). Research conducted during the past decade has shown that these autoantibodies activate AT(1) receptors on a variety of cell types and provoke biological responses that are relevant to the pathophysiology of preeclampsia. The introduction of these autoantibodies into pregnant mice results in hypertension, proteinuria and a variety of other features of preeclampsia including small fetuses and placentas. These findings demonstrate the pathophysiological role of these autoantibodies in preeclampsia. The biological properties of these autoantibodies can be blocked by a 7-amino acid peptide that corresponds to a specific sequence associated with the second extracellular loop of the AT(1) receptor. The fact that autoantibodies from different individuals are directed to a common epitope provides obvious diagnostic and therapeutic opportunities. Research reviewed here raises the intriguing possibility that preeclampsia may be a pregnancy-induced autoimmune condition characterized by the presence of disease-causing angiotensin receptor activating autoantibodies.

Topics: Adoptive Transfer; Angiogenesis Inducing Agents; Animals; Autoantibodies; Autoimmune Diseases; Female; Humans; Pre-Eclampsia; Pregnancy; Reactive Oxygen Species; Receptor, Angiotensin, Type 1; Thrombophilia; Thromboplastin

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

Thrombotic complications in patients with hematologic malignancies are as frequent as in those with solid tumors and significantly affect morbidity and mortality. In acute leukemia, thrombosis and bleeding manifestations may occur concomitantly as a part of the same thrombo-hemorrhagic syndrome. In patients with Philadelphia chromosome-negative chronic myeloproliferative disorders (i.e., essential thrombocythemia [ET] and polycythemia vera [PV]), a thrombosis rate as high as 40% has been recorded. A hypercoagulable state is present in virtually all of these patients, even without clinical manifestations. In this review, we focus on the pathogenic mechanisms underlying the hypercoagulable state of these two hematologic malignancies. Although the pathogenesis of hypercoagulability is complex, a central role is played by the fundamental molecular changes of both the leukemic cells and of the progeny arising from the hematopoietic progenitor cells that have undergone clonal rearrangement. These cells overexpress procoagulant factors, as well as adhesion molecules and cytokines capable of inducing procoagulant changes in the vascular wall and stimulating increased cellular interactions. Recent molecular studies in experimental models of human tumors have demonstrated for the first time that oncogene- and repressor gene-mediated neoplastic transformation induces activation of blood coagulation. Similarly, in cells from patients with acute promyelocytic leukemia, the T15-17 translocation induces hyperexpression of tissue factor (TF) and renders the patient hypercoagulable. Furthermore, in blood cells from patients with PV or ET, the presence of the JAK2V617F mutation translates into activation of hemostasis, with increased expression of platelet-associated TF microparticles and the formation of increased platelet/neutrophil aggregates. Understanding the pathophysiology of hypercoagulability is critical to the design of appropriate measures for intervention in these hematologic disorders to prevent thromboembolic complications.

Topics: Hematologic Neoplasms; Hemostasis; Humans; Leukemia, Promyelocytic, Acute; Myeloproliferative Disorders; Neovascularization, Pathologic; Polycythemia Vera; Thrombocythemia, Essential; Thrombophilia; Thromboplastin; Up-Regulation

Cellular microparticles (MP) are small membrane vesicles that are released from cells upon activation or apoptosis. They constitute a heterogeneous population of submicron elements differing in cellular origin, number, size, antigenic composition and functional properties. Circulating MP provide an additional procoagulant phospholipid surface enabling the assembly of the clotting enzyme complexes and thrombin generation. Their procoagulant properties rely on the exposure of phosphatidylserine and on the possible presence of tissue factor, the main initiator of blood coagulation. Microparticles constitute the main reservoir of blood-borne tissue factor. Derived from various cells, most notably platelets, erythrocytes, leucocytes and endothelial cells, circulating MP are detectable in the circulation of healthy subjects. Elevated levels are encountered in diseases with vascular involvement and hypercoagulability such as disseminated intravascular coagulation, diabetes, immune-mediated thrombosis, kidney diseases, acute coronary syndromes or systemic inflammatory disease, where they appear indicative of a poor clinical outcome. Converging evidence from experimental and clinical data underlines an involvement of procoagulant MP in the initiation/dissemination of procoagulant and inflammatory responses. In these clinical settings, the pharmacological modulation of MP level or activity provides challenging issues.

Topics: Cell Membrane Structures; Hemostatics; Humans; Particle Size; Thrombophilia; Thromboplastin; Vascular Diseases

Antiphospholipid antibodies (Abs) are associated with thrombosis and are a risk factor for recurrent pregnancy loss and obstetric complications in patients with the antiphospholipid syndrome. It is generally accepted that the major autoantigen for aPL Abs is beta (2) glycoprotein I, which mediates the binding of aPL Abs to target cells (i.e., endothelial cells, monocytes, platelets, trophoblasts, etc.) leading to thrombosis and fetal loss. This article addresses molecular events triggered by aPL Abs on endothelial cells, platelets, and monocytes and complement activation, as well as a review of the current knowledge with regard to the putative receptor(s) recognized by aPL Abs on target cells as well as novel mechanisms that involve fibrinolytic processes. A section is devoted to the description of thrombotic and inflammatory processes that lead to obstetric complications mediated by aPL Abs. Based on experimental evidence using in vitro and in vivo models, new targeted therapies for treatment and/or prevention of thrombosis and pregnancy loss in antiphospholipid syndrome are proposed.

Topics: Abortion, Habitual; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Autoantigens; beta 2-Glycoprotein I; Blood Platelets; Complement Activation; Endothelial Cells; Female; Fibrinolysis; Humans; Inflammation Mediators; Monocytes; Placenta; Pregnancy; Pregnancy Complications, Hematologic; Thrombin; Thrombophilia; Thromboplastin; Trophoblasts; Venous Thrombosis

The antiphospholipid syndrome (APS) is characterized by clinical manifestations such as venous and arterial thrombosis, thrombocytopenia and/or recurrent pregnancy loss, as well as the persistent presence of laboratory markers of antiphospholipid (aPL) antibodies detected in laboratory assays. Though it is generally accepted that aPL antibodies, such as anticardiolipin (aCL), anti-beta2 glycoprotein I (anti-beta2GPI), and lupus anticoagulants (LA) contribute to the pathogenesis of APS, precise mechanism(s) are yet to be fully described. It is probable that aPL antibodies bind to a range of cellular targets (e.g., platelets, endothelial cells, and monocytes), leading to thrombosis and obstetric complications. There is now increasing evidence that alterations to the tissue factor (TF) pathway of blood coagulation contribute toward hypercoagulability in patients with aPL antibodies. This article reviews current evidence that suggests changes and/or interference to the major pathway of blood coagulation may represent a novel mechanism that contributes to the development of APS.

Topics: Abortion, Habitual; Antibodies, Antiphospholipid; Antibody Specificity; Antiphospholipid Syndrome; Autoantibodies; beta 2-Glycoprotein I; Endothelial Cells; Female; Humans; Immunoglobulin G; Lipoproteins; Lupus Coagulation Inhibitor; Models, Biological; Pregnancy; Pregnancy Complications, Hematologic; Thrombin; Thrombophilia; Thromboplastin; Venous Thrombosis

Topics: Blood Coagulation Factors; Cardiovascular Diseases; Disease Susceptibility; Fibrinogen; Fibrinolysis; Humans; Polymorphism, Single Nucleotide; Prothrombin; Risk Factors; Thromboembolism; Thrombophilia; Thromboplastin; Thrombosis

Protease-activated receptors (PARs) are G-protein-coupled receptors (GPCRs) that are activated by a unique proteolytic mechanism. Besides the important role of blood coagulation factors in preventing bleeding after vascular injury, these serine proteinases actively engage target cells thereby fulfilling critical functions in cell biology. Cellular responses triggered by coagulation factor-induced PAR activation suggest that PARs play an important role in proliferation, survival and/or malignant transformation of tumor cells. Indeed, PAR expression correlates with cancer malignancy and clinical studies show that anticoagulant treatment is beneficial in cancer patients. In this review, we provide an overview on the PAR family, their mode of activation and mechanisms by which PAR signaling is terminated. In addition, we discuss the relationship between blood coagulation and cancer biology focusing on the potential role of PAR-induced modulation of cell survival, apoptosis and tumor growth.

Topics: Amino Acid Sequence; Animals; Apoptosis; Blood Coagulation Factors; Caspases; Cell Division; Cell Transformation, Neoplastic; Conserved Sequence; Enzyme Activation; Heterotrimeric GTP-Binding Proteins; Humans; Mice; Mice, Knockout; Models, Molecular; Molecular Sequence Data; Neoplasm Proteins; Neoplasms; Protein Conformation; Protein Structure, Tertiary; Receptors, Proteinase-Activated; Rhodopsin; Sequence Alignment; Sequence Homology, Amino Acid; Signal Transduction; Thrombophilia; Thromboplastin

Tissue factor (TF), the key regulator of haemostasis and angiogenesis, is also involved in the pathology of several diseases, including cardiovascular, inflammatory and neoplastic conditions. In the latter, TF is upregulated by cancer cells, as well as by certain host cells, and it is the interactions between these distinct pools of TF-expressing cells that likely influence tumour progression in several ways. Furthermore, the release of TF microparticles into the circulation is thought to contribute to the systemic coagulopathies commonly observed in cancer patients. The direct regulation of TF by oncogenic events has provided a plausible explanation for the relatively common overexpression of TF in various cancers and its involvement in tumour growth, angiogenesis, metastasis and coagulopathy. However, this constitutive influence is modified by the tumour microenvironment, cellular interactions and host factors rendering TF expression patterns complex and heterogeneous. It appears that in many biological contexts TF plays a central role in disease progression and thereby potentially constitutes an attractive therapeutic target, especially in scenarios where the risk of bleeding can be avoided by selecting appropriate medications, refined dosing or by targeting the signalling component of TF activity. The efficacy and safety of such approaches still awaits clinical verification.

Topics: Animals; Antineoplastic Agents; Blood Coagulation Factors; Blood Platelets; Cell Transformation, Neoplastic; Cell-Derived Microparticles; Drug Delivery Systems; Genes, Tumor Suppressor; Hemostasis; Humans; Mice; Mice, Knockout; Models, Biological; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; Neovascularization, Pathologic; Oncogenes; Organ Specificity; Platelet Activation; Thrombophilia; Thromboplastin

Inherited thrombophilia can be defined as a genetically determined predisposition to the development of thromboembolic complications. Since the discovery of activated protein C resistance in 1993, several additional disorders have been described and, at present, it is possible to identify an inherited predisposition in about 60 to 70% of patients with such complications. These inherited prothrombotic risk factors include qualitative or quantitative defects of coagulation factor inhibitors, increased levels or function of coagulation factors, defects of the fibrinolytic system, altered platelet function, and hyperhomocysteinemia. In this review, the main inherited prothrombotic risk factors are analyzed from epidemiological, laboratory, clinical, and therapeutic points of view. Finally, we discuss the synergism between genetic and acquired prothrombotic risk factors in particular conditions such as childhood and pregnancy.

Topics: Afibrinogenemia; Blood Coagulation Factor Inhibitors; Blood Coagulation Factors; Child, Preschool; Coagulation Protein Disorders; Female; Fibrinolysis; Genetic Predisposition to Disease; Heparin Cofactor II; Humans; Male; Pregnancy; Risk Factors; Thrombophilia; Thromboplastin

The 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitors (statins) have been shown to exhibit several vascular protective effects, including antithrombotic properties, that are not related to changes in lipid profile. There is growing evidence that treatment with statins can lead to a significant downregulation of the blood coagulation cascade, most probably as a result of decreased tissue factor expression, which leads to reduced thrombin generation. Accordingly, statin use has been associated with impairment of several coagulant reactions catalyzed by this enzyme. Moreover, evidence indicates that statins, via increased thrombomodulin expression on endothelial cells, may enhance the activity of the protein C anticoagulant pathway. Most of the antithrombotic effects of statins are attributed to the inhibition of isoprenylation of signaling proteins. These novel properties of statins, suggesting that these drugs might act as mild anticoagulants, may explain, at least in part, the therapeutic benefits observed in a wide spectrum of patients with varying cholesterol levels, including subjects with acute coronary events. The HMG-CoA reductase inhibitors (statins) have been shown to exhibit several vascular protective effects, including antithrombotic properties, that are not related to changes in lipid profile. Treatment with statins can lead to a significant downregulation of the blood coagulation cascade, most probably as a result of decreased tissue factor expression, which leads to reduced thrombin generation.

Topics: Animals; Anticholesteremic Agents; Anticoagulants; Arteriosclerosis; Blood Coagulation; Blood Coagulation Factors; Blood Proteins; Factor VII; Fibrinogen; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Models, Biological; Protein Prenylation; Protein Processing, Post-Translational; Staphylococcal Protein A; Thrombin; Thrombomodulin; Thrombophilia; Thromboplastin; Venous Thrombosis

The hypercoagulability exhibited by most cancer patients leads to serious complications such as venous thromboembolism and contributes to the pathogenesis of tumor growth and metastasis by promoting angiogenesis. The key player in this vicious cycle is tissue factor (TF), the initiator of blood coagulation. Although TF normally safeguards vascular integrity by inducing hemostasis upon injury, abnormal expression of TF in different tumors and related vascular endothelial cells contributes to unnecessary clot formation in cancer patients. Clotting-dependent induction of tumor angiogenesis is primarily mediated by TF-induced generation of thrombin and subsequent deposition of cross-linked fibrin. A cross-linked fibrin network provides a provisional proangiogenic matrix that facilitates blood vessel infiltration. Clotting-independent mechanisms of TF-induced tumor angiogenesis have also been described, mediated primarily by the cytoplasmic tail of the TF receptor. TF activation could contribute to the venous thromboembolism that has been reported as a complication of the use of novel antiangiogenic agents in combination with chemotherapy. Anticoagulants, such as low-molecular-weight heparin, may act to prevent these complications both by interfering with TF-mediated activation of clotting and by directly down-regulating angiogenesis. Thus, TF may prove to be a novel target for cancer therapy.

Topics: Animals; Fibrin; Humans; Neoplasms; Neovascularization, Pathologic; Thrombophilia; Thromboplastin

The purpose of this paper is to review the rationale for the development of coagulation-reactive drugs for the experimental therapy of gliomas. Numerous reactants familiar to students of blood coagulation have been shown to contribute to neoplastic proliferation, invasion and metastasis. Recently, considerable progress has been made in demonstrating the ability of drugs capable of inhibiting these reactants to alter cancer progression. Biological features of gliomas within the realm of blood coagulation suggest that clinical trials of such drugs warrant consideration. This approach offers the prospect of a novel treatment for this devastating tumour type that does not share the toxicities of conventional cancer therapies.

Topics: Anticoagulants; Aprotinin; Blood Coagulation; Brain Neoplasms; Factor Xa; Glioma; Heparin; Heparin, Low-Molecular-Weight; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lipoproteins; Thrombin; Thrombomodulin; Thrombophilia; Thromboplastin; Urokinase-Type Plasminogen Activator

There is evidence of activation of both blood coagulation and platelets in sickle cell disease. For example, plasma samples obtained in the steady state and during painful crisis demonstrate high levels of thrombin generation, depletion of anticoagulant proteins, and abnormal activation of the fibrinolytic system. Similarly, exposure of surface markers such as CD62P and CD40L, along with increased circulating levels of thrombospondin, signal platelet activation. In addition to its effects on the cleavage of fibrinogen and its ability to activate platelets, the increase in circulating thrombin levels, with its wide-ranging effects on endothelial cells and blood vessels, may be important in the pathophysiology of sickle cell disease. Therefore, treatments that could decrease thrombin generation or platelet activation may be beneficial in both the treatment of sickle cell disease and the prevention of complications that characterize this genetic disorder. This review discusses hypercoagulability in the various forms of sickle cell disease, including homozygous sickle cell anemia, hemoglobin SC disease, hemoglobin SD disease, and sickle cell-beta-thalassemia.

Topics: Anemia, Sickle Cell; Anticoagulants; Blood Coagulation; Endothelium, Vascular; Humans; Necrosis; Platelet Activation; Platelet Aggregation Inhibitors; Thrombophilia; Thromboplastin; Thrombosis

Exposure of blood to tissue factor (TF) sets off the coagulation cascade. TF is a transmembrane protein that serves as an essential cofactor for activated coagulation factor VII (FVIIa). TF may be exposed locally by vascular injury (such as balloon angioplasty) or by spontaneous rupture of an atherosclerotic plaque. Expression of TF may also be induced on monocytes and endothelial cells in conditions like sepsis and cancer, causing a more generalised activation of clotting. TF may thus play a central role in thrombosis in a number of settings, and attention has turned to blocking TF as a means to prevent thrombosis. Inhibiting the inducible expression of TF by monocytes can be achieved by 'deactivating' cytokines, such as interleukin (IL)-4, -10 and -13, or by certain prostanoids; by drugs that modify signal transduction, such as pentoxifylline, retinoic acid or vitamin D(3), or by antisense oligonucleotides. Such approaches are for the most part at a preclinical stage. The function of TF can be blocked by antibodies that prevent the binding of FVIIa to TF; by active site-inhibited FVIIa, which competes with native FVIIa for binding; by antibodies or small molecules that block the function of the TF/FVIIa complex; and by molecules, such as TF pathway inhibitor or nematode anticoagulant peptide C2, which inhibit the active site of FVIIa in the TF/FVIIa complex after first binding to activated factor X. The latter two agents have entered Phase II clinical trials. Perhaps most intriguing is the use of anti-TF agents locally, which holds the promise of stopping thrombosis at a specific site of injury without the bleeding risk associated with systemic anticoagulation.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Antibodies, Monoclonal; Arteriosclerosis; Blood Coagulation; Clinical Trials as Topic; Cytokines; Drug Design; Endothelium, Vascular; Enzyme Activation; Factor VIIa; Fibrinolytic Agents; Glycoproteins; Helminth Proteins; Hemorrhage; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lipoproteins; Mice; Monocytes; Multiprotein Complexes; Neoplasms; Oligonucleotides, Antisense; Prostaglandins; Signal Transduction; Swine; Swine, Miniature; Thrombophilia; Thromboplastin; Thrombosis

Tissue factor is considered to be the physiologic trigger of the blood clotting system in normal hemostasis and in many--perhaps most--thrombotic diseases. A wealth of new knowledge is available regarding the structure and assembly of the TF:VIIa complex and the role of factor VIIa and tissue factor in hypercoagulable states. The exciting recent finding that tissue factor can function as a signaling receptor, and suggestions that tissue factor may have important, non-hemostatic roles, will be the subject of much additional study in the near future.

Topics: Blood Coagulation; Factor VIIa; Humans; Protein Binding; Protein Conformation; Thrombophilia; Thromboplastin

Tumor cells produce tissue factor, cancer procoagulant, plasminogen activators and other factors that interact with the coagulation system, the fibrinolytic system and vascular or blood cells such that they can upset the normal homeostasis and balance between activation and inhibition of the coagulation and fibrinolytic systems. These activities play a role in tumor cell growth and metastasis, vascular wall function, and hemostasis. Proteases and their inhibitors are intimately involved in all aspects of the hemostatic, cell proliferation and cellular signalling systems. This review provides a brief examination of recent observations in this complex interaction of cellular and hemostatic factors.

Topics: Blood Coagulation; Blood Coagulation Factors; Humans; Neoplasms; Thrombophilia; Thromboplastin

Human tissue factor pathway inhibitor (TFPI) is a modular protein comprised of three Kunitz type domains flanked by peptide segments that are less structured. The sequential order of the elements are: an N-terminal acidic region followed by the first Kunitz domain (K1), a linker region, a second Kunitz domain (K2), a second linker region, the third Kunitz domain (K3), and the C-terminal basic region. The K1 domain inhibits factor VIIa complexed to tissue factor (TF) while the K2 domain inhibits factor Xa. No direct protease inhibiting functions have been demonstrated for the K3 domain. Importantly, the Xa-TFPI complex is a much more potent inhibitor of the VIIa-TF than TFPI by itself. Furthermore, the C-terminal basic region of TFPI is required for rapid physiologic inhibition of coagulation and is needed for the inhibition of smooth muscle cell proliferation. Although a number of additional targets for attachment have been reported, the C-terminal basic region appears to play an important role in binding of TFPI to cell surfaces. A primary site of TFPI synthesis is endothelium and the endothelium-bound TFPI contributes to the antithrombotic potential of the vascular endothelium. Further, increased levels of plasma TFPI under septic conditions may represent endothelial dysfunction. We have proposed that the extravascular cells that synthesize TF also synthesize TFPI providing dual components necessary for the regulation of clotting in their microenvironment. Like the TF synthesis in these cells is augmented by serum, so is the case with the TFPI gene expression. TFPI gene knock out mice reveal embryonic lethality suggesting a possible role of this protein in early development. Since TF-induced coagulation is thought to play a significant role in many disease states, including disseminated intravascular clotting, sepsis, acute lung injury and cancer, recombinant TFPI may be a beneficial therapeutic agent in these disease states to attenuate pathologic clotting. The purpose of this review is to outline recent developments in the field related to the structural specificity and biology of TFPI.

Topics: Acute Disease; Amino Acid Sequence; Amino Acids; Antiphospholipid Syndrome; Blood Coagulation; Cardiovascular Diseases; Endothelium, Vascular; Humans; Lipoproteins; Lung Diseases; Models, Biological; Models, Molecular; Molecular Sequence Data; Neoplasm Metastasis; Neoplasms; Protein Conformation; Protein Structure, Tertiary; Sepsis; Sequence Alignment; Sequence Homology, Amino Acid; Structure-Activity Relationship; Thrombophilia; 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

There is considerable evidence that the hemostatic system is involved in the growth and spread of malignant disease. There is an increased incidence of thromboembolic disease in patients with cancers and hemostatic abnormalities are extremely common in such patients. Antihemostatic agents have been successfully used to treat a variety of experimental tumors, and several clinical trials in humans have been initiated. Although metastasis is undoubtedly multifactorial, intravascular coagulation activation and peritumor fibrin deposition seem to be important. The mechanisms by which hemostatic activation facilitates the malignant process remain to be completely elucidated. Of central importance may be the presence on malignant cells of tissue factor and urokinase receptor. Recent studies have suggested that these proteins, and others, may be involved at several stages of metastasis, including the key event of neovascularization. Tissue factor, the principal initiator of coagulation, may have additional roles, outside of fibrin formation, that are central to the biology of some solid tumors.

Topics: Animals; Anticoagulants; Antineoplastic Agents; Biomarkers; Blood Coagulation Tests; Cell Adhesion; Cysteine Endopeptidases; Disseminated Intravascular Coagulation; Factor Xa; Fibrin; Fibrinolysis; Hemostasis; Heparin; Humans; Monocytes; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; Neoplasms, Experimental; Neoplastic Cells, Circulating; Neovascularization, Pathologic; Platelet Activation; Platelet Aggregation Inhibitors; Receptors, Thrombin; Thrombophilia; Thrombophlebitis; Thromboplastin; Vitamin K

For many years, the laboratory investigation of patients with thrombophilia has lagged behind that of patients with bleeding diathesis. Improved understanding of the mechanisms that control and regulate coagulation, and the resultant recognition of new defects, have greatly stimulated clinical laboratory interest in this area. Assays to detect resistance to activated protein C; deficiencies of antithrombin, protein C, and protein S; and the presence of antiphospholipid antibodies are widely available and should form part of the investigation of patients that present with idiopathic thrombosis. Such a work-up will likely provide an explanation for thrombosis in 40 to 60% of patients. Abnormalities of fibrinogen and fibrinolysis may explain still more, although such defects are currently considered rare. In addition, presently unrecognized defects almost certainly exist, and the identification of such individuals will undoubtedly improve our understanding of the hemostatic mechanism. Laboratory tests to define the hypercoagulable state are continually being developed. They include whole blood coagulation and platelet function tests and novel activation markers. However, acceptance of these approaches by clinical laboratories has been slow.

Topics: Afibrinogenemia; Antiphospholipid Syndrome; Antithrombins; Blood Coagulation Tests; Factor V; Fibrinolysis; Homocysteine; Humans; Lipoproteins; Lupus Coagulation Inhibitor; Monocytes; Platelet Activation; Protein C; Protein S; Protein S Deficiency; Prothrombin; Thrombomodulin; Thrombophilia; Thromboplastin

It is widely hypothesized that autoantibodies directly contribute to the prothrombotic state in the antiphospholipid syndrome (APS). The discovery that antiphospholipid autoantibodies are specific for phospholipid-binding plasma proteins (beta2-glycoprotein I, prothrombin, etc.) has allowed a much more precise investigation of the interactions of autoantibodies and antigens, and the effects of these interaction on hemostasis. Recent studies suggest that two types of interactions may be important in the pathophysiology of APS: (1) antibody cross-linking of membrane bound antigens may alter the kinetics of phospholipid-dependent reactions; and (2) antibody cross-linking of antigens bound to cell surface receptors may trigger signal transduction and cellular activation. In light of these findings, previous reports implicating various mechanisms of autoantibody-mediated thrombosis are being re-evaluated.

Topics: Antibodies, Anticardiolipin; Antigen-Antibody Reactions; Antiphospholipid Syndrome; Autoantibodies; Autoantigens; Autoimmune Diseases; beta 2-Glycoprotein I; Cell Adhesion; Epitopes; Glycoproteins; Hemostasis; Humans; Lupus Coagulation Inhibitor; Membrane Lipids; Models, Immunological; Monocytes; Phospholipids; Plasminogen Activator Inhibitor 1; Thrombophilia; Thromboplastin; Thrombosis

Cerebral injury and brain death is associated with apparent hypercoagulation and poor organ outcome. This experimental study challenges the hypotheses that i) brain death causes hypercoagulation and microvascular thrombosis and that ii) neutralizing systemic tissue factor (TF) by in vitro addition of a TF inhibitor (recombinant active site-inhibited factor VIIa (ASIS)) can reverse the hypercoagulable profile.. Using a validated pig model of intracranial hemorrhage and brain death, 20 pigs were randomized to either control or brain death. The primary endpoints were coagulation parameters measured with whole blood thromboelastometry (ROTEM), thrombin generation and a porcine TF-sensitive plasma clotting time assay. In vitro spiking experiments with ASIS were performed in parallel with the latter two assessments. The kidneys were examined histologically for microvascular thromboses.. Brain death induced hypercoagulation, as demonstrated with ROTEM, thrombin generation, and reduced TF-sensitive plasma clotting time. In vitro inhibition of TF with ASIS did not reverse the hypercoagulation. No microvascular thromboses were found in the kidneys.. Brain death causes hypercoagulation; however, inhibition of TF does not reverse the coagulopathy. Thus, TF release does not seem to be the primary cause of this hypercoagulation. Minor changes in the levels of protein C suggest that the protein C pathway may be linked to the observed coagulopathy.

Topics: Animals; Blood Coagulation; Blood Coagulation Tests; Brain Death; Cerebral Hemorrhage; Disease Models, Animal; Random Allocation; Swine; Thrombelastography; Thrombophilia; Thromboplastin

The effects of statins on platelet activation markers, chemokines and adiponectin, were investigated in 135 patients with hyperlipidemia. Of the 135 hyperlipidemic patients, 63 were allocated to the simvastatin group, treated with simvastatin at the dose of 10 mg daily, and the remaining 72 were allocated to the pitavastatin group, treated with pitavastatin at the dose of 2 mg daily. Plasma levels of platelet-derived microparticles (PDMP), cell adhesion molecules (sCD40L and sP-selectin), chemokines [monocyte chemoattractant protein-1 (MCP-1) and regulated on activation normally T-cell expressed and secreted] and adiponectin were measured at the baseline and after 6 months of treatment in both the groups. In addition, we carried out a basic study to investigate the MCP-1-dependent induction of tissue factor expression on a histiocytic cell line (U937 cells). The plasma levels of PDMP, sCD40L, sP-selectin, regulated on activation normally T-cell expressed and secreted and MCP-1 were higher, whereas those of adiponectin were lower, in the hyperlipidemic patients than in the normolipidemic controls. Plasma PDMP and sCD40L were positively correlated, whereas plasma adiponectin was negatively correlated, with the plasma levels of MCP-1. No significant differences in the plasma levels of PDMP, sCD40L, sP-selectin, regulated on activation normally T-cell expressed and secreted and MCP-1 measured before and after treatment were observed in either the simvastatin or pitavastatin group. A significant increase of the plasma adiponectin levels was observed after 6 months of treatment with pitavastatin but not after an equal duration of treatment with simvastatin. When pitavastatin-treated patients were divided into two groups according to the adiponectin response to pitavastatin treatment, significant decreases of the plasma MCP-1, PDMP and sCD40L levels were observed after pitavastatin treatment in the responder group. In the aforementioned basic study, MCP-1 by itself did not induce the expression of tissue factor on the U937 cells. However, the recombinant sCD40L-induced expression of tissue factor on U937 was enhanced by the addition of MCP-1. These findings suggest that PDMP, sCD40L and MCP-1 may participate in the development of atherothrombosis in patients with hyperlipidemia and that pitavastatin may exert an adiponectin-dependent antiatherothrombotic effect in hyperlipidemic patients.

Topics: Adiponectin; Adult; Aged; Atherosclerosis; CD40 Ligand; Cell-Derived Microparticles; Chemokine CCL2; Female; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hyperlipidemias; Male; Middle Aged; P-Selectin; Quinolines; T-Lymphocytes; Thrombophilia; Thromboplastin; U937 Cells

Men have a higher incidence of cardiovascular disease (CVD) than women of similar age, and it has been suggested that testosterone may influence the development of CVD. Recently, we demonstrated that elderly men with low testosterone levels had lower plasma levels of free tissue factor pathway inhibitor (TFPI) Ag associated with shortened tissue factor (TF)-induced coagulation initiation in a population based case-control study. Our hypothesis was that one year of testosterone treatment to physiological levels in elderly men would increase the levels of free TFPI Ag in plasma and have a favorable effect on TF-induced coagulation. Twenty-six men with low testosterone levels (< or =11.0 nM) were randomly assigned to treatment with intramuscular testosterone depot injections (testosterone undecanoate 1,000 mg) or placebo in a double-blinded study. Each participant received a total of five injections, at baseline, 6, 16, 28 and 40 weeks, and TF-induced thrombin generation ex vivo and plasma free TFPI Ag were measured after one year. At the end of the study total and free testosterone levels were significantly higher in the testosterone treated group (14.9 +/- 4.5 nM vs. 8.1 +/- 2.4 nM; p < 0.001, and 363.3 +/- 106.6 pM vs. 187.3 +/- 63.2 pM; p < 0.001, respectively). Testosterone treatment for one year did neither cause significant changes in TF-induced thrombin generation ex vivo nor changes in plasma levels of free TFPI Ag. In conclusion, normalising testosterone levels by testosterone treatment for 12 months in elderly men did not affect TF-induced coagulation or plasma TFPI levels. The potential antithrombotic role of testosterone therapy remains to be elucidated.

Topics: Aged; Antigens; Cardiovascular Diseases; Case-Control Studies; Delayed-Action Preparations; Double-Blind Method; Factor VII; Follow-Up Studies; Humans; Hypogonadism; Injections, Intramuscular; Lipids; Lipoproteins; Male; Middle Aged; Risk Factors; Testosterone; Thrombin; Thrombophilia; Thromboplastin; Time Factors

A prothrombotic state may contribute to the elevated cardiovascular risk in patients with obstructive sleep apnea (OSA). We investigated the relationship between apnea severity and hemostasis factors and effect of continuous positive airway pressure (CPAP) treatment on hemostatic activity. We performed full overnight polysomnography in 44 OSA patients (mean age 47+/-10 years), yielding apnea-hypopnea index (AHI) and mean nighttime oxyhemoglobin saturation (SpO2) as indices of apnea severity. For treatment, subjects were double-blind randomized to 2 weeks of either therapeutic CPAP (n = 18), 3 l/min supplemental nocturnal oxygen (n = 16) or placebo-CPAP (<1 cm H2O) (n = 10). Levels of von Willebrand factor antigen (VWF:Ag), soluble tissue factor (sTF), D-dimer, and plasminogen activator inhibitor (PAI)-1 antigen were measured in plasma pre- and posttreatment. Before treatment, PAI-1 was significantly correlated with AHI (r = 0.47, p = 0.001) and mean nighttime SpO2 (r = -0.32, p = 0.035), but these OSA measures were not significantly related with VWF:Ag, sTF, and D-dimer. AHI was a significant predictor of PAI-1 (R2 = 0.219, standardized beta = 0.47, p = 0.001), independent of mean nighttime SpO2, body mass index (BMI), and age. A weak time-by-treatment interaction for PAI-1 was observed (p = 0.041), even after adjusting for age, BMI, pre-treatment AHI, and mean SpO2 (p = 0.046). Post hoc analyses suggested that only CPAP treatment was associated with a decrease in PAI-1 (p = 0.039); there were no changes in VWF:Ag, sTF, and D-dimer associated with treatment with placebo-CPAP or with nocturnal oxygen. Apnea severity may be associated with impairment in the fibrinolytic capacity. To the extent that our sample size was limited, the observation that CPAP treatment led to a decrease in PAI-1 in OSA must be regarded as tentative.

Topics: Adult; Antigens; Continuous Positive Airway Pressure; Double-Blind Method; Female; Fibrin Fibrinogen Degradation Products; Fibrinolysis; Humans; Male; Middle Aged; Oxyhemoglobins; Plasminogen Activator Inhibitor 1; Polysomnography; Reference Values; Sleep Apnea, Obstructive; Statistics as Topic; Thrombophilia; Thromboplastin; Treatment Outcome; von Willebrand Factor

In acute myocardial infarction (AMI), increased Tissue Factor (TF) expression on circulating monocytes and microparticles (MP) may contribute to thrombotic events. Because surfacebound Tissue Factor Pathway Inhibitor-1 (TFPI) inhibits TF activity on monocytes and endothelial cells decreased TFPI expression may reinforce the procoagulant activity of circulating MP. Aim of the study was to analyze TFPI expression and TF activity after stenting and thrombolysis inAMI. Thirty-nine patients of a randomized study comparing intravenous thrombolysis (n=19) and stenting (n=20) were included. Before and after therapy blood samples for analysis of MPs, TF antigen and activity, prothrombin fragment F1+2 and D-dimer were obtained. TFPI expression on TF positive MPs was decreased after thrombolysis but not after stenting. In contrast, TF plasma levels and TF positive MP remained unchanged in both treatment groups. After thrombolysis increased D-dimer and F1+2 plasma concentrations indicated activation of fibrinolysis and coagulation. Significance of MPTFPI for inhibition of TF activity was measured using inhibitory TFPI antibodies. Membrane-associated TFPI inhibited TF activity on circulating MPs. After thrombolysis inhibition of TF activity by TFPI was decreased as compared to stenting. Correlation of circulating TF with F1+2 only after thrombolysis, suggests a role for TF-induced activation of coagulation after thrombolysis. Enhanced TF activity on circulating MPs in AMI is inhibited by endogenous surface-boundTFPI. After thrombolysis but not after stenting MPTFPI is degraded and may induce thrombin generation due to unopposed tissue factor activity. Anti-TF therapies during thrombolysis may reduce thrombin generation in AMI.

Topics: Aged; Blood Coagulation; Female; Fibrin Fibrinogen Degradation Products; Humans; Lipoproteins; Male; Middle Aged; Myocardial Infarction; Particle Size; Peptide Fragments; Prothrombin; Stents; Thrombolytic Therapy; Thrombophilia; Thromboplastin; Thrombosis; Treatment Failure

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

Tissue factor (TF; an initiator of coagulation) and vascular endothelial growth factor (VEGF; a marker of angiogenesis) are involved in the hypercoagulable state associated with malignancy. We investigated their roles in chronic atrial fibrillation (AF), a condition also associated with increased risk of stroke and thromboembolism, as well as a prothrombotic or hypercoagulable state.. We studied 25 patients with AF (20 men; mean+/-SD age, 62+/-13 years) who were compared with 2 control groups in sinus rhythm: 30 healthy control subjects (17 men; mean age, 60+/-9 years) and 35 patient control subjects with coronary artery disease (CAD; 27 men; mean age, 60+/-12 years). Plasma levels of TF, VEGF, and the VEGF receptor sFlt-1 were measured by enzyme-linked immunosorbent assay.. VEGF, sFlt-1, and TF were significantly different between the 3 groups, with abnormal levels in AF and CAD patients compared with control subjects (P<0.001, P=0.022, and P=0.008, respectively). Among the AF patients, TF levels were significantly correlated with VEGF (Spearman's r=0.65, P<0.001) and sFlt (r=0.54, P=0.006) levels. Only TF and VEGF levels were significantly correlated in CAD patients (r=0.39, P=0.02). There were no significant correlations among the healthy control subjects.. Patients with chronic AF have high TF levels, in keeping with the prothrombotic state associated with this arrhythmia. The relationships between TF and VEGF and its receptor sFlt-1 in AF suggest a possible role for VEGF in the hypercoagulable state found in AF, as seen in malignancy and atherosclerosis.

Topics: Anticoagulants; Atrial Fibrillation; Blood Pressure; Case-Control Studies; Chronic Disease; Coronary Artery Disease; Cross-Sectional Studies; Demography; Endothelial Growth Factors; Female; Humans; Lymphokines; Male; Middle Aged; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Thrombophilia; Thromboplastin; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factors; Warfarin

Factor V (FV) plays pivotal roles in both procoagulant and anticoagulant mechanisms. Genetic mutations, FV-W1920R (FVNara) and FV-A2086D (FVBesançon), in the C1 and C2 domains of FV light chain, respectively, seem to be associated with deep vein thrombosis. However, the detailed mechanism(s) through which these mutations are linked to thrombophilia remains to be fully explored. The aim of this study was to clarify thrombotic mechanism(s) in the presence of these FV abnormalities. Full-length wild-type (WT) and mutated FV were prepared using stable, human cell lines (HEK293T) and the piggyBac transposon system. Susceptibility of FVa-A2086D to activated protein C (APC) was reduced, resulting in significant inhibition of APC-catalyzed inactivation with limited cleavage at Arg306 and delayed cleavage at Arg506. Furthermore, APC cofactor activity of FV-A2086D in APC-catalyzed inactivation of FVIIIa through cleavage at Arg336 was impaired. Surface plasmon resonance-based assays demonstrated that FV-A2086D bound to Glu-Gly-Arg-chloromethylketone active site-blocked APC and protein S (P) with similar affinities to that of FV-WT. However, weakened interaction between FVa-A2086D and phospholipid membranes was evident through the prothrombinase assay. Moreover, addition of FVa-A2086D to plasma failed to inhibit tissue factor (TF)-induced thrombin generation and reduce prothrombin times. This inhibitory effect was independent of PC, PS, and antithrombin. The coagulant and anticoagulant characteristics of FV(a)-W1920R were similar to those of FV(a)-A2086D. FV-A2086D presented defects in the APC mechanisms associated with FVa inactivation and FV cofactor activity, similar to FV-W1920R. Moreover, both FV proteins that were mutated in the light chain impaired inhibition of TF-induced coagulation reactions. These defects were consistent with congenital thrombophilia.

Topics: Anticoagulants; Factor V; HEK293 Cells; Humans; Mutation; Thrombophilia; Thromboplastin; Venous Thrombosis

Convallatoxin (CNT) is a natural cardiac glycoside extracted from lily of the valley ( Convallaria majalis ). Although it is empirically known to cause blood coagulation disorders, the underlying mechanism remains unclear. CNT exerts cytotoxicity and increases tissue factor (TF) expression in endothelial cells. However, the direct action of CNT on blood coagulation remains unclear. Therefore, herein, we investigated the effects of CNT on whole blood coagulation system and TF expression in monocytes.. Blood samples were collected from healthy volunteers to measure plasma thrombin-antithrombin complex (TAT) concentration using ELISA and to perform rotational thromboelastometry (ROTEM) and whole-blood extracellular vesicle (EV)-associated TF (EV-TF) analysis. The effects of CNT were also investigated using the monocytic human cell line THP-1. Quantitative real-time PCR and western blotting were performed, and PD98059, a mitogen-activated protein kinase (MAPK) inhibitor, was used to elucidate the action mechanism of CNT-mediated TF production.. CNT treatment increased EV-TF activity, shortened the whole blood clotting time in rotational thromboelastometry analysis, and increased TAT levels, which is an index of thrombin generation. Furthermore, CNT increased TF mRNA expression in THP-1 cells and EV-TF activity in the cell culture supernatant. Therefore, CNT may induce a hypercoagulable state with thrombin generation, in which elevated EV-TF activity derived from monocytes might be involved. These procoagulant effects of CNT were reversed by PD98059, suggesting that CNT-induced TF production in monocytes might be mediated by the MAPK pathway.. The findings of the present study have further clarified the procoagulant properties of CNT.

Topics: Endothelial Cells; Extracellular Vesicles; Humans; Monocytes; Protein Kinase Inhibitors; Thrombin; Thrombophilia; Thromboplastin

The coagulopathy of traumatic brain injury (TBI) remains poorly understood. Contradictory descriptions highlight the distinction between systemic and local coagulation, with descriptions of systemic hypercoagulability despite intracranial hypocoagulopathy. This perplexing coagulation profile has been hypothesized to be due to tissue factor release. The objective of this study was to assess the coagulation profile of TBI patients undergoing neurosurgical procedures. We hypothesize that dura violation is associated with higher tissue factor and conversion to a hypercoagulable profile and unique metabolomic and proteomic phenotype.. This is a prospective, observational cohort study of all adult TBI patients at an urban, Level I trauma center who underwent a neurosurgical procedure from 2019 to 2021. Whole blood samples were collected before and then 1 hour following dura violation. Citrated rapid and tissue plasminogen activator (tPA) thrombelastography (TEG) were performed, in addition to measurement of tissue factory activity, metabolomics, and proteomics.. Overall, 57 patients were included. The majority (61%) were male, the median age was 52 years, 70% presented after blunt trauma, and the median Glasgow Coma Score was 7. Compared with pre-dura violation, post-dura violation blood demonstrated systemic hypercoagulability, with a significant increase in clot strength (maximum amplitude of 74.4 mm vs. 63.5 mm; p < 0.0001) and a significant decrease in fibrinolysis (LY30 on tPAchallenged TEG of 1.4% vs. 2.6%; p = 0.04). There were no statistically significant differences in tissue factor. Metabolomics revealed notable increases in metabolites involved in late glycolysis, cysteine, and one-carbon metabolites, and metabolites involved in endothelial dysfunction/arginine metabolism/responses to hypoxia. Proteomics revealed notable increase in proteins related to platelet activation and fibrinolysis inhibition.. A systemic hypercoagulability is observed in TBI patients, characterized by increased clot strength and decreased fibrinolysis and a unique metabolomic and proteomics phenotype independent of tissue factor levels.

Topics: Adult; Blood Coagulation Disorders; Brain Injuries, Traumatic; Cohort Studies; Female; Humans; Male; Middle Aged; Proteomics; Thrombelastography; Thrombophilia; Thromboplastin; Tissue Plasminogen Activator

COVID-19 causes hypercoagulability, but the association between coagulopathy and hypoxemia in critically ill patients has not been thoroughly explored. This study hypothesized that severity of coagulopathy would be associated with acute respiratory distress syndrome severity, major thrombotic events, and mortality in patients requiring intensive care unit-level care.. Viscoelastic testing by rotational thromboelastometry and coagulation factor biomarker analyses were performed in this prospective observational cohort study of critically ill COVID-19 patients from April 2020 to October 2020. Statistical analyses were performed to identify significant coagulopathic biomarkers such as fibrinolysis-inhibiting plasminogen activator inhibitor 1 and their associations with clinical outcomes such as mortality, extracorporeal membrane oxygenation requirement, occurrence of major thrombotic events, and severity of hypoxemia (arterial partial pressure of oxygen/fraction of inspired oxygen categorized into mild, moderate, and severe per the Berlin criteria).. In total, 53 of 55 (96%) of the cohort required mechanical ventilation and 9 of 55 (16%) required extracorporeal membrane oxygenation. Extracorporeal membrane oxygenation-naïve patients demonstrated lysis indices at 30 min indicative of fibrinolytic suppression on rotational thromboelastometry. Survivors demonstrated fewer procoagulate acute phase reactants, such as microparticle-bound tissue factor levels (odds ratio, 0.14 [0.02, 0.99]; P = 0.049). Those who did not experience significant bleeding events had smaller changes in ADAMTS13 levels compared to those who did (odds ratio, 0.05 [0, 0.7]; P = 0.026). Elevations in plasminogen activator inhibitor 1 (odds ratio, 1.95 [1.21, 3.14]; P = 0.006), d-dimer (odds ratio, 3.52 [0.99, 12.48]; P = 0.05), and factor VIII (no clot, 1.15 ± 0.28 vs. clot, 1.42 ± 0.31; P = 0.003) were also demonstrated in extracorporeal membrane oxygenation-naïve patients who experienced major thrombotic events. Plasminogen activator inhibitor 1 levels were significantly elevated during periods of severe compared to mild and moderate acute respiratory distress syndrome (severe, 44.2 ± 14.9 ng/ml vs. mild, 31.8 ± 14.7 ng/ml and moderate, 33.1 ± 15.9 ng/ml; P = 0.029 and 0.039, respectively).. Increased inflammatory and procoagulant markers such as plasminogen activator inhibitor 1, microparticle-bound tissue factor, and von Willebrand factor levels are associated with severe hypoxemia and major thrombotic events, implicating fibrinolytic suppression in the microcirculatory system and subsequent micro- and macrovascular thrombosis in severe COVID-19.

Topics: Blood Coagulation Disorders; COVID-19; Critical Illness; Fibrinolysis; Humans; Hypoxia; Microcirculation; Oxygen; Plasminogen Activator Inhibitor 1; Prospective Studies; Respiratory Distress Syndrome; Retrospective Studies; Thrombophilia; Thromboplastin; Thrombosis

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

Absolute or relative protein (P)C pathway abnormalities (PC deficiency, PS deficiency, antiphospholipid syndrome (APS), factor (F)V-abnormality, and high FVIII level) cause thrombophilia. Although screening assays for these thrombophilias are available, one utilizing clot waveform analysis (CWA) remains unknown. We aimed to establish a CWA-based screening assay to distinguish PC pathway abnormality-related thrombophilia.. Samples were reacted with tissue factor (TF)/phospholipids and recombinant thrombomodulin (rTM; optimal 20 nM), followed by CWA measurement. The peak ratio (with/without rTM) of the first derivative curve of clot waveform was calculated.. The peak ratio in healthy plasmas (n = 35) was 0.36 ± 0.13; hence, the cutoff value was set to 0.49. The peak ratios in plasmas with PC deficiency, PS deficiency, high-FVIII (spiked 300 IU/dl), and APS were higher than the cutoff values (0.79/0.97/0.50/0.93, respectively). PC-deficient plasma or PS-deficient plasma mixed with normal plasma (25%/50%/75%/100% PC or PS level) showed dose-dependent decreases in the peak ratios (PC deficient: 0.85/0.64/0.44/0.28; PS deficient: 0.69/0.53/0.40/0.25), suggesting that the peak ratio at ≤50% of PC or PS level exceeded the cutoff value. The peak ratio in FV deficiency with FV ≤25% was higher than the cutoff value. FV-deficient plasma spiked with 40 IU/dl rFV-R506Q (FV. rTM-mediated TF-triggered CWA might be useful for screening PC pathway abnormality-related thrombophilia.

Topics: Blood Coagulation Tests; Humans; Protein C; Thrombomodulin; Thrombophilia; Thromboplastin; Thrombosis

Trauma induced coagulopathy (TIC) is common after severe trauma, increasing transfusion requirements and mortality among patients. TIC has several phenotypes, with primary hyperfibrinolysis being among the most lethal. We aimed to investigate the contribution of hypercoagulation, hemodilution, and fibrinolytic activation to the hyperfibrinolytic phenotype of TIC, by examining fibrin formation in a plasma-based model of TIC. We hypothesized that instabilities arising from TIC will be due primarily to increased fibrinolytic activation rather than hemodilution or tissue factor (TF) induced hypercoagulation.. The influence of TF, hemodilution, fibrinogen consumption, tissue plasminogen activator (tPA), and the antifibrinolytic tranexamic acid (TXA) on plasma clot formation and structure were examined using rheometry, optical properties, and confocal microscopy. These were then compared to plasma samples from trauma patients at risk of developing TIC.. Combining TF-induced clot formation, 15 % hemodilution, fibrinogen consumption, and tPA-induced fibrinolysis, the clot characteristics and hyperfibrinolysis were consistent with primary hyperfibrinolysis. TF primarily increased fibrin polymerization rates and reduced fiber length. Hemodilution decreased clot optical density but had no significant effect on mechanical clot stiffness. TPA addition induced primary clot lysis as observed mechanically and optically. TXA restored mechanical clot formation but did not restore clot structure to control levels. Patients at risk of TIC showed increased clot formation, and lysis like that of our simulated model.. This simulated TIC plasma model demonstrated that fibrinolytic activation is a primary driver of instability during TIC and that clot mechanics can be restored, but clot structure remains altered with TXA treatment.

Topics: Blood Coagulation Disorders; Fibrin; Fibrinogen; Hemodilution; Hemostatics; Humans; Thrombophilia; Thromboplastin; Tissue Plasminogen Activator; Tranexamic Acid

Cancer-associated thrombosis is the second-leading cause of mortality in patients with cancer and presents a poor prognosis, with a lack of effective treatment strategies. NAD(P)H quinone oxidoreductase 1 (NQO1) increases the cellular nicotinamide adenine dinucleotide (NAD

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Disease Models, Animal; Extracellular Traps; Female; Mice; Mice, Inbred BALB C; NAD; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Sirtuin 1; Thrombophilia; Thromboplastin; Thrombosis

Pancreatic cancer patients have an elevated risk of suffering from venous thrombosis. Among several risk factors that contribute to hypercoagulability of this malignancy, platelets possess a key role in the initiation of clot formation. Although single mechanisms of platelet activation are well-known in principle, combinations thereof and their potential synergy to mediate platelet activation is, in the case of pancreatic cancer, far from being clear. Applying an inhibitor screening approach using light transmission aggregometry, dense granule release, and thrombin formation assays, we provide evidence that a combination of tissue factor-induced thrombin formation by cancer cells and their platelet P-selectin binding is responsible for AsPC-1 and Capan-2 pancreatic cancer cell-mediated platelet activation. While the blockade of one of these pathways leads to a pronounced inhibition of platelet aggregation and dense granule release, the simultaneous blockade of both pathways is inevitable to prevent platelet aggregation completely and minimize ATP release. In contrast, MIA PaCa-2 pancreatic cancer cells express reduced levels of tissue factor and P-selectin ligands and thus turn out to be poor platelet activators. Consequently, a simultaneous blockade of thrombin and P-selectin binding seems to be a powerful approach, as mediated by heparin to crucially reduce the hypercoagulable state of pancreatic cancer patients.

Topics: Blood Platelets; Cell Line, Tumor; Humans; Ligands; P-Selectin; Pancreatic Neoplasms; Platelet Activation; Platelet Adhesiveness; Platelet Aggregation; Risk Factors; Thrombin; Thrombophilia; Thromboplastin; Venous Thrombosis

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with the hypercoagulable state. Tissue factor (TF) is the primary cellular initiator of coagulation. Most of the TF expressed on cell surfaces remains cryptic. Sphingomyelin (SM) is responsible for maintaining TF in the encrypted state, and hydrolysis of SM by acid sphingomyelinase (ASMase) increases TF activity. ASMase was shown to play a role in virus infection biology. In the present study, we investigated the role of ASMase in SARS-CoV-2 infection-induced TF procoagulant activity. Infection of human monocyte-derived macrophages (MDMs) with SARS-CoV-2 spike protein pseudovirus (SARS-CoV-2-SP-PV) markedly increased TF procoagulant activity at the cell surface and released TF+ extracellular vesicles. The pseudovirus infection did not increase either TF protein expression or phosphatidylserine externalization. SARS-CoV-2-SP-PV infection induced the translocation of ASMase to the outer leaflet of the plasma membrane, which led to the hydrolysis of SM in the membrane. Pharmacologic inhibitors or genetic silencing of ASMase attenuated SARS-CoV-2-SP-PV-induced increased TF activity. Inhibition of the SARS-CoV-2 receptor, angiotensin-converting enzyme-2, attenuated SARS-CoV-2-SP-PV-induced increased TF activity. Overall, our data suggest that SARS-CoV-2 infection activates the coagulation by decrypting TF through activation of ASMase. Our data suggest that the US Food and Drug Administration-approved functional inhibitors of ASMase may help treat hypercoagulability in patients with COVID-19.

Topics: Angiotensin-Converting Enzyme 2; Cell-Derived Microparticles; COVID-19; Enzyme Activation; Humans; Hydrolysis; Macrophages; Membrane Proteins; Molecular Targeted Therapy; Plasmids; Protein Transport; Receptors, Virus; RNA Interference; RNA, Small Interfering; SARS-CoV-2; Sphingomyelin Phosphodiesterase; Sphingomyelins; Spike Glycoprotein, Coronavirus; Thrombophilia; Thromboplastin

Alveolar hypercoagulation and fibrinolysis inhibition were associated with the refractory hypoxemia and the high mortality in patient with acute respiratory distress syndrome (ARDS), and NF-κB pathway was confirmed to contribute to the process. Triptolide (TP) significantly inhibited NF-κB pathway and thus depressed accessive inflammatory response in ARDS. We speculate that TP could improve alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS via NF-κB inactivation.. The aim of this experiment was to explore the efficacy and potential mechanism of TP on alveolar hypercoagulation and fibrinolysis inhibition in LPS-induced ARDS in mice.. 50 μl of LPS (5 mg/ml) was inhalationally given to C57BL/6 mice to set up ARDS model. Male mice were randomly accepted with LPS, LPS + TP (1 μg/kg, 10 μg/kg, 50 μg/kg respectively), or with NEMO Binding domain peptide (NBD), an inhibitor of NF-κB. TP (1 μg/kg, 10 μg/kg, 50 μg/kg) were intraperitoneally injected or 10 μg/50 μl of NBD solution were inhaled 30 min before LPS inhalation. A same volume of normal saline (NS) substituted for TP in mice in control. The endpoint of experiment was at 8 hours after LPS stimulation. Pulmonary tissues were taken for hematoxylin-eosin (HE) staining, wet / dry ratio and for lung injury scores (LIS). Tissue factor (TF) and plasminogen activator inhibitor (PAI)-1 in lung tissue were detected by Western-blotting and by quantitative Real-time PCR(qPCR) respectively. Concentrations of TF, PAI-1, thrombin-antithrombin complex (TAT), procollagen peptide type Ⅲ (PⅢP) and activated protein C (APC) in bronchoalveolar lavage fluid (BALF) were measured by ELISA. NF-κB activation and p65-DNA binding activity in pulmonary tissue were simultaneously determined.. LPS stimulation resulted in pulmonary edema, neutrophils infiltration, obvious alveolar collapse, interstitial congestion, with high LIS, which were all dose-dependently ameliorated by Triptolide. LPS also dramatically promoted the expressions of TF and PAI-1 either in mRNA or in protein in lung tissue, and significantly stimulated the secretions of TF, PAI-1, TAT, PⅢP but inhibited APC production in BALF, which were all reversed by triptolide treatment in dose-dependent manner. TP dose-dependently inhibited the activation of NF-κB pathway induced by LPS, indicated by the changes of phosphorylations of p65 (p-p65), p-IKKα/β and p-IκBα, and weakened p65-DNA binding activity. TP and NBD had same efficacies either on alveolar hypercoagulation and fibrinolysis inhibition or on NF-κB signalling pathway in ARDS mice.. TP dose-dependently improves alveolar hypercoagulation and fibrinolysis inhibition in ARDS mice through inhibiting NF-κB signaling pathway. Our data demonstrate that TP is expected to be an effective selection in ARDS.

Topics: Animals; Disease Models, Animal; Diterpenes; Epoxy Compounds; Fibrinolysis; Lipopolysaccharides; Lung; Lung Injury; Male; Mice; Mice, Inbred C57BL; NF-kappa B; Phenanthrenes; Respiratory Distress Syndrome; Signal Transduction; Thrombophilia; Thromboplastin

Small cell lung cancer (SCLC) patients have augmented risk of developing venous thromboembolism, but the mechanisms triggering this burden on the coagulation system remain to be understood. Recently, cell-derived microparticles carrying procoagulant phospholipids (PPL) and tissue factor (TF) in their membrane have attracted attention as possible contributors to the thrombogenic processes in cancers. The aims of this study were to assess the coagulation activity of platelet-poor plasma from 38 SCLC patients and to provide a detailed procoagulant profiling of small and large extracellular vesicles (EVs) isolated from these patients at the time of diagnosis, during and after treatment compared to 20 healthy controls. Hypercoagulability testing was performed by thrombin generation (TG), procoagulant phospholipid (PPL), TF activity, Protein C, FVIII activity and cell-free deoxyribonucleic acid (cfDNA), a surrogate measure for neutrophil extracellular traps (NETs). Our results revealed a coagulation activity that is significantly increased in the plasma of SCLC patients when compared to age-related healthy controls, but no substantial changes in coagulation activity during treatment and at follow-up. Although EVs in the patients revealed an increased PPL and TF activity compared with the controls, the TG profiles of EVs added to a standard plasma were similar for patients and controls. Finally, we found no differences in the coagulation profile of patients who developed VTE to those who did not, i.e. the tests could not predict VTE. In conclusion, we found that SCLC patients display an overall increased coagulation activity at time of diagnosis and during the disease, which may contribute to their higher risk of VTE.

Topics: Adult; Aged; Aged, 80 and over; Blood Coagulation Tests; Carcinoma, Small Cell; Centrifugation; Cysteine Endopeptidases; DNA; Extracellular Vesicles; Female; Humans; Lung Neoplasms; Male; Microscopy, Immunoelectron; Middle Aged; Nanoparticles; Neoplasm Proteins; Pulmonary Embolism; Risk Factors; Thrombin; Thrombophilia; Thromboplastin; Venous Thromboembolism

We have explored the relationship between possible hemostatic changes and clinical manifestation of the systemic lupus erythematosus (SLE) as a function of greater or lesser disease activity according to Systemic Lupus Erythematosus Disease Activity Index-2000 (SLEDAI-2K) criteria. Endothelial injury and hypercoagulability were investigated in patients with SLE by measuring thrombomodulin (TM), D-dimer (DDi) and thrombin generation (TG) potential. A total of 90 participants were distributed into three groups: 1) women with SLE presenting with low disease activity (laSLE) (SLEDAI-2K ≤ 4), 2) women with SLE presenting with moderate to high disease activity (mhaSLE) (SLEDAI-2K > 4), and 3) a control group comprising healthy women. Levels of TM and DDi were higher both in the laSLE and mhaSLE groups compared to controls and in mhaSLE compared to the laSLE group. With respect to TG assay, lagtime and endogen thrombin potential, low concentrations of tissue factor provided the best results for discrimination among groups. Analysis of these data allow us to conclude that TM, DDi and TG are potentially useful markers for discriminating patients with very active from those with lower active disease. Higher SLE activity may cause endothelial injury, resulting in higher TG and consequently a hypercoagulability state underlying the picture of thrombosis common in this inflammatory disease.

Topics: Adult; Biomarkers; Case-Control Studies; Cross-Sectional Studies; Endothelium, Vascular; Female; Fibrin Fibrinogen Degradation Products; Humans; Lupus Erythematosus, Systemic; Male; Middle Aged; Severity of Illness Index; Thrombomodulin; Thrombophilia; Thromboplastin; Young Adult

High plasma levels of activated Factor VII-Antithrombin complex (FVIIa-AT) have been associated with an increased risk of cardiovascular mortality in patients with stable coronary artery disease (CAD).. To investigate if FVIIa-AT levels are associated with activated factor X generation (FXaG) in modified assays.. Forty CAD patients were characterized for FVIIa-AT levels by ELISA and for FXaG in plasma. Novel fluorogenic FXaG assays, based on aptamers inhibiting thrombin and/or tissue factor pathway inhibitor (TFPI), were set up.. High FVIIa-AT plasma levels were associated with increased FXaG. Hypercoagulability features were specifically detectable in the coagulation initiation phase, which may have implications for cardiovascular risk prediction by either FVIIa-AT complex measurement or modified FXaG assays.

Topics: Coronary Artery Disease; Factor VIIa; Factor Xa; Humans; Thrombin; Thrombophilia; Thromboplastin

Within tumors, the coagulation-inducing protein tissue factor (TF), a major initiator of blood coagulation, has been shown to play a critical role in the hematogenous metastasis of tumors, due to its effects on tumor hypercoagulability and on the mediation of interactions between platelets and tumor cells. Targeting tumor-associated TF has therefore great therapeutic potential for antimetastasis therapy and preventing thrombotic complication in cancer patients. Herein, we reported a novel peptide-based nanoparticle that targets delivery and release of small interfering RNA (siRNA) into the tumor site to silence the expression of tumor-associated TF. We showed that suppression of TF expression in tumor cells blocks platelet adhesion surrounding tumor cells

Topics: Animals; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Lung Neoplasms; Mice, Nude; Nanoparticles; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms, Experimental; Neoplastic Cells, Circulating; RNA, Small Interfering; Thrombophilia; Thromboplastin; Thrombosis

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

Topics: Adolescent; Adult; Aged; Cohort Studies; Female; Foramen Ovale, Patent; Humans; Incidence; Male; Methylenetetrahydrofolate Reductase (NADPH2); Middle Aged; Migraine with Aura; Mutation; Predictive Value of Tests; Surveys and Questionnaires; Thrombophilia; Thromboplastin; Ultrasonography, Doppler, Transcranial; Young Adult

The hypercoagulable state associated with pancreatic adenocarcinoma (PDA) results in increased risk of venous thromboembolism, leading to substantial morbidity and mortality. Recently, neutrophil extracellular traps (NETs), whereby activated neutrophils release their intracellular contents containing DNA, histones, tissue factor, high mobility group box 1 (HMGB1) and other components have been implicated in PDA and in cancer-associated thrombosis.. Utilizing an orthotopic murine PDA model in C57/Bl6 mice and patient correlative samples, we studied the role of NETs in PDA hypercoagulability and targeted this pathway through treatment with the NET inhibitor chloroquine. PAD4 and RAGE knockout mice, deficient in NET formation, were used to study the role of NETs in platelet aggregation, release of tissue factor and hypercoagulability. Platelet aggregation was assessed using collagen-activated impedance aggregometry. Levels of circulating tissue factor, the initiator of extrinsic coagulation, were measured using ELISA. Thromboelastograms (TEGs) were performed to assess hypercoagulability and changes associated with treatment. Correlative data and samples from a randomized clinical trial of preoperative gemcitabine/nab-paclitaxel with and without hydroxychloroquine were studied and the impact of treatment on venous thromboembolism (VTE) rate was evaluated.. The addition of NETs to whole blood stimulated platelet activation and aggregation. DNA and the receptor for advanced glycation end products (RAGE) were necessary for induction of NET associated platelet aggregation. PAD4 knockout tumor-burdened mice, unable to form NETs, had decreased aggregation and decreased circulating tissue factor. The NET inhibitor chloroquine reduces platelet aggregation, reduces circulating tissue factor and decreases hypercoagulability on TEG. Review of correlative data from patients treated on a randomized protocol of preoperative chemotherapy with and without hydroxychloroquine demonstrated a reduction in peri-operative VTE rate from 30 to 9.1% with hydroxychloroquine that neared statistical significance (p = 0.053) despite the trial not being designed to study VTE.. NETs promote hypercoagulability in murine PDA through stimulation of platelets and release of tissue factor. Chloroquine inhibits NETs and diminishes hypercoagulability. These findings support clinical study of chloroquine to lower rates of venous thromboembolism in patients with cancer.. This study reports correlative data from two clinical trials that registered with clinicaltrials.gov, NCT01128296 (May 21, 2010) and NCT01978184 (November 7, 2013).

Topics: Adenocarcinoma; Animals; Chloroquine; DNA; Extracellular Traps; Female; Humans; Hydrolases; Hydroxychloroquine; Mice; Mice, Inbred C57BL; Pancreatic Neoplasms; Platelet Aggregation; Protein-Arginine Deiminase Type 4; Receptor for Advanced Glycation End Products; Thrombelastography; Thrombophilia; Thromboplastin; Venous Thromboembolism

Patients with chronic liver disease and cirrhosis have a dysregulated coagulation system and are prone to thrombosis. The basis for this hypercoagulable state is not completely understood. Tissue factor (TF) is the primary initiator of coagulation in vivo. Patients with cirrhosis have increased TF activity in white blood cells and circulating microparticles. The aim of our study was to determine the contribution of TF to the hypercoagulable state in a mouse model of chronic liver injury.. We measured levels of TF activity in the liver, white blood cells and circulating microparticles, and a marker of activation of coagulation (thrombin-antithrombin complexes (TATc)) in the plasma of mice subjected to bile duct ligation for 12days. We used wild-type mice, mice with a global TF deficiency (low TF mice), and mice deficient for TF in either myeloid cells (TF(flox/flox),LysMCre mice) or in hepatocytes (TF(flox/flox),AlbCre).. Wild-type mice with liver injury had increased levels of white blood cell, microparticle TF activity and TATc compared to sham mice. Low TF mice and mice lacking TF in hepatocytes had reduced levels of TF in the liver and in microparticles and exhibited reduced activation of coagulation without a change in liver fibrosis. In contrast, mice lacking TF in myeloid cells had reduced white blood cell TF but no change in microparticle TF activity or TATc.. Hepatocyte TF activates coagulation in a mouse model of chronic liver injury. TF may contribute to the hypercoagulable state associated with chronic liver diseases in patients.

Topics: Animals; Cells, Cultured; Chronic Disease; Disease Models, Animal; Hepatocytes; Humans; Liver Diseases; Male; Mice; Mice, Inbred C57BL; Thrombophilia; Thromboplastin

Hypercoagulability in type 2 diabetes mellitus (T2DM) patients increases their risk of cardiovascular diseases.. The aim of this work was to investigate the hypercoagulation mechanism in T2DM patients in terms of circulating tissue factor (TF).. Whole blood coagulation tests by damped oscillation rheometry and dielectric blood coagulometry (DBCM) were performed.. The average coagulation time was significantly shorter for T2DM patients than for healthy controls. In vitro addition of either anti-TF or anti-activated factor VII (FVIIa) antibody to hypercoagulable blood samples prolonged coagulation times for one group of patients, while coagulation times remained short for another group. The levels of circulating TF were estimated in the former group by measuring the coagulation times for blood samples from healthy subjects with addition of various concentrations of TF and comparing them with the coagulation times for the group. The results indicated that the levels of circulating TF were on the order of subpicomolar at most.. Circulating TF is at least partially responsible for a hypercoagulable group of T2DM patients, while an abnormality in the intrinsic coagulation pathway probably occurs in the other group.

Topics: Blood Coagulation; Blood Coagulation Tests; Diabetes Complications; Diabetes Mellitus, Type 2; Dielectric Spectroscopy; Humans; Thrombophilia; Thromboplastin

The role of circulating microparticles (MP) of different origin and tissue factor (TF)-bearing in overweight and obese patients with and without metabolic syndrome is still a matter of debate. In a case-control study, the presence of hypercoagulability was evaluated in overweight and obese patients by measuring MP, thrombin generation (TG) and FVIIa-AT complexes. Twenty overweight patients (body mass index [BMI] range 25-29.9 kg/m²), 20 with I degree (30-34.9 kg/m²), 20 with II degree (35-39.9 kg/m²) and 20 with III degree obesity (≥ 40 kg/m²) were enrolled and compared to 40 age and gender-matched normal weight individuals. A significant increase in median levels of all MP subtypes was observed in the three degrees of obese patients compared to controls. Overweight patients had higher levels of annexin V-MP (AMP), endothelial-derived, leukocyte-derived and TF-bearing MP than controls. Obese patients had a significantly shorter median lag time (p< 0.05), higher median peak thrombin (p< 0.01) and increased median endogenous thrombin potential [ETP] (p< 0.001) compared to controls. Overweight subjects had significantly increased ETP compared to controls (p< 0.05). Both AMP levels and ETP were found to positively correlate with BMI, waist circumference, and inflammatory parameters. No significant increase in FVIIa-AT complex was seen in cases compared to controls. We conclude that obesity is associated with overproduction of procoagulant MP and increase TG. Interestingly, hypercoagulability is found in overweight patients free of metabolic syndrome and increases with the severity of obesity. Assessment of MP and TG may be helpful in the early characterisation of the prothrombotic state in obese patients.

Topics: Adult; Antithrombins; Biomarkers; Blood Coagulation; Blood Coagulation Tests; Body Mass Index; Case-Control Studies; Cell-Derived Microparticles; Cross-Sectional Studies; Factor VIIa; Female; Humans; Inflammation Mediators; Male; Middle Aged; Obesity; Overweight; Risk Assessment; Risk Factors; Severity of Illness Index; Thrombin; Thrombophilia; Thromboplastin; Thrombosis; Waist Circumference

Hemoglobin SC disease is a very prevalent hemoglobinopathy; however, very little is known about this condition specifically. There appears to be an increased risk of thromboembolic events in hemoglobin SC disease, but studies evaluating the hemostatic alterations are lacking. We describe the findings of a cross-sectional observational study evaluating coagulation activation markers in adult patients with hemoglobin SC, comparing them with those in sickle cell anemia patients and healthy controls. A total of 56 hemoglobin SC and 39 sickle cell anemia patients were included in the study, all in steady state, and 27 healthy controls. None of the patients was taking hydroxyurea. Hemoglobin SC patients had a significantly up-regulated relative expression of tissue factor, as well as elevations in thrombin-antithrombin complex and D-dimer, in comparison to controls (P<0.01). Hemoglobin SC patients had lower tissue factor expression, and thrombin-antithrombin complex and D-dimer levels when compared to sickle cell anemia patients (P<0.05). Markers of endothelial activation (soluble thrombomodulin and soluble vascular cell adhesion molecule-1) and inflammation (tumor necrosis factor-alpha) were both significantly elevated in hemoglobin SC patients when compared to controls, being as high as the levels seen in patients with sickle cell anemia. Overall, in hemoglobin SC patients, higher hemolytic activity and inflammation were associated with a more intense activation of coagulation, and hemostatic activation was associated with two very prevalent chronic complications seen in hemoglobin SC disease: retinopathy and osteonecrosis. In summary, our results demonstrate that hemoglobin SC patients have a hypercoagulable state, although this manifestation was not as intense as that seen in sickle cell anemia.

Topics: Adult; Biomarkers; Blood Coagulation; Cross-Sectional Studies; Endothelial Cells; Female; Gene Expression; Hemoglobin SC Disease; Hemolysis; Humans; Inflammation Mediators; Leukocytes; Male; Middle Aged; Thrombophilia; Thromboplastin

Patients with schizophrenia are at increased risk of venous thromboembolism. The mechanisms underlying this association are poorly understood.. We investigated whether there is a global hypercoagulable state in patients with schizophrenia utilising the overall haemostatic potential (OHP) assay which assesses overall coagulation potential (OCP), haemostatic potential (OHP) and fibrinolytic potential (OFP).. Citrated plasma was collected for OHP assays from patients with schizophrenia on long-term antipsychotic treatment and compared with healthy age- and sex-matched controls. Time courses of fibrin formation and degradation were measured by spectrophotometry (absorption of 405nm) after the addition of tissue factor and tissue plasminogen activator to plasma.. Ninety patients with schizophrenia (antipsychotic treatment-15.9±9.7years) and 30 controls were recruited. Patients with schizophrenia had higher rates of smoking and levels of inflammatory markers (high-sensitivity C-reactive protein and neutrophil-to-lymphocyte ratio) than controls. Whilst D-dimer, fibrinogen and platelet count did not differ between patients with schizophrenia and controls, the OCP (54.0±12.6 vs 45.9±9.1, p=0.002) and OHP (12.6±5.8 vs 7.2±3.7, p<0.001) were higher, and OFP was lower (76.6±9.8% vs 84.9±6.4%, p<0.001) in patients with schizophrenia, implying both a hypercoagulable and hypofibrinolytic state in these patients. Importantly, abnormalities in overall coagulation were independently predicted by levels of plasminogen-activator-inhibitor-1, fibrinogen, platelet count, inflammatory markers and plasma triglycerides, suggesting a multifactorial aetiology.. Patients with schizophrenia have evidence of a global hypercoagulable and hypofibrinolytic state which may contribute to their increased risk of venous thromboembolism.

Topics: Adult; Antipsychotic Agents; Blood Coagulation; Female; Fibrin; Humans; Male; Plasma; Schizophrenia; Spectrophotometry; Thrombophilia; Thromboplastin; Tissue Plasminogen Activator

Patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV) have a high frequency of venous thromboembolic events and a hypercoagulable state. As C5a-primed neutrophils play an important role in the development of AAV, we investigated whether C5a-induced neutrophil tissue factor (TF)-expressing microparticles (MPs) and neutrophil extracellular traps (NETs) might promote hypercoagulability in AAV.. TF-expressing MPs were measured by flow cytometry. TF-expressing NETs were assessed by confocal microscopy. Levels of thrombin-antithrombin complexes were determined by enzyme-linked immunosorbent assay. The effect of C5a in sera from AAV patients was evaluated by treating neutrophils with C5a receptor antagonist before incubation with sera from AAV patients with active disease.. Treatment of C5a-primed neutrophils with antineutrophil cytoplasmic antibody (ANCA)-positive IgG resulted in the release of TF-bearing MPs and NETs. Neutrophils from healthy donors treated with sera from patients with active AAV released TF-bearing MPs and NETs, which were abolished by treatment with C5a receptor antagonist. Involvement of TF in MP- or NET-dependent thrombin generation was indicated by the findings of antibody neutralization studies. NETs with thrombin-generating capacity were demonstrated by DNase I treatment.. C5a-primed neutrophils produce TF-expressing MPs and NETs after stimulation with ANCAs, indicating a mechanism for hypercoagulability in AAV that was not previously recognized.

Topics: Adult; Aged; Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis; Antibodies, Antineutrophil Cytoplasmic; Case-Control Studies; Cell-Derived Microparticles; Cells, Cultured; Complement C5a; Extracellular Traps; Female; Humans; Male; Microscopy, Confocal; Middle Aged; Neutrophils; Nucleosomes; Thrombin; Thrombophilia; Thromboplastin

Topics: Adult; Blood Coagulation; Blood Coagulation Factors; Blood Coagulation Tests; Blood Donors; Coumarins; Enzyme Activation; Fibrin; Fluorometry; Humans; Immobilized Proteins; Male; Microscopy, Video; Middle Aged; Oligopeptides; Plasma; Plasmapheresis; Thrombelastography; Thrombin; Thrombophilia; Thromboplastin; Young Adult

Recent epidemiologic data suggest that sickle cell trait (HbAS; AS) is a risk factor for venous thromboembolism. We conducted an exploratory study of healthy subjects with AS under baseline conditions to determine whether a chronic basal hyperactivation of coagulation exists, and if so, what mechanism(s) contribute to this state. Eighteen healthy AS individuals were compared to 22 African-American controls with a normal haemoglobin profile (HbAA; AA) and 17 patients with sickle cell disease (HbSS; SS). Plasma thrombin-antithrombin complexes and D-dimer levels were elevated in AS relative to AA patients (P = 0·0385 and P = 0·017, respectively), and as expected, were much higher in SSversusAA (P < 0·0001 for both). Thrombin generation in platelet poor plasma was indistinguishable between AA and AS subjects, whereas a paradoxical decrease in endogenous thrombin potential was observed in SS (P ≤ 0·0001). Whole blood tissue factor was elevated in SS compared to AA (P = 0·005), but did not differ between AA and AS. Plasma microparticle tissue factor activity was non-significantly elevated in AS (P = 0·051), but was clearly elevated in SS patients (P = 0·004) when compared to AA controls. Further studies in larger cohorts of subjects with sickle cell trait are needed to confirm the results of this preliminary investigation.

Topics: Adult; Anemia, Sickle Cell; Antithrombin III; Black or African American; Case-Control Studies; Cell-Derived Microparticles; Cytokines; Female; Fibrin; Fibrin Fibrinogen Degradation Products; Humans; Inflammation Mediators; Male; Middle Aged; Peptide Hydrolases; Plasma; Sickle Cell Trait; Thrombin; Thrombophilia; Thromboplastin; Venous Thromboembolism

Antineutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV) is characterised by neutrophil activation. An elevated prevalence of venous thromboembolic events has been reported in AAV. Because of the critical role of neutrophils in inflammation associated thrombosis, we asked whether neutrophil tissue factor (TF) may be implicated in the thrombotic diathesis in AAV.. Neutrophils from four patients and sera from 17 patients with ANCA associated vasculitis with active disease and remission were studied. TF expression was assessed by immunoblotting and confocal microscopy. Circulating DNA levels were evaluated. TF expressing microparticles (MPs) were measured by flow cytometry and thrombin-antithrombin complex levels by ELISA.. Peripheral blood neutrophils from four patients with active disease expressed elevated TF levels and released TF expressing neutrophil extracellular traps (NETs) and MPs. TF positive NETs were released by neutrophils isolated from the bronchoalveolar lavage and were detected in nasal and renal biopsy specimens. Elevated levels of circulating DNA and TF expressing neutrophil derived MPs were further observed in sera from patients with active disease. Induction of remission attenuated the aforementioned effects. Control neutrophils treated with sera from patients with active disease released TF bearing NETs and MPs which were abolished after IgG depletion. Treatment of control neutrophils with isolated IgG from sera from patients with active disease also resulted in the release of TF bearing NETs. TF implication in MP dependent thrombin generation was demonstrated by antibody neutralisation studies.. Expression of TF in NETs and neutrophil derived MPs proposes a novel mechanism for the induction of thrombosis and inflammation in active AAV.

Topics: Adult; Aged; Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis; Antibodies, Antineutrophil Cytoplasmic; Bronchoalveolar Lavage Fluid; Case-Control Studies; Cell-Derived Microparticles; Extracellular Traps; Female; Humans; Immunoglobulin G; Male; Middle Aged; Neutrophil Activation; Neutrophils; Remission Induction; Thrombophilia; Thromboplastin; Tumor Necrosis Factor-alpha

Patients with hematological malignancies have a 28-fold increased risk of venous thromboembolism (VTE). Among patients with acute myelogenous leukemia (AML), the 2-year cumulative incidence of VTE is 5.2%. Several studies suggest that microvesicles (MVs) harboring TF may play a role in VTE and disseminated intravascular coagulation (DIC) in acute promyelocytic leukemia (APL). The aim of this study was to assess the capacity of untreated (APL) cells to shed procoagulant MVs. APL cells (NB4 and HL-60 cell lines) and MVs were separated by filtration (0.1-0.22-0.45-0.65 μm). The procoagulant activity (PCA) was assessed by thrombin generation assay (TGA). Alternatively, MVs were incubated with anti-Tissue Factor (TF) antibodies, with annexin V to assess the contribution of TF and phospholipids (PL) to the PCA, respectively. NB4 cells had a high PCA mainly triggered by MVs of size under 0.45 μm. The PCA of MVs was related to the expression of active TF and PL. HL-60 cells had a weaker PCA since TF is mostly present in its inactive form. Moreover, HL-60 do not produce MVs<0.65 μm associated with PCA. MVs could have a predicting value for VTE and DIC in patients with acute promyelocytic leukemia and could inform physicians about the optimal use of a thromboprophylaxis.

Topics: Antibiotics, Antineoplastic; Cell Differentiation; Cell Growth Processes; Cell Line, Tumor; Cell-Derived Microparticles; Daunorubicin; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Microscopy, Electron, Transmission; Thrombophilia; Thromboplastin; Thrombosis; Tumor Cells, Cultured; Venous Thromboembolism

Neoadjuvant chemotherapy (NACT) improves the prognosis of patients with esophageal cancer who respond, but it is not effective in nonresponders. Therefore, it is crucial to establish a reliable method of predicting response before initiation of chemotherapy. Hypercoagulability, which is thought to be because of upregulation of tissue factor (TF) in cancer cells, was reported to be associated with chemoresistance. The aim of this study was to investigate the association between TF expression and response to NACT in esophageal cancer.. In 67 patients with advanced esophageal cancer, TF expression in pretreatment biopsy samples was evaluated immunohistochemically and correlated with clinicopathologic factors and response to chemotherapy.. TF was expressed by 43.3% of the tumors, but there were no correlations observed with any clinicopathologic parameters examined. Clinical and histologic responses to chemotherapy were significantly worse in TF-positive patients compared with TF-negative patients. Multivariate analysis revealed that TF expression was significantly associated with a poor clinical response (P = 0.0431). TF expression was also independently associated with poor progression-free survival (P = 0.0353).. TF expression levels in pretreatment biopsy samples are useful for predicting response to NACT in advanced esophageal cancer. Further studies of mechanisms underlying the relationship between TF expression and chemosensitivity are needed.

Topics: Aged; Biopsy; Carcinoma, Squamous Cell; Disease Progression; Disease-Free Survival; Drug Resistance, Neoplasm; Esophageal Neoplasms; Esophagus; Female; Humans; Male; Middle Aged; Neoadjuvant Therapy; Neoplasm Grading; Predictive Value of Tests; Prognosis; Thrombophilia; Thromboplastin

Increased hypercoagulability has been reported with low doses of direct thrombin inhibitors but not with direct factor Xa inhibitors.. To compare the effects of rivaroxaban with those of melagatran and dabigatran on thrombin generation (TG) and tissue factor-induced hypercoagulability and to explore the possible involvement of the thrombin-thrombomodulin/activated protein C system.. In normal human plasma and in protein C-deficient plasma, TG was investigated in vitro in the presence and absence of recombinant human soluble thrombomodulin (rhs-TM). TG was determined by calibrated automated thrombography and an ELISA for prothrombin fragments 1+2 (F1+2 ). In an in vivo rat model, hypercoagulability was induced by tissue factor; levels of thrombin-antithrombin (TAT) and fibrinogen and the platelet count were determined.. Rivaroxaban inhibited TG in a concentration-dependent manner. In the absence of rhs-TM, melagatran and dabigatran also inhibited TG concentration dependently. However, in the presence of rhs-TM, lower concentrations of melagatran (119-474 nmol L(-1) ) and dabigatran (68-545 nmol L(-1) ) enhanced endogenous thrombin potential, peak TG, and F1+2 formation in normal plasma but not in protein C-deficient plasma. In vivo, rivaroxaban dose-dependently inhibited TAT generation, whereas melagatran showed a paradoxical effect, with an increase in TAT and a small decrease in fibrinogen and platelet count at lower doses.. Low concentrations of the direct thrombin inhibitors melagatran and dabigatran enhanced TG and hypercoagulability, possibly via inhibition of the protein C system. In contrast, rivaroxaban reduced TG and hypercoagulability under all conditions studied, suggesting that it does not suppress this negative-feedback system.

Topics: Animals; Antithrombins; Azetidines; Benzylamines; Blood Coagulation; Blood Platelets; Enzyme-Linked Immunosorbent Assay; Factor Xa Inhibitors; Humans; Male; Morpholines; Plasma; Platelet Count; Protein C; Prothrombin; Rats; Rats, Wistar; Rivaroxaban; Thiophenes; Thrombelastography; Thrombin; Thrombomodulin; Thrombophilia; Thromboplastin

Preeclampsia (P-EC) is a multisystem disorder of pregnancy whose cause and pathogenesis remain poorly understood. However, abnormal haemostasis and endothelial dysfunction are thought to be implicated. Women with a past medical history of P-EC have a baseline hypercoagulable state postpregnancy. The aim of this study is to examine the relationship between tissue factor (TF) and TF pathway inhibitor (TFPI) in women who have had P-EC within the last 3 years (more than 6 months postpartum) and their normal counterparts. Blood specimens were collected from women known to have had P-EC within the last 3 years (n = 26) and aged-matched healthy women without past history of P-EC in previous pregnancy (n = 26). Plasma TF and TFPI levels were measured using ELISAs. Women who have had P-EC showed increased TF levels compared with their normal counterparts, whereas TFPI levels were reduced. Neither parameter differed significantly when the groups were tested against each other. Interestingly, the TF/TFPI ratio was significantly increased (P = 0.024) when the two groups were compared. In summary, there was a trend towards increased TF and reduced TFPI levels in the P-EC group. Such a tendency was not statistically significant. However, the TF/TFPI ratio was significantly increased when the groups were compared. Our findings suggest an imbalance between TF/TFPI levels in women with past history of P-EC postpregnancy. This may contribute to the development of maternal hypercoagulable states and may predispose women with a history of P-EC to cardiovascular risks later in life.

Topics: Adult; Case-Control Studies; Female; Humans; Lipoproteins; Middle Aged; Pre-Eclampsia; Pregnancy; Thrombophilia; Thromboplastin; Young Adult

Outer membrane vesicles (OMVs) were previously shown to be capable of initiating the inflammatory response seen in the transition of an infection to sepsis. However, another tenet of sepsis is the development of a hypercoagulable state and the role of OMVs in the development of this hypercoagulability has not been evaluated. The objective of this study was to evaluate the ability of OMVs to elicit endothelial mediators of coagulation and inflammation and induce platelet activation.. Human umbilical vein endothelial cells (HUVECs) were incubated with OMVs and were analyzed for the expression of tissue factor (TF), thrombomodulin, and the adhesion molecules P-selectin and E-selectin. Supernatants of OMV-treated HUVECs were mixed with whole blood and assessed for prothrombotic monocyte-platelet aggregates (MPA).. OMVs induce significantly increased expression of TF, E-selectin, and P-selectin, whereas, the expression of thrombomodulin by HUVECs is significantly decreased (P < 0.05). The lipopolysaccharide inhibitor clearly inhibited the expression of E-selectin following incubation with OMVs, although its impact on TF and thrombomodulin expression was nominal. Incubation of whole blood with supernatant from HUVECs exposed to OVMs resulted in increased MPAs.. This study demonstrates that, at the cellular level, OMVs from pathogenic bacteria play a complex role in endothelial activation. Although OMV-bound lipopolysaccharide modulates inflammatory proteins, including E-selectin, it has a negligible effect on the tested coagulation mediators. Additionally, endothelial activation by OMVs facilitates platelet activation as indicated by increased MPAs. By influencing the inflammatory and coagulation cascades, OMVs may contribute to the hypercoagulable response seen in sepsis.

Topics: Bacterial Outer Membrane Proteins; Blood Coagulation; Cell-Derived Microparticles; Cytoplasmic Vesicles; E-Selectin; Escherichia coli; Human Umbilical Vein Endothelial Cells; Humans; Lipopolysaccharides; Monocytes; P-Selectin; Platelet Activation; Sepsis; Thrombomodulin; Thrombophilia; Thromboplastin

Topics: Adult; Aged; Biomarkers; Blood Coagulation Tests; Case-Control Studies; Female; Humans; Liver Cirrhosis; Male; Middle Aged; Predictive Value of Tests; Reproducibility of Results; Severity of Illness Index; Thrombin; Thrombomodulin; Thrombophilia; Thromboplastin

The association between cancer and thrombogenesis has been recognized since 1865, and tissue factor (TF) is important at various stages in the natural history of the disease. It is involved in cancer angiogenesis, growth and metastasis. TF pathway inhibitor (TFPI), being the major physiological regulator of the TF-dependent coagulation pathway, is also important in establishing net procoagulant potential. In this study, we determine TF and TFPI levels in three prostate epithelial cell lines, one of normal and two of malignant origin. Cells were grown in standard maintenance conditions and harvested at more than 90% confluence. These were fractionated into cytosol, membrane and nuclei for analysis. Microparticles secreted into the culture medium were also analysed. TF and TFPI levels were determined using an ELISA. TF expression in these cells was also visualized using immunocytochemistry. There was absence of TF and TFPI in nuclei of all cell lines. TF expression was higher in subcellular fractions and microparticles of normal prostate cells than cancer cells. In contrast, levels of TFPI (structurally resembling a secreted, rather than transmembrane protein) in microparticles of normal prostate cells were much lower than tumour cells. In conclusion, the activity of prostate cancer cells themselves is unlikely to be the source of hypercoagulability in patients, but might precipitate chains of events that would produce such an effect.

Topics: Cell Line, Tumor; Cell Membrane; Cell Nucleus; Cell-Derived Microparticles; Cytosol; Gene Expression; Humans; Lipoproteins; Male; Prostate; Prostatic Neoplasms; Thrombophilia; Thromboplastin; Thrombosis

What is the effect of estrogen on heparanase procogulant activity?. Estrogen increases heparanase procoagulant activity.. Estrogen therapy increases the risk of thrombosis and was previously found to up-regulate heparanase expression. Heparanase is involved in angiogenesis and metastasis, and has been shown to form a complex with tissue factor (TF) and also shown to enhance the generation of factor Xa.. A case-control study. Thirty-four healthy women using oral contraceptives (OC) and 41 women not using hormonal therapy and not pregnant per history were enrolled, over a 5-month period, at the Rambam Medical Center, Haifa, Israel. In vitro, estrogen receptor-positive (MCF-7) and -negative (MDA-231) cell lines were incubated with estrogen, tamoxifen and ICI-182.780 a pure estrogen receptor antagonist. The cell medium was evaluated for TF/heparanase complex activity, TF activity and heparanase procoagulant activity by chromogenic substrate.. Exclusion criteria included age <18 years, post-menopausal women, concomitant medications other than supplement minerals and vitamins, acute or chronic illness.. The study demonstrates increased risk of high heparanase procoagulant activity in OC users. When a cutoff level of 0.25 (absorbance 405-490 nm) was set, the odds ratio was 131 (P < 0.0001). When all results were studied by quartiles, in quartiles 3 and 4 the results were almost exclusively of the OC users (P < 0.0001). In cell cultures, estrogen and tamoxifen increased heparanase procoagulant activity in the medium of estrogen receptor-positive (MCF-7) cells.. The main limitation of the current study is that the two estrogens given to the women and cell cultures, ethinyl estradiol (EE) and 17-β-estradiol (E2), respectively, may have different effects on the coagulation system, although an increase in heparanase procoagulant activity was demonstrated in both of them. Although the sample size of the study group was limited, significant differences in the activation of the extrinsic coagulation pathway were demonstrated.. The clinical relevance of the heparanase procoagulant activity assay as a screening tool in thrombophilia work-up should further be elucidated.

Topics: Adult; Blood Coagulation; Case-Control Studies; Cell Line; Contraceptives, Oral; Estradiol; Estrogen Antagonists; Estrogens; Female; Fulvestrant; Glucuronidase; Humans; Israel; Mammary Glands, Human; Receptors, Estrogen; Risk; Selective Estrogen Receptor Modulators; Signal Transduction; Tamoxifen; Thrombophilia; Thromboplastin; Young Adult

Patients with lung adenocarcinoma undergoing surgery are in high risk for VTE and receive routine post-operative thromboprophylaxis with LWMH.. We investigated markers of hypercoagulability in patients with primary localized adenocarcinoma and the modifications induced by lobectomy and postoperative administration of enoxaparin.. Patients suffering from localised primary lung adenocarcinoma (n=15) scheduled for lobectomy were studied. The control group consisted of 15 healthy age and sex-matched individuals. Blood was collected before anaesthesia induction and after surgery, at several intervals until the 7th post-operative day. Samples were assessed for thrombin generation, phosphatidylserin expressing platelet derived microparticles expressing (Pd-MP/PS(+)), tissue factor activity (TFa), FVIIa and TFPI levels, procoagulant phospholipid dependent clotting time and anti-Xa activity.. At baseline, patients showed increased thrombin generation and Pd-MP/PS(+). After lobectomy thrombin generation significantly decreased. Administration of enoxaparin attenuated thrombin generation. In about 50% of samples collected post-operatively an increase of thrombin generation occurred despite the presence of the expected anti-Xa activity in plasma. At the 7th post-operative day, 3 out of 15 patients showed a significant increase of thrombin generation.. In patients with localized lung adenocarcinoma, hypercoagulability is characterized by high thrombin generation and increased concentration of Pd-MP/PS(+). Tumor mass resection is related with attenuation of thrombin generation, which is inhibited by postoperative thromboprophylaxis with enoxaparin. The response to enoxaparin is not predicted by the concentration of the anti-Xa activity in plasma. The assessment of thrombin generation during prophylaxis with enoxaparin allows to identify patients with high residual plasma hypercoagulability.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Aged; Anticoagulants; Blood Coagulation; Blood Coagulation Tests; Blood Platelets; Cell-Derived Microparticles; Enoxaparin; Factor Xa Inhibitors; Female; Humans; Lung; Lung Neoplasms; Male; Middle Aged; Postoperative Period; Thrombin; Thrombophilia; Thromboplastin

Concanavalin A (Con A) treatment induces severe hepatitis in mice in a manner dependent on T cells, interferon (IFN)-gamma, and tumor necrosis factor (TNF). Treatment with the anticoagulant heparin protects against hepatitis, despite healthy production of IFN-γ and TNF. Here, we investigated molecular and cellular mechanisms for hypercoagulation-mediated hepatitis. After Con A challenge, liver of wild-type (WT) mice showed prompt induction of Ifnγ and Tnf, followed by messenger RNA expression of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1), which initiate blood coagulation and inhibit clot lysis, respectively. Mice developed dense intrahepatic fibrin deposition and massive liver necrosis. In contrast, Ifnγ(-/-) mice and Ifnγ(-/-) Tnf(-/-) mice neither induced Pai1 or Tf nor developed hepatitis. In WT mice TF blockade with an anti-TF monoclonal antibody protected against Con A-induced hepatitis, whereas Pai1(-/-) mice were not protected. Both hepatic macrophages and sinusoidal endothelial cells (ECs) expressed Tf after Con A challenge. Macrophage-depleted WT mice reconstituted with hematopoietic cells, including macrophages deficient in signal transducer and activator of transcription-1 (STAT1) essential for IFN-γ signaling, exhibited substantial reduction of hepatic Tf and of liver injuries. This was also true for macrophage-depleted Stat1(-/-) mice reconstituted with WT macrophages. Exogenous IFN-γ and TNF rendered T-cell-null, Con A-resistant mice deficient in recombination-activating gene 2, highly susceptible to Con A-induced liver injury involving TF.. Collectively, these results strongly suggest that proinflammatory signals elicited by IFN-γ, TNF, and Con A in both hepatic macrophages and sinusoidal ECs are necessary and sufficient for the development of hypercoagulation-mediated hepatitis.

Topics: Animals; Concanavalin A; Endothelial Cells; Fibrin; Hepatitis, Animal; Interferon-gamma; Liver; Macrophages; Mice; Mice, Knockout; Mitogens; Necrosis; Plasminogen Activator Inhibitor 1; Signal Transduction; STAT1 Transcription Factor; T-Lymphocytes; Thrombophilia; Thromboplastin; Tumor Necrosis Factor-alpha

Major trauma is an independent risk factor for developing venous thromboembolism. While increases in thrombin generation and/or procoagulant microparticles have been detected in other patient groups at greater risk for venous thromboembolism, such as cancer or coronary artery disease, this association has yet to be documented in trauma patients. This pilot study was designed to characterize and quantify thrombin generation and plasma microparticles in individuals early after traumatic injury.. Blood was collected in the trauma bay from 52 blunt injured patients (cases) and 19 uninjured outpatients (controls) and processed to platelet poor plasma to allow for (1) isolation of microparticles for identification and quantification by flow cytometry, and (2) in vitro thrombin generation as measured by calibrated automatic thrombography. Data collected are expressed as either mean ± standard deviation or median with interquartile range.. Among the cases, which included 39 men and 13 women (age, 40 ± 17 years), the injury severity score was 13 ± 11, the international normalized ratio was 1.0 ± 0.1, the thromboplastin time was 25 ± 3 seconds, and platelet count was 238 ± 62 (thousands). The numbers of total (cell type not specified) procoagulant microparticles, as measured by Annexin V staining, were increased compared to nontrauma controls (541 ± 139/μL and 155 ± 148/μL, respectively; P < .001). There was no significant difference in the amount of thrombin generated in trauma patients compared to controls; however, peak thrombin was correlated to injury severity (Spearman correlation coefficient R, 0.35; P = .02).. Patients with blunt trauma have greater numbers of circulating procoagulant microparticles and increased in vitro thrombin generation. Future studies to characterize the cell-specific profiles of microparticles and changes in thrombin generation kinetics after traumatic injury will determine whether microparticles contribute to the hypercoagulable state observed after injury.

Topics: Adult; Annexin A5; Biomarkers; Case-Control Studies; Cell-Derived Microparticles; Cohort Studies; Female; Humans; Male; Middle Aged; Partial Thromboplastin Time; Pilot Projects; Prospective Studies; Prothrombin Time; Risk Factors; Thrombin; Thrombophilia; Thromboplastin; Trauma Severity Indices; Venous Thromboembolism; Wounds and Injuries

Complement activation plays a role in pathogenesis of the antiphospholipid syndrome (APS), but the involvement of the C5b-9 membrane attack complex (MAC) is unknown. Here we studied the effects of human polyclonal antiphospholipid (aPL) antibodies on thrombosis and tissue factor (TF) up-regulation in C6 deficient (C6(-/-)) mice.. C6(-/-) mice or the wild-type C3H/HeJ (C6(+/+)) mice were injected twice with IgG-APS (n = 2) or IgM-APS (n = 1) isolated from APS patients or with the corresponding control immunoglobulins (Igs) of normal human serum, (NHS) (IgG-NHS or IgM-NHS). Then, the sizes of induced thrombi in the femoral vein were determined 72 hours after the first injection. Tissue factor was determined in homogenates of carotid arteries and in peritoneal macrophages.. Thrombus sizes were significantly larger in C6(+/+) treated with IgG-APS1 or with IgG-APS2 or with IgM-APS when compared with C6(+/+) mice treated with IgG-NHS or with IgM-NHS, respectively. The sizes of thrombi were significantly smaller in the C6(-/-) mice injected with IgG-APS1, IgG-APS2 or IgM-APS (p < 0.001), compared to their C6(+/+) counterparts showing an important abrogation of thrombus formation in mice lacking C6. The TF expression and activity in the C6(-/-) mice treated with IgG-APS or IgM-APS were diminished when compared to C3H/HeJ (C6(+/+)) mice treated with the same Igs. All mice injected with IgG-APS and IgM-APS had medium-high titers of anticardiolipin (aCL) and anti-β(2)glycoprotein I (aβ(2)GPI) antibodies.. These data indicate that the C6 component of the complement system mediates aPL-thrombogenic effects, underscoring an important pathogenic mechanism and indicating the possibility of inhibiting complement to ameliorate APS-related manifestations.

Topics: Adult; Animals; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Carotid Arteries; Complement C6; Female; Femoral Vein; Humans; Immunoglobulin G; Immunoglobulin M; Macrophages, Peritoneal; Male; Mice; Mice, Inbred C3H; Mice, Knockout; Middle Aged; Thrombophilia; Thromboplastin; Thrombosis; Up-Regulation

Topics: Cardiovascular Diseases; Factor VII; Factor VIIa; Humans; Plaque, Atherosclerotic; Thrombophilia; Thromboplastin; Thrombosis

Cytomegalovirus is associated with hypercoagulability, and is reported to increase the risk of venous thrombosis in human immunodeficiency virus (HIV)-infected patients. Progression to AIDS, however, is also associated with hypercoagulability and venous thrombosis, and may result in more comorbidities, such as reactivation of cytomegalovirus. It is therefore unknown whether active cytomegalovirus in HIV infection results in a procoagulant state or whether hypercoagulability is the result of HIV infection itself. In this cross-sectional study of 104 consecutive HIV-infected patients, active cytomegalovirus infection was associated with hypercoagulability independently of stage of HIV disease. This finding may deserve attention in preventative recommendations for use of thromboprophylaxis in HIV-infected patients.

Topics: Adult; Aged; AIDS-Related Opportunistic Infections; CD4-Positive T-Lymphocytes; Cross-Sectional Studies; Cytomegalovirus; Cytomegalovirus Infections; Female; HIV Infections; Humans; Lymphocyte Count; Male; Middle Aged; Protein S; Thrombophilia; Thromboplastin; Thrombosis

Xuezhikang, extract of red yeast rice, is a traditional Chinese medicine with multiple cardioprotective effect. It contains a family of naturally occurring statins, such as lovastatin. Tissue factor (TF) is overexpressed in macrophages of lipid core plaques, which display high procoagulant activity and seem to be a potentially target for anti-atherothrombosis. Therefore, the purpose of this study was to explore the effect and possible molecular mechanisms of xuezhikang on inhibiting TF expression and hypercoagulable state and the differences compared with lovastatin. Our results showed that xuezhikang significantly suppressed oxidized low-density lipoprotein-induced TF expression in macrophages in a concentration-dependent manner. Xuezhikang reduced nicotinamide adenine dinucleotide phosphate oxidase activity by decreasing membrane translocation of p47 through inhibition of extracellular signal-regulated kinase 1/2 activation. Nicotinamide adenine dinucleotide phosphate inhibitor (diphenyleneiodonium) also inhibited the oxidized low-density lipoprotein-induced TF expression, similar to the effects of xuezhikang. Furthermore, consistent with the severity of aortic atherosclerosis, xuezhikang (300 mg·kg·d) significantly reduced blood coagulation activation and TF expression in high-cholesterol diet-induced atherosclerotic rats. In addition, xuezhikang was more potent than lovastatin on inhibiting the expression of TF and nicotinamide adenine dinucleotide phosphate oxidase activation. These observations provide evidences that inhibition of xuezhikang on hypercoagulation and TF expression may partly account for its cardioprotective benefits.

Topics: Animals; Aorta; Biological Products; Blood Coagulation Tests; Cell Survival; Cholecalciferol; Cholesterol; Diet, High-Fat; Disease Models, Animal; Drugs, Chinese Herbal; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lipoproteins, LDL; Lovastatin; Macrophages; Male; Mice; NADPH Oxidases; Oryza; Random Allocation; Rats; Rats, Wistar; Reactive Oxygen Species; Thrombophilia; Thromboplastin; Vitamins

Shedding of microvesicles (MVs) by cancer cells is implicated in a variety of biological effects, including the establishment of cancer-associated hypercoagulable states. However, the mechanisms underlying malignant transformation and the acquisition of procoagulant properties by tumour-derived MVs are poorly understood. Here we investigated the procoagulant and prothrombotic properties of MVs produced by a melanocyte-derived cell line (melan-a) as compared to its tumourigenic melanoma counterpart Tm1. Tumour cells exhibit a two-fold higher rate of MVs production as compared to melan-a. Melanoma MVs display greater procoagulant activity and elevated levels of the clotting initiator protein tissue factor (TF). On the other hand, tumour- and melanocyte-derived MVs expose similar levels of the procoagulant lipid phosphatidylserine, displaying identical abilities to support thrombin generation by the prothrombinase complex. By using an arterial thrombosis model, we observed that melanoma- but not melanocyte-derived MVs strongly accelerate thrombus formation in a TF-dependent manner, and accumulate at the site of vascular injury. Analysis of plasma obtained from melanoma-bearing mice showed the presence of MVs with a similar procoagulant pattern as compared to Tm1 MVs produced in vitro. Remarkably, flow-cytometric analysis demonstrated that 60% of ex vivo MVs are TF-positive and carry the melanoma-associated antigen, demonstrating its tumour origin. Altogether our data suggest that malignant transformation in melanocytes increases the production of procoagulant MVs, which may contribute for a variety of coagulation-related protumoural responses.

Topics: Animals; Blood Coagulation; Cell Line, Tumor; Cell Transformation, Neoplastic; Cell-Derived Microparticles; Coagulants; Humans; Melanocytes; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Plasma; Skin Neoplasms; Thrombophilia; Thromboplastin; Thrombosis; Tumor Microenvironment

Elevated factor (F)XI is associated with an increased risk for ischemic stroke. Activated FXI (FXIa) and tissue factor (TF) have not been studied following stroke. The aim of the current study was to evaluate circulating FXIa and TF in patients with prior cerebrovascular events.. We studied 241 patients, including 162 after ischemic stroke and 79 after transient ischemic attack (TIA), recruited 6 months to 4 years (median, 36 months) after the events. Plasma TF and FXIa activity following the index event were determined in clotting assays by measuring the response to inhibitory monoclonal antibodies.. Active TF was detected in 25 (10.4%) of the patients, while FXIa activity (median, 37.5 [IQR 397] pM) was found in 64 (26.7%) of the patients (p<0.01). The prevalence of active TF and FXIa was higher in subjects with previous stroke compared with those with a history of TIA (13% vs 5.1%, p=0.05, and 34% vs 11.4%, p<0.0001, respectively). Patients with circulating FXIa were younger and had higher fibrinogen and interleukin-6 compared to the remainder. Patients with detectable TF or FXIa activity had higher NIHSS score, higher modified Rankin scale and lower Barthel Index than the remaining subjects (all p<0.05).. Circulating active TF and FXIa can occur in patients with cerebrovascular ischemic events ≥6 months after the events. The presence of these factors is associated with worse functional outcomes, which highlights the role of persistent hypercoagulable state in cerebrovascular disease.

Topics: Adult; Age Factors; Blood Coagulation Tests; Brain Ischemia; Factor XIa; Female; Fibrinogen; Humans; Interleukin-6; Ischemic Attack, Transient; Male; Middle Aged; Predictive Value of Tests; Prognosis; Stroke; Thrombophilia; Thromboplastin; Young Adult

Cigarette smoking is associated with plasmatic hypercoagulability, and carbon monoxide has been demonstrated to enhance coagulation by binding to a fibrinogen-bound heme. Our objective was to design and test a redox-based method to detect carboxyhemefibrinogen. Normal, pooled, citrated plasma was exposed to 0-100  μmol/l carbon monoxide releasing molecule-2 (tricarbonyldichlororuthenium (II) dimer; CORM-2) before or after exposure to the organic reductant phenylhydroxylamine (PHA, 0-30  mmol/l), a compound that rapidly converts Fe(+2) to Fe(+3) in heme. Addition of calcium and tissue factor activation in disposable thrombelastographic cups was performed, followed by data collection at 37°C for 15  min. Elastic modulus (G, dynes/cm(2)) was the primary endpoint. CORM-2 significantly increased G values by 67.8% compared to unexposed plasma; pretreatment with 10  mmol/l PHA significantly decreased G values in CORM-2-exposed plasma by 77.1%, whereas 30  mmol/l PHA was required to significantly decrease G values by 64.0% in plasma following CORM-2 pre-exposure. G values were not significantly different between unexposed plasma and plasma exposed to CORM-2 followed by 30  mmol/l PHA addition. Conversion of fibrinogen-bound to the metheme state alone decreased G by 34.3-38.9% following exposure to 10-30  mmol/l PHA. Conversion of fibrinogen-bound heme Fe(+2) to Fe(+3) with PHA abrogated carbon monoxide-mediated increases in clot strength. Clinical trials are planned to investigate smoking individuals to mechanistically link carboxyhemefibrinogen formation with in-vitro hypercoagulability.

Topics: Blood Coagulation; Calcium; Carbon Monoxide; Dose-Response Relationship, Drug; Elastic Modulus; Fibrinogen; Heme; Humans; Hydroxylamines; Organometallic Compounds; Oxidation-Reduction; Plasma; Protein Binding; Smoking; Thrombelastography; Thrombophilia; Thromboplastin

Monocyte- and microparticle (MP)-associated tissue factor (TF) is upregulated in diabetes. Lipopolysaccharide (LPS) induces expression of TF and alternatively spliced TF (asTF) and increases MP release from monocytes. Using LPS-stimulated TF-bearing human monocytes, we examined whether glibenclamide, a sulfonylurea used to treat diabetes type 2, might possess anticoagulant properties.. We studied the effects of glibenclamide on cell- and supernatant-associated procoagulant activity (Factor Xa-generating assay and clot formation assay), on expression of TF and asTF (flow cytometry, RT-qPCR, western blot) and on cell viability and MP release (flow cytometry).. Glibenclamide dose-dependently decreased procoagulant activity of cells and supernatants. The reduction in cellular procoagulant activity coincided with reduced expression of TF and asTF in cells, whereas cell viability remained almost unchanged. The glibenclamide-induced reduction in procoagulant activity of supernatants appeared to be associated with a decreased number of released MPs.. Reduction of monocyte- and supernatant-associated procoagulant activity by glibenclamide is associated with decreased expression of TF and asTF and possibly with a reduced MP number. Our data indicate that glibenclamide reduces the prothrombotic state in LPS-stimulated monocytes in vitro. Glibenclamide might therefore also have an anticoagulant effect in vivo, but this needs to be further evaluated.

Topics: Anticoagulants; Blood Coagulation Tests; Cell Survival; Cell-Derived Microparticles; Cells, Cultured; Glyburide; Humans; Hypoglycemic Agents; Lipopolysaccharides; Monocytes; Thrombophilia; Thromboplastin

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; DNA; Doxorubicin; Endothelial Cells; Epirubicin; Female; Histones; Humans; Monocytes; Neoplasms; Protein C; Thrombin; Thrombomodulin; Thrombophilia; Thromboplastin; Thrombosis

Whereas procoagulation abnormalities in acute stress are well established, little is known about the mechanism of hypercoagulation in chronic stress, such as post-traumatic stress disorder (PTSD). This is crucial, given the fact that chronic coagulation disturbances have been associated with increased morbidity and premature mortality due to thromboembolism and cardiovascular disorders, complications recently described in PTSD patients.. To explore the mechanisms of hypercoagulation in chronic PTSD.. Thirty patients diagnosed with chronic PTSD were enrolled and compared with a control group matched for age, gender and ethnicity. Hypercoagulation state was evaluated by levels of fibrinogen, D-dimer, prothrombin fragment F 1+2, von Willebrand factor (vWF) antigen, factor VIII activity, activated protein C resistance, ProC Global assay, and tissue factor antigen. Psychiatric evaluation was performed using the Mini-International Neuropsychiatric Interview and Clinician Administered PTSD Scale (CAPS).. vWF antigen levels were significantly higher in patients with chronic PTSD compared with the controls (121.3 +/- 42 vs. 99.7 +/- 23, respectively, P = 0.034). Higher levels of vWF antigen and factor VIII activity were found in patients with severe chronic PTSD (CAPS > 80), compared to controls and patients with chronic PTSD and less severe symptoms (CAPS < or = 80). However, no differences were observed in any other studied coagulation parameters between patients and controls.. Increased levels of vWF antigen and factor VIII activity were documented in severe chronic PTSD. These findings suggest that the higher risk of arterial and venous thromboembolic events in PTSD patients could be related to endothelial damage or endothelial activation.

Topics: Activated Protein C Resistance; Adult; Biomarkers; Blood Coagulation Factors; Chronic Disease; Factor VIII; Fibrin Fibrinogen Degradation Products; Fibrinogen; Humans; Israel; Male; Peptide Fragments; Protein Precursors; Prothrombin; Stress Disorders, Post-Traumatic; Surveys and Questionnaires; Thrombophilia; Thromboplastin; von Willebrand Factor

Amniotic fluid embolism (AFE) is a rare but catastrophic complication of pregnancy characterized by severe hypotension, cardiovascular collapse, and massive consumptive coagulopathy. Several animal models of this syndrome have been proposed, but most have yielded inconclusive results.. The objective of this study was to develop a suitable animal model of AFE.. Twelve rabbits in late gestation (25 days) were used. Amniotic fluid was collected from the fetal amniotic sacs after laparotomy, and autologous fluid was injected into 6 rabbits via the left auricular vein. Six other rabbits received saline (control group). Blood pressure, platelet counts, and coagulation variables were measured at baseline and at various intervals for 60 minutes after injection. The in vitro effect of amniotic fluid on coagulation was assessed by thrombelastographic (TEG) analysis.. Injection of amniotic fluid did not reproduce clinical signs of AFE and had no effect on activated partial thromboplastin time (aPTT), prothrombin time (PT), or Factor VIII activity. However, significant thrombocytopenia was observed 5 minutes after administration of amniotic fluid and resolved by 60 minutes. In vitro addition of amniotic fluid to blood resulted in accelerated clotting on TEG tracings.. The syndrome of AFE was not reproduced in this rabbit model. However, injection of autologous amniotic fluid induced a transient and severe thrombocytopenia. Moreover, TEG analysis indicated that amniotic fluid could initiate the coagulation cascade. Other factors such as the presence of meconium in amniotic fluid may be needed to provoke more severe clinical signs.

Topics: Amniotic Fluid; Animals; Blood Coagulation; Blood Coagulation Tests; Blood Pressure; Disease Models, Animal; Embolism, Amniotic Fluid; Female; Humans; Injections, Intravenous; Platelet Count; Pregnancy; Pregnancy Complications; Rabbits; Thrombelastography; Thrombocytopenia; Thrombophilia; Thromboplastin; Time Factors

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

Acute promyelocytic leukaemia (APL) confers an increased risk of thrombosis and bleeding. Current treatments are insufficient to inhibit these complications. We recently showed that a combined immunoinhibitory and immunostimulatory short interference (si) RNA effectively inhibited leukaemic growth and metastasis in rats with APL. We now asked if the reported anti-leukaemic effects of siRNA treatment could be explained by inhibition of hypercoagulability. We measured markers of coagulation and fibrinolysis in plasma collected from APL rats with overt leukaemia using conventional assays. Coagulopathy developed in untreated leukaemic rats evidenced by increase in several haemostatic markers. Treatment of leukaemic rats with the siRNA reduced (p < 0.05) the concentration of thrombin-anti-thrombin complex (a marker of coagulation) by 40% compared with rats treated with an inactive, control siRNA. Substantial reductions (p < 0.05) were also obtained for two markers of fibrinolysis: D-dimer (72%) and plasminogen activator inhibitor type 1 (51%). The activity of tissue factor, the main initiator of coagulation, was not increased (p > 0.05) in untreated leukaemic rats compared with healthy rats, and did not change (p > 0.05) upon treatment with the siRNA. The bifunctional siRNA reduces the hypercoagulable state in APL in addition to its direct anti-leukaemic properties, supporting testing of this small molecule in human APL.

Topics: Animals; Antithrombin III; Biomarkers; Blood Coagulation; Cells, Cultured; Dendritic Cells; Disease Models, Animal; Fibrin Fibrinogen Degradation Products; Fibrinogen; Genetic Therapy; Interleukin-10; Leukemia, Promyelocytic, Acute; Male; Peptide Hydrolases; Plasminogen Activator Inhibitor 1; Rats; Rats, Inbred BN; RNA Interference; RNA, Small Interfering; Thrombophilia; Thromboplastin; Transfection

Racial differences in coagulation are poorly understood. While some studies suggest a 'prothrombotic' coagulation profile in blacks compared with whites, others report an increased bleeding risk for blacks in various clinical settings. Moreover, preclinical data suggest a link between the Duffy antigen (=DARC, Duffy antigen receptor of chemokines) and coagulation.. Based on our previous research in Duffy antigen negative Africans, we hypothesized that Africans have an attenuated procoagulant response compared with Caucasians in a model of lipopolysaccharide (LPS)-induced, tissue factor (TF)-triggered coagulation activation.. Healthy male volunteers (16 Duffy-negative Africans, 16 Duffy-positive Caucasians) received 2 ng kg(-1) LPS, and outcome parameters were measured using enzyme immunoassays and real-time polymerase chain reaction (RT-PCR, Taqman).. LPS increased microparticle (MP)-associated TF procoagulant activity (PCA) less in Africans than Caucasians. Africans had reduced in vivo thrombin formation compared with Caucasians: they generated less thrombin-antithrombin (TAT) complexes (10.4 pg mL(-1) vs. 23.0 pg mL(-1), P<0.0001) and less prothrombin fragments (F1+2) (337 pmol mL(-1) vs. 819 pmol mL(-1), P<0.0001). Consistently, Africans also had decreased fibrin formation (D-dimer: 0.3 pg mL(-1) vs. 0.5 pg mL(-1), P=0.02).. Duffy-negative subjects of African descent have a markedly reduced procoagulant response in a model of LPS-induced, TF-triggered coagulation activation compared with Duffy-positive healthy Caucasians.

Topics: Adult; Biomarkers; Black People; Blood Coagulation; Duffy Blood-Group System; Endotoxins; Humans; Lipopolysaccharides; Racial Groups; Thrombophilia; Thromboplastin; White People; Young Adult

Patients with end-stage renal disease (ESRD) exhibit features of a hypercoagulable state, which may contribute to atherosclerosis. Kynurenines are the metabolites of tryptophan degradation in mammals. We examined the relationship between coagulation activation and kynurenines in 92 patients with ESRD on maintenance haemodialysis (HD) or continuous ambulatory peritoneal dialysis (CAPD) and 20 healthy controls. We measured the plasma levels of: tissue factor (TF), its pathway inhibitor (TFPI), the marker of coagulation activation - prothrombin fragments 1+2 (F(1+2)), kynurenine (KYN) and its metabolites: kynurenic (KYNA), anthranilic (AA) and quinolinic (QA) acids. The ratio of KYNA to KYN (kyna/kyn), AA to KYN (aa/kyn) and QA to KYN (qa/kyn), reflecting intensified activity of enzymes which converted KYN to its metabolites, were also determined. Measured coagulation parameters and kynurenines were significantly elevated in ESRD patients compared to controls. TF, TFPI and F(1+2) were significantly associated with AA, aa/kyn, QA and qa/kyn ratio. Multiple regression analysis showed that fibrinogen (p<0.01) and above mentioned KYN metabolites (all p<0.05) were the independent variables significantly associated with increased F(1+2) levels, reflecting hypercoagulability in ESRD patients. In conclusion, this study represents the first to investigate both the coagulation system and KYN pathway in ESRD patients. The coagulation was enhanced in dialysed uraemic patients compared with the healthy controls demonstrated by increased TF, TFPI and F(1+2) levels. These changes were correlated with activation of the KYN pathway. Finally, fibrinogen and KYN metabolites are independently and significantly associated with the hypercoagulable state in uraemic patients on CAPD and HD treatment.

Topics: Adult; Aged; Blood Coagulation; Female; Humans; Kidney Failure, Chronic; Kynurenine; Lipoproteins; Male; Middle Aged; Peptide Fragments; Peritoneal Dialysis, Continuous Ambulatory; Protein Precursors; Prothrombin; Renal Dialysis; Thrombophilia; Thromboplastin; Uremia

Patients with haematological malignancies carry increased risk of venous thrombosis (VT). However, the mechanisms that link these malignancies to activated coagulation have not been fully identified. Since anti-haemostatic agents are studied in clinical trials for their potential to prolong survival in cancer patients, a detailed characterisation of haemostatic markers in cancer subtypes is needed. Hence, in this study, we measured the plasma concentrations and mRNA expression in blood mononuclear cells of haemostatic parameters in 93 patients with haematological neoplasias (acute myeloid leukaemia, chronic lymphatic leukaemia, multiple myeloma, and non-Hodgkin's lymphoma) before start and after completion of cancer therapy. At diagnosis we found activation of coagulation and fibrinolysis, especially in patients with acute myeloid leukaemia. This hypercoagulation was not associated with increased levels of tissue factor (TF) or factor VII (fVII) antigen or mRNA, or levels of activated fVII. In conclusion we found a hypercoagulable state in patients with haematological malignancy that did not seem to be initiated by TF.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Blood Coagulation; Case-Control Studies; Factor VII; Female; Fibrin Fibrinogen Degradation Products; Glycoproteins; Hematologic Neoplasms; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Myeloid, Acute; Lipoproteins; Longitudinal Studies; Lymphoma, Non-Hodgkin; Male; Middle Aged; Multiple Myeloma; Norway; Peptide Fragments; Prothrombin; RNA, Messenger; Thrombophilia; Thromboplastin; Venous Thrombosis

To examine the role of Fibrinogen-like protein 2 (fgl2)/fibroleukin in tumor development. Fgl2 has been reported to play a vital role in the pathogenesis in MHV-3 (mouse hepatitis virus) induced fulminant and severe hepatitis, spontaneous abortion, allo- and xeno- graft rejection by mediating "immune coagulation".. Tumor tissues from 133 patients with six types of distinct cancers and the animal tumor tissues from human hepatocellular carcinoma (HCC) model on nude mice (established from high metastasis HCC cell line MHCC97LM6) were obtained.. Hfgl2 was detected in tumor tissues from 127 out of 133 patients as well as tumor tissues collected from human HCC nude mice. Hfgl2 was highly expressed both in cancer cells and interstitial inflammatory cells including macrophages, NK cells, and CD8(+) T lymphocytes and vascular endothelial cells. Hfgl2 mRNA was localized in cells that expressed hfgl2 protein. Fibrin (nogen) co-localization with hfgl2 expression was determined by dual immunohistochemical staining. In vitro, IL-2 and IFN-gamma increased hfgl2 mRNA by 10-100 folds and protein expression in both THP-1 and HUVEC cell lines. One-stage clotting assays demonstrated that THP-1 and HUVEC cells expressing hfgl2 had increased procoagulant activity following cytokines stimulation.. The hfg12 contributes to the hypercoagulability in cancer and may induce tumor angiogenesis and metastasis via cytokine induction.

Topics: Adult; Aged; Animals; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Disease Models, Animal; Endothelial Cells; Female; Fibrinogen; Humans; Interferon-gamma; Interleukin-2; Leukemia, Monocytic, Acute; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; RNA, Messenger; Thrombophilia; Thromboplastin

Anti-vimentin antibodies (AVA) are associated with autoimmunity and solid organ transplantation, conditions associated with vascular disease, but their contribution to disease pathogenesis is unknown. Here, we have examined interactions between AVA (mAb and serum from patients) and various leukocyte populations using whole blood and flow cytometry. Normal blood treated with patient sera containing high AVA-IgM titers or with a vimentin-specific monoclonal IgM led to activation of platelets and other leukocytes, as demonstrated by induced expression of P-selectin, fibrinogen, tissue factor, and formation of platelet:leukocyte (P:L) conjugates and a reduction in platelet counts. This activity was antigen (vimentin)-specific and was not mediated by irrelevant IgM antibodies. Flow cytometry demonstrated that AVA do not bind directly to resting platelets in whole blood, but they bind to approximately 10% of leukocytes. Supernatant, derived from AVA-treated leukocytes, induced platelet activation, as measured by the generation of platelet microparticles, when added to platelet-rich plasma. When AVA were added to whole blood in the presence of CV-6209, a platelet-activating factor (PAF) receptor inhibitor, platelet depletion was inhibited. This suggests that PAF is one of the mediators released from AVA-activated leukocytes that leads to P:L conjugation formation and platelet activation. In summary, AVA bind to leukocytes, resulting in release of a PAF and prothrombotic factor that exert a paracrine-activating effect on platelets. Overall, this proposed mechanism may explain the pathogenesis of thrombotic events in autoimmune diseases associated with AVA.

Topics: Apoptosis; Autoantibodies; Autoimmune Diseases; Blood Platelets; Cell Adhesion; Complement C3d; Culture Media, Conditioned; Fibrinogen; Humans; Immunoglobulin M; Immunosorbent Techniques; Leukocytes; P-Selectin; Platelet Activating Factor; Platelet Activation; Pyridinium Compounds; Recombinant Proteins; Thrombophilia; Thromboplastin; Vimentin

Patients with obstructive sleep apnea (OSA) are at increased risk of atherothrombosis independent of the Framingham risk factors. Studies on hemostasis factors in OSA are scarce and inconsistent. We sought to understand the variation in atherothrombotic propensity as a function of apoptotic circulating endothelial cells (CECs) in OSA by investigating the relationship between CEC apoptosis and plasma levels of hemostatic factors tissue factor (TF) and von Willebrand Factor (vWF) in apneic subjects. Apoptotic CECs were detected by flow cytometry in 35 male subjects free of cardiovascular diseases (AHI range 8-43) and 12 healthy male controls (AHI range 2-5) before and after 8 weeks of nasal continuous positive airway pressure (nCPAP). Quantitative determination of TF and vWF was performed using an enzyme-linked immunosorbent assay (ELISA) kit. The mean levels of TF (66.78 +/- 41.59 pg/ml) and vWF (189.70 +/- 69.24 IU/dl) were significantly higher in OSA patients compared with those in healthy subjects (42.83 +/- 14.18 pg/ml; and 124.48 +/- 31.43 IU/dl). Apoptotic CECs were elevated in patients with OSA and correlated strongly with TF and vWF levels (p = 0.02 and p < 0.001; respectively). There were no correlations between TF, vWF and apnea hypopnea index, or arousal index. Only the percentage of time spent <90% oxygen saturation was inversely associated with TF (r = 0.38; p = 0.02). Following nCPAP therapy, there was significant decrease in TF levels that correlated with decrease in apoptotic CECs. In patients with OSA, increased prothrombotic factors are strongly determined by apoptotic CECs. Treatment with nCPAP may alleviate the coagulation propensity.

Topics: Adult; Apoptosis; Atherosclerosis; Continuous Positive Airway Pressure; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Hemostasis; Humans; Male; Oxygen; Polysomnography; Reference Values; Risk Factors; Sleep Apnea, Obstructive; Thrombophilia; Thromboplastin; Thrombosis; von Willebrand Factor

Orthostatic stress causes significant plasma shift and raises transmural pressure in lower extremities, resulting in an increase in endothelial activation and plasma proteins concentrations, possibly including coagulation factors. This may lead to activation of the coagulation system during standing. To test this hypothesis, we recruited 18 healthy volunteers (9 females and 9 males; mean age: 25+/-1.2 years; body mass index: 21.7+/-0.5 kg/m(2)). Hemodynamics, plasma shift (extrapolated from sequential hematocrit concentration), plasma proteins, and coagulation tests, including procoagulants; fibrinogen, factor V, and factor VIII activity; prothrombin fragments 1 and 2; and endothelial activation-related factors (tissue factor and von Willebrand factor), as well as protein C global pathway, were determined at rest supine and at 15 minutes, 30 minutes, and 60 minutes of still standing. Thirty minutes of standing caused a decrease in plasma volume by 12.0+/-0.5% and an increase in plasma protein by 13.0+/-0.7%. Fibrinogen, factor V, and factor VIII activity rose by 12.0+/-1.2%, 13.0+/-1.0%, and 40.0+/-6.0% (P<0.002 for all), respectively. Prothrombin fragments 1 and 2 were elevated by 150.0+/-30.0%. Tissue factor and von Willebrand factor increased by 30.0+/-9.0% and 17.4+/-51.0% (P<0.02 for both), respectively. However, protein C assay results decreased from 0.95+/-0.20 to 0.83+/-0.16 (P<0.001). We hereby introduce a novel physiological mechanism, "orthostatic procoagulation," that should be considered during coagulation tests. Furthermore, it could be extrapolated to the pathophysiology of stasis and venous thromboembolism.

Topics: Adult; Blood Coagulation; Blood Pressure; Endothelium, Vascular; Factor V; Factor VIII; Female; Fibrinogen; Hematocrit; Humans; Male; Peptide Fragments; Plasma Volume; Posture; Protein C; Protein Precursors; Prothrombin; Thrombophilia; Thromboplastin; Venous Thromboembolism; von Willebrand Factor

Thromboelastography (TEG) evaluates the visco-elastic properties of whole blood to assess clot formation and hemostasis. When blood cannot be analyzed immediately, it is stored in citrated tubes to be analyzed after recalcification. In this study, we evaluated the results of TEG analysis performed on citrated blood and compared these results to values obtained from activated (kaolin and tissue factor) and non activated, fresh blood samples, obtained at various time intervals (one, two, and three hours).. Four blood samples were collected from each of ten healthy volunteers. The following TEG analyses were performed on each sample: reaction time (r), k time (k), alpha angle (alpha), and maximum amplitude (MA). Studies were done using fresh, non citrated blood, obtained within five minutes of collection, and using citrated blood, one, two, and three hours after collection. Samples were analyzed, with and without activation, using kaolin and tissue factor.. Tissue factor activated and non activated, citrated samples had shorter r and k times (P=0.03, P=0.008, P<0.0001, and P<0.0001, respectively) and higher alpha angle and MA values (P<0.0001, P<0.0001, P=0.79, and P=0.03, respectively) compared to fresh, non citrated samples. These findings were consistent with a hypercoagulable state. Conversely, citrated samples, activated with kaolin, yielded results similar to those obtained from fresh, non citrated samples. The TEG measurements were similar among citrated samples stored from one to three hours.. Our results demonstrate that TEG measures, performed on citrated blood samples, yield results that are consistent with a hyperocoagulable state. Using kaolin to activate citrated samples, on the other hand, yields results similar to those obtained from non citrated, fresh blood samples.

Topics: Adult; Blood Coagulation; Blood Specimen Collection; Citrates; Female; Humans; Kaolin; Male; Thrombelastography; Thrombophilia; Thromboplastin; Time Factors; Treatment Outcome; Whole Blood Coagulation Time

Patients with chronic kidney disease exhibit features of a hypercoagulable state and have endothelial dysfunction, which may contribute to their increased cardiovascular risk. We examined the relationship between coagulation activation and vascular function in patients with chronic kidney disease.. We measured parameters of the tissue factor pathway of blood coagulation (tissue factor, factor VIIc and factor X); natural inhibitors (tissue factor pathway inhibitor, protein C, free and total protein S, antithrombin III) and markers of coagulation activation (thrombin-antithrombin complexes, prothrombin fragment 1+2) in 66 stage 4&5 chronic kidney disease patients and 36 healthy controls. Their relationship with markers of vascular function (flow mediated dilatation, soluble E-selectin and thrombomodulin) and a mediator of inflammation (interleukin-6) was determined.. Up-regulation of the tissue factor pathway (increased tissue factor and factor VIIc), increased prothrombin fragment 1+2 and significant reductions in antithrombin III and the ratio of free protein S: total protein S were found in patients compared to healthy controls. Increased tissue factor antigen was significantly and independently correlated with creatinine and interleukin-6 (P<0.001). Factor X and antithrombin III were both reduced in chronic kidney disease and correlated (r=0.58; P<0.001). Changes in coagulation and anti-coagulation were independent of all measures of endothelial function.. Significant activation of the TF pathway of coagulation and depletion or reduction of some natural anticoagulants in chronic kidney disease was correlated with the degree of renal dysfunction, but not correlated with the abnormalities of vascular function. These data are consistent with a hypercoagulable state in chronic kidney disease that may be independent of endothelial based regulation but associated with an inflammatory state.

Topics: Aged; Antigens; Antithrombin III; Biological Phenomena; Biomarkers; Blood Coagulation; Creatinine; E-Selectin; Endothelium, Vascular; Factor VII; Factor X; Female; Humans; Interleukin-6; Kidney Failure, Chronic; Male; Middle Aged; Peptide Fragments; Protein S; Prothrombin; Renal Dialysis; Solubility; Thrombomodulin; Thrombophilia; Thromboplastin

We investigated the effects of viral infection on Tissue Factor (TF) expression and activity in mice within the myocardium to understand increased thrombosis during myocarditis. Mice were infected with coxsackie virus B3 (CVB3) and the hearts were collected at day 4, 8 and 28 post infection (p.i.). Myocardial TF expression and cellular activity as well as plasma activity were analyzed from CVB3 infected mice by Western blot, chromogenic Factor Xa generation assay, in situ staining for active TF and immunohistochemistry. In addition to TF expression, hemodynamic parameters were measured during the time course of infection. Furthermore, we analyzed myocardial tissues from patients with suspected inflammatory cardiomyopathy. TF protein expression was maximally 5-fold elevated 8 days p.i. in mice and remained increased on day 28 p.i. (P<0.001 vs. non-infected controls). Alterations in TF expression were associated with fibrin deposits within the myocardium. The TF pathway inhibitor protein expression in the myocardium was not altered during myocarditis. Active cellular TF co-localized with CD3 positive cells and VCAM-1 positive endothelial cells in the myocardium. The TF expression was positively correlated with the amount of infiltrating CD3 and Mac3 positive cells (Spearman-Rho rho=0.749 P<0.0001 for CD3(+) and rho=0.775 P<0.0001 for Mac3(+); N=35). Increased myocardial TF expression was associated with a 2-fold elevated plasma activity (P<0.05 vs. non-infected controls). In the human hearts, the TF expression correlated positively with an endothelial cell activation marker (rho=0.523 P<0.0001 for CD62E; N=54). Viral myocarditis is a hypercoagulative state which is associated with increased myocardial TF expression and activity. Upregulation of TF contributes to a systemic activation of the coagulation cascade.

Topics: Animals; Antigens, Differentiation; Blood Coagulation; CD3 Complex; Coxsackievirus Infections; Enterovirus; Fibrin; Hemodynamics; Humans; Mice; Myocarditis; Thrombophilia; Thromboplastin; Time Factors; Vascular Cell Adhesion Molecule-1

Topics: Blood Cells; Humans; Neoplasms; Particle Size; Thrombophilia; Thromboplastin

The pathogenesis of hypercoagulability in cancer is not entirely understood. We hypothesized that in cancer patients circulating tissue factor-positive microparticles (TF (+) MPs) are increased and associated with hemostatic system activation. In 20 patients with advanced colorectal cancer and in 20 age- and sex-matched controls, number and cellular origin of TF (+) MPs were determined in plasma by flow cytometry. D-dimer was determined as an indicator of hemostatic system activation. Compared to controls, the median (interquartile range) number of TF (+) MPs was two-fold higher in cancer patients: 25.9 (15.4 - 42.0) x 10 (3) /ml plasma versus 13.1 (11.9 - 19.7) x 10 (3) /ml plasma, p = 0.007. This was mainly due to a higher amount of TF (+) MPs from platelets (13.4 [5.0 - 17.4] x 10 (3) /ml plasma vs. 5.8 [4.5 - 7.5] x 10 (3) /ml plasma, p = 0.017). TF (+) MPs correlated with D-dimer ( ? = 0.48, p = 0.002). High levels of TF (+) MPs in cancer patients and their correlation with D-dimer suggest that TF (+) MPs might be involved in hemostasis activation in cancer patients.

Topics: Blood Coagulation; Cell Membrane; Colorectal Neoplasms; Female; Fibrin Fibrinogen Degradation Products; Flow Cytometry; Hemostasis; Humans; Male; Middle Aged; Particle Size; Thrombophilia; Thromboplastin

C-reactive protein (CRP) and serum amyloid A (SAA) increase in the blood of patients with inflammatory conditions and CRP-induced monocyte tissue factor (TF) may contribute to inflammation-associated thrombosis. This study demonstrates that SAA is a potent and rapid inducer of human monocyte TF. SAA induced TF mRNA in PBMC within 30 min and optimal procoagulant activity within 4 h, whereas CRP (25 mug/ml)-induced activity was minimal at this time. Unlike CRP, SAA did not synergize with LPS. Procoagulant activity was inhibited by anti-TF and was dependent on factors VII and X, and TF Ag levels were elevated on CD14(+) monocytes. Responses were optimal with lymphocytes, although these were not obligatory. Inhibitor studies indicate activation of NF-kappaB through the ERK1/2 and p38 MAPK pathways; the cyclo-oxygenase pathway was not involved. SAA-induced TF was partially inhibited by high-density lipoprotein, but not by low-density lipoprotein or by apolipoprotein A-I. SAA is a ligand for the receptor for advanced glycation end products (RAGE), and TF generation was suppressed by approximately 50% by a RAGE competitor, soluble RAGE, and by approximately 85% by anti-RAGE IgG. However, another RAGE ligand, high mobility group box-1 protein, capable of inducing monocyte chemotactic protein-1 mRNA in 2 h, did not induce TF within 24 h. Cross-linking studies confirmed SAA binding to soluble RAGE. Elevated SAA is a marker of disease activity in patients with rheumatoid arthritis, and PBMC from patients with rheumatoid arthritis were more sensitive to SAA than normals, suggesting a new link between inflammation and thrombosis.

Topics: Aged; Arthritis, Rheumatoid; C-Reactive Protein; Cells, Cultured; Female; Gene Expression Regulation; Humans; Lipoproteins; Male; Middle Aged; Monocytes; NF-kappa B; Receptor for Advanced Glycation End Products; Receptors, Immunologic; RNA, Messenger; Serum Amyloid A Protein; Signal Transduction; Thrombophilia; Thromboplastin

Stromal cell-derived factor-1 (SDF-1) is a CXC chemokine that activates and directs the migration of leukocytes that have CXCR4, which is the unique receptor for SDF-1. Although SDF-1/CXCR4 interaction has been implicated in various inflammatory conditions, its role in modulating coagulation has not been determined. We studied the plasma SDF-1 levels in 90 patients with suspected disseminated intravascular coagulation (DIC) and we found that circulating SDF-1 was significantly increased in the overt DIC patients and was also increased in overt DIC patients who have a poor outcome. We then tested in vitro whether SDF-1 can affect the expression of monocyte tissue factor (TF) and endothelial thrombomodulin (TM), and both of these play important roles in coagulopathy. SDF-1 did not affect the expression of surface TF protein and its function and the TF mRNA level in both monocytes and the monocytic leukemia cell line THP-1. SDF-1 also did not change the surface TM expression of endothelial cells. SDF-1 could enhance low-dose ADP induced platelet aggregation, although it failed by itself to induce aggregation. These findings suggest that plasma SDF-1 might be closely associated with hypercoagulability though its action as a platelet activator.

Topics: Cells, Cultured; Chemokine CXCL12; Chemokines, CXC; Disseminated Intravascular Coagulation; Endothelial Cells; Humans; Monocytes; Platelet Function Tests; Prognosis; Thrombomodulin; Thrombophilia; Thromboplastin

A study was undertaken to investigate the in vivo pathogenic role of Toll-like receptor 4 (TLR-4) in the antiphospholipid syndrome (APS) by studying the thrombogenic antiphospholipid (aPL) activity in lipopolysaccharide (LPS) non-responsive (LPS-/-) mice and the association between tlr4 gene polymorphisms and APS in patients.. IgGs from two patients with APS, one with aPL negative systemic lupus erythematosus (SLE) and one with normal human serum (NHS), were evaluated for thrombosis, tissue factor (TF) activity and endothelial cell activation in LPS-/- mice displaying a tlr4 spontaneous mutation vs LPS responsive (LPS+/+) mice. Human tlr4 Asp299Gly and Thr399Ile polymorphisms were evaluated by allele-specific PCR in 110 patients with APS with arterial/venous thrombosis and in 220 controls of the same ethnic origin.. IgG-APS produced significantly larger thrombi and more leucocytes (WBC) adhering to endothelial cells in the cremaster muscle microcirculation of LPS+/+ mice than IgG-NHS or aPL negative SLE-IgG. These effects were abrogated after absorption of the anti-beta(2)glycoprotein I activity by an affinity column. The two IgG-APS induced significantly smaller thrombi and fewer WBC adhering to endothelial cells in LPS-/- mice than in LPS+/+ mice. IgG-APS induced higher TF activity in carotid artery homogenates of LPS+/+ mice than in LPS-/- mice. The prevalence of Asp299Gly and Thr399Ile tlr4 polymorphisms was significantly lower than in controls.. These findings in LPS-/- mice and the reduction in the "protective" polymorphism in patients with APS with thrombosis suggest that TLR-4 is involved in the interaction of aPL with endothelial cells in vivo.

Topics: Adult; Animals; Antibodies, Anticardiolipin; Antibodies, Antiphospholipid; Anticoagulants; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Cell Adhesion; Endothelial Cells; Female; Humans; Immunoglobulin G; Leukocytes; Male; Mice; Mice, Inbred C3H; Polymorphism, Genetic; Thrombophilia; Thromboplastin; Thrombosis; Toll-Like Receptor 4

Subjective sleep disturbances have been associated with increased risk of coronary artery disease (CAD). We hypothesized that disrupted sleep as verified by polysomnography is associated with increased levels of prothrombotic hemostasis factors previously shown to predict CAD risk.. Full-night polysomnography was performed in 135 unmedicated men and women (mean age +/- SD, 36.8 +/- 7.8 years) without a history of sleep disorders. Morning fasting plasma levels of von Willebrand Factor (VWF) antigen, soluble tissue factor (sTF) antigen, d-dimer, and plasminogen activator inhibitor (PAI)-1 antigen were determined. Statistical analyses were adjusted for age, gender, ethnicity, body mass index, BP, and smoking history.. Higher total arousal index (ArI) was associated with higher levels of VWF (beta = 0.25, p = 0.011, DeltaR(2) = 0.045), and longer wake after sleep onset was associated with higher levels of sTF (beta = 0.23, p = 0.023, DeltaR(2) = 0.038). More nighttime spent at mean oxygen saturation < 90% (beta = 0.20, p = 0.020, DeltaR(2) = 0.029) and higher apnea-hypopnea index (AHI) [beta = 0.19, p = 0.034, DeltaR(2) = 0.024] were associated with higher PAI-1. There was a trend for a relationship between mean oxygen desaturation < 90% and PAI-1 (p = 0.053), even after controlling for AHI. Total ArI (beta = 0.28, p = 0.005, DeltaR(2) = 0.056) and WASO (beta = 0.25, p = 0.017, DeltaR(2) = 0.042) continued to predict VWF and sTF, respectively, even after controlling for AHI.. Polysomnographically verified sleep disruptions were associated with prothrombotic changes. Measures of sleep fragmentation and sleep efficiency were related to VWF and sTF, respectively. Apnea-related measures were related to PAI-1. Our findings suggest that sleep disruptions, even in a relatively healthy population, are associated with potential markers of prothrombotic cardiovascular risk.

Topics: Adult; Antigens; Arousal; Coronary Thrombosis; Female; Fibrin Fibrinogen Degradation Products; Humans; Male; Middle Aged; Oxygen; Plasminogen Activator Inhibitor 1; Polysomnography; Risk Factors; Sleep Apnea, Obstructive; Sleep Deprivation; Sleep Stages; Thrombophilia; Thromboplastin; von Willebrand Factor

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

Alterations in blood coagulation may explain the poorer neurological outcome with diabetes mellitus and hyperglycemia after acute ischemic stroke. We studied the relationships between diabetes mellitus, hyperglycemia, whole blood tissue factor procoagulant activity (TF-PCA) and plasma factorVIIa (FVIIa) in ten patients with type 2 diabetes mellitus and 11 non-diabetic patients at baseline and 6, 12, 24, and 48 hours (h) after presentation for acute stroke. In addition, we examined plasma prothrombin fragment 1+2 (F1.2) and thrombin-antithrombin complexes (TAT) as markers of thrombin generation. Stroke severity, assessed by National Institute of Health Stroke Scale (NIHSS), was similar at baseline (p=0.26) but worse in diabetic (8.20+/-4.3) than nondiabetic patients (2.67+/-2.1, p=0.023) at 48 h. At presentation, diabetic patients had higher FVIIa (p=0.004) and lower TF-PCA (p=0.027) than non-diabetic patients but both were higher than in normal control subjects. FVIIa levels remained higher in diabetic patients at 6, 12 and 24 h after stroke. In diabetic patients, FVIIa (r=0.40, p=0.02) and TF-PCA (r=0.50, p=0.02) correlated with blood glucose; and, FVIIa correlated with plasma F1.2 (r=0.34, p=0.002) and TAT levels (r=0.62, p<0.0001). In non-diabetic patients, TF-PCA, but not FVIIa, correlated with F1.2 (r=0.402, p=0.010) and TAT (r=0.39, p=0.011). Combining both groups, NIHSS scores were positively related to FVIIa levels (r=0.50, p=0.021) and inversely related to TF-PCA levels (r=-0.498, p=0.02). Acute ischemic stroke patients with diabetes and hyperglycemia have a more intense procoagulant state compared with nondiabetic patients. This is related to glucose levels and provides a potential mechanism for the observed worse prognosis in such patients after acute stroke.

Topics: Aged; Aged, 80 and over; Biomarkers; Case-Control Studies; Diabetes Complications; Diabetes Mellitus, Type 2; Factor VIIa; Female; Humans; Hyperglycemia; Male; Middle Aged; Prognosis; Severity of Illness Index; Stroke; Thrombophilia; Thromboplastin

Topics: Endothelium, Vascular; Humans; Thrombin; Thrombophilia; Thromboplastin; Thrombosis

Elevated plasma C-reactive protein (CRP) levels predict coronary events, but it is unclear whether CRP plays a role in thrombosis associated with these events. We investigated tissue factor (TF) induction by CRP on peripheral blood mononuclear cells (PBMC) from patients with coronary disease.. PBMC from 35 patients with stable angina (SA) in study 1, 10 male patients with SA, 10 with unstable angina (UA) and 10 matched controls in study 2, and 25 patients with inflammatory disorders (ID) and 24 normal controls in study 3 were stimulated with CRP, interferon-gamma (IFN) or lipopolysaccharide (LPS), or their combination. PBMC from additional normal donors were also stimulated with CRP in adherent and non-adherent conditions, and TF activity, antigen and mRNA expression detected.. CRP (5-25 microg mL(-1)) dose dependently induced more TF on PBMC from SA patients than 42 contemporary controls (P = 0.001, study 1). Compared with controls, patients with SA or UA had higher basal, and much higher CRP- or CRP/LPS-induced monocyte TF activity although serum CRP levels were similar (study 2). IFN induced monocyte TF activity in patients with angina, but not in controls. Basal or CRP-induced TF levels did not differ between controls and ID, even though ID patients had much higher serum CRP levels (study 3). CRP-induced monocyte TF activity correlated with serum CRP levels in controls (P = 0.005) and ID (P = 0.007) in study 3, but not in patients with angina (P =0.84) in study 2. CRP induced more TF activity, protein and mRNA under adherent than non-adherent conditions implying that it may mainly target macrophages in lymphocyte-rich lesions.. Our results indicate that monocytes from patients with angina are preactivated and express TF but CRP is unlikely to be a major priming factor in vivo. IFN and CRP further increase TF levels that may contribute to the hypercoagulable state in coronary disease.

Topics: Adult; Aged; Angina Pectoris; C-Reactive Protein; Case-Control Studies; Cells, Cultured; Coronary Artery Disease; Dose-Response Relationship, Drug; Female; Gene Expression Regulation; Humans; Interferon-gamma; Leukocytes, Mononuclear; Lipopolysaccharides; Male; Middle Aged; Thrombophilia; Thromboplastin

To assess the hypercoagulability in PBC and its relationship with homocysteine (HCY) and various components of the haemostatic system.. We investigated 51 PBC patients (43F/8M; mean age: 63+/-13.9 yr) and 102 healthy subjects (86 women/16 men; 63+/-13 yr), and evaluated the haemostatic process in whole blood by the Sonoclot analysis and the platelet function by PFA-100 device. We then measured HCY (fasting and after methionine loading), tissue factor (TF), thrombin-antithrombin complexes (TAT), D-dimer (D-D), thrombomodulin (TM), folic acid, vitamin B6 and B12 plasma levels. C677T 5,10-methylenetetrahydrofolate reductase (MTHFR) polymorphism was analyzed.. Sonoclot RATE values of patients were significantly (P<0.001) higher than those of controls. Sonoclot time to peak values and PFA-100 closure times were comparable in patients and controls. TAT, TF and HCY levels, both in the fasting and post-methionine loading, were significantly (P<0.001) higher in patients than in controls. Vitamin deficiencies were detected in 45/51 patients (88.2%). The prevalence of the homozygous TT677 MTHFR genotype was significantly higher in patients (31.4%) than in controls (17.5%) (P<0.05). Sonoclot RATE values correlated significantly with HCY levels and TF.. In PBC, hyper-HCY is related to hypovitaminosis and genetic predisposing factors. Increased TF and HCY levels and signs of endothelial activation are associated with hypercoagulability and may have an important role in blood clotting activation.

Topics: Adult; Aged; Antithrombin III; Female; Folic Acid; Hemostasis; Homocysteine; Humans; Hyperhomocysteinemia; Liver Cirrhosis, Biliary; Male; Middle Aged; Peptide Hydrolases; Platelet Function Tests; Thrombomodulin; Thrombophilia; Thromboplastin; Vitamin B 12; Vitamin B 6

Recently, our laboratory devised a dynamic whole blood (WB) thrombelastographic coagulation model employing activation with minute amounts of tissue factor. A series of studies were conducted to validate the feasibility of the model to illustrate hypocoagulation in various bleeding disorders and its usefulness in detecting the haemostatic effect of pro-coagulants by ex vivo titration experiments. In this context, the present study hypothesized that the thrombelastographic model also can reveal hypercoagulation. Hence, the objective of the present study was to record dynamic WB coagulation profiles in a series of patients (n=76) who had previously suffered from a venous (n=34) or arterial (n=42) thrombo-embolic event and to compare the results with those of a group of healthy reference subjects (n=60).. Patients receiving vitamin K antagonist treatment were not enrolled in the study. Forty-four of the patients had no known thrombophilia risk factor and 32 patients had at least one thrombophilia risk factor.. The most commonly found risk factors were mild hyperhomocysteinaemia and heterozygosity for the factor V Leiden polymorphism. The data showed that, as compared with the healthy controls, patients with a history of venous or arterial thromboembolism had a significantly greater hypercoagulant WB coagulation clot signature as defined by a shortened clotting time together with an accelerated maximum velocity of clot propagation.. In future studies with ex vivo dose titration assessment of pro-coagulant components mixed with blood from a patient suffering from compromised haemostasis, observation of a significantly shortened clot initiation concomitant with a distinctly accelerated clot propagation is likely to indicate an increased risk of thrombosis.

Topics: Adolescent; Adult; Aged; Factor V; Feasibility Studies; Female; Humans; Hyperhomocysteinemia; Male; Middle Aged; Risk Factors; Thrombelastography; Thromboembolism; Thrombophilia; Thromboplastin

Procoagulant microparticles (MP) constitute valuable hallmarks of vascular cell damage at the crossroad of atherothrombosis processes. Detectable at low concentrations in the blood flow of healthy individuals, elevated levels of procoagulant microparticles are characteristic features of most cardiovascular risk factors. Circulating MP support cellular cross-talk leading to vascular inflammation, endothelial dysfunction, leukocyte adhesion and recruitment possibly contributing to plaque growth with consecutive development of local thrombosis and altered vasomotion. Within the plaque, MP shed by apoptotic monocytes and smooth muscle cells are major determinant of plaque thrombogenicity mainly through the presence of tissue factor (TF) activity. Besides this procoagulant potential, trapped MP could contribute to plaque vulnerability through multiple pathways including angiogenesis, extracellular matrix proteolysis, recruitment of inflammatory cells, smooth muscle cell and endothelial apoptosis. Having long been considered sufficient to initiate coagulation following plaque disruption, the role assigned to plaque-bound TF does not appear physically realistic at a macroscopic scale, the swift growth of the thrombus probably involving blood-borne TF conveyed by circulating MP. As participants in crucial steps of atherosclerotic disease, MP can now be viewed as "partners in crime" in acute ischemic syndromes.

Topics: Arteriosclerosis; Cell Communication; Cell Membrane; Humans; Particle Size; Thrombophilia; Thromboplastin; Thrombosis

There is currently no validated method to detect a prothrombotic phenotype. The question remains, can tissue factor (TF) induced thrombin generation (TG), as measured with the calibrated automated thrombinography (CAT) technique, according to Hemker et al., recognise a prothrombotic state either as such, or when the activated protein C (APC)-system is boosted with thrombomodulin (TM)? We determined the normal range of CAT-TG +/- TM in a group of 71 healthy blood donors, in 11 healthy women using oral contraceptives (OC), and in 89 patients with a history of venous thromboembolism (VTE), divided into a group of 50 in which a prothrombotic risk factor could be found (VTEprf+) and 39 others (VTEprf-). The endogenous thrombin potential (ETP) in the OC, VTEprf+ and VTEprf- group was significantly higher than for the controls. In the presence of TM, the differences were significantly higher than in its absence. The VTEprf+ group had a higher ETP, +/- TM than the VTEprf-group. In conclusion, TG, measured with the CAT technique in the presence of TM is capable of detecting the prothrombotic phenotype with a high sensitivity of 0.93 (95% confidence limits 0.82-0.99).

Topics: Adult; Aged; Automation; Blood Coagulation Tests; Calibration; Case-Control Studies; Female; Humans; Male; Middle Aged; Sensitivity and Specificity; Thrombin; Thrombomodulin; Thrombophilia; Thromboplastin; Venous Thrombosis

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

Hepatic veno-occlusive disease (VOD) is one of the most disastrous complications after allogeneic hematopoetic stem cell transplantation (HSCT). Thrombocytopenia with refractoriness to platelet transfusions suggests an increased platelet consumption in these patients. Interactions between platelets and endothelial cells might contribute to the hypercoagulable state at the sinusoidal endothelium as a central mechanism in the pathogenesis of VOD.. The influence of activated platelets on cultured human endothelial cells was investigated in vitro. We focused on the release of plasminogen activator inhibitor-1 (PAI-1) from endothelial cells which has earlier been found to be significantly elevated in plasma of VOD patients. Endothelial cells isolated from human umbilical cords (HUVEC) were incubated with activated platelets. The release of PAI-1 in the presence or absence of specific antibodies was determined by ELISA technique. Tissue factor (TF) expression on endothelial cells was observed by flowcytometric analysis.. HUVEC incubated with activated platelets were found to release significantly more PAI-1 compared to untreated cultures. The endothelial PAI-1-secretion after incubation of HUVEC with activated platelets was completely inhibited by an IgG monoclonal antibody against human transforming growth factor beta-1 (TGF beta-1). In contrast, PAI-1 production was not suppressed after inhibition of HUVEC-platelet-interaction by an IgG monoclonal antibody against CD154 (CD40L) expressed on the surface of activated platelets. An increased release of PAI-1 and an increased expression of tissue factor (TF) on the endothelial cell surface were observed after stimulation with TGF beta-1.. TGF beta-1 released from activated platelets contributes to the hemostatic imbalance at the sinusoidal endothelium in patients with hepatic VOD by increase of endothelial cell PAI-1 production and TF expression. As a potent profibrotic cytokine, TGF beta-1 might further be involved in phlebosclerosis and sinusoidal fibrosis occurring in VOD.

Topics: Blood Platelets; Cell Communication; Cells, Cultured; Coculture Techniques; Endothelium, Vascular; Hematopoietic Stem Cell Transplantation; Hepatic Veno-Occlusive Disease; Humans; Models, Biological; Plasminogen Activator Inhibitor 1; Platelet Activation; Thrombophilia; Thromboplastin; Transforming Growth Factor beta; Transforming Growth Factor beta1; Umbilical Cord

Mice with a targeted truncation in the gene encoding tissue factor of blood coagulation (TF) to eliminate the cytosolic domain and carrying a neo(R) cassette in intron 5 unexpectedly displayed severe spontaneous thrombosis in various vascular beds. Thrombosis was observed in heterozygous TF(+/neo) mice, causing death of over 50% of adults within 36 weeks of birth, and fulminantly exacerbating in pregnant females. Homozygous TF(neo/neo) mice were more severely affected and died within 7 weeks after birth. These TF(neo) mice primarily synthesized a mutant mRNA aberrantly spliced from exon 5 to neo(R), encoding an apparently non-vesicle-binding soluble TF lacking both the transmembrane and cytosolic domain, but still capable of blood coagulation induction. This severe thrombotic phenotype associated with the presence of a non-anchored soluble TF variant underscores the recently recognized significance of circulating TF for thrombus formation and development.

Topics: Animals; Cell Membrane; Cytosol; Gene Silencing; Genetic Predisposition to Disease; Mice; Mice, Transgenic; Mutagenesis, Site-Directed; Phenotype; Protein Structure, Tertiary; Survival Rate; Thrombophilia; Thromboplastin

Topics: Adolescent; Adult; Aged; Female; Humans; Male; Middle Aged; Monocytes; Polymerase Chain Reaction; Reference Values; RNA, Messenger; Thrombophilia; Thromboplastin

Topics: Arteriosclerosis; Coronary Thrombosis; Death, Sudden, Cardiac; Disease Progression; Endothelium, Vascular; Female; Glutathione; Humans; Hypochlorous Acid; Inflammation Mediators; Macrophages; Male; Models, Biological; Peroxidase; Rupture, Spontaneous; Thrombophilia; Thromboplastin

Measurement of thrombin generation by calibrated automated thrombography (CAT) could fulfill the requirements of a global test of coagulability and is potentially applicable to routine clinical laboratory practice. The purpose of this study was to determine if corn trypsin inhibitor (CTI) could be used to abolish contact factor activation in this assay, thus allowing accurate measurement of low tissue factor (TF) concentration-triggered thrombin generation on samples taken in a routine clinical setting.. The endogenous thrombin potential (ETP) was measured by CAT.. The study demonstrated that addition of CTI after plasma separation is not sufficient and blood must be drawn into tubes containing CTI if in-vitro contact factor-activated thrombin generation is to be abolished. Contact factor-activated thrombin generation is completely inhibited at a CTI concentration of 18.3 microg mL(-1) whole blood. Increasing the CTI concentration above this level does not lead to suppression of the TF-triggered ETP. At a TF concentration of 2 pmol, ETPs were significantly lower in the presence of CTI (P < 0.001). The difference (no CTI minus CTI) between results ranged from - 1 to 2159 nM min(-1) (median - 754). Whilst the low concentration TF-ETP assay was not optimized to distinguish degrees of coagulability between patient samples, there was a significant difference in ETP between normal and hemophilia samples and samples from patients with a clinical prothrombotic tendency.. CTI can be applied to ETP measurement by CAT. This permits the use of CAT in a low TF-triggered thrombin generation assay without concern for the effect of interference from in-vitro contact factor activation and the optimum reagent conditions for using CAT as a global test of coagulability in clinical practice can now be defined.

Topics: Adult; Aged; Aged, 80 and over; Automation; Blood Coagulation Tests; Calibration; Clinical Laboratory Techniques; Hemophilia A; Humans; Middle Aged; Plant Proteins; Reproducibility of Results; Thrombin; Thrombophilia; Thromboplastin

We have developed a model of a pre-thrombotic state in rats based on venous stasis induced by partial ligature of the inferior vena cava. The degree of stenosis was calibrated by using variations in upstream venous pressure. Different degrees of stasis were tested in order to obtain a pre-thrombotic state. Increasing doses of thromboplastin were infused. The thrombogenic potential of this model was evaluated by measuring thrombus weight and by the increase in levels of thrombin-antithrombin complexes. A pre-thrombotic state was induced by 2 h of exposure to a 40% stasis obtained by increasing by 40% the upstream venous pressure (mean thrombus weight, 0.2 +/- 0.6 mg). In these conditions of stasis, low doses of thromboplastin induced venous thrombosis (mean weight, 23 +/- 20 mg; P < 0.05). The increase in thrombus size was correlated to the rise in thrombin-antithrombin levels (r = 0.53, P < 0.001). In conclusion, we have developed the first animal model in which venous stasis can be calibrated by varying the degree of stenosis of the inferior vena cava. This model could be used to study the kinetics of biological markers of hypercoagulability, to study the pathogeny of thrombosis or to evaluate the therapeutic efficacy of new drugs in pre-clinical trials.

Topics: Animals; Blood Pressure; Calibration; Constriction, Pathologic; Disease Models, Animal; Dose-Response Relationship, Drug; Escherichia coli Proteins; Hemostasis; Hemostatics; Membrane Transport Proteins; Rats; Thrombophilia; Thromboplastin; Thrombosis; Veins; Vena Cava, Inferior

Procoagulant activity (PCA) of monocytes is known to play a pivotal role in a variety of physiologic and pathophysiologic processes, such as disseminated intravascular coagulation, atherosclerosis, arterial and venous thromboembolism, cancer-related hypercoagulability and immunopathologies. Until now, PCA has been studied by clotting assays of a whole cell population or at single cell level by analyzing tissue factor antigen, the protein that initiates PCA but does not always correlate with it. Here, we describe a new simple flow cytometric method that allows the PCA of monocytes to be studied at a single cell level by quantifying the fibrin formed around the cells in suspension.. Purified fibrinogen was tagged with FITC and added to a recalcified developer plasma containing suitable amounts of heparin in order to inhibit the expansion of clotting, thus limiting the formation of fibrin to the surface of cells with PCA. With appropiate amounts of heparin, in 10 min, large sea urchin-like cells with fibrin needles around some monocytes were formed and, after fixation, cytofluorimetrically analyzed.. Blood mononuclear cells isolated and immediately analyzed showed less than 0.1% sea urchin cells. Adherence alone, lipopolysaccharides or ionomycin stimulated expression of PCA in a dose- and time-dependent relationship: after 30 min, 1-3% of the MNC showed PCA, and after 20 h this reached 5-10%. Density separation of monocytes showed that different stimulators act on different maturation stages. Subjects with diabetes express more monocytes with PCA than normal subjects after 30 min stimulation.. This method allows PCA analysis of monocytes at single cell level and requires only a low number of cells. The signal produced by the fluorescent fibrin is strong and easily analyzed by flow cytometry. The method is suitable for analyzing blood from patients with different pathologies and many conditions under different stimuli.

Topics: Blood Coagulation; Fibrin; Flow Cytometry; Humans; Monocytes; Thrombophilia; Thromboplastin

Most animal models of venous thrombosis involve acute thrombosis with hypercoagulability in small rodents. To better replicate human disease, we developed two models in the pig, a species similar to humans in size and in vascular and coagulation reactivity. One model involves de-endothelialisation with 50% or 80% stenosis and the other replacement of a venous segment by a Gore-Tex vascular prosthesis. Both models were tested with and without acute induced hypercoagulability (thromboplastin infusion). Thrombi obtained without thromboplastin infusion were composed of a multilayered platelet and a fibrin meshwork structure similar to that usually found in humans. With thromboplastin infusion, the thrombi were homogeneous fibrin structures imprisoning red blood cells. The high incidence of thrombosis obtained with the 80% stenosis model would be useful for studying anticoagulant treatments, whereas the low incidence with 50% stenosis would be useful for evaluating procoagulant effects of conditions or treatments. These new models shed further light on the development of venous thrombi under conditions similar to those seen in humans and may prove useful for investigating anticoagulant and procoagulant effects.

Topics: Animals; Blood Vessel Prosthesis; Chronic Disease; Constriction, Pathologic; Disease Progression; Endothelium, Vascular; Fibrin; Hemorheology; Jugular Veins; Models, Animal; Swine; Thrombophilia; Thromboplastin; Venous Thrombosis

In a preliminary study, we have demonstrated that tissue factor (TF) is immunohistochemically stained in monocytes from patients with disseminated intravascular coagulation (DIC) but not from healthy volunteers, and that leukocytes from DIC patients induce enhanced activated factor X (FXa) generation in the presence of a mixture of FVIIa, FX and Ca(2+). Then, TF mRNA levels in leukocytes were measured to evaluate the role of TF in the pathophysiology of various diseases. TF mRNA levels in leukocytes were low in healthy volunteers but they were significantly increased in various diseases, especially in patients with infectious diseases, solid cancer, and hematopoietic tumors. TF mRNA levels in leukocytes were significantly higher in patients with high levels of C reactive protein (CRP) than in those with low CRP. TF mRNA levels were significantly higher in patients with DIC than in those without DIC. TF mRNA levels were well correlated with TF antigens in plasma and leukocytes. These findings suggest that the expression of TF mRNA in leukocytes is increased in various diseases and that this may play an important role in hypercoagulability or DIC.

Topics: Adult; Aged; C-Reactive Protein; Calcium; Disseminated Intravascular Coagulation; Factor VIIa; Factor X; Factor Xa; Female; Humans; Immunohistochemistry; Leukocytes; Male; Middle Aged; RNA, Messenger; Thrombophilia; Thromboplastin; Time Factors

During ovarian gonadotrophin stimulation for ovulation induction or in vitro fertilization, a clinical severe ovarian hyperstimulation syndrome (OHSS) may occur. Only few studies have investigated the mechanism responsible for the alterations of the haemostatic system in women affected by severe OHSS. The aim of the present study was to investigate the correlation between the magnitude of ovarian stimulation and the increase in fibrin formation and degradation in severe OHSS. Twenty-five patients (age range 23-43 years) who were hospitalized for severe OHSS, 25 women undergoing in vitro fertilization who did not develop OHSS (case-control group) and 25 healthy age-matched women (healthy control group) were investigated. On the day of admission a number of haemostatic markers, including D-dimer, thrombin-antithrombin complexes (TAT), prothrombin fragment 1 + 2 (F1 + 2), plasmin-antiplasmin complexes (PAP), tissue factor (TF), tissue factor pathway inhibitor (TFPI) and von Willebrand factor antigen (vWF), were examined. In patients with severe OHSS, TF, D-dimer, TAT, F1 + 2, PAP and vWF antigen plasma levels were significantly higher than those observed both in the case-control group and in healthy controls, whereas TFPI levels were significantly lower (P < 0.005) with respect to both case-controls and healthy controls. D-Dimer levels were related with serum oestradiol levels and oocyte number recovered (r = 0.45, P < 0.001 and r = 0.47, P < 0.001, respectively). D-Dimer and TAT levels were significantly (P < 0.05 and P < 0.005, respectively) higher in OHSS patients with unsuccessful pregnancy outcome (D-dimer, 226.5, 56-1449 ng/ml; TAT, 19.8, 3.1-82.6 microg/l) with respect to those with successful outcome of pregnancy (D-dimer, 145, 29-330 ng/ml; TAT, 5.0, 1.0-19.6 microg/l). Our data indicate that a marked hypercoagulability with alterations of TF and TFPI levels is detectable in patients with severe OHSS and that it is related to the clinical outcome.

Topics: Adult; Biomarkers; Case-Control Studies; Estradiol; Female; Fibrin; Humans; Lipoproteins; Ovarian Hyperstimulation Syndrome; Predictive Value of Tests; Pregnancy; Pregnancy Outcome; Thrombophilia; Thromboplastin

Immunological status, levels of pro-inflammatory cytokines of non-specific resistance and tumor expression factor have been studied in patients with cervical dysplasia or cancer versus stage. Slight and moderate dysplasia involved virtually no changes in interleukin-8 (Il-8) and tumor necrotic factor TNF-alpha concentrations whereas those of Il-1 alpha and Il-1 beta were 5 times as high. Monocyte-dependent expression of tissue factor was similar to that in healthy women. In cases of advanced dysplasia and cervical carcinoma, monocyte-dependent expression of tissue factor and production of Il-1 alpha, Il-1 beta, Il-8 and TNF-alpha were significantly enhanced. Patients with cervical carcinoma stage II and III revealed signs of depression of the cellular component of immunity as well as non-specific resistance. Hence, increased concentrations of cytokines induce monocyte-dependent expression of tissue factor in advanced dysplasia and cervical carcinoma by triggering-on of hypercoaggulation.

Topics: Adult; Cytokines; Female; Gene Expression Regulation, Neoplastic; Humans; Interleukin-1; Interleukin-8; Middle Aged; Neoplasm Staging; Thrombophilia; Thromboplastin; Tumor Necrosis Factor-alpha; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

The endogenous thrombin potential (ETP) represents the balance between pro- and anti-coagulant forces operating in plasma and can be used to investigate hyper- and hypo-coagulability. As a preliminary step to larger clinical studies we investigated the effect on ETP of phospholipids, tissue factor (TF) and residual platelets in frozen plasma.. We investigated platelet-poor and platelet-rich plasmas from healthy subjects, patients on oral anticoagulants (OA), or with hemophilia and women on oral contraceptives (OC), chosen as examples of the normal, hypo- and hyper-coagulable states in which ETP has been reported to be impaired.. Phospholipids had only a slight effect on ETP in all conditions except in women on OC, in whom the best diagnostic efficacy was observed at 0.5 microM. TF had only a slight effect in all conditions except hemophilia, in which an ETP impairment was observed only at low (1 pM) concentration. Residual platelets had considerable effects on ETP in frozen plasmas, but this was abrogated by filtration before freezing. ETP in platelet-rich plasma at 150x103/mm3 was similar to that obtained in filtered-plasma with 1.5 microM phospholipids in healthy subjects, patients on OA and patients with severe hemophilia, but not in those with mild- or moderate-hemophilia, where the ETP was higher in platelet-rich plasma.. The results suggest that the method can be used for investigations on the clinical value of ETP. Platelet-rich and platelet-poor plasma are suitable testing materials. The latter should be filtered before freezing to minimize the effect of residual platelets.

Topics: Anticoagulants; Blood Coagulation Disorders; Blood Coagulation Tests; Blood Platelets; Contraceptives, Oral; Dose-Response Relationship, Drug; Female; Freezing; Hemophilia A; Humans; Phospholipids; Reproducibility of Results; Thrombin; Thrombophilia; Thromboplastin

The surface of synthetic vascular grafts is thrombogenic, which implies a risk for their occlusion. The aim of the study was to evaluate expression of coagulation components in the polyester vascular grafts neointima. The study was carried out on 18 dogs, which underwent replacement of the abdominal aorta with a polyester prosthesis. Grafts were removed after 1, 4 and 12 months. Immunohistochemical labeling for von Willebrand factor, tissue factor, factor XII, tissue factor pathway inhibitor, thrombomodulin, protein C, protein S and prothrombin activation fragment F1 + 2 was performed. Increasing intensity of von Willebrand factor expression was found in successive periods of the study. Factor XII was shown in the whole neointima after 1 month, whereas in the following periods its presence was limited to the luminal surface. Tissue factor expression was demonstrated after 1 month and its intensity increased in later periods. Tissue factor pathway inhibitor and thrombomodulin expression was demonstrated after 4 and 12 months. Protein C and protein S were present in all observation periods, as well as prothrombin activation fragment F1 + 2. Results indicate a high thrombotic potential of the graft neointima early after prosthesis implantation, whereas in the late postoperative follow-up increasing expression of coagulation inhibitors reduces thrombotic properties of the graft neointima.

Topics: Animals; Aorta, Abdominal; Biomarkers; Blood Coagulation Factors; Blood Proteins; Blood Vessel Prosthesis; Dogs; Female; Hyperplasia; Lipoproteins; Male; Peptide Fragments; Polyesters; Postoperative Period; Prothrombin; Thrombomodulin; Thrombophilia; Thromboplastin; Tunica Intima; Wound Healing

Localized large vessel thrombosis in acute leukaemia is rare, haemorrhagic complications being more common.. We present a patient with acute promyelocytic leukaemia (APL) presenting with an acutely ischaemic lower limb. Large vessel thrombosis is a rare presentation of APL. We reviewed the literature on the coagulopathy of APL and discuss the pathology and current treatment options.. Disordered haemostasis is typical of acute promyelocytic leukaemia (FAB M3) and relates to the intrinsic properties of the blast cells as well as thrombocytopenia from bone marrow involvement. Expression of procoagulants, stimulation of cytokines and alterations in endothelial cell anticoagulant properties initiate a disseminated intravascular coagulation (DIC) resulting in the typical clinical and laboratory findings in APL. The promyelocytes are characterized by the balanced reciprocal translocation between chromosomes 15 and 17. All-trans-retinoic acid (ATRA) induces differentiation in these cells, revolutionizing the treatment of APL.. Unexpected limb ischaemia in a young, apparently healthy patient might be the presenting symptom of an underlying haematological disorder such as APL. A thorough haematological investigation should be performed prior to contemplating surgery. New treatment strategies based on knowledge of the molecular biology of APL has improved the prognosis of patients suffering from APL.

Topics: Amputation, Surgical; Antineoplastic Combined Chemotherapy Protocols; Arterial Occlusive Diseases; Cysteine Endopeptidases; Cytarabine; Daunorubicin; Female; Gangrene; Humans; Intermittent Claudication; Ischemia; Leg; Leukemia, Promyelocytic, Acute; Middle Aged; Neoplasm Proteins; Neoplastic Stem Cells; Popliteal Artery; Remission Induction; Smoking; Thioguanine; Thrombophilia; Thromboplastin; Thrombosis; Toes; Tretinoin

Increasing evidence points toward a prothrombotic state in hypertension and atherosclerosis, conditions associated with thrombosis-related complications, such as myocardial infarction and stroke. We hypothesized that this increased risk of thrombogenesis may be related to endothelial damage/dysfunction and abnormal angiogenesis, and thus, an increased risk of future cardiovascular disease. Thrombogenesis, endothelial damage/dysfunction, and angiogenesis can be assessed by measurement of tissue factor (TF), von Willebrand Factor (vWF), flow-mediated dilatation (FMD), and vascular endothelial growth factor (VEGF), respectively. To test this hypothesis, we measured TF, vWF, FMD, and VEGF in 76 patients with systemic hypertension (71 men; mean age 64; mean blood pressure 167/72 mm Hg), considered additional risk factors such as diabetes, and related them to the patient's 10-year cardiovascular and cerebrovascular risk score using the Framingham equation. Patients were compared with 48 healthy normotensive controls. In these patients, the effects of 6 months of intensified blood pressure and (where appropriate) lipid-lowering treatment were investigated. In our patients, TF, VEGF, and vWF levels were higher, but FMD was lower (all p <0.001) compared with the controls. All markers correlated with each other and with both cardiovascular and cerebrovascular risk scores (all p <0.001). After intensified blood pressure and hypercholesterolemia treatment, total cholesterol, blood pressure, TF, VEGF, and vWF levels all decreased, whereas FMD increased (all p <0.001). Thus, in subjects with hypertension and other risk factors, endothelial damage/dysfunction (and thus, atherogenesis), thrombogenesis, and angiogenesis are abnormal, correlate with overall cardiovascular risk, and importantly, can be related to each other in a "Birmingham Vascular Triangle." Furthermore, these processes are beneficially affected by intensive blood pressure and lipid treatment.

Topics: Arteriosclerosis; Endothelial Growth Factors; Endothelium, Vascular; Female; Humans; Hypercholesterolemia; Hypertension; Intercellular Signaling Peptides and Proteins; Lymphokines; Male; Middle Aged; Neovascularization, Pathologic; Risk Factors; Thrombophilia; Thromboplastin; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Vasodilation; von Willebrand Factor

Concentrations of non-cell-bound (NCB; soluble) tissue factor (TF) are elevated in blood collecting in the pericardial cavity of patients during cardiopulmonary bypass (CPB). Previously, we reported microparticles supporting thrombin generation in such blood samples. In this study we investigated the extent of microparticle association of the NCB form of TF in pericardial and systemic blood, and whether this microparticle-associated form is active in thrombin generation compared with non-microparticle-bound, (fluid-phase) TF.. Systemic and pericardial blood samples were collected before and during CPB from six patients undergoing cardiac surgery. Microparticles were isolated by differential centrifugation and their thrombin-generating capacity measured in a chromogenic assay. Microparticle-associated and fluid-phase forms of NCB TF were measured by ELISA. Microparticle-associated TF was visualized by flow cytometry.. In pericardial samples, 45-77% of NCB TF was microparticle-associated, and triggered factor VII (FVII)-mediated thrombin generation in vitro. Microparticles from systemic samples triggered thrombin generation independently of FVII, except at the end of bypass (P = 0.003). The fluid-phase form of TF did not initiate thrombin generation. Both forms of NCB TF were, at least in part, antigenically cryptic.. We demonstrate the occurrence of two forms of NCB TF. One form, which is microparticle-associated, supports thrombin generation via FVII. The other form, which is fluid-phase, does not stimulate thrombin formation. We hypothesize that the microparticle-associated form of NCB TF may be actively involved in postoperative thromboembolic processes when pericardial blood is returned into the patients.

Topics: Blood Circulation; Coronary Artery Bypass; Coronary Circulation; Factor VII; Humans; Octoxynol; Particle Size; Postoperative Complications; Solubility; Thrombin; Thromboembolism; Thrombophilia; Thromboplastin

On a previous model using Wessler's principle in the rat, we have demonstrated that a partial ligature of the inferior vena cava leading to a 40% increase in up-stream venous pressure was thrombogenic only in association with the infusion of low dose of thromboplastin (90 microg/kg). In these thrombogenic conditions, the infusion of pentasaccharide (Arixtra, fondaparinux) should lead to a strong inhibition of thrombus formation. Therefore, we performed on five groups of 10 rats: stasis alone (group S) with a 40% increase in up-stream venous pressure; stasis and thromboplastin (group ST90); and stasis, thromboplastin and pentasaccharide (groups SPT50, SPT100 and SPT250) at three different dosages (50, 100 and 250 microg/kg). The efficacy of pentasaccharide was measured according to the variations in up-stream venous pressure, thrombus weight and thrombin-antithrombin complexes levels. Only 250 microl/kg pentasaccharide significantly reduced the thrombus weight in comparison with group ST90 (5 mg versus 23.8 mg, P = 0.01) but it was not sufficient to induce a return to the basic state (5 mg versus 0.2 mg in group S, P = 0.049). Thrombin-antithrombin complex levels measured at the end of the experiment were significantly reduced in comparison with group ST90 (16.7 versus 57.8 mg, P = 0.01) and were not statistically different from group S (16.1 versus 16.6 mg, P = 0.65). In conclusion, in a very borderline model toward thrombogenesis, pentasaccharide was able to reduce thrombus weight and abolished biological hypercoagulability.

Topics: Animals; Antithrombin III; Blood Pressure; Disease Models, Animal; Dose-Response Relationship, Drug; Fondaparinux; Hemostasis; Male; Peptide Hydrolases; Polysaccharides; Rats; Rats, Sprague-Dawley; Thrombophilia; Thromboplastin; Thrombosis; Treatment Outcome

Antiphospholipid antibodies (aPLA) are associated with thrombophilia and recurrent pregnancy loss. They bind directly to anionic phospholipids or via phospholipid-binding proteins such as beta(2)-glycoprotein 1 (beta(2)GP1). The underlying mechanisms by which aPLA induce a thrombophilic phenotype are not well understood. The present work was done to determine whether antibodies to beta(2)GP1 activate endothelial cells (EC) and whether NFkappaB is involved in this activation. Incubation of EC with these antibodies resulted in a redistribution of NFkappaB from the cytoplasm to the nucleus after a delay of several hours. This was accompanied by an increased expression of tissue factor and of the leukocyte adhesion molecules ICAM-1, VCAM-1 and E-selectin. Inhibition of the nuclear translocation of NFkappaB abolished the response to these antibodies. In comparison to anti-beta(2)GP1 antibodies, incubation of EC with TNF resulted in a more rapid (within 30 minutes) redistribution of NFkappaB and a more pronounced expression of tissue factor and of the leukocyte adhesion molecules. The slower response to the antibodies as compared to TNF suggests that the NFkappaB response to anti-beta(2)GP1 antibodies is indirect. Taken together our results imply that NFkappaB is an essential intermediate in the activation of EC by anti-beta(2)GP1 antibodies.

Topics: Active Transport, Cell Nucleus; Antibodies; beta 2-Glycoprotein I; E-Selectin; Endothelium, Vascular; Glycoproteins; Humans; Intercellular Adhesion Molecule-1; NF-kappa B; Thrombophilia; Thromboplastin; Umbilical Veins; Up-Regulation

The GAIT (Genetic Analysis of Idiopathic Thrombophilia) Project is a family-based study dedicated to elucidating the genetic basis of hemostasis-related phenotypes and thrombosis risk. In this paper, we have examined several lesser-studied hemostasis-related phenotypes in the 21 GAIT families: levels of vitamin B 12, serum folate, whole blood folate, alpha2-antiplasmin, prekallikrein, beta2-glycoprotein I, soluble P-selectin, factor XIII A and B subunits and a new coagulation measurement based on thromboplastin time in the presence or absence of thrombomodulin. Using the variance component method, we estimated the relative contributions of genetic and environmental influences on these phenotypes. In addition, we calculated the genetic correlations between thrombosis risk and each of these phenotypes. All 12 phenotypes showed significant genetic contributions with genes accounting for 22% to 78% of the variance after correction for covariate effects. Four phenotypes (three traits involving thromboplastin-thrombomodulin mediated coagulation time and serum folate) exhibited significant genetic correlations with thrombosis. Thus, some of the genes that influence quantitative variation in these physiological phenotypes also influence the risk of thrombosis. The high heritabilities and significant genetic correlations between thrombosis and some risk factors suggest that joint consideration of correlated quantitative phenotypes will aid in identifying susceptibility genes.

Topics: Adult; alpha-2-Antiplasmin; beta 2-Glycoprotein I; Blood Coagulation Tests; Comorbidity; Contraceptives, Oral, Hormonal; Factor XIII; Female; Folic Acid; Genetic Predisposition to Disease; Glycoproteins; Humans; Male; Middle Aged; P-Selectin; Phenotype; Risk Factors; Smoking; Spain; Thrombomodulin; Thrombophilia; Thromboplastin; Vitamin B 12

Cytokines increase endothelial tissue factor (TF) and tissue plasminogen activator inhibitor type-1 (PAI-1) expression in vitro. Tissue factor interacts with factor VII to facilitate thrombosis and PAI-1 inhibits fibrinolysis by endogenous plasminogen activators. Because cytokine release is increased in children with sepsis-induced multiple organ failure (MOF), we hypothesized a cytokine associated increase in circulating TF and PAI-1 antigen release, and systemic activity in these patients.. One hundred and seven consecutive children, who met the criteria for sepsis, and 10 critically ill children without sepsis, were enrolled in the study. Plasma TF and PAI-1 antigen and activity levels, Interleukin-6 antigen levels (IL-6), nitrite + nitrate levels (marker of nitric oxide production) and number of organs failing were measured on days 1-3 of sepsis.. Increased TF and PAI-1 antigen, and PAI-1 activity levels were associated with increasing IL-6 and nitrite + nitrate levels (p <0.05), the development of MOF (p <0.05), and mortality (p <0.05). Increased systemic PAI-1 activity was associated with cardiovascular, renal. and hepatic failure (p <0.05). Increased systemic TF activity was associated with the development of coagulopathy (p <0.05) and tended to be associated with mortality (p = 0.06, power .77). A shift to an anti-fibrinolytic endothelium phenotype characterizes children who develop sepsis-induced MOF and mortality. Children with coagulopathy have a shift to a pro-coagulant phenotype. These findings support potential therapeutic roles for PAI-1 and TF pathway inhibitors in reversal of this devastating pathophysiologic process.

Topics: Adolescent; Child; Child, Preschool; Cytokines; Endothelium, Vascular; Female; Fibrinolysis; Humans; Infant; Infant, Newborn; Interleukin-6; Male; Multiple Organ Failure; Nitrates; Nitrites; Pennsylvania; Plasminogen Activator Inhibitor 1; Prospective Studies; Sepsis; Thrombophilia; Thromboplastin

The enhanced extrinsic coagulation in response to inflammation could contribute to disseminated intravascular coagulation, often manifesting cardiovascular complications. The complex mechanism remains unclear. Nor is the effective anticoagulation well established. The search for arresting hypercoagulation is of antithrombotic relevance. The ability of polybrene (PB) to inhibit tissue factor (TF)-initiated extrinsic blood coagulation was demonstrated at the protein and cellular levels as well as in human plasma samples. In a single-stage clotting assay, PB dose-dependently offset bacterial endotoxin (lipopolysaccharide)-induced monocytic TF (mTF) hypercoagulation and inhibited rabbit brain thromboplastin (rbTF) procoagulation. Consistent with these findings, the significantly prolonged prothrombin time indicated the depressed extrinsic coagulation by PB. However, PB showed no effect on thrombin time. We dissected the extrinsic pathway to further determine the inhibitory site(s) of PB. A two-stage chromogenic assay monitoring S-2288 hydrolysis showed that PB readily blocked mTF-dependent or rbTF-dependent FVII activation, which was verified by the diminished activated factor VII (FVIIa) formation derived from the proteolytic cleavage of its zymogen factor VII on Western blotting analyses. PB had no effect on FVIIa and activated factor X amidolytic activity. Nor was the dissected TF/FVIIa-catalyzed factor X activation affected. In conclusion, the preferential downregulation of factor VII activation was responsible for the depressed extrinsic coagulation. PB could present a novel anticoagulant antagonizing the extrinsic hypercoagulation for the prevention of thrombotic complication following sepsis and inflammations.

Topics: Anticoagulants; Blood Coagulation; Dose-Response Relationship, Drug; Drug Antagonism; Endotoxins; Factor VIII; Hexadimethrine Bromide; Humans; Lipopolysaccharides; Monocytes; Thrombophilia; Thromboplastin; Tumor Cells, Cultured

We have characterised the Procoagulant activity (PCA) of six well-established cell lines by assays of tissue factor (TF), thrombin and FXa generation, flow cytometry using Annexin V, and by binding studies with Factors VIII and IXa. The monocytic (THP-1 & U937) and promyelocytic (NB4) cells expressed high concentrations of TF antigen and activity, whereas TF in the lymphocytic cells (Molt 4, Jurkat & Nalm 6) was very low or absent. However the T-lymphoblastoid cells (Molt 4 & Jurkat) promoted the generation of large amounts of thrombin despite their low TF content, and these cells were also the most active in supporting Factor Xa generation. Molt 4 cells bound Factors VIII and IXa with high capacity and their activity was inhibited by Annexin V. These results indicate that the PCA of T-lymphoblastoid lines is due to expression of negatively charged phospholipids. Flow cytometry studies showed Annexin V binding to the major population of nonapoptotic Molt 4 cells and the PCA of Molt 4 was not increased when apoptosis was induced by staurosporine, indicating that PCA is independent of apoptotic status.

Topics: Anions; Apoptosis; Cell Membrane; Factor IXa; Factor VIII; Humans; Phosphatidylserines; Phospholipids; Protein Binding; T-Lymphocytes; Thrombophilia; Thromboplastin; Tumor Cells, Cultured

15 deoxy delta12,14 PGJ2 (15d-PGJ2), a high affinity ligand of peroxisome proliferator-activated receptor gamma (PPARgamma) has been proposed to act as a negative feedback regulator of the inflammatory response. We investigated the effect of 15d-PGJ2 on the anticoagulant property of endothelial cells. 15d-PGJ2 stimulated a moderate but sustained increase in tissue factor (TF) activity in HUVECs and EA.hy926 cells while causing a partial loss of thrombomodulin (TM) activity. When cells were co-treated with 15d-PGJ2 and TNF-alpha, the subsequent elevation of TF activity was synergistically increased over that of cells treated with TNF-alpha alone and the decline of TF activity after 24 h was less marked than TNF-alpha alone. The induction of TF by 15d-PGJ2 alone and in combination with TNF-alpha was reduced in the presence of PD 98059, suggesting the participation of the MEK/ERK pathway. The thiazolidinedione PPARgamma agonist ciglitazone had no effect on TF levels but reduced the expression of endothelial protein C receptor. The ability of 15d-PGJ2 to enhance a procoagulant phenotype arising from TNF-alpha suggests a pro-inflammatory role for the prostaglandin.

Topics: Cells, Cultured; Drug Synergism; Endothelium, Vascular; Humans; Inflammation Mediators; MAP Kinase Signaling System; Phenotype; Prostaglandin D2; Thrombomodulin; Thrombophilia; Thromboplastin; Tumor Necrosis Factor-alpha; Umbilical Cord

Topics: Amino Acid Substitution; Case-Control Studies; DNA Mutational Analysis; Humans; Mutation; Polymorphism, Single-Stranded Conformational; Thrombophilia; Thromboplastin; Venous Thrombosis

A new test for screening the procoagulant capacity of plasma is described and evaluated. This test is based on the coagulation of plasma initiated by thromboplastin (Tp) in the presence of thrombomodulin (TM). In a previous paper we reported that this test had a significant phenotypic and genetic correlation with thrombosis susceptibility. The present report describes the characteristics of the test and its sensitivity to the concentration of some hemostasis factors.. Plasma from normal subjects, from individuals with various disorders of hemostasis and plasma with different concentrations of factors II, V, VII, VIII, X, fibrinogen, protein C and protein S were studied. The thromboplastin-thrombomodulin-mediated time (Tp-TMT) is measured after mixing 100 mL of plasma diluted 1/10 at 37 C with 100 mL of a solution composed of 2 parts of thromboplastin, 1 part of thrombomodulin at 30 U/mL and 1 part of Owren's buffer. The results are expressed as the ratio of the patient's clotting time to that of the control. Values were compared with Student's t test and the Mann-Whitney test. Differences were considered statistically significant when p<0.05.. In the control group women showed significantly lower values than men. Raised levels of factors II, V, VII and X reduced the coagulation time obtained with Tp-TM. Elevated concentrations of fibrinogen and factor VIII did not influence the test. The Tp-TMT was sensitive to protein S deficiencies, but not to protein C deficiencies.. These results indicate that the effect of protein S on the test is through its anti-prothrombinase activity.. Tp-TMT, which is correlated with thrombosis susceptibility, is sensitive to raised levels of factors II, V, VII and X, as well as to low levels of protein S, and may be an indicator of thrombosis risk.

Topics: Blood Coagulation; Blood Coagulation Factors; Blood Coagulation Tests; Case-Control Studies; Female; Humans; Male; Protein S Deficiency; Sensitivity and Specificity; Thrombomodulin; Thrombophilia; Thromboplastin

Monocyte tissue factor expression was evaluated in 67 patients with hepatosplenic Schistosomiasis. They were classified as Child A (n = 15), Child B (n = 15), Child C (n = 12) and Bleeders (n = 10), in addition to 15 healthy controls. Mononuclear cells were cultured in vitro with and without lipopolysaccharide (LPS) to assess monocyte tissue factor (TF) antigen (Ag) and activity (Act) in cell lysate, in addition to measurement of prothrombin fragment 1 + 2 (F1 + 2) as a marker of in vivo thrombin generation. A significant increase in monocyte TF Ag and TF Act was noted in all stages of the disease compared with the control group, with marked accentuation during an acute attack of variceal bleeding. This enhanced monocyte expression was noted before the addition of LPS and became more obvious with addition of LPS. An increasing level of F1 + 2 was similarly noted. These findings constitute further evidence for an existing prothrombotic state in hepatosplenic Schistosomiasis, and also that monocytes are closely implicated in the haemostatic diathesis characterizing the disease.

Topics: Adult; Case-Control Studies; Hemorrhagic Disorders; Humans; Lipopolysaccharides; Liver Diseases; Monocytes; Peptide Fragments; Prothrombin; Schistosomiasis; Severity of Illness Index; Splenic Diseases; Thrombophilia; Thromboplastin

High cholesterol levels are a known risk factor for coronary events. The molecular links between high serum cholesterol and the increased thrombogenicity of the arterial wall are still matter of investigation. In the present study we investigate the relationship between plasma cholesterol, thrombus formation and TF expression in a atherosclerotic rabbit model. Hypercholesterolemic rabbits showed a pronounced TF staining as well as NF-kappaB activation in the aortic arch. A consistent vessel wall platelet deposition was also observed. Treatment with fluvastatin reduced lipid accumulation, TF overexpression (-60%), NF-kappaB activation, and platelet deposition (-56%). In vitro studies showed that the drug upregulated IkappaB alpha in unstimulated as well as in TNFalpha-stimulated cells and also impaired the TNFalpha-induced Cdc42 prenylation, indicating that fluvastatin interferes with the transcriptional activation of TF gene. These results indicate that the prothrombotic phenotype of arterial wall, associated with elevated serum cholesterol levels, is mediated by TF overexpression. Fluvastatin treatment reduces the prothrombotic tendency by inhibiting TF synthesis.

Topics: Animals; Anticholesteremic Agents; Anticoagulants; Aorta; cdc42 GTP-Binding Protein; Cells, Cultured; Cholesterol; Diet, Atherogenic; Endothelium, Vascular; Enoxaparin; Fatty Acids, Monounsaturated; Fluvastatin; Gene Expression Regulation; Hemorheology; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypercholesterolemia; I-kappa B Proteins; Indoles; Male; NF-kappa B; NF-KappaB Inhibitor alpha; Platelet Adhesiveness; Protein Prenylation; Protein Processing, Post-Translational; Rabbits; RNA, Messenger; Signal Transduction; Thrombophilia; Thromboplastin; Transcription, Genetic; Tumor Necrosis Factor-alpha; Umbilical Veins

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

Thrombosis is a life-threatening complication that occurs in a subset of patients with heparin-induced thrombocytopenia (HITT). The pathogenic mechanisms underlying the variable occurrence of thrombosis in HITT is poorly understood. It was hypothesized that monocyte activation leading to tissue factor expression may play a role in promoting a thrombogenic state in HITT. This study demonstrates that a human platelet factor 4 (PF4)/heparin-specific murine monoclonal antibody (KKO) binds to peripheral blood-derived human monocytes in a PF4-dependent manner. KKO and antibodies from patients with HITT induce monocytes to synthesize and secrete interleukin-8 and induce cell-surface procoagulant activity, which is abrogated following treatment with antihuman tissue factor antibody. The findings suggest a novel mechanism by which PF4/heparin antibodies may promote a hypercoagulable state in patients with HITT. (Blood. 2001;98:1252-1254)

Topics: Antibodies, Monoclonal; Autoantibodies; Coagulants; Heparin; Humans; Interleukin-8; Monocytes; Platelet Factor 4; Thrombocytopenia; Thrombophilia; Thromboplastin; Thrombosis

Blood coagulation is activated commonly in pancreatic carcinoma but the role of the tumor cell in this activation is undefined. Immunohistochemical procedures were applied to fixed sections of 22 cases of resected adenocarcinoma of the pancreas to determine the presence of components of coagulation and fibrinolysis pathways in situ. Tumor cell bodies stained for tissue factor: prothrombin: and factors VII, VIIIc, IX, X, XII, and subunit "a" of factor XIII. Fibrinogen existed throughout the tumor stroma, and tumor cells were surrounded by fibrin. Staining for tissue factor pathway inhibitor, and plasminogen activators was minimal and inconsistent. Plasminogen activator inhibitors -1, -2, and -3 were present in the tumor stroma, and on tumor cells and vascular endothelium. Extravascular coagulation activation exists associated with pancreatic carcinoma cells in situ that is apparently unopposed by naturally occurring inhibitors or the plasminogen activator-plasmin system. We postulate that such local coagulation activation may regulate growth of this malignancy. These findings provide a rationale for testing agents that modulate the blood coagulation/fibrinolytic system (that inhibit tumor growth in other settings) in pancreatic carcinoma.

Topics: Adenocarcinoma; Aged; Blood Coagulation Factors; Endothelium, Vascular; Female; Fibrin; Fibrinogen; Humans; Immunoenzyme Techniques; Male; Middle Aged; Neoplasm Proteins; Neoplastic Stem Cells; Pancreatic Neoplasms; Plasminogen Activator Inhibitor 1; Plasminogen Activator Inhibitor 2; Protein C; Protein S; Prothrombin; Stromal Cells; Thrombophilia; Thromboplastin

Atherosclerotic cardiovascular disease is the leading cause of the increased morbidity and mortality observed in uremic patients. Thrombosis is an important contributor to the evolution of atherosclerotic lesions. The physiologically-relevant blood clotting depends on binding of activated factor VII (FVIIa) to exposed tissue factor (TF) on activated/damaged cells.. A cross-sectional study was performed on three age- and sex-matched groups of individuals: one group of 50 patients on maintenance hemodialysis (D group), one of 50 patients with a non-dialysed renal insufficiency (ND group) and one of 50 healthy controls (HC group). We studied basal plasma concentrations of FVIIa, factor VII-related antigen (FVIIAg), soluble TF, tissue factor pathway inhibitor (TFPI), TF-dependent circulating monocytes procoagulant activity (TF-dMPA), tissue factor-dependent plasma reactivity to activated protein C (TF-aPC), D-dimers (D-Di), and circulating markers of cellular activation/injury: soluble thrombomodulin (sTM), circulating microparticles (microP), soluble leukocyte, endothelial and platelet selectins (sL-selectin, sE-selectin, sP-selectin), soluble intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 (sICAM-1 and sVCAM-1). Their variations induced, in hemodialysis patients, by a dialysis run were thereafter studied. Values of FVIIa, FVIIa/FVIIAg ratio, sTF, TFPI, TF-dMPA, D-Di, sTM, microP, sL, sE and sP selectins, sICAM-1 and sVCAM-1 increased all along the hierarchy HC group/ND group/D group. Microparticles were mainly of platelet origin, to a lesser extent of monocyte origin. Dialysis induced an increase of FVIIa, sTF, TF-dMPA and circulating markers of cellular activation/injury. Strong correlations were observed between FVIIa/FVIIAg ratio and serum creatinine levels, sTF, TF-dMPA, sTM, sE-selectin, sVCAM-1. The TF-aPC was impaired in the ND and the D group, and the lower values were, in the D group, associated with antecedents of vascular access thrombosis.. Renal insufficiency is associated to an activation of the tissue factor coagulation pathway, to a platelet, monocyte and endothelial activation/injury and to a deficient tissue-factor induced response to activated protein C which culminate in end-stage disease and are increased by hemodialysis runs. This contributes to linked coagulation and cellular conditions for an enhanced atherosclerosis progression. Due to the TF pathway activation, the therapeutic use of recombinant TFPI should be evaluated.

Topics: Activated Protein C Resistance; Adult; Aged; Blood Coagulation; Blood Coagulation Factors; Case-Control Studies; Cell Adhesion Molecules; Cross-Sectional Studies; Female; Humans; Male; Middle Aged; Renal Dialysis; Renal Insufficiency; Thrombophilia; Thromboplastin

Patients with meningococcal sepsis generally suffer from disseminated intravascular coagulation (DIC). The aim of this study was to address whether these patients have elevated numbers of circulating microparticles that contribute to the development of DIC. Plasma samples from 5 survivors, 2 nonsurvivors, and 5 healthy volunteers were analyzed for the presence of microparticles by flow cytometry. Ongoing coagulation activation in vivo was quantified by enzyme-linked immunosorbent assay of plasma prothrombin fragment F(1 + 2), and procoagulant properties of microparticles in vitro were estimated by thrombin-generation assay. On admission, all patients had increased numbers of microparticles originating from platelets or granulocytes when compared with controls (P =.004 and P =.008, respectively). Patients had elevated levels of F(1 + 2) (P =.004), and their microparticles supported thrombin generation more strongly in vitro (P =.003) than those of controls. Plasma from the patient with the most fulminant disease course and severe DIC contained microparticles that expressed both CD14 and tissue factor, and these microparticles demonstrated extreme thrombin generation in vitro. We conclude that patients with meningococcal sepsis have elevated numbers of circulating microparticles that are procoagulant. These findings may suggest a novel therapeutic approach to combat clinical conditions with excessive coagulation activation.

Topics: Adolescent; Adult; Biomarkers; Blood Coagulation Factors; Blood Platelets; Child; Child, Preschool; Disseminated Intravascular Coagulation; Female; Granulocytes; Humans; Infant; Lipopolysaccharide Receptors; Male; Meningococcal Infections; Particle Size; Sepsis; Survivors; Thrombin; Thrombophilia; Thromboplastin

Apoptotic microparticles are responsible for almost all tissue factor activity of the plaque lipid core. We hypothesized that elevated levels of procoagulant microparticles could also circulate in the peripheral blood of patients with recent clinical signs of plaque disruption and thrombosis.. We studied 39 patients with coronary heart disease, including 12 patients with stable angina and 27 patients with acute coronary syndromes (ACS), and 12 patients with noncoronary heart disease. We isolated the circulating microparticles by capture with annexin V and determined their procoagulant potential with a prothrombinase assay. The cell origins of microparticles were determined in an additional 22 patients by antigenic capture with specific antibodies. The level of procoagulant microparticles did not differ between stable angina patients and noncoronary patients (10.1+/-1.6 nmol/L phosphatidylserine [PS] equivalent versus 9.9+/-1.6 nmol/L PS equivalent, respectively). However, procoagulant microparticles were significantly elevated in patients with ACS (22.2+/-2.7 nmol/L PS equivalent) compared with other coronary (P<0.01) or noncoronary (P<0.01) patients. Microparticles of endothelial origin were significantly elevated in patients with ACS (P<0.01), which suggests an important role for endothelial injury in inducing the procoagulant potential.. High levels of procoagulant endothelial microparticles are present in the circulating blood of patients with ACS and may contribute to the generation and perpetuation of intracoronary thrombi.

Topics: Acute Disease; Angina Pectoris; Antigens, CD; Apoptosis; CD146 Antigen; Coronary Artery Disease; Coronary Thrombosis; Endothelium, Vascular; Female; Humans; Male; Membrane Glycoproteins; Middle Aged; Neural Cell Adhesion Molecules; Phosphatidylserines; Platelet Endothelial Cell Adhesion Molecule-1; Prospective Studies; Receptors, Cell Surface; Thrombophilia; Thromboplastin

Two human monoclonal antiphospholipid antibodies (APA) of the IgG type, HL-5B and RR-7F have been generated from a patient with primary antiphospholipid syndrome and recurrent cerebral microemboli (H.L.) and from a patient with SLE without evidence of recurrent thrombosis (R.R.). Both monoclonal APA have similar characteristics in ELISA tests. To further analyse the prothrombotic potential, their effect on human monocytes and platelets, and bovine aortic endothelial cells (BAEC) was investigated. Monocytes were isolated from buffy coats by standard techniques. They were incubated either with the respective monoclonal APA in different concentrations, or a control monoclonal IgG of the same subtype, or plasma of the patients, from whom the antibodies were isolated. Incubation with LPS served as positive control. BAEC were grown to confluence, and then incubated with the appropriate agonists. Procoagulant activity (PCA) was determined by a single stage clotting assay. PCA expression after incubation is given as the ratio of the coagulation times observed with media only divided by that observed with the agonist. A PCA ratio >1 indicates the induction of PCA by the agonist. At 1 microg/ml HL-5B yielded a PCA ratio of 1.63 +/- 0.16 while RR-7F induced no significant rise to 1.06 +/- 0.18. Dose response curves showed that RR-7F can induce PCA at higher concentrations. However, its effect is approx. 1/50 of HL-5B based on equimolar antibody concentration. Further analysis indicates that the majority of the PCA induced by monoclonal APA can be inhibited by a specific tissue factor antibody. Neither monoclonal antibody induced PCA in BAEC. Sera from both patients were able to induce PCA in monocytes. However, the PCA ratio of serum from H.L. was higher (1.78) than that of R.R. (1.44). Neither monoclonal APA had an effect on platelets as determined by flow cytometric analysis of CD62P, CD41, CD42b expression and fibrinogen binding with and without previous activation with 5 microM ADP or 15 microM TRAP-6. Similarly, there were no differences in platelet aggregation to different stimuli including submaximal activation. In summary, these data provide further evidence that induction of tissue factor in monocytes is one of the procoagulant effects of APA. Furthermore, the binding specificity of APA is perhaps not suited to predict the biological effects of the antibodies.

Topics: Adenosine Diphosphate; Animals; Antibodies, Antiphospholipid; Antibodies, Monoclonal; Antibody Specificity; Antiphospholipid Syndrome; Autoimmune Diseases; Biomarkers; Blood Coagulation Factors; Blood Platelets; Cattle; Cells, Cultured; Dose-Response Relationship, Immunologic; Endothelium, Vascular; Female; Fibrinogen; Humans; Immunoglobulin G; Intracranial Embolism; Lipopolysaccharides; Lupus Erythematosus, Systemic; Male; Middle Aged; Monocytes; Peptide Fragments; Platelet Activation; Recurrence; Thrombophilia; Thromboplastin

In ischemic heart disease (IHD) patients high plasma levels of Tissue Factor (TF), the trigger of coagulation cascade, are present. Homocysteine (Hcy) is a risk factor for coronary artery disease, and several different pathophysiological mechanisms by which Hcy may play a role in thrombus formation have been postulated in "in vitro" studies. We investigated the "in vivo" role of Hcy in affecting plasma levels of TF, its inhibitor Tissue Factor Pathway Inhibitor (TFPI) and hypercoagulability.. We investigated 119 IHD patients who underwent PTCA and compared them with 103 healthy subjects. TF, TFPI, Thrombin-Antithrombin complexes (TAT) and Hcy levels were significantly higher in the patients than in the controls. A positive correlation was found between Hcy and TF (r = 0.54; p < 0.0001), Hcy and TFPI (r = 0.26; p <0.05) as well as Hcy and TAT (r = 0.33; p <0.0001) levels. An inverse correlation existed between folate intake and Hcy levels (r = -0.28; p = 0.001). Hcy levels within the first quartile and in the highest quartile were associated with a lower (p < 0.001) and higher (p <0.0001) rate of clinical recurrences, respectively. Patients with TF values in the first quartile had a lower rate of angiographically documented clinical recurrences as compared to those in the fourth quartile (p <0.01); those in the highest quartile of TF showed a higher rate of recurrences (p = 0.001). Multivariate analysis confirmed these results (first quartile of Hcy: OR 0.02, C1 0.002-0.27; fourth quartile of Hcy: OR 36.5, C1 3.6-365/first quartile of TF: OR 0.006, C1 0.001-0.44; fourth quartile of TF: OR 16.4, C1 3.0 - 90.0), also after adjustment for risk factors and Hcy and TF respectively.. In this study we show that TF, TFPI and TAT levels are correlated with Hcy plasma levels in IHD patients, providing evidence of an "in vivo" pathophysiological mechanism of hyperhomocysteinemia. The observed association between angiographically documented clinical recurrences and TF and Hcy values awaits confirmation in studies designated to evaluate this issue on a larger number of patients.

Topics: Adult; Aged; Angina Pectoris; Angioplasty, Balloon, Coronary; Antithrombin III; Coronary Angiography; Female; Homocysteine; Humans; Hyperhomocysteinemia; Lipoproteins; Male; Middle Aged; Myocardial Ischemia; Peptide Hydrolases; Recurrence; Risk Factors; Thrombophilia; Thromboplastin; Vitamins

The study of the molecular bases of thrombophilia in a large family with 4 symptomatic members is reported. Three thrombophilic genetic components (FV R506Q, FV H1299R, and PT 20210G/A), all affecting the activity of the prothrombinase complex, were detected alone and in combination in various family members. In addition, a newly identified missense mutation (factor V [FV] Y1702C), causing FV deficiency, was also present in the family and appeared to enhance activated protein C (APC) resistance in carriers of FV R506Q or FV H1299R by abolishing the expression of the counterpart FV allele. The relationships between complex genotypes, coagulation laboratory findings, and clinical phenotypes were analyzed in the family. All symptomatic family members were carriers of combined defects and showed APC resistance and elevated F1 + 2 values. Evidence for the causative role of the FV Y1702C mutation, which affects a residue absolutely conserved in all 3 A domains of FV, factor VIII, and ceruloplasmin, relies on (1) the absolute cosegregation between the mutation and FV deficiency, both in the family and in the general population; (2) FV antigen and immunoblot studies indicating the absence of Y1702C FV molecules in plasma of carriers of the mutation, despite normal levels of the FV Y1702C messenger RNA; and (3) molecular modeling data that support a crucial role of the mutated residue in the A domain structure. These findings help to interpret the variable penetrance of thrombosis in thrombophilic families and to define the molecular bases of FV deficiency. (Blood. 2000;96:1443-1448)

Topics: Amino Acid Sequence; Female; Humans; Male; Middle Aged; Molecular Sequence Data; Mutation; Pedigree; Thrombophilia; Thromboplastin

We investigated the role of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in the lungs of patients with idiopathic pulmonary fibrosis (IPF). Bronchoalveolar lavage (BAL) fluid was obtained from 22 patients with IPF, and the levels of TF and TFPI antigen were measured by ELISA. The TF and TFPI levels in BAL fluid supernatant were significantly higher in IPF patients than in normal controls. In addition, both levels were significantly higher in advanced cases than in nonadvanced cases. There was a significant correlation between the TF and TFPI levels. Localization of TF and TFPI antigens was investigated by immunohistochemical staining. Both antigens were mainly localized in hyperplastic cuboidal epithelial cells, suggesting that the widespread distribution of these cells contributed to the increase of TF and TFPI antigen levels in the lungs of IPF patients. To assess whether TF activity is counterbalanced by TFPI in the lungs of IPF patients, we examined procoagulant activity and TF activity. It was found, however, that both procoagulant and TF activities were significantly higher in the BAL fluid supernatant of IPF patients than in that of normal controls, which suggested that TFPI was actually increased, but the increase was insufficient to counterbalance TF, leading to the development of a hypercoagulable state in the lungs of IPF patients.

Topics: Adult; Aged; Anticoagulants; Antigens; Blood Coagulation Tests; Bronchoalveolar Lavage Fluid; Epithelial Cells; Female; Fibrin; Fibrinolytic Agents; Humans; Immunohistochemistry; Lipoproteins; Lung; Male; Middle Aged; Pulmonary Fibrosis; Regression Analysis; Serine Proteinase Inhibitors; Thrombophilia; Thromboplastin

To determine the duration of vascular blood vessel dysfunction and coagulation abnormalities after administration of endotoxin in a nonlethal septic rabbit model.. Randomized, controlled, interventional trial.. University animal laboratory.. A total of 30 male New Zealand White rabbits, randomly assigned to one of two groups.. Male New Zealand White rabbits were randomly divided into control or lipopolysaccharide (LPS) (0.5 mg/kg iv bolus Escherichia coli endotoxin)-treated groups. Metabolic acidosis and coagulation activation confirmed the presence of septic shock. The abdominal aorta was removed at 24 hrs (day 1), day 5, or day 21 after LPS injection. Immunohistochemical staining for an endothelial cell marker (PECAM-1/CD31) was performed to assess endothelial injury. Endothelium-dependent vascular relaxation was analyzed by in vitro vascular reactivity studies. Responses to acetylcholine, to calcium ionophore (A-23187), and to sodium nitroprusside were studied. In addition, arterial blood samples were collected on day 1, day 5, and day 21 for measurement of clotting factors and tissue factor activity.. LPS injection resulted in endothelial injury, with loss of approximately 25% of the endothelial area on day 5, which disappeared on day 21. LPS injection also caused a significantly reduced relaxation response to acetylcholine (44.9% +/-9.9% vs. 76.5%+/-5.4% for the control group; p < .005), which was restored on day 21. In contrast, vascular relaxation in response to A-23187 and sodium nitroprusside was not altered. A significant decrease in the platelet count was observed on day 1, associated with a decrease in factors II and V. On day 5, platelet count and factors II and V were corrected in conjunction with an elevated monocyte tissue factor activity in LPS-injected rabbits. On day 21, coagulation abnormalities were corrected.. A single endotoxin injection in the rabbit was responsible for prolonged aortic endothelial cell dysfunction, as well as a prolonged procoagulant state. The latter is a potential trigger for disseminated intravascular coagulation. Importantly, these features are associated with normalization of conventional biological evidence of septic shock.

Topics: Animals; Blood Coagulation Factors; Disseminated Intravascular Coagulation; Endothelium, Vascular; Endotoxins; Escherichia coli; Male; Rabbits; Shock, Septic; Thrombophilia; Thromboplastin; Vascular Resistance

Microparticles (MPs) resulting from vesiculation of platelets and other blood cells have been extensively documented in vitro and have been found in increased numbers in several vascular diseases, but little is known about MPs of endothelial origin. The aim of this study was to analyze morphological, immunological, and functional characteristics of MPs derived from human umbilical vein endothelial cells (HUVECs) stimulated by TNF, and to investigate whether these MPs are detectable in healthy individuals and in patients with a prothrombotic coagulation abnormality. Electron microscopy evidenced bleb formation on the membrane of TNF-stimulated HUVECs, leading to increased numbers of MPs released in the supernatant. These endothelial microparticles (EMPs) expressed the same antigenic determinants as the corresponding cell surface, both in resting and activated conditions. MPs derived from TNF-stimulated cells induced coagulation in vitro, via a tissue factor/factor VII-dependent pathway. The expression of E-selectin, ICAM-1, alphavbeta3, and PECAM-1 suggests that MPs have an adhesion potential in addition to their procoagulant activity. In patients, labeling with alphavbeta3 was selected to discriminate EMPs from those of other origins. We provide evidence that endothelial-derived MPs are detectable in normal human blood and are increased in patients with a coagulation abnormality characterized by the presence of lupus anticoagulant. Thus, MPs can be induced by TNF in vitro, and may participate in vivo in the dissemination of proadhesive and procoagulant activities in thrombotic disorders.

Topics: Antiphospholipid Syndrome; Autoimmune Diseases; Cell Adhesion Molecules; Cells, Cultured; Endothelium, Vascular; Factor VII; Flow Cytometry; Humans; Infections; Lupus Coagulation Inhibitor; Lupus Erythematosus, Systemic; Microscopy, Confocal; Neoplasms; Receptors, Vitronectin; Thrombophilia; Thromboplastin; Tumor Necrosis Factor-alpha; Umbilical Veins

Procoagulant activity after cardiopulmonary bypass (CPB) in infants may predispose to thrombotic and bleeding complications. The induction of tissue factor and prothrombinase activity on endothelial cell membranes is a primary step in the activation of the extrinsic clotting cascade. The purpose of this study is to characterize the fibrinolytic and endothelial procoagulant state in infants undergoing congenital cardiac repairs with and without CPB.. Fourteen infants (aged 1 to 12 weeks) underwent repair of congenital cardiac defects. Two patients had closed procedures (controls) and 12 had open cardiac procedures. Serum samples were taken before and after CPB, 1, 4, and 24 hours after CPB. Tissue plasminogen activator, plasminogen activator inhibitor-1, interleukin-1beta, interleukin-6, plasma tissue factor, and factor V levels were measured. Human umbilical vein endothelial cell cultures were incubated with serum taken from the above time points and assayed for induction of tissue factor and prothrombinase activity.. Control patients had no change from preoperative values in any of the parameters examined. In experimental patients, tissue plasminogen activator levels peaked at 1 hour after CPB and then decreased to normal by 24 hours. Plasminogen activator inhibitor-1 levels peaked at 4 hours after CPB and returned to baseline by 24 hours. The plasma of all patients had no intrinsic tissue factor activity. Induction of tissue factor activity on umbilical vein endothelial cells peaked immediately and again at 24 hours, whereas prothrombinase activity peaked early and stayed elevated. Serum factor V levels were significantly reduced after CPB, but returned to near baseline levels by 24 hours.. Cardiopulmonary bypass is associated with derangement of the coagulation and fibrinolytic systems in infants. The serum of these patients promotes the induction of endothelial procoagulant activity, suggesting that there may be a hypercoagulable state in the postbypass period.

Topics: Blood Coagulation Factors; Cardiopulmonary Bypass; Endothelium, Vascular; Female; Fibrinolysis; Heart Defects, Congenital; Hemostasis; Humans; Infant; Infant, Newborn; Male; Postoperative Hemorrhage; Reference Values; Risk Factors; Thrombophilia; Thromboplastin

Topics: Abortion, Habitual; Adult; Animals; Antibodies, Antiphospholipid; Anticoagulants; Antiphospholipid Syndrome; Autoimmune Diseases; Drug Monitoring; Female; Humans; International Normalized Ratio; Intracranial Embolism; Intracranial Thrombosis; Male; Middle Aged; Pregnancy; Recombinant Proteins; Recurrence; Risk; Seizures; Thromboembolism; Thrombophilia; Thrombophlebitis; Thromboplastin; Warfarin

The involvement of coagulation and fibrinolysis in the development of chronic subdural haematoma (CSH) from subdural effusion was investigated. Subdural fluid and venous blood samples were obtained from 34 patients with CSH and 9 patients with subdural effusion, and analyzed using enzyme-linked immunosorbent assays for thrombin-antithrombin III complex (TAT), prothrombin fragment F1 + 2 (F1 + 2), tissue factor, tissue factor pathway inhibitor (TFPI) and D-dimer. CSH was classified into the layering type, believed to be active, and other types according to x-ray computed tomography. All markers in the blood of both patient groups were similar to the values of normal subjects. Levels of TAT and F1 + 2 were much higher in the subdural fluid than in the blood of patients with CSH (P < 0.001, P < 0.001) and with subdural effusion (P < 0.05, P < 0.05). The level of D-dimer in the subdural fluid was significantly higher than in the blood (P < 0.001) in patients with CSH, but not in patients with subdural effusion. All markers in the subdural fluid of layering type CSH, except TFPI, were significantly higher than in the other types (P < 0.05). Local hypercoagulative activity in the subdural space is present in subdural effusion and precedes hyperfibrinolytic activity in CSH. Thrombin generation as indicated by TAT and F1 + 2 might be involved in the development of CSH. Propagation of CSH may be modulated by the coagulation system including the extrinsic pathway and fibrinolysis.

Topics: Adult; Aged; Aged, 80 and over; Antithrombin III; Blood Coagulation Factors; Blood Coagulation Tests; Chronic Disease; Female; Fibrin Fibrinogen Degradation Products; Fibrinolysis; Hematoma, Subdural; Humans; Lipoproteins; Male; Middle Aged; Peptide Fragments; Peptide Hydrolases; Prothrombin; Subdural Effusion; Thrombophilia; Thromboplastin

Multiple stimuli converge in cardiopulmonary bypass to create a tremendous prothrombotic stimulus. The ideal anticoagulant for cardiopulmonary bypass should selectively target only the intravascular stimuli, thereby eliminating pathologic clotting in the bypass circuit while preserving hemostasis in the thoracic cavity. We propose the inhibition of factor IX as such a targeted anticoagulant strategy.. We prepared an inhibitor of activated factor IX and applied it to a primate model of cardiopulmonary bypass to confirm the anticoagulant efficacy of activated factor IX in this setting and to assess more subtle markers of thrombin generation, macrophage procoagulant activity, and cellular tissue factor expression. Seven baboons that received activated factor IX (460 microg/kg) and 7 that received heparin (300 IU/kg) and protamine underwent cardiopulmonary bypass for 90 minutes and were followed after the operation for 3 hours.. Analysis of plasma factor IX activity demonstrated adequate inhibition (<20%) of factor IX throughout cardiopulmonary bypass. Activated factor IX-treated baboons demonstrated similar circuit patency to heparin-treated baboons but had significantly diminished intraoperative blood loss. Preservation of extravascular hemostasis was further demonstrated in activated factor IX-treated animals by (1) significantly increased levels of thrombin-antithrombin III complex and prothrombin activation peptide (F1+2) without intravascular thrombosis, (2) significantly greater macrophage procoagulant activity in pericardial-derived monocytes, and (3) immunohistochemical evidence of tissue factor expression in pericardial mesothelial cells and macrophages.. Anticoagulation with activated factor IX allows for intravascular anticoagulation with maintenance of extravascular hemostasis. These findings suggest activated factor IX as an agent that not only exemplifies a targeted approach to selective anticoagulation in cardiac surgery but also further characterizes the procoagulant milieu during cardiopulmonary bypass.

Topics: Animals; Anticoagulants; Blood Coagulation Tests; Blood Loss, Surgical; Cardiopulmonary Bypass; Factor IX; Factor IXa; Heparin; Humans; Macrophages; Microscopy, Electron, Scanning; Papio; Surface Properties; Thrombin; Thrombophilia; Thromboplastin

Topics: Antiphospholipid Syndrome; Autoimmune Diseases; Female; Humans; Leukocytes, Mononuclear; Male; Models, Biological; Thrombophilia; Thromboplastin

Diabetes is associated with a hypercoagulable state that contributes to macrovascular complications, including cardiovascular events. The glycation reaction, a consequence of chronic hyperglycemia, has also been implicated in the pathogenesis of diabetic complications. Glycated proteins have receptors on monocytes and generate reactive oxygen species that can regulate the expression of a number of genes. As abnormal monocyte expression of tissue factor (TF), the main initiator of the coagulation cascade, is responsible for thrombosis in a number of clinical settings, we studied the effect of glycated albumin on monocyte TF expression. Mononuclear cells were incubated with glycated albumin for 24 hours, and monocyte TF activity was measured with a plasma recalcification time assay; TF antigen was measured by ELISA and TF mRNA by RT-PCR. Glycated albumin induced blood monocyte expression of the procoagulant protein TF at the mRNA level. Oxidative stress appeared to be involved in this effect, as the antioxidant N-acetylcysteine diminished TF mRNA accumulation in stimulated monocytes. Hydroxyl radicals, which may be generated inside cells from H2O2 via the Fenton reaction, also appeared to be involved in this effect, as hydroxyl radical scavengers downregulated TF activity and antigen levels (but not TF mRNA). Finally, the involvement of activated protein tyrosine kinase in the transmission of the signal from the membrane to the nucleus was suggested by the inhibitory effect of herbimycin A. These results point to a new mechanism for the hypercoagulability often described in diabetic patients and suggest that antioxidants or protein tyrosine kinase inhibitors might be of therapeutic value in this setting.

Topics: Acetylcysteine; Antioxidants; Benzoquinones; Diabetes Complications; Enzyme Activation; Enzyme Inhibitors; Free Radical Scavengers; Gene Expression Regulation; Glycation End Products, Advanced; Glycosylation; Humans; Hydroxyl Radical; Lactams, Macrocyclic; Monocytes; Oxidative Stress; Phosphorylation; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Quinones; Rifabutin; RNA, Messenger; Serum Albumin; Serum Albumin, Human; Signal Transduction; Thrombophilia; Thromboplastin

Topics: Blood Coagulation Tests; Humans; Plasma; Thrombophilia; Thromboplastin; Thrombosis

Topics: Adrenal Cortex Hormones; Anticoagulants; Arteriosclerosis; Blood Platelet Disorders; Coronary Disease; Fats; Female; Humans; Intracranial Embolism; Intracranial Embolism and Thrombosis; Neoplasms; Pharmacology; Postoperative Complications; Pregnancy; Pregnancy Complications; Pregnancy Complications, Hematologic; Thrombophilia; Thromboplastin; Thrombosis

Topics: Animals; Blood Coagulation Disorders; Burns; Hematocrit; Heparin; Mice; Research; Skin; Thrombophilia; Thromboplastin; Tissue Extracts

Topics: Blood Coagulation; Humans; Intercellular Signaling Peptides and Proteins; Peptides; Thrombophilia; Thromboplastin

Topics: Cardiovascular Diseases; Hematologic Diseases; Humans; Prothrombin Time; Research; Sclerosis; Thrombophilia; Thromboplastin

Topics: Arteries; Hemostatics; Humans; Niacin; Nicotinic Acids; Prothrombin; Thrombophilia; Thromboplastin