thromboplastin and Disease-Models--Animal

Endothelial nitric oxide synthase (eNOS) dysfunction is known to exacerbate the progression and prognosis of diabetic kidney disease (DKD). One of the mechanisms through which this is achieved is that low eNOS levels are associated with hypercoagulability, which promotes kidney injury. In the extrinsic coagulation cascade, the tissue factor (factor III) and downstream coagulation factors, such as active factor X (FXa), exacerbate inflammation through activation of the protease-activated receptors (PARs). Recently, it has been shown that the lack of or reduced eNOS expression in diabetic mice, as a model of advanced DKD, increases renal tissue factor levels and PAR1 and 2 expression in their kidneys. Furthermore, pharmaceutical inhibition or genetic deletion of coagulation factors or PARs ameliorated inflammation in DKD in mice lacking eNOS. In this review, we summarize the relationship between eNOS, coagulation, and PARs and propose a novel therapeutic option for the management of patients with DKD.

Topics: Animals; Antibodies, Neutralizing; Blood Coagulation; Diabetic Nephropathies; Disease Models, Animal; Factor Xa Inhibitors; Humans; Kidney; Mice; Mice, Knockout; Nitric Oxide Synthase Type III; Receptors, Proteinase-Activated; Signal Transduction; Thromboplastin

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era in which heparin and its derivatives-the currently recommended therapy for cancer-associated thrombosis-might be superseded by direct oral anticoagulants, novel data from mouse models of cancer-associated thrombosis suggest the possibility of future personalised therapeutic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

Cancer-associated venous thromboembolism (VTE) constitutes the second cause of death after cancer. Many risk factors for cancer-associated VTE have been identified, among them soluble tissue factor and microparticles (MPs). Few data are available about the implication of MPs in cancer associated-VTE through animal model of cancer. The objective of the present review was to report the state of the current literature about MPs and cancer-associated VTE in animal model of cancer. Fourteen series have reported the role of MPs in cancer-associated VTE, through three main mouse models: ectopic or orthotopic tumor induction, experimental metastasis by intravenous injection of tumor cells into the lateral tail vein of the mouse. Pancreatic cancer is the most used animal model, due to its high rate of cancer-associated VTE. All the series reported that tumor cell-derived MPs can promote thrombus formation in TF-dependent manner. Some authors reported also the implication of phosphatidylserine and PSGL1 in the generation of thrombin. Moreover, MPs seem to be implicated in cancer progression through a coagulation-dependent mechanism secondary to thrombocytosis, or a mechanism implicating the regulation of the immune response. For these reasons, few authors have reported that antiplatelet and anticoagulant treatments may prevent tumor progression and the formation of metastases in addition of coagulopathy.

Topics: Animals; Anticoagulants; Blood Coagulation; Cell-Derived Microparticles; Disease Models, Animal; Disease Progression; Humans; Neoplasm Metastasis; Neoplasms; Platelet Aggregation Inhibitors; Risk Factors; Thromboplastin; Thrombosis; Venous Thromboembolism

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

Hyperlipidemia affects millions of people worldwide and is a major risk factor for cardiovascular disease. People with hyperlipidemia have elevated levels of serum cholesterol and an increased risk of thrombosis. Studies have suggested that oxidized lipoproteins, such as oxidized low-density lipoprotein (oxLDL), contribute to the development of a pro-thrombotic state. In this review, we discuss our recent studies demonstrating a role for hematopoietic cell-derived tissue factor (TF) expression in the activation of coagulation and increased thrombosis associated with hyperlipidemia. In addition, we investigated the effect of simvastatin on TF expression and coagulation. We found that simvastatin reduced leukocyte TF expression, TF⁺ microparticles, and coagulation. These results and earlier studies suggest that the anti-coagulant activity of statins is due, in part, to their ability to reduce monocyte TF expression in patients with cardiovascular disease.

Topics: Animals; Blood Coagulation; Disease Models, Animal; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hyperlipidemias; Simvastatin; Thromboplastin; Thrombosis

Thrombosis is one of the major causes of human death worldwide. Identification of the cellular and molecular mechanisms leading to thrombus formation is thus crucial for the understanding of the thrombotic process. To examine thrombus formation in a living mouse, new technologies have been developed. Digital intravital microscopy allows to visualize the development of thrombosis and generation of fibrin in real-time within living animal in a physiological context. This specific system allowed the identification of new cellular partners involved in platelet adhesion and activation. Furthermore, it improved, especially, the knowledge of the early phase of thrombus formation and fibrin generation in vivo. Until now, platelets used to be considered the sole central player in thrombus generation. However, recently, it has been demonstrated that leukocytes, particularly neutrophils, play a crucial role in the activation of the blood coagulation cascade leading to thrombosis. In this review, we summarized the mechanisms leading to thrombus formation in the microcirculation according to the method of injury in mice with a special focus on the new identified roles of neutrophils in this process.

Topics: Animals; Arteries; Blood Proteins; Chlorides; Computer Systems; Cytoplasmic Granules; Disease Models, Animal; Endothelium, Vascular; Extracellular Matrix; Ferric Compounds; Lasers; Mice; Microcirculation; Neutrophil Infiltration; Neutrophils; Platelet Activation; Thromboplastin; Thrombosis

Topics: Animals; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Disease Models, Animal; Factor XI; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Immunity, Innate; Lupus Coagulation Inhibitor; Mice; Oxidation-Reduction; Thromboplastin; Thrombosis

Topics: Animals; Antithrombin III; Blood Coagulation; Brain; Cerebral Hemorrhage; Disease Models, Animal; Epoprostenol; Hemostasis; Humans; Microcirculation; Nitric Oxide; Plasminogen Activator Inhibitor 1; Protease Nexins; Stroke; Thrombomodulin; Thromboplastin; Thrombosis; Tissue Plasminogen Activator

Deep vein thrombosis (DVT) is a major health problem that requires improved prophylaxis and treatment. Inflammatory conditions such as infection, cancer, and autoimmune diseases are risk factors for DVT. We and others have recently shown that extracellular DNA fibers produced in inflammation and known as neutrophil extracellular traps (NETs) contribute to experimental DVT. NETs stimulate thrombus formation and coagulation and are abundant in thrombi in animal models of DVT. It appears that, in addition to fibrin and von Willebrand factor, NETs represent a third thrombus scaffold. Here, we review how NETs stimulate thrombosis and discuss known and potential interactions of NETs with endothelium, platelets, red blood cells, and coagulation factors and how NETs could influence thrombolysis. We propose that drugs that inhibit NET formation or facilitate NET degradation may prevent or treat DVT.

Topics: Animals; Blood Coagulation; Blood Platelets; Disease Models, Animal; Endothelium, Vascular; Erythrocytes; Humans; Immunity, Innate; Mechanical Thrombolysis; Neutrophils; Thromboplastin; Venous Thrombosis

Venous thromboembolism (VTE) is a leading cause of morbidity and mortality worldwide. However, the mechanisms by which clots are formed in the deep veins have not been determined. Tissue factor (TF) is the primary initiator of the coagulation cascade and is essential for hemostasis. Under pathological conditions, TF is released into the circulation on small-membrane vesicles termed microparticles (MPs). Recent studies suggest that elevated levels of MP TF may trigger thrombosis. This review provides an overview of the role of TF in VTE.

Topics: Animals; Biomarkers; Blood Coagulation; Cell-Derived Microparticles; Disease Models, Animal; Humans; Mice; Neoplasms; Thromboplastin; Venous Thrombosis; Wounds and Injuries

Recurrent fetal loss affects 1-5% of women of childbearing age. Immunological mechanisms may account for 40% of recurrent miscarriages, and in particular, the antiphospholipid syndrome (APS) appears to be implicated in 7-25% of the cases. Because antiphospholipid (aPL) antibodies have thrombogenic properties, fetal loss in patients with APS has been ascribed to thrombosis of placental vessels. However, we have shown that inflammation, specifically activation of complement with generation of the anaphylotoxin C5a, is an essential trigger of fetal injury. Thrombosis and inflammation are linked in many clinical conditions. Tissue factor (TF), the major cellular initiator of the coagulation protease cascade, plays important roles in both thrombosis and inflammation, and its expression is increased in patients with APS. Here we describe how TF, acting as a proinflammatory molecule, induces trophoblast injury and fetal death in a mouse model of APS. Importantly, we will discuss how TF contributes to C5a-induced oxidative burst in neutrophils leading to trophoblasts and fetal injury in APS. The finding that TF is an important effector in aPL-induced inflammation may allow the development of new therapies to abrogate the inflammatory loop caused by tissue factor and improve pregnancy outcomes in patients with aPL antibodies. Statins downregulate TF-induced inflammation and rescued the pregnancies in aPL-treated mice, suggesting they may be a good treatment for women with aPL-induced pregnancy complications.

Topics: Abortion, Habitual; Animals; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Complement Activation; Disease Models, Animal; Embryo, Mammalian; Factor VIIa; Female; Fetal Death; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Mice; Neutrophil Activation; Pregnancy; Receptor, PAR-2; Thromboplastin; Thrombosis

Binding of factor VIIa (FVIIa) to tissue factor (TF) and the subsequent initiation of the clotting cascade is essential for hemostasis. However, the aberrant expression of FVIIa-TF contributes to thrombosis. Despite the tremendous progress made in the past 25years in understanding the molecular mechanisms involved in the interaction between FVIIa and TF, there is less known about the cell biology of these proteins. Availability of hemophilic mice (by specific knock-out of FVIII or FIX genes) and novel TF transgenic mice has allowed us in recent years to investigate the importance of TF-FVIIa-induced coagulation from wound healing to sepsis. This supplement explores new aspects of TF-FVIIa biology, with a particular focus on structural biology, cell biology and animal models.

Topics: Animals; Cell-Derived Microparticles; Disease Models, Animal; Factor IX; Factor VIIa; Hemophilia A; Hemostasis; Humans; Mice; Mice, Knockout; Sepsis; Thromboplastin; Wound Healing

Wound healing involves a number of physiologic mechanisms including coagulation, inflammation, formation of granulation tissue, and tissue remodeling. Coagulation with robust thrombin generation leading to fibrin formation is necessary for wound healing. It is less clear if there is a requirement for ongoing coagulation to support tissue remodeling. We have studied wound healing in mice with defects in both the initiation (low tissue factor) and propagation (hemophilia B) phases. In hemophilia B mice, dermal wound healing is delayed; this delay is associated with bleeding into the granulation tissue. Mice can be treated with replacement therapy (factor IX) or bypassing agents (factor VIIa) to restore thrombin generation. If treated just prior to wound placement, mice will have normal hemostasis in the first day of wound healing. As the therapeutic agents clear, the mice will revert to hemophilic state. If the primary role of coagulation in wound healing is to provide a stable platelet/fibrin plug that is loaded with thrombin, then treating hemophilic animals just prior to wound placement should restore normal wound healing. The results from this study did not support that hypothesis. Instead the results show that restoring thrombin generation only at the time of wound placement did not improve the delayed wound healing. In preliminary studies on low tissue factor mice, there also appears to be a delay in wound healing with evidence of bleeding into the granulation tissue. The current data suggests that ongoing coagulation function needs to be maintained to support a normal wound healing process.

Topics: Animals; Blood Coagulation; Disease Models, Animal; Factor VIIa; Hemophilia A; Hemophilia B; Mice; Mice, Transgenic; Models, Biological; Signal Transduction; Thrombin; Thromboplastin; Wound Healing

There has recently been intense interest in the clinical measurement of tissue factor (TF)-positive microparticles (MPs) in clinical disease states. This interest has been driven by the demonstration of an putative role for circulating TF-positive MPs in animal models of thrombus propagation. Both immunological and functional assays for MP-TF have been described. While each approach has its own advantages and drawbacks, neither has yet been truly established as the 'gold standard'. Heterogeneity of TF-bearing MPs, such as the variable co-expression of surface phosphatidylserine, may determine not only their procoagulant potential, but also additional properties including rate of clearance from the circulation.

Topics: Animals; Biomarkers; Blood Coagulation; Cell-Derived Microparticles; Disease Models, Animal; Humans; Immune System; Models, Biological; Particle Size; Phosphatidylserines; Thrombin; Thromboplastin; Thrombosis

Antiphospholipid syndrome (APS) is an acquired autoimmune disorder defined by the presence of an antiphospholipid antibody (aPL) and the occurrence of at least one associated clinical condition that includes venous thrombosis, arterial thrombosis or pregnancy morbidity. The aPL detected in APS have long been thought to have a direct prothrombotic effect in vivo. However, the pathophysiology underlying their coagulopathic effect has not been defined. Emerging data suggest a role for the procoagulant protein tissue factor (TF). In this review we provide an overview of TF, describe mouse models used in the evaluation of the role of TF in thrombosis, as well as summarize recent work on TF and APS.

Topics: Animals; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Disease Models, Animal; Female; Humans; Mice; Pregnancy; Pregnancy Complications; Thromboplastin; Thrombosis

Mouse models of thrombosis have extended our understanding of the role of tissue factor (TF) in thrombogenesis. Because tissue factor deficiency is embryonic lethal in mice, inventive genetic models are required to probe the role of TF in thrombosis. TF is expressed by different cell types, including vascular smooth muscle cells, cardiomyocytes, fibroblasts, and monocytes. Platelets and endothelial cells also express TF under certain conditions, but the importance of this TF remains controversial. Animal models are commonly used to evaluate the contribution of TF from each cell type to thrombogenesis. Although a variety of well-established injury techniques are used to induce thrombosis, it is likely that the sources of TF that drive thrombosis are model dependent. Therefore, rigorous controls are needed before thrombogenesis can be attributed to TF from a particular cell type. This review summarizes data from mouse models that have attempted to delineate the role of TF in thrombus formation in response to various types of vascular injury. We have consolidated this information to generate unifying concepts that require testing in future studies.

Topics: Animals; Blood Coagulation; Blood Vessels; Chlorides; Disease Models, Animal; Evidence-Based Medicine; Ferric Compounds; Genotype; Mice; Mice, Transgenic; Phenotype; Reproducibility of Results; Thromboplastin; Thrombosis

Thrombosis, or complications from thrombosis, currently occupies the top three positions in the cardiovascular causes of morbidity and mortality in the developed world. There are a limited number of safe and effective drugs to prevent and treat thrombosis. Animal models of thrombosis are necessary to better understand the complex components and interactions involved in the formation of a clot. Tissue factor (TF) is required for the initiation of blood coagulation and likely plays a key role in both arterial and venous thrombosis. Understanding the role of TF in thrombosis may permit the development of new antithrombotic drugs. This review will focus on the role of TF in in vivo models of thrombosis.

Topics: Animals; Arterial Occlusive Diseases; Blood Coagulation; Disease Models, Animal; Disseminated Intravascular Coagulation; Fibrinolytic Agents; Humans; Microcirculation; Thromboplastin; Thrombosis; Venous Thrombosis

Tissue factor (TF) is a transmembrane glycoprotein that localizes the coagulation serine protease factor VII/VIIa (FVII/VIIa) to the cell surface. The primary function of TF is to activate the clotting cascade. The TF:FVIIa complex also activates cells by cleavage of a G-protein coupled receptor called protease-activated receptor 2 (PAR2). TF is expressed by tumor cells and contributes to a variety of pathologic processes, such as thrombosis, metastasis, tumor growth, and tumor angiogenesis. For instance, tumor cells release TF-positive procoagulant microparticles into the circulation and these may trigger venous thromboembolism in patients with cancer. TF on circulating tumor cells also leads to the coating of the cells with fibrin that traps them within the microvasculature and facilitates hematogenous metastasis. In addition, TF:FVIIa-dependent activation of PAR2 on tumor cells increases tumor growth via an undefined mechanism. One possibility is that PAR2-dependent signaling increases the expression of proangiogenic proteins. Other studies have reported that endothelial cells in the tumor vasculature express TF and this may enhance angiogenesis. These results suggest that inhibition of TF should reduce several pathologic pathways that increase tumor growth and metastasis. This would represent a novel approach to anticancer therapy. Initial studies using inhibitors of the TF:FVIIa complex in mouse tumor models have produced encouraging results. Nevertheless, additional studies are needed to determine if this strategy can be successfully translated to the treatment of cancer patients.

Topics: Animals; Biomarkers, Tumor; Disease Models, Animal; Disease Progression; Factor VIIa; Female; Humans; Male; Mice; Neoplasm Invasiveness; Neoplasms; Neovascularization, Pathologic; Prognosis; Receptor, PAR-2; Sampling Studies; Severity of Illness Index; Survival Analysis; Thromboplastin; Tumor Burden; Venous Thrombosis

The hemostatic process is a host defense mechanism to preserve the integrity of the closed high pressure circulatory system. This process must remain inactive but poised to minimize extravasation of blood from the vasculature following tissue injury. Given the complexity of the hemostatic mechanism, paradigms developed from biochemical and cell biological approaches have been revisited by studying thrombus formation in a live animal by intravital microscopy. Many of these paradigms have proven accurate, but others need to be reconsidered given the results of whole animal experiments.

Topics: Animals; Blood Coagulation Factors; Cell-Derived Microparticles; Chlorides; Collagen; Disease Models, Animal; Ferric Compounds; Fibrin; Integrin beta3; Lasers; Mesentery; Mice; Mice, Knockout; Microscopy, Fluorescence; Muscle, Skeletal; Platelet Aggregation; Protein Disulfide-Isomerases; Thromboplastin; Thrombosis

Tissue factor (TF) is the primary initiator of the coagulation cascade and plays an essential role in hemostasis. TF also contributes to many diseases, including cancer. The correlation between thrombosis and cancer has been recognized for more than a century. However, it is only in the past two decades that we have begun to understand the role of TF in tumor biology. TF expression is upregulated on both tumor and host cells in cancer patients as well as in the circulation. Clinical observations indicate a direct correlation between the levels of tumor cell TF expression and poor prognosis for cancer patients. The role of TF in tumor biology has been extensively studied using various mouse tumor models. It has been demonstrated that tumor cell TF contributes to tumor metastasis, growth, and angiogenesis. The role of host TF in tumor progression is less clear. Recently developed mouse models with altered levels of TF may be useful in further analysis of the role of host cell TF in cancer.

Topics: Animals; Disease Models, Animal; Mice; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms; Neovascularization, Pathologic; Thromboplastin; Up-Regulation

During normal pregnancy, the renin-angiotensin system (RAS) plays a vitally important role in salt balance and subsequent well-being of mother and fetus. In this balance, one must consider not only the classical renal RAS but also that of the uteroplacental unit, where both maternal and fetal tissues contribute to the signaling cascade. Many studies have shown that in normal pregnancy there is an increase in almost all of the components of the RAS. In derangements of pregnancy this delicate equilibrium can become unbalanced. Preeclampsia is one such case. It is a disorder of pregnancy characterized by hypertension, proteinuria and placental abnormalities associated with shallow trophoblast invasion and impaired spiral artery remodeling. Despite being a leading cause of maternal death and a major contributor to maternal and perinatal morbidity, the mechanisms responsible for the pathogenesis of preeclampsia are poorly understood. Immunological mechanisms and the RAS have been long considered to be involved in the development of preeclampsia. Numerous recent studies demonstrate the presence of the angiotensin II type I receptor agonistic autoantibody (AT1-AA). This autoantibody can induce many key features of the disorder and upregulate molecules involved in the pathogenesis of preeclampsia. Here we review the functional role of the RAS during pregnancy and the impact of AT1-AA on preeclampsia.

Topics: Angiotensin II; Animals; Autoantibodies; Calcium; Disease Models, Animal; Female; Humans; Peptidyl-Dipeptidase A; Placenta; Placenta Diseases; Plasminogen Activator Inhibitor 1; Pre-Eclampsia; Pregnancy; Reactive Oxygen Species; Renin-Angiotensin System; Thromboplastin; Vascular Endothelial Growth Factor Receptor-1

Fetal loss in patients with antiphospholipid antibodies (aPL) has been ascribed to thrombosis of placental vessels. However, we have shown that inflammation, specifically complement activation with generation of the anaphylotoxin C5a, is an essential mediator of fetal injury. We have analysed the role of tissue factor (TF) in a mouse model of aPL-induced pregnancy loss. TF is the major cellular activator of the coagulation cascade but also has cell signaling activity. Mice that received aPL-IgG showed strong TF staining throughout the decidua and on embryonic debris. This TF staining was not associated with either fibrin staining or thrombi in deciduas. The absence of fibrin deposition and thrombi suggests that TF-dependent activation of coagulation does not mediate aPL-induced pregnancy loss.We found that either blockade of TF with a monoclonal antibody in wild type mice or a genetic reduction of TF prevented aPL-induced inflammation and pregnancy loss indicated a pathogenic role for TF in aPL-induced pregnancy complications. In response to aPL-generated C5a, neutrophils express TF potentiating inflammation in the deciduas and leading to miscarriages. Importantly, we showed that TF in myeloid cells, but not fetal-derived cells (trophoblasts), was associated with fetal injury, suggesting that the site for pathologic TF expression is neutrophils. We found that TF expression in neutrophils contributes to respiratory burst and subsequent trophoblast injury and pregnancy loss induced by aPL. The identification of TF, acting as an important pro-inflammatory mediator in aPL-induced fetal injury, provides a new target for therapy to prevent pregnancy loss in the aPL syndrome.

Topics: Animals; Antibodies, Antiphospholipid; Antigen-Antibody Reactions; Antiphospholipid Syndrome; Disease Models, Animal; Female; Fetal Death; Humans; Mice; Pregnancy; Thromboplastin

The classic intrinsic pathway of coagulation is triggered by contact activation of the plasma protease factor (F)XII, followed by sequential proteolytic activation of FX1 and FIX. While a key mechanism for initiating coagulation in some clinically useful in vitro assays, the absence of abnormal bleeding associated with congenital FXII deficiency indicates that the intrinsic pathway is not important for normal blood coagulation in vivo. However, recent work with mice lacking FXII or FXI suggest that these proteases make important contributions to formation of pathologic intravascular thrombi. In models of arterial injury, FXII or FXI null mice are protected from formation of platelet rich occlusive thrombi to a degree similar to that seen in FIX deficient mice (a model for the severe bleeding disorder hemophilia B) or to wild type mice treated with high dose heparin. FXII or FXI deficiency does not appear to prevent the initiation of thrombus formation in these models, but instead causes significant thrombus instability that prevents occlusion of the vessel. These findings raise the possibility that a pathway similar or identical to the intrinsic pathway may operate in vivo under some circumstances. Furthermore, the disproportionate importance of FXII and FXI to occlusive thrombus formation compared to normal hemostasis makes these proteases attractive candidates for therapeutic inhibitors to treat or prevent thromboembolic disorders.

Topics: Animals; Anticoagulants; Blood Coagulation; Disease Models, Animal; Factor XI; Factor XII; Humans; Mice; Models, Cardiovascular; Thromboembolism; Thromboplastin

During the last 15 years, transgenic mice have been generated that carry defective and/or mutant alleles of the natural anticoagulant pathways and display a spontaneous thrombotic phenotype. With the generation of these mouse lines, better opportunities became available for investigating both existing and novel risk factors for venous thrombosis. In addition, these models could serve as a tool for evaluating novel antithrombotic strategies. This review summarizes these mouse models and evaluates whether they have fulfilled the expectations.

Topics: Animals; Disease Models, Animal; Factor V; Factor Xa; Humans; Mice; Mice, Knockout; Mice, Transgenic; Protein C; Thrombomodulin; Thromboplastin; Venous Thrombosis

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

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

Topics: Animals; Disease Models, Animal; Mice; Microcirculation; Microscopy, Video; Platelet Activation; Thromboplastin; Thrombosis

The development of anticoagulants for treating patients with atherothrombotic disorders of the arterial circulatory system has focused, either directly or indirectly, on thrombin - a pleuripotential effector enzyme with prothrombotic and proinflammatory properties. The pivotal role of factor (f) Xa in thrombin generation, coupled with its direct cellular effects and widely recognized limitations of currently available anticoagulants, has led to the development of pharmacologic inhibitors of this important protease. The following review focuses on DX-9065a - first in a class of direct, selective and reversible fXa antagonists - and its potential applications in the management of patients with cardiovascular disease.

Topics: Animals; Anticoagulants; Area Under Curve; Blood Coagulation; Cardiovascular Diseases; Clinical Trials as Topic; Coronary Artery Disease; Disease Models, Animal; Dose-Response Relationship, Drug; Factor Xa; Factor Xa Inhibitors; Humans; International Normalized Ratio; Mice; Models, Biological; Models, Chemical; Naphthalenes; Pilot Projects; Platelet Aggregation Inhibitors; Propionates; Rats; Thrombin; Thromboplastin; Thrombosis; Time Factors

Reperfusion of the ischemic heart is necessary to prevent irreversible injury of the myocardium, which leads to permanent organ dysfunction. However, reperfusion in itself leads to myocardial ischemia/reperfusion (I/R) injury, which is characterized by an acute inflammatory response mediated by activated inflammatory cells, chemokines, cytokines, and adhesion molecules. The molecular mechanisms of myocardial I/R injury are not completely known. Tissue factor (TF) and thrombin, two potent procoagulant and proinflammatory mediators, are recognized to play significant roles in myocardial I/R injury. To investigate the role of TF and thrombin in myocardial I/R injury, we used rabbit and murine in situ coronary artery ligation models. Increased TF mRNA, antigen, and activity were found in ischemic cardiomyocytes. Administration of an inhibitory antirabbit TF monoclonal antibody before or during the onset of ischemia resulted in a significant reduction in infarct size. Functional inhibition of thrombin with hirudin also reduced the infarct size. However, defibrinogenating rabbits with ancrod had no effect on infarct size, suggesting a requirement of thrombin generation but not fibrin deposition in myocardial I/R injury.

Topics: Ancrod; Animals; Antibodies, Monoclonal; Disease Models, Animal; Fibrinolytic Agents; Hirudins; Mice; Mice, Knockout; Myocardial Reperfusion Injury; Myocardium; Rabbits; Receptor, PAR-1; Receptors, Thrombin; RNA, Messenger; Thrombin; Thromboplastin

Tissue factor (TF) is a transmembrane glycoprotein, currently considered as being the major regulator of the coagulation cascade and the initiator of thrombogenesis in vivo. When TF comes in contact with blood, it forms a high-affinity complex with factors VII/VIIa, activating factors IX and X and thus leading to the formation of an insoluble fibrin clot. The regulation of TF-VIIa activity plays a key role in blood-vessel wall interactions. Selective patterns of cellular expression of TF are observed in tissues. TF is constitutively localized only on the surface of cells anatomically separated from the blood, where it plays an essential role in hemostasis by limiting hemorrhage after vessel wall injury. A number of pathophysiologic stimuli are capable of inducing TF transcription and activity in endothelial cells and monocytes. An aberrant TF expression in contact with blood is implicated in thrombotic complications of atherosclerosis, including acute myocardial infarction. Recent findings have demonstrated cell-derived microparticles containing TF in the circulating blood of patients with acute coronary syndromes, capable of triggering and propagating thrombus growth. This observation suggests a new view of thrombosis that does not necessarily require the exposure of vessel wall-derived TF at the site of vascular injury to initiate and propagate thrombosis.

Topics: Angina, Unstable; Animals; Arteriosclerosis; Blood Coagulation; Cardiovascular Diseases; Disease Models, Animal; Factor IX; Factor VII; Factor VIIa; Factor X; Fibrinolytic Agents; Helminth Proteins; Hemostasis; Humans; Myocardial Infarction; Syndrome; Thromboplastin; Thrombosis

There is now considerable evidence that the blood coagulation system plays an important role in the biology of malignant tumors. This evidence has been derived from a combination of clinical, biochemical, histological, and pharmacological observations that point to the possibility of favorably affecting the course of malignant disease with agents that interfere with blood coagulation pathways. For a number of years our laboratory has used experimental models of blood-borne metastasis to study the events that follow the introduction of procoagulant-bearing tumor cells into the circulating blood. This article summarizes our experience with these models, which suggests that intravascular coagulation is a necessary prelude to lung tumor formation and that interruption of coagulation pathways in various ways may be an effective antimetastatic strategy. We have shown that anticoagulation with commonly used agents such as unfractionated heparin and warfarin (Coumadin) prevent tumor formation by limiting the ability of tumor cells to be retained in the pulmonary microvasculature. Binding of fibrin-coated tumor cells to activated platelets is essential for this retention and, therefore, treatment with potent antiplatelet agents such as abciximab is also effective. The predominant tumor procoagulant is tissue factor (TF), and direct targeting of this protein with concanavalin A, monoclonal antibodies, and tissue factor pathway inhibitor (TFPI) has provided compelling evidence that TF is an important determinant of tumor seeding in these experimental models. Collectively, our data provide strong support for the concept that some form of anticoagulant therapy would be a useful adjunct to existing cancer treatments.

Topics: Animals; Anticoagulants; Blood Coagulation Factors; Disease Models, Animal; Hemostasis; Humans; Neoplasm Metastasis; Neoplasms, Experimental; Thromboplastin

In the pathogenesis of disseminated intravascular coagulation, dysfunctional natural anticoagulant pathways appear to play a pivotal role. In this article, we will address the mechanisms that contribute to this defect in the regulation of coagulation activation. Furthermore, we will explore the experimental and clinical evidence that restoration of these anticoagulant pathways results in clinical improvement.. We have searched and reviewed published articles on experimental studies of disseminated intravascular coagulation models in animals and clinical studies in patients with disseminated intravascular coagulation.. All three major anticoagulant pathways, that is, the antithrombin pathway, the protein C system, and tissue factor pathway inhibitor, are defective in sepsis and disseminated intravascular coagulation. Several mechanisms contribute to this defect. Restoration of these pathways, in principle, by administration of coagulation inhibitor concentrates or recombinant anticoagulant factors, appears to ameliorate the coagulation disorder and, more important, result in improvement of clinically relevant outcomes, such as a reduction of organ failure and mortality.. Restoration of disrupted physiologic anticoagulant pathways in disseminated intravascular coagulation is not only a logical point of impact in patients with sepsis and an activated coagulation system, but also is associated with an improved outcome in experimental and (initial) clinical studies.

Topics: Animals; Anticoagulants; Cytokines; Disease Models, Animal; Disseminated Intravascular Coagulation; Humans; Multiple Organ Failure; Protein C; Sepsis; Thrombin; Thromboplastin; Treatment Outcome

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

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

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

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

Acute lung injury (ALI) is characterized by fibrin deposition in the tissue and vascular spaces. Coagulation is activated after exposure to endotoxin or bacteria, and a procoagulant environment rapidly develops in the vascular, interstitial, and alveolar spaces of the lung. These changes are tissue factor (TF)-dependent and associated with increases in inflammatory cytokines. Procoagulant changes also occur in the lungs of patients with the acute respiratory distress syndrome (ARDS), suggesting that epithelial inflammation activates the extrinsic pathway. Many inflammatory mediators have specific effects on coagulation; however, the role of TF in regulation of pulmonary inflammatory responses is less clear. Here we report initial data on blockade of TF-initiated coagulation in baboons with Escherichia coli sepsis-induced ALI, using active site-inactivated FVIIa (FVIIai ASIS). Treatment with FVIIai prevented plasma fibrinogen depletion and attenuated fibrin deposition in the tissues. The drug also decreased systemic cytokine responses and inflammatory changes in the lung, including neutrophil infiltration, and decreased edema. Coagulation blockade with FVIIai improved lung function by preserving gas exchange and compliance, decreased pulmonary hypertension, and enhanced renal function. These results show that TF-FVIIa complex is an important regulatory site for the pathologic response of the lung to sepsis.

Topics: Animals; Disease Models, Animal; Humans; Papio; Respiratory Distress Syndrome; Thromboplastin

An occlusive thrombus in the coronary arteries is the critical pathological event that immediately precedes most cases of myocardial infarction. Often the thrombus originates with a bleed from a fissured atheroma. Atheroma formation, therefore, creates risk of thrombosis; asymptomatic episodes of thrombosis and healing contribute to the pathogenesis of atherosclerosis and the development of atherosclerotic plaques. Based largely on in vitro and animal model evidence, infectious agents and their products can activate the coagulation cascade enzymatically or by up-regulating tissue factor. By initiating a procoagulant response, infectious agents can indirectly trigger a prothrombotic response. Alternatively, some microbes can directly trigger platelet aggregation in vitro and in animal models, suggesting direct prothrombotic potential in human cardiovascular disease. Activation of coagulation and thrombosis characterizes the pathological response to infectious agents in human disseminated intravascular coagulation and infective endocarditis. Given the underlying biological plausibility, the cumulative lifetime burden of chronic pathogens may be expected to create risk of atherosclerosis and thrombosis, and, indirectly, signs of cardiovascular disease.

Topics: Animals; Antigens, Bacterial; Bacteremia; Bacterial Proteins; Blood Coagulation; Cardiovascular Diseases; Collagen; Coronary Artery Disease; Coronary Thrombosis; Disease Models, Animal; Disseminated Intravascular Coagulation; Endocarditis, Bacterial; Humans; Platelet Aggregation; Risk Factors; Thromboplastin; Thrombosis

Abnormal expression of fibrinolytic genes [e.g., tissue-type and urokinase-type plasminogen activators (t-PA and u-PA) and their specific inhibitor (PAI-1)] and of the procoagulant molecule tissue factor (TF), has been reported in various types of renal diseases. In this review, the expression pattern of these genes was demonstrated in two murine models of renal disease. One is acute renal failure due to microthrombosis under septic conditions, using endotoxin-treated mice, and the other one is lupus nephritis observed in female MRL lpr/lpr mice. Quantitative reverse transcription-polymerase chain reaction analysis and in situ hybridization were employed to investigate the expression of their mRNAs in tissues from endotoxin-treated mice or from MRL lpr/lpr mice. A dramatic increase in PAI-1 activity in plasma and in PAI-1 mRNA in the kidneys was observed in both models, and this increase appeared to correlate with fibrin deposition in the renal microvasculature and with the progression of lupus nephritis. In addition to these changes in PAI-1, decreases in u-PA mRNA and increases in TF mRNA were demonstrated in the kidneys from lupus-prone mice as a function of age. Similar changes were also observed in the kidneys from endotoxin-treated mice. The induction of PAI-1 and TF, and the decrease in u-PA expression in the kidneys of lupus-prone or of endotoxemic mice may promote the formation of renal microthrombi and thus contribute to the progression of renal damage in these models.

Topics: Aging; Animals; Disease Models, Animal; Female; Fibrinolysis; Gene Expression Regulation; Humans; Kidney; Kidney Diseases; Mice; Plasminogen Activator Inhibitor 1; Thromboplastin; Urokinase-Type Plasminogen Activator

The coagulation cascade is initiated by binding of plasma activated or non-activated factor VII [FVII(a)] to cell surface tissue factor (TF). TF-induced coagulation plays a primary role not only in haemostasis but also in the pathogenesis of various thrombotic disorders. Recent studies with animal model systems showed that the administration of active site-inhibited FVIIa (FVIIai) blocked TF-FVIIa-induced fibrin and thrombus formation. These data suggest that FVIIai competes with plasma FVII(a) for a limited number of TF sites expressed on cells either constitutively or induced after the perturbation. To obtain insights into the mechanism(s) by which FVIIai is effective in inhibiting TF-FVIIa induced coagulation in vivo, we compared the interaction of FVIIai and FVIIa with TF using a variety of competition assays and direct binding assays. The TF-FVIIa amidolytic activity competition assay showed that FVIIai bound with a threefold higher affinity than that of FVIIa to TF relipidated in phosphatidylcholine (PC) vesicles, whereas no significant differences were found between FVIIa and FVIIai binding to TF if it had been relipidated in mixed phospholipid vesicles containing PC and phosphatidylserine (PS). When FVIIa and FVIIai binding to TF was analysed in a FXa generation assay, we found that FVIIai bound to TF in PCPS vesicles with two- to fivefold higher affinity than that of FVIIa, whereas the affinity of FVIIai for TF in PC vesicles was seven- to 10-fold higher than that of FVIIa. Direct binding analysis to TF, immobilized on a sensor chip or on a cell surface, showed a faster association and a slower dissociation of FVIIai to TF compared with that of FVIIa. Equilibrium binding to cell surface TF showed that the affinity of FVIIai was fivefold higher than that of FVIIa to non-functional TF, whereas both FVIIa and FVIIai bound functional TF with the same high affinity. The enhanced affinity of FVIIai to TF, particularly to non-functional TF, would make FVIIai a valuable reagent to block TF-induced coagulation before it is triggered by cell injury or a pathological stimuli.

Topics: Binding Sites; Disease Models, Animal; Factor VIIa; Fibrinolytic Agents; Humans; Protein Binding; Thromboplastin

Topics: Animals; Basement Membrane; Blood Coagulation; Disease Models, Animal; Glomerulonephritis; Humans; Kidney Diseases; Kidney Glomerulus; Macrophages; Thromboplastin

Thrombosis is the most frequent complication and the second cause of death in patients with overt malignant diseases. Increasing evidence suggests that thrombotic episodes may also precede the diagnosis of cancer by months or years thus representing a potential marker for occult malignancy. Recently, emphasis has been given to the potential risk of cancer therapy (both surgery and chemotherapy) in enhancing the risk for thromboembolic disease. Post-operative deep-vein thrombosis is indeed more frequent in patients operated for malignant diseases than for other disorders. On the other hand, both chemotherapy and hormone therapy are associated with an increased thrombotic risk, which can be prevented by low-dose oral anticoagulation. Possible contributory causes for thromboembolic disease in cancer include the capacity of tumor cells and their products to interact with platelets, clotting and fibrinolytic systems, as well as their interactions with endothelial cells and tumor-associated macrophages. In particular, procoagulant activities of tumor cells have been extensively studied; one of these, cancer procoagulant, could represent a novel marker of malignancy in both solid tumors and acute promyelocytic leukemia (APL). In solid tumors, CP, a vitamin K dependent enzyme could represent the selective target of the antimetastatic effects of warfarin treatment. In APL, CP may contribute to trigger the well known intravascular coagulation syndrome accompanying the early manifestations of the disease and is depressed by all-trans-retinoic acid, an agent capable to determine complete remission with a rapid amelioration of the bleeding syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Antineoplastic Agents; Cysteine Endopeptidases; Disease Models, Animal; Disease Progression; Hormones; Humans; Leukemia, Promyelocytic, Acute; Mice; Mice, Transgenic; Neoplasm Proteins; Neoplasms; Neoplasms, Experimental; Neoplasms, Unknown Primary; Plasminogen Activator Inhibitor 1; Thrombophlebitis; Thromboplastin

The baboon model of E. coli sepsis illustrates three concepts with respect to the host response and vascular endothelium. First, the endothelium is the primary target. E. coli sepsis is an acute inflammatory disease of the vascular endothelium. Second, the endothelium is not a passive target. Initially it regulates both the inflammatory and coagulopathic aspects of E. coli sepsis through membrane associated regulatory receptor/plasma protein assemblies including protein C/thrombomodulin, activated protein C/protein S, C4bBP/protein S, tissue factor pathway inhibitor/Xa, antithrombin III/glycosaminoglycans. Third, when overridden by inflammatory events, the endothelium can change its anticoagulant phenotype and mount a massive procoagulant fibrinolytic counter-attack on its luminal side through the expression of tissue factor and release of tissue plasminogen activator. Fourth, again when overridden by inflammatory events, the endothelium can change its antioxidant phenotype and produce a "distal" tissue hypoxia on its abluminal side through induction of free radical generation and peroxidation of mitochondrial lipid membranes of those tissues with high metabolic rates. It has become increasingly clear that the so-called anticoagulant systems which act on the proximal factors of the clotting cascade (protein C, TFPI, AT-III, PGI2) also attenuate the amplification of the inflammatory response. Aspects of the mechanism by which this occurs are coming to light. This includes the attenuation of Il-6 response by TFPI and the attenuation of the complement effects by C4bBP/PS. The specifics of these observations in the E. coli sepsis model will be reviewed.

Topics: Animals; Antibodies; Anticoagulants; Carrier Proteins; Complement Inactivator Proteins; Disease Models, Animal; Disseminated Intravascular Coagulation; Endothelium, Vascular; Escherichia coli Infections; Glycoproteins; Interleukin-6; Lipoproteins; Papio; Protein C; Protein S; Thromboplastin; Tumor Necrosis Factor-alpha

Reversible acute disseminated intravascular coagulation (DIC) has been induced in dogs by intravenous injection of homologous tissue thromboplastin. There was no measurable consumption of antithrombin III and heparin cofactor II even if fibrinogen was reduced during DIC by more than 80% of its baseline. The prothrombin level remained practically constant. These data correspond to the generation of a few nanomoles of thrombin in vivo with subsequent pseudo-first order inactivation by the major thrombin inhibitors. An ex vivo measure of the pseudo-first order rate constant (dynamic thrombin inhibitory capacity, DTIC) was a sensitive probe of circulating heparin. There was no change of DTIC during DIC in the absence of exogenous heparin suggesting that heparin-like endogenous glycosaminoglycans were not released in substantial amounts. Pretreatment with heparin efficiently inhibited the development of tissue thromboplastin induced DIC. This animal model may serve as a tool for the study of glycosaminoglycan anticoagulants in vivo.

Topics: Animals; Antithrombin III; Disease Models, Animal; Disseminated Intravascular Coagulation; Dogs; Female; Fibrinogen; Glycoproteins; Glycosaminoglycans; Heparin; Heparin Cofactor II; Male; Thrombin; Thromboplastin

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

Lower extremity deep venous thrombosis (LEDVT) is a venous reflux disorder caused by abnormal coagulation of blood. LEDVT can obstruct the lumen and LEDVT is the third vascular disease after cerebrovascular diseases and coronary artery diseases. miRNAs are associated with thrombosis, and miR-185 was reported to affect the proliferation and apoptosis of vascular endothelial cells by regulating receptor of advanced glycation end products (RAGE). However, no study has reported the effect of miR-185 on LEDVT. Here, we studied the effects of miR-185 on the PI3K/AKT and MAPK signaling pathways in the LEDVT cells. The results showed that miR-185 promotes cell proliferation through activating the PI3K/AKT and MAPK signaling pathways and then inhibits tissue factor and fibrin expression to reduce thrombosis. In short, our study provides new ideas and a theoretical basis for research on the prevention, diagnosis, and treatment of LEDVT.

Topics: Animals; Cell Proliferation; Cells, Cultured; Chromones; Disease Models, Animal; Endothelial Cells; Fibrin; Male; MAP Kinase Signaling System; Mice; MicroRNAs; Morpholines; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Thromboplastin; Venous Thrombosis

Mesenchymal stromal cells (MSCs) express surface tissue factor (TF), which may affect hemostasis and detract from therapeutic outcomes of MSCs if administered intravenously. In this study, we determine a safe dose of MSCs for intravenous (IV) administration and further demonstrate the impact of IV-MSC on acute traumatic coagulopathy (ATC) in rats.. Tissue factor expression of rat bone marrow-derived mesenchymal stromal cell (BMSC) or adipose-derived mesenchymal stromal cell (AMSC) was detected by immunohistochemistry and enzyme-linked immunosorbent assay. The coagulation properties were measured in MSC-treated rat whole blood, and blood samples were collected from rats after IV administration of MSCs. Acute traumatic coagulopathy rats underwent polytrauma and 40% hemorrhage, followed by IV administration of 5 or 10 million/kg BMSCs (BMSC-5, BMSC-10), or vehicle at 1 hour after trauma.. Rat MSCs expressed TF, and incubation of rat BMSCs or AMSCs with whole blood in vitro led to a significantly shortened clotting time. However, a dose-dependent prolongation of prothrombin time with reduction in platelet counts and fibrinogen was found in healthy rat treated with IV-MSCs. Bone marrow-derived mesenchymal stromal cells at 5 million/kg or less led to minimal effect on hemostasis. Mesenchymal stromal cells were not found in circulation but in the lungs after IV administration regardless of the dosage. Acute traumatic coagulopathy with prolonged prothrombin time was not significantly affected by 5 or 10 million/kg BMSCs. Intravenous administration of 10 million/kg BMSCs led to significantly lower fibrinogen and platelet counts, while significantly higher levels of lactate, wet/dry weight ratio, and leukocyte infiltration in the lung were present compared with BMSC-5 or vehicle. No differences were seen in immune or inflammatory profiles with BMSC treatment in ATC rats, at least in the acute timeframe.. Intravenous administration of MSCs leads to a risk of coagulopathy associated with a dose-dependent reduction in platelet counts and fibrinogen and is incapable of restoring hemostasis of rats with ATC after polytrauma and hemorrhagic shock.

Topics: Administration, Intravenous; Animals; Blood Coagulation Disorders; Blood Coagulation Tests; Disease Models, Animal; Mesenchymal Stem Cell Transplantation; Multiple Trauma; Platelet Count; Rats; Shock, Hemorrhagic; Thromboplastin

Tissue factor (TF) is a critical initiator of extrinsic coagulation that sometimes causes thromboembolism. Diallyl trisulphide (DATS) is a secondary metabolite of allicin generated in crushed garlic, with various pharmacological effects. This study aimed to clarify the effect of DATS on the extrinsic coagulation elicited by TF and arteriosclerosis. TF activity was measured using a clotting assay in TF-expressing HL60 cells. DATS inhibited TF activity in a dose-dependent manner. TF expression in TNF-α-stimulated human umbilical vein endothelial cells was examined using real-time PCR and western blotting. DATS inhibited TF mRNA and protein expression induced by TNF-α

Topics: Allyl Compounds; Animals; Antioxidants; Apoptosis; Disease Models, Animal; Garlic; Male; Mice; Sulfides; Thromboplastin

Topics: Animals; Bacterial Infections; Disease Models, Animal; Mice; Mice, Inbred C57BL; Rats; Spleen; Thromboplastin

Pulmonary hypertension (PAH) is a proliferative disease of pulmonary blood vessels, but the pathogenesis of pulmonary hypertension is still unclear. This article explores the role of tumor necrosis factor-

Topics: Animals; Blood Coagulation; Disease Models, Animal; Drug Carriers; Hypertension, Pulmonary; Monocrotaline; Nanoparticles; Pulmonary Artery; Rats; Sildenafil Citrate; Thromboplastin; Tumor Necrosis Factor-alpha

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

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

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

Topics: Animals; Blood Coagulation; Cell Line, Tumor; Disease Models, Animal; Factor XI; Humans; Mice; Pancreatic Neoplasms; Thromboplastin; Thrombosis

Endotoxin-induced lung injury is one of the major causes of death induced by endotoxemia, however, few effective therapeutic options exist. Hydrogen inhalation has recently been shown to be an effective treatment for inflammatory lung injury, but the underlying mechanism is unknown. In the current study we aim to investigate how hydrogen attenuates endotoxin-induced lung injury and provide reference values for the clinical application of hydrogen. LPS was used to establish an endotoxin-induced lung injury mouse model. The survival rate and pulmonary pathologic changes were evaluated. THP-1 and HUVECC cells were cultured

Topics: Acute Lung Injury; Animals; Anti-Inflammatory Agents; Coculture Techniques; Disease Models, Animal; Down-Regulation; Human Umbilical Vein Endothelial Cells; Humans; Hydrogen; Interleukin-6; Lipopolysaccharides; Lung; Macrophages; Male; Matrix Metalloproteinase 9; Mice, Inbred ICR; Neutrophil Infiltration; Pulmonary Edema; Signal Transduction; Thioredoxins; THP-1 Cells; Thromboplastin

Lipopolysaccharide (LPS) and tissue factor (TF) have frequently been used to induce disseminated intravascular coagulation (DIC) in experimental animal models. We have previously reported that the pathophysiology of DIC differs according to the inducing agents. However, inflammatory status and bleeding symptoms have not been fully compared between rat models of the two forms of DIC. We attempted to evaluate detailed characteristic features of LPS- and TF-induced DIC models, especially in regard to inflammatory status and bleeding symptoms, in addition to selected hemostatic parameters and pathologic findings in the kidneys. The degree of hemostatic activation in both types of experimental DIC was identical, based on the results of thrombin-antithrombin complex levels. Markedly elevated tumor necrosis factor, interleukin-6, and high-mobility group box-1 concentrations were observed with severe organ dysfunction and marked fibrin deposition in the kidney on administration of LPS, whereas markedly elevated D-dimer concentration and bleeding symptoms were observed with TF administration. Pathophysiology such as fibrinolytic activity, organ dysfunction, inflammation status, and bleeding symptom differed markedly between LPS- and TF-induced DIC models in rats. We, therefore, recommend that these disease models be assessed carefully as distinct entities to determine the implications of their experimental and clinical use.

Topics: Animals; Biomarkers; Blood Coagulation; Blood Coagulation Tests; Disease Models, Animal; Disease Susceptibility; Disseminated Intravascular Coagulation; Hemorrhage; Humans; Lipopolysaccharides; Male; Prognosis; Rats; Thromboplastin

It has recently been shown that expression levels of tissue factor (TF) are high in the serum and peripheral blood mononuclear cells of patients with asthma. However, whether TF impacts airway inflammation and remodelling in asthma remains unknown. The aim of this study was to investigate the effect of TF in asthma airway inflammation and remodelling using a house dust mite (HDM)-induced chronic asthma model and human bronchial epithelial (16HBE) cells. A chronic asthma model was constructed in BALB/c mice by the intranasal instillation of HDM. Mice were treated with short hairpin TF (shTF), and airway inflammation and remodelling features of asthma and epithelial-mesenchymal transition (EMT) were assessed. 16HBE cells were induced by transforming growth factor-β1 (TGF-β1) and HDM in the presence or absence of shTF; then, EMT markers and invasion and migration ability were determined. TF expression increased in the lung tissue and 16HBE cells when exposed to HDM. TF downregulation in the lung significantly reduced airway hyperresponsiveness, eosinophil inflammation, the EMT process, and levels of interleukin (IL)-4, IL-6, IL-13, and TGF-β1 in bronchoalveolar lavage fluid of asthmatic mice. Moreover, TF downregulation inhibited migration and incursion and decreased the expression levels of fibronectin 1 and TGF-β1, but increased the expression of E-cadherin in HDM- and TGF-β1-stimulated 16HBE cells. These results demonstrated that TF promoted airway pathological features by enhancing the EMT of bronchial epithelial cells both in vitro and in mice with house dust mite-induced asthma.

Topics: Airway Remodeling; Allergens; Animals; Asthma; Bronchi; Bronchoalveolar Lavage Fluid; Dermatophagoides pteronyssinus; Disease Models, Animal; Epithelial Cells; Epithelial-Mesenchymal Transition; HEK293 Cells; Humans; Mice; Specific Pathogen-Free Organisms; Thromboplastin; Up-Regulation

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

We investigated the molecular mechanism of paraoxonase-2 (PON-2) in regulating blood coagulation activation in rats with haemorrhagic shock through endothelial tissue factor (TF). Thirty adult Sprague Dawley rats were randomly divided into three groups: healthy control group (group A), the haemorrhagic shock PON-2 treatment group (group B), and the haemorrhagic shock group (group C). After the model was established, blood was withdrawn from the inferior vena cava of all rats. The difference in plasma thrombomodulin (TM) levels of the three groups was determined by Western blotting. The expression of transcription factors Egr-1 and Sp1 was detected by Western blotting assays. reverse transcription-polymerase chain Reaction (RT-PCR) was used to determine the mRNA expression of t-PA, PAI-1, TM, and PON-2 in the serum of three groups of rats. Endothelial TF was measured by enzyme linked immunosorbent assay (ELISA), and coagulation assay was used to detect the activity of coagulation factor VIII. Histopathological examination of the arteries of the rats was performed. The molecular mechanism of PON-2 in regulating blood coagulation activation in haemorrhagic shock model rats by endothelial tissue factor was analysed. The expression of thrombin was determined by electrophoresis. Compared with the healthy control group, the expression of TM in groups B and C decreased, both 188.64 ± 12.47 and 137.48 ± 9.72, respectively, with a significant difference. The mRNA expression of TM and PON was determined by RT-PCR. The mRNA expression of TM and PON in group B was 0.97 ± 0.07 and 1.14 ± 0.09, compared with the control group, and the mRNA expression of TM and PON in group C was 0.86 ± 0.38 and 1.12 ± 0.41, both of which increased, and there were significant differences. By measuring the expression of endothelial TF, the expression of TF in groups B and C was elevated to 12.69 ± 1.07 and 11.59 ± 0.87, with significant differences. The enzyme activities of PON-2 in groups B and C, which were 110.34 ± 14.37 and 52.37 ± 8.06, respectively, were increased compared with the healthy control group and there were significant differences. PON-2 regulates the activation of coagulation in rats with haemorrhagic shock by regulating the expression of endothelial tissue-related genes such as plasma TM and endothelial TF under hypoxic and ischaemic conditions.

Topics: Animals; Aryldialkylphosphatase; Blood Coagulation; Disease Models, Animal; Early Growth Response Protein 1; Female; Male; Rats; Rats, Sprague-Dawley; RNA, Messenger; Shock, Hemorrhagic; Sp1 Transcription Factor; Thrombomodulin; Thromboplastin

Topics: Acute Lung Injury; Animals; Biomarkers; Biopsy; Cysteine Endopeptidases; Disease Models, Animal; Immunophenotyping; JNK Mitogen-Activated Protein Kinases; Lipopolysaccharides; Macrophages; Mice; Mice, Knockout; Monocytes; Phosphorylation; Protein Kinase Inhibitors; Reactive Oxygen Species; Thromboplastin

Venous thromboembolism (VTE) carries a high risk of morbidity and mortality. Understanding the mechanisms of venous thrombus formation and resolution is critical for improving VTE management. AKT2 kinase is essential for platelet activation and arterial thrombosis. In this study, we examined the role of AKT2 in venous thrombosis in a mouse model of venous thrombosis induced by inferior vena cava (IVC) ligation. We observed an induction of AKT2 expression in the ligated IVC of wild-type (WT) mice. Interestingly, although the initial thrombus size of the ligated IVC was similar between Akt2

Topics: Animals; Apoptosis; Blood Coagulation; Disease Models, Animal; Endothelial Cells; Homeostasis; Mice; Mice, Knockout; Proto-Oncogene Proteins c-akt; Signal Transduction; Thrombomodulin; Thromboplastin; Thrombosis; Venous Thrombosis

Tissue factor (TF) is the primary initiator of blood coagulation in vivo and the only blood coagulation factor for which a human genetic defect has not been described. As there are no routine clinical assays that capture the contribution of endogenous TF to coagulation initiation, the extent to which reduced TF activity contributes to unexplained bleeding is unknown. Using whole genome sequencing, we identified a heterozygous frameshift variant (p.Ser117HisfsTer10) in F3, the gene encoding TF, causing premature termination of TF (TFshort) in a woman with unexplained bleeding. Routine hematological laboratory evaluation of the proposita was normal. CRISPR-edited human induced pluripotent stem cells recapitulating the variant were differentiated into vascular smooth muscle and endothelial cells that demonstrated haploinsufficiency of TF. The variant F3 transcript is eliminated by nonsense-mediated decay. Neither overexpression nor addition of exogenous recombinant TFshort inhibited factor Xa or thrombin generation, excluding a dominant-negative mechanism. F3+/- mice provide an animal model of TF haploinsufficiency and exhibited prolonged bleeding times, impaired thrombus formation, and reduced survival following major injury. Heterozygous TF deficiency is present in at least 1 in 25,000 individuals and could limit coagulation initiation in undiagnosed individuals with abnormal bleeding but a normal routine laboratory evaluation.

Topics: Animals; Base Sequence; Blood Coagulation Disorders, Inherited; Codon, Nonsense; Disease Models, Animal; Female; Frameshift Mutation; Gene Editing; Haploinsufficiency; Heterozygote; Humans; Induced Pluripotent Stem Cells; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Peptide Chain Termination, Translational; Phenotype; Thromboplastin

This study aimed to explore thrombolysis therapy based on ultrasound combined with urokinase and Arg-Gly-Asp sequence (RGDS)-targeted microbubbles by evaluating the histological changes in a thrombotic rabbit model. Forty-two New Zealand rabbits featuring platelet-rich thrombi in the femoral artery were randomized to (n = 6/group): ultrasound alone (US); urokinase alone (UK); ultrasound plus non-targeted microbubbles (US + M); ultrasound plus RGDS-targeted microbubbles (US + R); RGDS-targeted microbubbles plus urokinase (R + UK); ultrasound, non-targeted microbubbles and urokinase (US + M + UK); and ultrasound, RGDS-targeted microbubbles and urokinase (US + R + UK) groups. Diagnostic ultrasound was used transcutaneously over the thrombus for 30 min. We evaluated the thrombolytic effect based on ultrasound thrombi detection, blood flow, and histological observations. Among all study groups, complete recanalization was achieved in the US + R + UK group. Hematoxylin and eosin staining showed that the thrombi were completely dissolved. Scanning electron microscopy examination demonstrated that the fiber network structure of the thrombi was damaged. Transmission electron microscopy showed that the thrombus was decomposed into high electron-dense particles. Histology for von Willebrand factor and tissue factor were both negative in the US + R + UK group. This study revealed that a thrombolytic therapy consisting of diagnostic ultrasound together with RGDS-targeted and urokinase coupled microbubbles.

Topics: Animals; Blood Platelets; Contrast Media; Disease Models, Animal; Female; Femoral Artery; Male; Microbubbles; Muscle, Skeletal; Oligopeptides; Particle Size; Rabbits; Regional Blood Flow; Thrombolytic Therapy; Thromboplastin; Ultrasonography; Urokinase-Type Plasminogen Activator; von Willebrand Factor

Chemotherapeutic enteritis is a major dose-limiting adverse reaction to chemotherapy, with few effective drugs in clinic. Intestinal ischemic injury plays prominent role in chemotherapeutic enteritis clinically. However, mechanism is not clear. In this article, irinotecan (CPT-11) was used to establish chemotherapeutic enteritis mice model. Western blotting, gelatin zymography, immunohistochemistry (IHC), Laser Doppler flowmetry (LDF) were used to detect the pathogenesis of ischemia-hypoxia injury. CPT-11 increased levels of tissue factor (TF) both in the blood and in intestines, and decreased the intestinal blood flow in mice. Interestingly, the elevation of TF in the blood displayed "double-peak," which was consistent with the intestinal mucosal "double-strike" injury trend. Intestinal microthrombus and mixed thrombus formation were detectable in chemotherapeutic enteritis. Furthermore, ozone therapy relieved chemotherapeutic enteritis in mice. Ozone inhibited TF expression induced by CPT-11 via activating AMPK/SOCS3, and effectively ameliorated the intestinal mucosal injury in mice. Moreover, ozone autotransfusion therapy effectively attenuated chemotherapeutic enteritis and the blood hypercoagulability in patients. For the first time, we proposed that TF-induced thrombotic intestinal ischemic injury is a core trigger pathological mechanism of chemotherapeutic enteritis, and provided a new treatment strategy, ozone therapy, to suppress TF expression and treat chemotherapeutic enteritis.

Topics: Aged; AMP-Activated Protein Kinases; Animals; Disease Models, Animal; Enteritis; Female; Humans; Intestinal Mucosa; Irinotecan; Male; Mice; Middle Aged; Ozone; Reperfusion Injury; Thromboplastin

Topics: Administration, Topical; Animals; Blood Loss, Surgical; Disease Models, Animal; Female; Hemostasis, Surgical; Hemostatics; Hepatectomy; Humans; Liver; Male; Rats; Recombinant Proteins; Swine; Thromboplastin

Activated factor VII (FVIIa) is pertinent to the initiation of blood coagulation. Proteolytic and amidolytic activity of FVIIa are greatly enhanced by its cofactor, tissue factor (TF).. We aimed to generate a single-domain antibody (sdAb) that recognizes free FVIIa rather than TF-bound FVIIa.. A llama-derived phage library was used to screen for anti-FVIIa sdAbs.. This observation is compatible with the view that FVIIa functions independently of TF under these conditions. In conclusion, we have generated a sdAb that specifically blocks TF-independent activity of FVIIa. This antibody can be used to gain insight into the roles of TF-bound and TF-free FVIIa.

Topics: Animals; Anticoagulants; Blood Coagulation; Coagulants; Disease Models, Animal; Factor VIIa; Factor VIII; Female; Hemophilia A; Humans; Male; Mice, Inbred C57BL; Protein Binding; Single-Domain Antibodies; Thromboplastin

In situ thrombus formation is one of the major pathological features of pulmonary hypertension (PH). The mechanism of in situ thrombosis has not been clearly identified. Fibrinogen-like protein 2 (FGL2) prothrombinase is an immune coagulant that can cleave prothrombin to thrombin, which then converts fibrinogen into fibrin. This mechanism triggers in situ thrombus formation directly, bypassing both the intrinsic and extrinsic coagulation pathways. FGL2 prothrombinase is mainly expressed in endothelial cells and mediates multiple pathological processes. This implies that it may also play a role in PH. In this study, we examined the expression of FGL2 in idiopathic pulmonary arterial hypertension (IPAH) patients, and in monocrotaline-induced rat and hypoxia-induced mouse PH models.

Topics: Aged; Animals; Apoptosis; Disease Models, Animal; Down-Regulation; Endothelial Cells; Endothelium, Vascular; Fibrin; Fibrinogen; Humans; Hypertension, Pulmonary; Male; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Rats; Rats, Sprague-Dawley; Signal Transduction; Thromboplastin; Up-Regulation

Topics: Animals; Antibodies, Monoclonal, Humanized; Disease Models, Animal; Factor VIIa; Female; Hemophilia A; Hemorrhage; Hemostasis; Rabbits; Thromboplastin

Topics: Animals; Disease Models, Animal; Evolution, Molecular; Mice; Neoplasms; Receptors, Aryl Hydrocarbon; Thromboplastin

Patients with malignancy are at 4- to 7-fold higher risk of venous thromboembolism (VTE), a potentially fatal, yet preventable complication. Although general mechanisms of thrombosis are enhanced in these patients, malignancy-specific triggers and their therapeutic implication remain poorly understood. Here we examined a colon cancer-specific VTE model and probed a set of metabolites with prothrombotic propensity in the inferior vena cava (IVC) ligation model. Athymic mice injected with human colon adenocarcinoma cells exhibited significantly higher IVC clot weights, a biological readout of venous thrombogenicity, compared with the control mice. Targeted metabolomics analysis of plasma of mice revealed an increase in the blood levels of kynurenine and indoxyl sulfate (tryptophan metabolites) in xenograft-bearing mice, which correlated positively with the increase in the IVC clot size. These metabolites are ligands of aryl hydrocarbon receptor (AHR) signaling. Accordingly, plasma from the xenograft-bearing mice activated the AHR pathway and augmented tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1) levels in venous endothelial cells in an AHR-dependent manner. Consistent with these findings, the endothelium from the IVC of xenograft-bearing animals revealed nuclear AHR and upregulated TF and PAI-1 expression, telltale signs of an activated AHR-TF/PAI-1 axis. Importantly, pharmacological inhibition of AHR activity suppressed TF and PAI-1 expression in endothelial cells of the IVC and reduced clot weights in both kynurenine-injected and xenograft-bearing mice. Together, these data show dysregulated tryptophan metabolites in a mouse cancer model, and they reveal a novel link between these metabolites and the control of the AHR-TF/PAI-1 axis and VTE in cancer.

Topics: Animals; Colonic Neoplasms; Disease Models, Animal; Female; Humans; Male; Metabolome; Mice; Mice, Nude; Plasminogen Activator Inhibitor 1; Receptors, Aryl Hydrocarbon; Signal Transduction; Thromboplastin; Tryptophan; Venous Thromboembolism; Xenograft Model Antitumor Assays

Severe sepsis is critical to health and can result in acute renal failure (ARF). Tissue factor (TF) and thrombomodulin (TM) play key roles in vascular endothelial functions by helping maintain microcirculation in the kidney. Budding uninhibited by benzimidazole-1 (Bub1) plays a role in Akt and JNK signaling, which control TF and TM, respectively. We hypothesized that Bub1 could control vascular endothelial function in sepsis. The aim of this study was to determine the role of Bub1 in septic ARF. We used Mouse cecum ligation and puncture (CLP) using low Bub1 expressing (Bub1) and wild-type (Bub1) mice in vivo and lipopolysaccharide (LPS) stimulation of human aortic endothelial cell (HAEC) in vitro. Bub1 mice had a higher survival rate after CLP than Bub1. Bub1 mice had more severe ARF after CLP than Bub1 with blood biochemical and pathological analyses. TF expression in Bub1 mice and control HAEC (control) significantly increased in the septic model compared with Bub1 and Bub1 silenced HAEC (siBub1). TM expression in the control significantly decreased after LPS stimulation compared with siBub1. Akt and JNK phosphorylation of siBub1 were attenuated after LPS stimulation. Associations of Bub1 with Akt or JNK after LPS stimulation of HAEC were detected using immunoprecipitation, suggesting that Bub1 is involved in the phosphorylation of Akt and JNK after LPS stimulation. Bub1 insufficiency attenuates TF expression and reduces TM suppression by blocking Akt and JNK phosphorylation, respectively, thus leading to the prevention of ARF and death caused by sepsis.

Topics: Acute Kidney Injury; Animals; Disease Models, Animal; Endothelial Cells; Humans; Mice; Mice, Mutant Strains; Protein Serine-Threonine Kinases; Sepsis; Thrombomodulin; Thromboplastin

Essentials The coagulation initiator, tissue factor (TF), is on the herpes simplex virus 1 (HSV1) surface. HSV1 surface TF was examined in mice as an antiviral target since it enhances infection in vitro. HSV1 surface TF facilitated infection of all organs evaluated and anticoagulants were antiviral. Protease activated receptor 2 inhibited infection in vivo and its pre-activation was antiviral. SUMMARY: Background Tissue factor (TF) is the essential cell surface initiator of coagulation, and mediates cell signaling through protease-activated receptor (PAR) 2. Having a diverse cellular distribution, TF is involved in many biological pathways and pathologies. Our earlier work identified host cell-derived TF on the envelope covering several viruses, and showed its involvement in enhanced cell infection in vitro. Objective In the current study, we evaluated the in vivo effects of virus surface TF on infection and on the related modulator of infection PAR2. Methods With the use of herpes simplex virus type 1 (HSV1) as a model enveloped virus, purified HSV1 was generated with or without envelope TF through propagation in a TF-inducible cell line. Infection was studied after intravenous inoculation of BALB/c, C57BL/6J or C57BL/6J PAR2 knockout mice with 5 × 10

Topics: Animals; Anticoagulants; Antiviral Agents; Disease Models, Animal; Female; Herpes Simplex; Herpesvirus 1, Human; Host-Pathogen Interactions; Injections, Intravenous; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Receptor, PAR-2; Th1 Cells; Thromboplastin; Viral Envelope Proteins

Atherosclerosis is the pathological process in arteries due to the plaque formation that is responsible for several diseases like heart disease, stroke and peripheral arterial disease. In this study, we performed in vitro and in vivo assays to evaluate the potential anti-atherosclerosis activity of peach kernel oil. For the in vitro assay, we incubated human umbilical vein endothelial cells (HUVEC) with tumor necrosis factor-α (TNF-α) to induce tissue factors (TF, an essential mediator of hemostasis and trigger of thrombosis) elevation. We found that TNF-α-induced TF elevation was suppressed by peach kernel oil in a dose-dependent manner at both mRNA and protein levels. Peach kernel oil can significantly improve HUVEC viability, protect the endothelial cells, which achieved the goal of prevention of thrombotic diseases. For the in vivo assay, we investigated the effect and mechanism of peach kernel oil on preventing atherosclerotic lesion formation in ApoE knockout mice. Results show that peach kernel oil could reduce total cholesterol, triglyceride, low-density lipoprotein cholesterol levels, elevate the high-density lipoprotein cholesterol level in serum, and reduce the area of the aortic atherosclerotic lesions in high-fat diet fed ApoE knockout mice. Moreover, peach kernel oil treatment can significantly down regulate the expression of TF protein to inhibit the formation of atherosclerotic plaque. In conclusion, peach kernel oil may be a potential health food to prevent atherosclerosis in cardiovascular diseases.

Topics: Animals; Aorta; Atherosclerosis; Cell Line; Cell Survival; Disease Models, Animal; Fatty Acids; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; Mice; Mice, Knockout, ApoE; Models, Biological; Phytochemicals; Plant Oils; Plaque, Atherosclerotic; Prunus persica; RNA, Messenger; Thromboplastin; Tumor Necrosis Factor-alpha

To investigate the expression changes of high mobility group box-1 (HMGB-1) and tissue factor (TF) and their correlation in the serum of sepsis rat models.. 30 rats were divided into the sham-operated group, 15 rats were in the control group. The cecal ligation and puncture method was used to make the animal model with abdominal infection induced by sepsis. There were 15 rats in the sepsis group among which they were divided into 3 subgroups at different time points after modeling (after 6 hours, 12 hours, 24 hours). Cardiac function indicators of the rats in each subgroup were monitored, including heart rate (HR), left ventricular end-diastolic pressure (LVEDP) and left ventricular developed pressure (LVDP), and enzyme-linked immunosorbent assay (ELISA) was used to test the changes of the expression levels of HMGB-1 and TF in the serum of the rats after 6 hours, 12 hours, 24 hours. Pearson correlation analysis was used to analyze the correlation between HMGB-1 and TF.. HR and LVEDP of the rats in the sepsis group were significantly higher than those of the rats in the control group. The differences were statistically significant (p<0.050). LVDP of the rats in the sepsis group was markedly lower than that of the rats in the control group. The differences were statistically significant (p<0.050). The expressions of HMGB-1 and TF of the rats in the subgroups of the sepsis group were higher than those of the rats in the control group after 6 hours, 12 hours, 24 hours; the expression levels of HMGB-1 and TF of the rats with sepsis increased with time. The differences were statistically significant (p<0.050). When the expressions of HMGB-1 and TF of the rats in the sepsis group were compared with each other within the group the differences were significantly different (p<0.050). The expressions of HMGB-1 and TF in the subgroups at the 24th hour were significantly higher than those at the 6th hour. The differences were statistically significant (p<0.050). The differences of the expression of TF of the rats in the control group were not statistically significant (p>0.050). There was a significant positive correlation between HMGB-1 and TF of the rats in the sepsis group (r=0.772, p=0.002).. The expression levels of HMGB-1 and TF of the rats with sepsis gradually increased with time, and the level of HMGB-1 was positively correlated with the level of TF.

Topics: Animals; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; HMGB1 Protein; Rats; Rats, Wistar; Sepsis; Thromboplastin

Antiphospholipid antibodies (aPL) promote endothelial dysfunction, inflammation and procoagulant state. We investigated the effect of hydroxychloroquine (HCQ) on prothrombotic state and endothelial function in mice and in human aortic endothelial cells (HAEC). Human aPL were injected to C57BL/6 mice treated or not with HCQ. Vascular endothelial function and eNOS were assessed in isolated mesenteric arteries. Thrombosis was assessed both in vitro by measuring thrombin generation time (TGT) and tissue factor (TF) expression and in vivo by the measurement of the time to occlusion in carotid and the total thrombosis area in mesenteric arteries. TGT, TF, and VCAM1 expression were evaluated in HAEC. aPL increased VCAM-1 expression and reduced endothelium dependent relaxation to acetylcholine. In parallel, aPL shortened the time to occlusion and extended thrombus area in mice. This was associated with an overexpression of TF and an increased TGT in mice and in HAEC. HCQ reduced clot formation as well as TGT, and improved endothelial-dependent relaxations. Finally, HCQ increased the p-eNOS/eNOS ratio. This study provides new evidence that HCQ improves procoagulant status and vascular function in APS by modulating eNOS, leading to an improvement in the production of NO.

Topics: Animals; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Disease Models, Animal; Endothelial Cells; Endothelium, Vascular; Female; Humans; Hydroxychloroquine; Male; Mice; Nitric Oxide Synthase Type III; Thrombin; Thromboplastin; Thrombosis; Vascular Cell Adhesion Molecule-1

Tissue factor, coagulation factor XII, platelets, and neutrophils are implicated as important players in the pathophysiology of (experimental) venous thrombosis (VT). Their role became evident in mouse models in which surgical handlings were required to provoke VT. Combined inhibition of the natural anticoagulants antithrombin (

Topics: Animals; Antithrombin III; Blood Platelets; Disease Models, Animal; Factor XII; Female; Mice; Mice, Inbred C57BL; Neutrophils; Protein C; Thromboplastin; Venous Thrombosis

Inflammasome activation and subsequent pyroptosis are critical defense mechanisms against microbes. However, overactivation of inflammasome leads to death of the host. Although recent studies have uncovered the mechanism of pyroptosis following inflammasome activation, how pyroptotic cell death drives pathogenesis, eventually leading to death of the host, is unknown. Here, we identified inflammasome activation as a trigger for blood clotting through pyroptosis. We have shown that canonical inflammasome activation by the conserved type III secretion system (T3SS) rod proteins from Gram-negative bacteria or noncanonical inflammasome activation by lipopolysaccharide (LPS) induced systemic blood clotting and massive thrombosis in tissues. Following inflammasome activation, pyroptotic macrophages released tissue factor (TF), an essential initiator of coagulation cascades. Genetic or pharmacological inhibition of TF abolishes inflammasome-mediated blood clotting and protects against death. Our data reveal that blood clotting is the major cause of host death following inflammasome activation and demonstrate that inflammasome bridges inflammation with thrombosis.

Topics: Animals; Bacterial Infections; Biomarkers; Blood Coagulation; Caspases; Cell-Derived Microparticles; Disease Models, Animal; Humans; Inflammasomes; Lipopolysaccharides; Macrophages; Mice; Monocytes; Pyroptosis; Signal Transduction; Thromboplastin; Thrombosis

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

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

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

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

Cancer patients are at high risk of developing deep venous thrombosis (DVT) and venous thromboembolism, a leading cause of mortality in this population. However, it is largely unclear how malignant tumors drive the prothrombotic cascade culminating in DVT.. Here, we addressed the pathophysiology of malignant DVT compared with nonmalignant DVT and focused on the role of tumor microvesicles as potential targets to prevent cancer-associated DVT. We show that microvesicles released by pancreatic adenocarcinoma cells (pancreatic tumor-derived microvesicles [pcMV]) boost thrombus formation in a model of flow restriction of the mouse vena cava. This depends on the synergistic activation of coagulation by pcMV and host tissue factor. Unlike nonmalignant DVT, which is initiated and propagated by innate immune cells, thrombosis triggered by pcMV was largely independent of myeloid leukocytes or platelets. Instead, we identified externalization of the phospholipid phosphatidylethanolamine as a major mechanism controlling the prothrombotic activity of pcMV. Disrupting phosphatidylethanolamine-dependent activation of factor X suppressed pcMV-induced DVT without causing changes in hemostasis.. Together, we show here that the pathophysiology of pcMV-associated experimental DVT differs markedly from innate immune cell-promoted nonmalignant DVT and is therefore amenable to distinct antithrombotic strategies. Targeting phosphatidylethanolamine on tumor microvesicles could be a new strategy for prevention of cancer-associated DVT without causing bleeding complications.

Topics: Adenocarcinoma; Animals; Bacteriocins; Blood Coagulation; Cell Line, Tumor; Cell-Derived Microparticles; Disease Models, Animal; Drug Design; Factor Xa; Fibrinolytic Agents; Humans; Mice; Mice, Inbred C57BL; Mice, Transgenic; Molecular Targeted Therapy; Pancreatic Neoplasms; Peptides; Phosphatidylethanolamines; Signal Transduction; Thromboplastin; Vena Cava, Inferior; Venous Thrombosis

Sirtuin 3 (Sirt3) is a mitochondrial, nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that reduces oxidative stress by activation of superoxide dismutase 2 (SOD2). Oxidative stress enhances arterial thrombosis. This study investigated the effects of genetic Sirt3 deletion on arterial thrombosis in mice in an inflammatory setting and assessed the clinical relevance of these findings in patients with ST-elevation myocardial infarction (STEMI).. Using a laser-induced carotid thrombosis model with lipopolysaccharide (LPS) challenge, in vivo time to thrombotic occlusion in Sirt3-/- mice (n = 6) was reduced by half compared to Sirt3+/+ wild-type (n = 8, P < 0.01) controls. Ex vivo analyses of whole blood using rotational thromboelastometry revealed accelerated clot formation and increased clot stability in Sirt3-/- compared to wild-type blood. rotational thromboelastometry of cell-depleted plasma showed accelerated clotting initiation in Sirt3-/- mice, whereas overall clot formation and firmness remained unaffected. Ex vivo LPS-induced neutrophil extracellular trap formation was increased in Sirt3-/- bone marrow-derived neutrophils. Plasma tissue factor (TF) levels and activity were elevated in Sirt3-/- mice, whereas plasma levels of other coagulation factors and TF expression in arterial walls remained unchanged. SOD2 expression in bone marrow -derived Sirt3-/- neutrophils was reduced. In STEMI patients, transcriptional levels of Sirt3 and its target SOD2 were lower in CD14+ leukocytes compared with healthy donors (n = 10 each, P < 0.01).. Sirt3 loss-of-function enhances experimental thrombosis in vivo via an increase of neutrophil extracellular traps and elevation of TF suggesting thrombo-protective effects of endogenous Sirt3. Acute coronary thrombosis in STEMI patients is associated with lower expression levels of SIRT3 and SOD2 in CD14+ leukocytes. Therefore, enhancing SIRT3 activity by pan-sirtuin activating NAD+-boosters may provide a novel therapeutic target to prevent or treat thrombotic arterial occlusion in myocardial infarction or stroke.

Topics: Animals; Blood Coagulation; Carotid Artery Diseases; Case-Control Studies; Disease Models, Animal; Extracellular Traps; Genetic Predisposition to Disease; Humans; Mice, Inbred C57BL; Mice, Knockout; Neutrophils; Phenotype; Prospective Studies; Sirtuin 3; ST Elevation Myocardial Infarction; Superoxide Dismutase; Thromboplastin; Thrombosis; Time Factors

Triple-negative breast cancer (TNBC) is a leading cause of breast cancer death and is often associated with

Topics: Animals; Antineoplastic Agents, Immunological; Biomarkers, Tumor; Cell Line, Tumor; CHO Cells; Cricetulus; Disease Models, Animal; Female; Gene Expression; Humans; Immunoconjugates; Immunohistochemistry; Molecular Targeted Therapy; Mutation; Thromboplastin; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays

The anticoagulant and antithrombotic properties of three structurally correlated sea urchin-derived 3-linked sulfated α-glycans and their low molecular-weight derivatives were screened comparatively through various in vitro and in vivo methods. These methods include activated partial thromboplastin time, the inhibitory activity of antithrombin over thrombin and factor Xa, venous antithrombosis, the inhibition of platelet aggregation, the activation of factor XII, and bleeding. While the 2-sulfated fucan from

Topics: Adult; Animals; Anticoagulants; Disease Models, Animal; Drug Evaluation, Preclinical; Factor Xa; Factor XII; Female; Fibrinolytic Agents; Healthy Volunteers; Humans; Male; Molecular Structure; Molecular Weight; Partial Thromboplastin Time; Polysaccharides; Rabbits; Rats; Rats, Wistar; Sea Urchins; Structure-Activity Relationship; Sulfates; Thromboplastin; Venous Thrombosis; Young Adult

Double-stranded DNA (dsDNA) constitutes a potent activator of innate immunity, given its ability to bind intracellular pattern recognition receptors during viral infections or sterile tissue damage. While effects of dsDNA in immune cells have been extensively studied, dsDNA signalling and its pathophysiological implications in non-immune cells, such as the vascular endothelium, remain poorly understood. The aim of this study was to characterize prothrombotic effects of dsDNA in vascular endothelial cells. Transfection of cultured human endothelial cells with the synthetic dsDNA poly(dA:dT) induced upregulation of the prothrombotic molecules tissue factor and PAI-1, resulting in accelerated blood clotting in vitro, which was partly dependent on RIG-I signalling. Prothrombotic effects were also observed upon transfection of endothelial cells with hepatitis B virus DNA-containing immunoprecipitates as well human genomic DNA. In addition, dsDNA led to surface expression of von Willebrand factor resulting in increased platelet-endothelium-interactions under flow. Eventually, intrascrotal injection of dsDNA resulted in accelerated thrombus formation upon light/dye-induced endothelial injury in mouse cremaster arterioles and venules in vivo. In conclusion, we show that viral or endogenous dsDNA induces a prothrombotic phenotype in the vascular endothelium. These findings represent a novel link between pathogen- and danger-associated patterns within innate immunity and thrombosis.

Topics: Animals; Blood Coagulation; Cells, Cultured; Disease Models, Animal; DNA; Endothelial Cells; Endothelium, Vascular; Humans; Mice; Plasminogen Activator Inhibitor 1; Thromboplastin; Thrombosis; von Willebrand Factor

Essentials Tissue factor (TF) represents a central link between hemostasis and inflammation. We studied the roles of myeloid and airway epithelial TF in acid-caused acute lung injury (ALI). TF on myeloid cells displays a non-coagulatory role regulating the inflammatory response in ALI. Airway epithelial TF contributes to hemostatic functions, but is dispensable in ALI pathogenesis.. Introduction Acute lung injury (ALI) is a life-threatening condition characterized by damaged alveolar-capillary structures and activation of inflammatory and hemostatic processes. Tissue factor (TF) represents a crucial link between inflammation and coagulation, as inflammatory mediators induce myeloid TF expression, and TF initiates extrinsic coagulation. Objective As pulmonary inflammation stimulates TF expression and TF modulates immune responses, we aimed to elucidate its impact on ALI. In particular, we wanted to distinguish the contributions of TF expressed on airway epithelial cells and TF expressed on myeloid cells. Methods Mice with different cell type-specific TF deficiency and wild-type littermates were intratracheally treated with hydrochloric acid, and leukocyte recruitment, cytokine levels, thrombin-antithrombin (TAT) complexes and pulmonary protein-rich infiltrates were analyzed. Results Our data demonstrate that a lack of epithelial TF did not influence acute responses, as bronchoalveolar neutrophil accumulation 8 h after ALI induction was unaltered. However, it led to mild, prolonged inflammation, as pulmonary leukocyte and erythrocyte numbers were still increased after 24 h, whereas those in wild-type mice had returned to basal levels. In contrast, myeloid TF was primarily involved in regulating the acute phase of ALI without affecting local coagulation, as indicated by increased bronchoalveolar neutrophil infiltration, pulmonary interleukin-6 levels, and edema formation, but equal TAT complex formation, 8 h after ALI induction. This augmented inflammatory response associated with myeloid TF deficiency was confirmed in vitro, as lipopolysaccharide-stimulated TF-deficient alveolar macrophages released increased levels of chemokine (C-X-C motif) ligand 1 and tumor necrosis factor-α as compared with wild-type macrophages. Conclusion We conclude that myeloid TF dampens inflammation in acid-induced ALI.

Topics: Acute Lung Injury; Animals; Antithrombin III; Blood Coagulation; Cells, Cultured; Chemotaxis, Leukocyte; Cytokines; Disease Models, Animal; Epithelial Cells; Genetic Predisposition to Disease; Hydrochloric Acid; Inflammation Mediators; Lung; Macrophages, Alveolar; Mice, Inbred C57BL; Mice, Knockout; Neutrophil Infiltration; Peptide Hydrolases; Phenotype; Pneumonia; Pulmonary Edema; Thromboplastin; Time Factors

Topics: Anemia, Sickle Cell; Animals; Antibodies, Monoclonal; Biomarkers; Bone Marrow Transplantation; Cell Aggregation; Disease Models, Animal; Early Growth Response Protein 1; Endothelial Cells; Endothelium, Vascular; Etanercept; Heart Function Tests; Humans; Inflammation Mediators; Leukocytes, Mononuclear; Mice; Mice, Knockout; Mice, Transgenic; Molecular Targeted Therapy; Monocytes; NF-kappa B; Phenotype; Protein Kinase Inhibitors; Signal Transduction; Thromboplastin; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Essentials Tumor-bearing mice have larger venous clots than controls. Human tissue factor is present in clots in tumor-bearing mice. Inhibition of human tissue factor reduces clot size in tumor-bearing mice. This new mouse model may be useful to study mechanisms of cancer-associated thrombosis.. Background Pancreatic cancer patients have a high rate of venous thromboembolism. Human pancreatic tumors and cell lines express high levels of tissue factor (TF), and release TF-positive microvesicles (TF

Topics: Animals; Antibodies, Monoclonal; Blood Coagulation; Blood Platelets; Cell Line, Tumor; Cell-Derived Microparticles; Disease Models, Animal; Fibrin Fibrinogen Degradation Products; Fibrinolytic Agents; Heterografts; Humans; Male; Mice, Nude; Neoplasm Transplantation; Neutrophils; Pancreatic Neoplasms; Thromboplastin; Venous Thrombosis

Recombinant factor VIIa (FVIIa) variants with increased activity offer the promise to improve the treatment of bleeding episodes in patients with inhibitor-complicated hemophilia. Here, an approach was adopted to enhance the activity of FVIIa by selectively optimizing substrate turnover at the membrane surface. Under physiological conditions, endogenous FVIIa engages its cell-localized cofactor tissue factor (TF), which stimulates activity through membrane-dependent substrate recognition and allosteric effects. To exploit these properties of TF, a covalent complex between FVIIa and the soluble ectodomain of TF (sTF) was engineered by introduction of a nonperturbing cystine bridge (FVIIa Q64C-sTF G109C) in the interface. Upon coexpression, FVIIa Q64C and sTF G109C spontaneously assembled into a covalent complex with functional properties similar to the noncovalent wild-type complex. Additional introduction of a FVIIa-M306D mutation to uncouple the sTF-mediated allosteric stimulation of FVIIa provided a final complex with FVIIa-like activity in solution, while exhibiting a two to three orders-of-magnitude increase in activity relative to FVIIa upon exposure to a procoagulant membrane. In a mouse model of hemophilia A, the complex normalized hemostasis upon vascular injury at a dose of 0.3 nmol/kg compared with 300 nmol/kg for FVIIa.

Topics: Allosteric Regulation; Animals; Biological Therapy; Blood Coagulation; Disease Models, Animal; Factor VIIa; Female; Hemophilia A; Humans; Kinetics; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Dynamics Simulation; Protein Engineering; Recombinant Proteins; Thromboplastin

Venous thromboembolism (VTE), the third leading cardiovascular complication, requires more understanding at molecular levels. Here, we have identified miR-145 as a key molecule for regulating thrombus formation in venous thrombosis (VT) employing network based bioinformatics approach and in vivo experiments. Levels of miR-145 showed an inverse correlation with thrombus load determined by coagulation variables. MiRNA target prediction tools and in vitro study identified tissue factor (TF) as a target gene for miR-145. The restoration of miR-145 levels in thrombotic animals via in vivo miR-145 mimic delivery resulted in decreased TF level and activity, accompanied by reduced thrombogenesis. MiR-145 levels were also reduced in VT patients and correlated with increased TF levels in patients, thereby, confirming our preclinical findings. Our study identifies a previously undescribed role of miRNA in VT by regulating TF expression. Therefore, restoration of miR-145 levels may serve as a promising therapeutic strategy for management of VT.

Topics: Animals; Blood Coagulation; Disease Models, Animal; Gene Expression Regulation; Humans; MicroRNAs; Rats; Thromboplastin; Thrombosis; Venous Thromboembolism; Venous Thrombosis

Hydroxychloroquine (HCQ) has been used for decades to treat patients with rheumatic diseases, for example, systemic lupus erythematosus (SLE), rheumatoid arthritis or the antiphospholipid syndrome (APS). We hypothesise that HCQ might target endosomal NADPH oxidase (NOX), which is involved in the signal transduction of cytokines as well as antiphospholipid antibodies (aPL).. For in vitro experiments, monocytic cells were stimulated with tumour necrosis factor α (TNFα), interleukin-1β (IL-1β) or a human monoclonal aPL and the activity of NOX was determined by flow cytometry. The expression of genes known to be induced by these stimuli was quantified by quantitative reverse transcription PCR. Live cell imaging was performed by confocal laser scanning microscopy. Finally, the effects of HCQ on NOX-induced signal transduction were analysed in an in vivo model of venous thrombosis.. HCQ strongly reduces or completely prevents the induction of endosomal NOX by TNFα, IL-1β and aPL in human monocytes and MonoMac1 cells. As a consequence, induction of downstream genes by these stimuli is reduced or abrogated. This effect of HCQ is not mediated by direct interference with the agonists but by inhibiting the translocation of the catalytic subunit of NOX2 (gp91phox) into the endosome. In vivo, HCQ protects mice from aPL-induced and NOX2-mediated thrombus formation.. We describe here a novel mechanism of action of HCQ, that is, interference with the assembly of endosomal NOX2. Since endosomal NOX2 is involved in many inflammatory and prothrombotic signalling pathways, this activity of HCQ might explain many of its beneficial effects in rheumatic diseases including the APS.

Topics: Adult; Aged; Animals; Antibodies, Antiphospholipid; Antirheumatic Agents; Cells, Cultured; Disease Models, Animal; Endosomes; Enzyme Induction; Female; Gene Expression; Humans; Hydroxychloroquine; Immunoglobulin G; Interleukin-1beta; Intravital Microscopy; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Middle Aged; Monocytes; NADPH Oxidase 2; NADPH Oxidases; NF-kappa B; Protein Transport; Reactive Oxygen Species; Signal Transduction; Thromboplastin; Tumor Necrosis Factor-alpha; Vena Cava, Inferior; Venous Thrombosis; Young Adult

Here we demonstrate a novel application of 'surface enhanced resonance Raman scattering nanoparticles' (SERRS NPs) for imaging breast cancer lung metastases with much higher precision than currently feasible. A breast cancer lung metastasis mouse model was established by intravenous injection of LM2 cells. These mice were intravenously administered SERRS NPs conjugated with ALT-836, an anti-tissue factor (TF) monoclonal antibody, and subjected to Raman imaging to visualize the expression of TF both in vivo and ex vivo. Raman imaging indicated marked uptake of αTF-SERRS-NPs by the lung metastases compared to isotype and blocking controls. Conversely, little uptake of αTF-SERRS-NPs was observed in the lungs of healthy mice. Successful detection and delineation of pulmonary micrometastatic lesions as small as 200 μm, corroborated by histology, immunohistochemistry, and bioluminescence imaging confirmed the suitability of both TF as a target and αTF-SERRS-NPs as an effective contrast agent for imaging breast cancer lung metastases. Further advancements of this technique in the form of Raman endoscopes coupled with ultrabright SERRS NPs developed in this work could lead to minimally invasive detection and resection of lung metastases.

Topics: Animals; Disease Models, Animal; Lung Neoplasms; Mice; Nanoparticles; Neoplasm Micrometastasis; Spectrum Analysis, Raman; Thromboplastin

The P2Y. Ticagrelor, unlike clopidogrel, exhibits endothelial-specific antithrombotic properties and blunts arterial thrombus formation. The additional antithrombotic properties displayed by ticagrelor may explain its greater efficacy in reducing thrombotic events in clinical trials. These findings may provide the basis for new indications for ticagrelor.

Topics: Adenosine; Animals; Blood Coagulation; Blood Platelets; Carotid Artery Injuries; Cells, Cultured; Clopidogrel; Disease Models, Animal; Down-Regulation; Endothelial Cells; Equilibrative Nucleoside Transporter 1; Fibrinolytic Agents; Humans; Male; Mice, Inbred C57BL; Platelet Aggregation Inhibitors; Proteasome Endopeptidase Complex; Proteolysis; Purinergic P2Y Receptor Antagonists; Receptors, Purinergic P2Y12; Thromboplastin; Thrombosis; Ticagrelor; Ticlopidine; Time Factors; Tumor Necrosis Factor-alpha

Celecoxib, a selective cyclooxygenase-2 inhibitor, produces thrombotic events in patients predisposed to cardiovascular risk factors. One theory reported an increase in endothelial expression of tissue factor (TF) as a predisposing factor. This work explored the effect of evening primrose oil (EPO), a source of prostaglandin E1, and forskolin (a cyclic adenosine monophosphate stimulator) against the prothrombotic effect of celecoxib in mice. Lipopolysaccharide mouse model of endotoxemia was used to induce an upregulation of TF activity. Male mice received celecoxib (25 mg/kg), celecoxib plus EPO, or celecoxib plus forskolin for 4 weeks and then subjected to a prothrombotic challenge in the form of an intraperitoneal injection of lipopolysaccharide. Results showed an increase in plasma TF activity, endothelial TF expression, and thrombin-antithrombin (TAT) but lower antithrombin III (ATIII) level in mice that received celecoxib in comparison to those that received the vehicle. Adding EPO or forskolin to celecoxib regimen significantly decreased the prothrombotic effect of celecoxib. A positive correlation (r = 0.8501) was found between TF activity and TAT. Co-administration of EPO or forskolin decreased the activity of TF and mitigated the prothrombotic effect of celecoxib. Therefore, these combinations may have the utility to abrogate the prothrombotic adverse effect of celecoxib in clinical setting.

Topics: Animals; Antithrombin III; Blood Coagulation; Celecoxib; Colforsin; Cyclooxygenase 2 Inhibitors; Disease Models, Animal; Endotoxemia; Fibrinolytic Agents; gamma-Linolenic Acid; Linoleic Acids; Lipopolysaccharides; Lung; Male; Mice; Oenothera biennis; Peptide Hydrolases; Plant Oils; Thromboplastin; Thrombosis; Up-Regulation

The lectin-like oxLDL receptor-1 (LOX-1) promotes endothelial uptake of oxidized low-density lipoprotein (oxLDL) and plays an important role in atherosclerosis and acute coronary syndromes (ACS). However, its role in arterial thrombus formation remains unknown. We investigated whether LOX-1 plays a role in arterial thrombus formation in vivo at different levels of oxLDL using endothelial-specific LOX-1 transgenic mice (LOX-1TG) and a photochemical injury thrombosis model of the carotid artery.. In mice fed a normal chow diet, time to arterial occlusion was unexpectedly prolonged in LOX-1TG as compared to WT. In line with this, tissue factor (TF) expression and activity in carotid arteries of LOX-1TG mice were reduced by half. This effect was mediated by activation of octamer transcription factor 1 (Oct-1) leading to upregulation of the mammalian deacetylase silent information regulator-two 1 (SIRT1) via binding to its promoter and subsequent inhibition of NF-κB signaling. In contrast, intravenous injection of oxLDL as well as high cholesterol diet for 6 weeks led to a switch from the Oct-1/SIRT1 signal transduction pathway to the ERK1/2 pathway and in turn to an enhanced thrombotic response with shortened occlusion time.. Thus, LOX-1 differentially regulates thrombus formation in vivo depending on the degree of activation by oxLDL. At low oxLDL levels LOX-1 activates the protective Oct-1/SIRT1 pathway, while at higher levels of the lipoprotein switches to the thrombogenic ERK1/2 pathway. These findings may be important for arterial thrombus formation in ACS and suggest that SIRT1 may represent a novel therapeutic target in this context.

Topics: Animals; Binding Sites; Blood Coagulation; Carotid Arteries; Carotid Artery Injuries; Cholesterol, Dietary; Disease Models, Animal; Genetic Predisposition to Disease; Humans; Lipoproteins, LDL; Male; Mice, Inbred C57BL; Mice, Transgenic; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-kappa B; Octamer Transcription Factor-1; Phenotype; Phosphorylation; Promoter Regions, Genetic; Scavenger Receptors, Class E; Signal Transduction; Sirtuin 1; Thromboplastin; Thrombosis; Time Factors

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

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

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

ESSENTIALS: The role of tissue factor (TF) in recombinant factor VIIa (rFVIIa) therapy in hemophilia is unclear. An acquired mouse hemophilia model with very low or normal levels of human TF was used in the study. rFVIIa is equally effective in correcting the bleeding in mice expressing low or normal levels of TF. Pharmacological doses of rFVIIa restore hemostasis in hemophilia independent of TF.. Recombinant factor VIIa (rFVIIa) has been used widely for treating hemophilia patients with inhibitory autoantibodies against factor VIII or IX. Its mechanism of action is not entirely known. A majority of in vitro studies suggested that pharmacological concentrations of rFVIIa restore hemostasis in hemophilia in a phospholipid-dependent manner, independent of tissue factor (TF). However, a few studies suggested that a TF-dependent mechanism has a primary role in correction of bleeding by rFVIIa in hemophilia patients. Here, we investigated the potential contribution of TF in rFVIIa-induced hemostasis in hemophilia employing a model system of FVIII antibody-induced hemophilia in TF transgenic mice.. Mice expressing low levels of human TF (LTF mice), mice expressing relatively high levels of human TF (HTF mice) and wild-type mice (WT mice) had neutralizing anti-FVIII antibodies administered in order to induce hemophilia in these mice. The mice were then treated with varying concentrations of rFVIIa. rFVIIa-induced hemostasis was evaluated with the saphenous vein bleeding model.. Administration of FVIII inhibitory antibodies induced the hemophilic bleeding phenotype in all three genotypes. rFVIIa administration rescued the bleeding phenotype in all three genotypes. No significant differences were observed in rFVIIa-induced correction of bleeding between LTF and HTF mice that had FVIII antibodies administered.. Our results provide strong evidence supporting the suggestion that the hemostatic effect of pharmacological doses of rFVIIa stems from a TF-independent mechanism.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Coagulants; Disease Models, Animal; Dose-Response Relationship, Drug; Factor VIIa; Factor VIII; Genotype; Hemophilia A; Hemostasis; Humans; Mice, Inbred C57BL; Mice, Transgenic; Phenotype; Recombinant Proteins; Thromboplastin

Coagulation factor XI (FXI) has been shown to contribute to thrombus formation on collagen or tissue factor-coated surfaces in vitro and in vivo by enhancing thrombin generation. Whether the role of the intrinsic pathway of coagulation is restricted to the local site of thrombus formation is unknown. This study was aimed to determine whether FXI could promote both proximal and distal platelet activation and aggregate formation in the bloodstream.. Pharmacological blockade of FXI activation or thrombin activity in blood did not affect local platelet adhesion, yet reduced local platelet aggregation, thrombin localization, and fibrin formation on immobilized collagen and tissue factor under shear flow, ex vivo. Downstream of the thrombus formed on immobilized collagen or collagen and 10 pmol/L tissue factor, platelet CD62P expression, microaggregate formation, and progressive platelet consumption were significantly reduced in the presence of FXI function-blocking antibodies or a thrombin inhibitor in a shear rate- and time-dependent manner. In a non-human primate model of thrombus formation, we found that inhibition of FXI reduced single platelet consumption in the bloodstream distal to a site of thrombus formation.. This study demonstrates that the FXI-thrombin axis contributes to distal platelet activation and procoagulant microaggregate formation in the blood flow downstream of the site of thrombus formation. Our data highlight FXI as a novel therapeutic target for inhibiting distal platelet consumption without affecting proximal platelet adhesion.

Topics: Animals; Blood Coagulation; Blood Platelets; Collagen; Disease Models, Animal; Factor XI; Factor XIa; Fibrin; Humans; Male; Mechanotransduction, Cellular; P-Selectin; Papio anubis; Platelet Activation; Platelet Aggregation; Regional Blood Flow; Stress, Mechanical; Thrombin; Thromboplastin; Thrombosis; Time Factors

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

Tissue factor (TF) is a critical mediator of direct acute lung injury (ALI) with global TF deficiency resulting in increased airspace inflammation, alveolar-capillary permeability, and alveolar hemorrhage after intra-tracheal lipopolysaccharide (LPS). In the lung, TF is expressed diffusely on the lung epithelium and intensely on cells of the myeloid lineage. We recently reported that TF on the lung epithelium, but not on myeloid cells, was the major source of TF during intra-tracheal LPS-induced ALI. Because of a growing body of literature demonstrating important pathophysiologic differences between ALI caused by different etiologies, we hypothesized that TF on myeloid cells may have distinct contributions to airspace inflammation and permeability between direct and indirect causes of ALI. To test this, we compared mice lacking TF on myeloid cells (TF(∆mye), LysM.Cre(+/-)TF(flox/flox)) to littermate controls during direct (bacterial pneumonia, ventilator-induced ALI, bleomycin-induced ALI) and indirect ALI (systemic LPS, cecal ligation and puncture). ALI was quantified by weight loss, bronchoalveolar lavage (BAL) inflammatory cell number, cytokine concentration, protein concentration, and BAL procoagulant activity. There was no significant contribution of TF on myeloid cells in multiple models of experimental ALI, leading to the conclusion that TF in myeloid cells is not a major contributor to experimental ALI.

Topics: Acute Lung Injury; Animals; Cytokines; Disease Models, Animal; Female; Lipopolysaccharides; Macrophages; Male; Mice; Mice, Transgenic; Myeloid Cells; Permeability; Phagocytosis; Pneumonia; Respiratory Distress Syndrome; Thromboplastin

Essentials Disorders of hemostasis can lead to delayed and defective wound healing. In hemophilia B (HB) mice, 7 days of Factor (F)IX or VIIa are needed to normalize wound healing. One dose of a highly active FVIIa variant (DVQ) restored normal wound closure time in HB mice. Coagulation factors with enhanced activity may acquire biological effects not due to hemostasis.. Introduction We have previously reported that hemophilia B (HB) mice have delayed healing of cutaneous wounds and alterations in wound histology. Administration of a single dose of either factor IX or recombinant activated FVII (rFVIIa) (NovoSeven) prior to wounding did not improve wound closure time or histology. The FVIIa analog DVQ (V158D, E296V and M298Q mutations) was designed to have higher tissue factor-independent activity than rVIIa. We hypothesized that a single dose of DVQ would be more effective in restoring wound healing in HB mice. Methods Cutaneous punch wounds were made on the backs of HB and wild-type mice, and the time to wound closure was monitored. HB mice were treated with a dose of rFVIIa (10 mg kg(-1) ) or DVQ (1 mg kg(-1) ) that corrected the tail bleeding time. Skin samples were taken at various time points after wounding, fixed, and stained, and the histology was examined. Results As previously reported, wound closure times in HB mice given one dose of rFVIIa were not improved over those in untreated HB mice. Surprisingly, healing times in HB mice treated with an equally hemostatic dose of DVQ were normalized to that in wild-type mice. However, DVQ did not correct all histologic abnormalities in HB mice. Conclusions As the doses of DVQ and rFVIIa were chosen to support comparable levels of hemostasis, our data suggest that the improved healing seen with DVQ is not solely attributable to its hemostatic activity. It is possible that the improved wound healing arises through the effect of DVQ on cell signaling mechanisms.

Topics: Administration, Topical; Animals; Bleeding Time; Disease Models, Animal; Factor IX; Factor VIIa; Genetic Variation; Hemophilia B; Hemostasis; Humans; Mice; Mutation; Recombinant Proteins; Thromboplastin; Wound Healing

Pancreatic adenocarcinoma is a highly aggressive cancer, currently treated with limited success and dismal outcomes. New diagnostic and treatment strategies offer the potential to reduce cancer mortality. Developing highly specific noninvasive imaging probes for pancreatic cancer is essential to improving diagnostic accuracy and monitoring therapeutic intervention.. A bispecific heterodimer was synthesized by conjugating an anti-tissue factor (TF) Fab with an anti-CD105 Fab, via the bio-orthogonal "click" reaction between tetrazine (Tz) and trans-cyclooctene (TCO). The heterodimer was labeled with (64)Cu for PET imaging of nude mice bearing BXPC-3 xenograft and orthotopic pancreatic tumors.. PET imaging of BXPC-3 (TF/CD105(+/+)) xenograft tumors with (64)Cu-labeled heterodimer displayed significantly enhanced tumor uptake (28.8 ± 3.2 %ID/g; n = 4; SD) at 30 hours postinjection, as compared with each of their monospecific Fab tracers (12.5 ± 1.4 and 7.1 ± 2.6 %ID/g; n = 3; SD). In addition, the activity-concentration ratio allowed for effective tumor visualization (tumor/muscle ratio 75.2 ± 9.4 at 30 hours postinjection.; n = 4; SD). Furthermore, (64)Cu-NOTA-heterodimer enabled sensitive detection of orthotopic pancreatic tumor lesions with an uptake of 17.1 ± 4.9 %ID/g at 30 hours postinjection and tumor/muscle ratio of 72.3 ± 46.7.. This study demonstrates that dual targeting of TF and CD105 provided synergistic improvements in binding affinity and tumor localization of the heterodimer. Dual-targeted imaging agents of pancreatic and other cancers may assist in diagnosing pancreatic malignancies as well as reliable monitoring of therapeutic response. Clin Cancer Res; 22(15); 3821-30. ©2016 AACR.

Topics: Animals; Biomarkers; Cell Line, Tumor; Disease Models, Animal; Female; Flow Cytometry; Humans; Immunoglobulin Fab Fragments; Mice; Neprilysin; Pancreatic Neoplasms; Positron-Emission Tomography; Protein Multimerization; Radiopharmaceuticals; Thromboplastin; Tissue Distribution

Recent trials suggest that Aspirin (ASA) reduces the incidence of venous thromboembolism in human. However, the molecular mechanisms underlying this effect are still unclear. In this study we assessed the effects of ASA in venous thrombosis mouse model induced by inferior vena cava (IVC) ligation and we investigated the mechanisms responsible for this effect. ASA (3mg/kg daily for 2 days) treatment decreased the thrombus size, the amounts of tissue factor activity in plasma microvesicles (TF-MP) and the levels of 2,3-dinor Thromboxane B2 (TXB-M) in urine compared to control mice. Interestingly, the thrombus size positively correlated with both TF-MP activity and TXB-M. In addition, positive correlation was observed between TF-MP activity and TXB-M. A reduced number of neutrophils and monocytes, and of TF-positive cells accompanied to a lower amount of fibrin and neutrophil extracellular traps (NETs) were also found in thrombi of ASA-treated mice. Similar results were obtained when mice were treated 24h before IVC ligation with SQ29548 (1mg/kg), a selective thromboxane receptor antagonist. In addition, transfusion of platelets in SQ29548 treated-mice excluded the likelihood of a redundant role of platelet-TP receptor in this context. Finally, incubation of macrophages and neutrophils with SQ29548 prevented TF activity and/or NETs formation induced by supernatant of activated platelets or by IBOP, a selective thromboxane analogue. In conclusion, ASA, suppressing TXA2, prevents macrophages and neutrophils activation and markedly reduces thrombus size with a mechanism most likely dependent of the inhibition of TF activity and NETs formation. These results provide a new link between platelet-produced thromboxane and the occurrence of venous thrombosis.

Topics: Animals; Aspirin; Blood Platelets; Cell-Derived Microparticles; Disease Models, Animal; Male; Mice; Neutrophils; Platelet Activation; Platelet Aggregation Inhibitors; Thromboplastin; Thromboxanes; Vena Cava, Inferior; Venous Thrombosis

Few reports have examined tissue factor (TF) and forkhead box transcription factor O-1 (FoxO1) expression in chronic thromboembolic pulmonary hypertension (CTEPH) animal models. To investigate the role of TF and FoxO1 and their interactions during CTEPH pathogenesis in a rat model. Autologous blood clots were repeatedly injected into the pulmonary arteries through right jugular vein to induce a rat model of CTEPH. Hemodynamic parameters, histopathology, and TF and FoxO1expression levels were detected. The mean pulmonary arterial pressure (mPAP), pulmonary vascular resistance and vessel wall area/total area (WA/TA) ratio in the experiment group increased significantly than sham group (P < 0.05). The cardiac output in the 1-, 2-, and 4-week groups decreased significantly (P < 0.05) when compared to sham group. TF mRNA expression levels in the experiment group increased significantly than sham group (P < 0.05). FoxO1 mRNA and protein expression levels were lower in the experiment group than sham group (P < 0.05). The mPAP had a positive correlation with WA/TA ratio (r = 0.45, P = 0.01). TF mRNA expression had a positive correlation with WA/TA ratio (r = 0.374, P = 0.035) and a positive correlation with mPAP (r = 0.48, P= 0.005). FoxO1 mRNA expression had a negative correlation trend with the WA/TA ratio (r = -0.297, P = 0.099) and a negative correlation trend with mPAP (r = -0.34, P = 0.057). TF mRNA expression had a negative correlation with FoxO1 mRNA expression (r = -0.62, P < 0.001). A rat model of CTEPH can be successfully established by the injection of autologous blood clots into the pulmonary artery. TF and FoxO1 may play a key role in vascular remodeling during CTEPH pathogenesis.

Topics: Animals; Disease Models, Animal; Gene Expression Regulation; Hypertension, Pulmonary; Male; Nerve Tissue Proteins; Pulmonary Embolism; Rats; Rats, Sprague-Dawley; Thromboplastin; Vascular Resistance

Mice with a complete absence of tissue factor (TF) die during embryonic development whereas mice with low levels of TF (Low-TF mice) survive to adulthood. Low-TF mice exhibit spontaneous hemorrhage in various organs, including the lung. In contrast, mice can survive without protease-activated receptor (PAR)-4, which is the major thrombin receptor on mouse platelets. We determined the effect of combining a deficiency PAR-4 (primary hemostasis) with a deficiency in TF (secondary hemostasis) on embryonic development and survival of adult mice.. Low-TF mice (mTF. We observed the expected number of Low-TF,PAR-4. Low-TF,PAR-4

Topics: Animals; Disease Models, Animal; Hemorrhage; Humans; Lung Diseases; Mice; Mice, Transgenic; Receptors, Proteinase-Activated; Thromboplastin

Venous thromboembolism constitutes one of the main causes of death during the progression of a cancer. We previously demonstrated that tissue factor (TF)-bearing cancer cell-derived microparticles accumulate at the site of injury in mice developing a pancreatic cancer. The presence of these microparticles at the site of thrombosis correlates with the size of the platelet-rich thrombus. The objective of this study was to determine the involvement of TF expressed by cancer cell-derived microparticles on thrombosis associated with cancer. We observed that pancreatic cancer cell derived microparticles expressed TF, its inhibitor tissue factor pathway inhibitor (TFPI) as well as the integrins αvβ1 and αvβ3. In mice bearing a tumor under-expressing TF, a significant decrease in circulating TF activity associated with an increase bleeding time and a 100-fold diminished fibrin generation and platelet accumulation at the site of injury were observed. This was mainly due to the interaction of circulating cancer cell-derived microparticles expressing TFPI with activated platelets and fibrinogen. In an ectopic model of cancer, treatment of mice with Clopidogrel, an anti-platelet drug, decreased the size of the tumors and restored hemostasis by preventing the accumulation of cancer cell-derived microparticles at the site of thrombosis. In a syngeneic orthotopic model of pancreatic cancer Clopidogrel also significantly inhibited the development of metastases. Together, these results indicate that an anti-platelet strategy may efficiently treat thrombosis associated with cancer and reduce the progression of pancreatic cancer in mice.

Topics: Animals; Blood Coagulation; Blotting, Western; Cell-Derived Microparticles; Clopidogrel; Disease Models, Animal; Flow Cytometry; Fluorescent Antibody Technique; Integrins; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; P-Selectin; Pancreatic Neoplasms; Platelet Activation; Platelet Aggregation Inhibitors; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thromboplastin; Thrombosis; Ticlopidine; Tumor Cells, Cultured

The efficiency of cellular uptake of triplex‑forming oligodexinucleotides (TFO), and the inhibition of tissue factor (TF) is low. The aim of the present study was to improve the absorption of TFO, and increase the inhibition of TF induced by shear stress both in vitro and in vivo, by using an ultrasound‑targeted microbubble destruction (UTMD)‑based delivery system. TFO‑conjugated lipid ultrasonic microbubbles (TFO‑M) were first constructed and characterised. The absorption of TFO was observed by a fluorescence‑based method, and the inhibition of TF by immunofluorescence and quantitative polymerase chain reaction. ECV304 human umbilical vein endothelial cells were subjected to fluid shear stress for 6 h after treatment with TFO conjugated lipid ultrasonic microbubbles without sonication (TFO‑M group); TFO alone; TFO conjugated lipid ultrasonic microbubbles, plus immediate sonication (TFO+U group and TFO‑M+U group); or mock treated with 0.9% NaCl only (SSRE group). The in vivo experiments were established in a similar manner to the in vitro experiments, except that TFO or TFO‑M was injected into rats through the tail vein. Six hours after the preparation of a carotid stenosis model, the rats were humanely sacrificed. The transfection efficiency of TFO in the TFO‑M+U group was higher as compared with the TFO‑M and TFO+U group (P<0.01). The protein and mRNA expression of TF in the TFO‑M+U group was significantly decreased both in vitro and in vivo (P<0.01), as compared with the TFO‑M, TFO+U and SSRE groups. The UTMD‑based TFO delivery system promoted the -absorption of TFO and the inhibition of TF, and was therefore considered to be favorable for preventing thrombosis induced by shear stress.

Topics: Animals; Carotid Arteries; Carotid Stenosis; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Fluorescein-5-isothiocyanate; Gene Expression; Human Umbilical Vein Endothelial Cells; Humans; Lipids; Microbubbles; Oligodeoxyribonucleotides; Rats; Rats, Sprague-Dawley; RNA, Messenger; Shear Strength; Sonication; Thromboplastin; Transfection

Therapeutic angiogenesis is a promising strategy for treating ischemia. Our previous work showed that endogenous endothelial tissue factor (TF) expression induces intracrine signaling and switches-on angiogenesis in microvascular endothelial cells (mECs). We have hypothesized that activated mECs could exert a further paracrine regulation through the release of TF-rich microvascular endothelial microparticles (mEMPs) and induce neovascularization of ischemic tissues.. Here, we describe for the first time that activated mECs are able to induce reparative neovascularization in ischemic zones by releasing TF-rich microparticles. We show in vitro and in vivo that mEMPs released by both wild-type and TF-upregulated-mECs induce angiogenesis and collateral vessel formation, whereas TF-poor mEMPs derived from TF-silenced mECs are not able to trigger angiogenesis. Isolated TF-bearing mEMPs delivered to nonperfused adductor muscles in a murine hindlimb ischemia model enhance collateral flow and capillary formation evidenced by MRI. TF-bearing mEMPs increase angiogenesis operating via paracrine regulation of neighboring endothelial cells, signaling through the β1-integrin pathway Rac1-ERK1/2-ETS1 and triggering CCL2 (chemokine [C-C motif] ligand 2) production to form new and competent mature neovessels.. These findings demonstrate that TF-rich mEMPs released by microvascular endothelial cells can overcome the consequences of arterial occlusion and tissue ischemia by promoting postischemic neovascularization and tissue reperfusion.

Topics: Animals; Cell Line; Cell-Derived Microparticles; Collateral Circulation; Disease Models, Animal; Endothelial Cells; Hindlimb; Humans; Integrin beta1; Ischemia; Magnetic Resonance Angiography; Male; Mice, Nude; Microvessels; Muscle, Skeletal; Neovascularization, Physiologic; Paracrine Communication; RNA Interference; Signal Transduction; Thromboplastin; Time Factors; Tissue Culture Techniques; Transfection

Hemostasis is a rapid response by the body to stop bleeding at sites of vessel injury. Both platelets and fibrin are important for the formation of a hemostatic plug. Mice have been used to uncover the molecular mechanisms that regulate the activation of platelets and coagulation under physiologic conditions. However, measurements of hemostasis in mice are quite variable, and current methods do not quantify platelet adhesion or fibrin formation at the site of injury.. We describe a novel hemostasis model that uses intravital fluorescence microscopy to quantify platelet adhesion, fibrin formation and time to hemostatic plug formation in real time. Repeated vessel injuries of ~ 50-100 μm in diameter were induced with laser ablation technology in the saphenous vein of mice.. Hemostasis in this model was strongly impaired in mice deficient in glycoprotein Ibα or talin-1, which are important regulators of platelet adhesiveness. In contrast, the time to hemostatic plug formation was only minimally affected in mice deficient in the extrinsic tissue factor (TF(low)) or the intrinsic factor IX coagulation pathways, even though platelet adhesion was significantly reduced. A partial reduction in platelet adhesiveness obtained with clopidogrel led to instability within the hemostatic plug, especially when combined with impaired coagulation in TF(low) mice.. In summary, we present a novel, highly sensitive method to quantify hemostatic plug formation in mice. On the basis of its sensitivity to platelet adhesion defects and its real-time imaging capability, we propose this model as an ideal tool with which to study the efficacy and safety of antiplatelet agents.

Topics: Animals; Bleeding Time; Blood Coagulation; Blood Platelets; Clopidogrel; Disease Models, Animal; Factor IX; Fibrin; Hemostasis; Intravital Microscopy; Laser Therapy; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Fluorescence; Microscopy, Video; Platelet Adhesiveness; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIb-IX Complex; Saphenous Vein; Talin; Thromboplastin; Ticlopidine; Time Factors; Vascular System Injuries

Biological and physical factors interact to modulate blood response in a wounded vessel, resulting in a hemostatic clot or an occlusive thrombus. Flow and pressure differential (ΔP) across the wound from the lumen to the extravascular compartment may impact hemostasis and the observed core/shell architecture. We examined physical and biological factors responsible for regulating thrombin-mediated clot growth.. Using factor XIIa-inhibited human whole blood perfused in a microfluidic device over collagen/tissue factor at controlled wall shear rate and ΔP, we found thrombin to be highly localized in the P-selectin(+) core of hemostatic clots. Increasing ΔP from 9 to 29 mm Hg (wall shear rate=400 s(-1)) reduced P-selectin(+) core size and total clot size because of enhanced extravasation of thrombin. Blockade of fibrin polymerization with 5 mmol/L Gly-Pro-Arg-Pro dysregulated hemostasis by enhancing both P-selectin(+) core size and clot size at 400 s(-1) (20 mm Hg). For whole-blood flow (no Gly-Pro-Arg-Pro), the thickness of the P-selectin-negative shell was reduced under arterial conditions (2000 s(-1), 20 mm Hg). Consistent with the antithrombin-1 activity of fibrin implicated with Gly-Pro-Arg-Pro, anti-γ'-fibrinogen antibody enhanced core-localized thrombin, core size, and overall clot size, especially at venous (100 s(-1)) but not arterial wall shear rates (2000 s(-1)). Pathological shear (15 000 s(-1)) and Gly-Pro-Arg-Pro synergized to exacerbate clot growth.. Hemostatic clotting was dependent on core-localized thrombin that (1) triggered platelet P-selectin display and (2) was highly regulated by fibrin and the transclot ΔP. Also, γ'-fibrinogen had a role in venous but not arterial conditions.

Topics: Animals; Arteries; Blood Flow Velocity; Collagen Type I; Disease Models, Animal; Fibrin; Fibrinogens, Abnormal; Hemostasis; Humans; Lab-On-A-Chip Devices; Male; Mechanotransduction, Cellular; Mice; P-Selectin; Polymerization; Pressure; Regional Blood Flow; Stress, Mechanical; Thrombin; Thromboplastin; Thrombosis; Time Factors; Vascular System Injuries; Veins

The hypothesis that hypertension induces a hypercoagulable state arises from the complications associated with hypertension: stroke and myocardial infarction. Here, we determine whether hypertension causes changes in the thrombin-generating capacity of the vascular wall.. We used spontaneously hypertensive rats (SHR) compared with Wistar rats. The addition of thoracic aortic rings of SHR to a Wistar or SHR plasma pool resulted in a greater increase in thrombin generation compared with equivalent rings from Wistar. This increase occurred in 12- but not 5-week-old rats and was prevented by an angiotensin II-converting enzyme inhibitor, indicating that established hypertension is required to induce increased thrombin generation within the vessel wall. Whereas no difference was observed for endothelial cells, thrombin formation was higher on aortic smooth muscle cells (SMCs) from SHR than on those from Wistar. Exposure of negatively charged phospholipids was higher on SHR than on Wistar rings, as well as on cultured SMCs. Tissue factor activity was higher in SHR SMCs. Twelve-week-old SHR exhibited accelerated FeCl3-induced thrombus formation in carotid arteries, and the resulting occlusive thrombi were disaggregated by blockade of glycoprotein Ibα-von Willebrand factor interactions. SHR SMCs were more sensitive to thrombin-induced proliferation than Wistar SMCs. This effect was totally abolished by a protease-activated receptor 1 inhibitor.. The prothrombotic phenotype of the SHR vessel wall was due to the ability of SMCs to support greater thrombin generation and resulted in accelerated occlusive thrombus formation after arterial injury, which was sensitive to glycoprotein Ibα-von Willebrand factor inhibitors.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Antihypertensive Agents; Aorta, Thoracic; Blood Coagulation; Blood Pressure; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Fibrinolytic Agents; Hypertension; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phenotype; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIb-IX Complex; Rats, Inbred SHR; Rats, Wistar; Receptor, PAR-1; Thrombin; Thromboplastin; Thrombosis; Time Factors; Vascular Remodeling; von Willebrand Factor

Hemophilia B is an inherited X-linked recessive bleeding disorder, due to a defect in human factor IX (FIX). The main treatment for hemophilia B is replacement therapy using FIX concentrates. Prophylactic treatment in severe hemophilia B is very effective but is limited by cost issues. Production of a recombinant FIX (rFIX) with enhanced clotting activity, offering the possibility of fewer infusions and fewer costs with similar efficacy, is one of the current challenges for hemophilia B treatment. The present study focused on an important amino acid sequence known to be involved in the interaction of activated FIX (FIXa) with its cofactor, activated factor VIII (FVIIIa).. Using site-directed mutagenesis of glutamate E410 (c240, chymotrypsin numbering), four recombinant FIX-E410 (E410H, A, L and N) mutants were developed and produced by the human hepatoma cell line Huh-7.. The in-vitro clotting activity of mutant FIX molecules was 3 to 5-fold higher than wild-type recombinant FIX (FIX-WT). FIX-E410H compound showed the highest in-vitro procoagulant activity. Enhanced specific activity was confirmed using thrombin generation assay. FIX-E410H induced 5.2-fold higher thrombin generation than FIX-WT. In hemophilia B mice, we observed significantly higher in-vivo clotting activity and thrombin generating capacity with FIX-E410H compared to FIX-WT. We demonstrated that increased procoagulant activity of FIX-E410H was mainly explained by 2.5- fold enhanced affinity of the mutant for human FVIIIa.. We have engineered and characterized four improved FIX proteins with enhanced in-vitro and in-vivo activity. Future studies are required to evaluate the immunogenicity of FIX-E410.

Topics: Amino Acid Substitution; Animals; Blood Coagulation; Blood Coagulation Tests; Blood Platelets; Cell Line; Disease Models, Animal; Factor IX; Factor VIIa; Factor VIIIa; Factor XIa; Gene Expression; Hemophilia B; Humans; In Vitro Techniques; Male; Mice; Mice, Mutant Strains; Mutagenesis, Site-Directed; Recombinant Proteins; Thromboplastin

Atherosclerotic lesions represent a hypoxic milieu. However, the significance of this milieu in atherothrombosis has not been established. We aimed to assess the hypothesis that vascular wall hypoxia promotes arterial thrombus formation. We examined the relation between vascular wall hypoxia and arterial thrombus formation using a rabbit model in which arterial thrombosis was induced by 0.5 %-cholesterol diet and repeated balloon injury of femoral arteries. Vascular wall hypoxia was immunohistochemically detected by pimonidazole hydrochloride, a hypoxia marker. Rabbit neointima and THP-1 macrophages were cultured to analyse prothrombotic factor expression under hypoxic conditions (1 % O2). Prothrombotic factor expression and nuclear localisation of hypoxia-inducible factor (HIF)-1α and nuclear factor-kappa B (NF-κB) p65 were immunohistochemically assessed using human coronary atherectomy plaques. Hypoxic areas were localised in the macrophage-rich deep portion of rabbit neointima and positively correlated with the number of nuclei immunopositive for HIF-1α and NF-κB p65, and tissue factor (TF) expression. Immunopositive areas for glycoprotein IIb/IIIa and fibrin in thrombi were significantly correlated with hypoxic areas in arteries. TF and plasminogen activator inhibitor-1 (PAI-1) expression was increased in neointimal tissues and/or macrophages cultured under hypoxia, and both were suppressed by inhibitors of either HIF-1 or NF-κB. In human coronary plaques, the number of HIF-1α-immunopositive nuclei was positively correlated with that of NF-κB-immunopositive nuclei and TF-immunopositive and PAI-1-immunopositive area, and it was significantly higher in thrombotic plaques. Vascular wall hypoxia augments the thrombogenic potential of atherosclerotic plaque and thrombus formation on plaques via prothrombotic factor upregulation.

Topics: Aged; Angioplasty, Balloon; Animals; Atherosclerosis; Cell Hypoxia; Cell Line; Cholesterol, Dietary; Coronary Artery Disease; Disease Models, Animal; Female; Femoral Artery; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Macrophages; Male; Middle Aged; Neointima; Plaque, Atherosclerotic; Plasminogen Activator Inhibitor 1; Rabbits; Risk Factors; Signal Transduction; Thromboplastin; Thrombosis; Tissue Culture Techniques; Transcription Factor RelA; Vascular System Injuries

Non-muscle myosin heavy chain IIA (NMMHC IIA) has been shown to be involved in thrombus formation and inflammatory microparticle release in endothelial cells. However, the role of NMMHC IIA in regulating the expression of tissue factor (TF) and deep venous thrombosis remains to be elucidated. In the present study, endothelial cells were stimulated with tumour necrosis factor-α (TNF-α) to induce TF expression. Pretreatment with the NMMHC II inhibitor blebbistatin suppressed the mRNA and protein expressions as well as the procoagulant activity of TF in a dose-dependent manner. Blebbistatin enhanced Akt and GSK3β phosphorylation and inhibited NF-κB p65 nuclear translocation and IκBα degradation. These observations were similar to the effect of CHIR99021, a GSK3β inhibitor. TF downregulation by blebbistatin was antagonised by the PI3K inhibitor, wortmannin. Furthermore, siRNA knockdown of NMMHC IIA, but not IIB or IIC, inhibited TF expression, activated Akt/GSK3β and suppressed NF-κB signalling pathways, whereas the overexpression of NMMHC IIA increased TF expression. The binding of NMMHC IIA and TNF receptor 2 mediated signal internalisation in TNF-α-stimulated endothelial cells. Importantly, blebbistatin decreased endothelium NMMHC IIA and TF expression, deactivated GSK3β by inducing its phosphorylation, suppressed p65 nuclear translocation, and inhibited thrombus formation in a mouse deep venous thrombosis model.Our findings provide solid evidence that inhibition of NMMHC II, most likely NMMHC IIA, impedes TF expression and venous thrombosis via Akt/GSK3β-NF-κB signalling pathways in the endothelium both in vitro and in vivo. NMMHC IIA might be a potential novel target for the treatment of thrombotic disorders.

Topics: Active Transport, Cell Nucleus; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelial Cells; Fibrinolytic Agents; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HEK293 Cells; Heterocyclic Compounds, 4 or More Rings; Humans; I-kappa B Proteins; Mice, Inbred C57BL; Molecular Motor Proteins; Myosin Heavy Chains; NF-kappa B; Nonmuscle Myosin Type IIA; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Receptors, Tumor Necrosis Factor, Type II; RNA Interference; Signal Transduction; Thromboplastin; Transcription Factor RelA; Transfection; Tumor Necrosis Factor-alpha; Venous Thrombosis

Tissue factor (TF) initiates the extrinsic coagulation cascade in response to tissue injury, leading to local fibrin deposition. Low levels of TF in mice are associated with increased severity of acute lung injury (ALI) after intratracheal LPS administration. However, the cellular sources of the TF required for protection from LPS-induced ALI remain unknown. In the current study, transgenic mice with cell-specific deletions of TF in the lung epithelium or myeloid cells were treated with intratracheal LPS to determine the cellular sources of TF important in direct ALI. Cell-specific deletion of TF in the lung epithelium reduced total lung TF expression to 39% of wild-type (WT) levels at baseline and to 29% of WT levels after intratracheal LPS. In contrast, there was no reduction of TF with myeloid cell TF deletion. Mice lacking myeloid cell TF did not differ from WT mice in coagulation, inflammation, permeability, or hemorrhage. However, mice lacking lung epithelial TF had increased tissue injury, impaired activation of coagulation in the airspace, disrupted alveolar permeability, and increased alveolar hemorrhage after intratracheal LPS. Deletion of epithelial TF did not affect alveolar permeability in an indirect model of ALI caused by systemic LPS infusion. These studies demonstrate that the lung epithelium is the primary source of TF in the lung, contributing 60-70% of total lung TF, and that lung epithelial, but not myeloid, TF may be protective in direct ALI.

Topics: Acute Lung Injury; Animals; Blood Coagulation; Capillary Permeability; Disease Models, Animal; Epithelial Cells; Gene Expression; Hemorrhage; Lipopolysaccharides; Mice; Mice, Knockout; Myeloid Cells; Pulmonary Alveoli; Respiratory Distress Syndrome; Respiratory Mucosa; Thromboplastin

The risk of thrombotic complications such as deep vein thrombosis (DVT) during tumor development is well known. Tumors release into the circulation procoagulant microparticles (MPs) that can participate in thrombus formation following vessel injury. The importance of this MP tissue factor (TF) in the initiation of cancer-associated DVT remains uncertain.. To investigate how pancreatic cancer MPs promote DVT in vivo.. We combined a DVT mouse model in which thrombosis is induced by flow restriction in the inferior vena cava with one of subcutaneous pancreatic cancer in C57BL/6J mice. We infused high-TF and low-TF tumor MPs to determine the importance of TF in experimental cancer-associated DVT.. Both tumor-bearing mice and mice infused with tumor MPs subjected to 3 h of partial flow restriction developed an occlusive thrombus; fewer than one-third of the control mice did. We observed that MPs adhered to neutrophil extracellular traps (NETs), which are functionally important players during DVT, whereas neither P-selectin nor glycoprotein Ib were required for MP recruitment in DVT. The thrombotic phenotype induced by MP infusion was suppressed by hirudin, suggesting the importance of thrombin generation. TF carried by tumor MPs was essential to promote DVT, as mice infused with low-TF tumor MPs had less thrombosis than mice infused with high-TF tumor MPs.. TF expressed on tumor MPs contributes to the increased incidence of cancer-associated venous thrombosis in mice in vivo. These MPs may adhere to NETs formed at the site of thrombosis.

Topics: Animals; Antithrombins; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell-Derived Microparticles; Disease Models, Animal; Extracellular Traps; Hirudins; Ligation; Male; Mice, Inbred C57BL; Mice, Knockout; P-Selectin; Pancreatic Neoplasms; Platelet Glycoprotein GPIb-IX Complex; Regional Blood Flow; Thromboplastin; Vena Cava, Inferior; Venous Thrombosis

Tissue factor (TF) is a membranous glycoprotein that activates the coagulation system when blood vessels or tissues are damaged. TF was up-regulated in monocrotaline (MCT)/lipopolysaccharide (LPS) hepatotoxicity model. The present study aimed to test the hypothesis that TF-dependent fibrin deposition occurs in liver toxicity induced by CCl4 in mice. Pericentral deposition of TF and fibrin is induced after CCl4-induced liver toxicity. The toxicity was evaluated by determination of serum activities of ALT, AST and ALP as well as GSH content and histopathological changes. The results showed that injection of mice with TF-antisense deoxyoligonucleotide (TF-AS) prevented the accumulation of TF and fibrin in the hepatic tissues. Furthermore, it significantly restored blood biochemical parameters, GSH content and distorted histopathological features caused by CCl4. The current study demonstrates that TF activation is associated with CCl4-induced liver injury. Furthermore, administration of TF-AS successfully prevented this type of liver injury.

Topics: Animals; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; Disease Models, Animal; DNA, Antisense; Fibrin; Gene Expression Regulation, Enzymologic; Glutathione; Male; Mice; Thromboplastin; Transaminases

To enhance the regional antitumor activity of the vascular-targeting agent truncated tissue factor (tTF)-NGR by combining the therapy with low-energy ultrasound (US) treatment.. For the in vitro US exposure of human umbilical vein endothelial cells (HUVECs), cells were put in the focus of a US transducer. For analysis of the US-induced phosphatidylserine (PS) surface concentration on HUVECs, flow cytometry was used. To demonstrate the differences in the procoagulatory efficacy of TF-derivative tTF-NGR on binding to HUVECs with a low versus high surface concentration of PS, we performed factor X activation assays. For low-energy US pretreatment, HT1080 fibrosarcoma xenotransplant-bearing nude mice were treated by tumor-regional US-mediated stimulation (ie, destruction) of microbubbles. The therapy cohorts received the tumor vessel-infarcting tTF-NGR protein with or without US pretreatment (5 minutes after US stimulation via intraperitoneal injection on 3 consecutive days).. Combination therapy experiments with xenotransplant-bearing nude mice significantly increased the antitumor activity of tTF-NGR by regional low-energy US destruction of vascular microbubbles in tumor vessels shortly before application of tTF-NGR (P < .05). Mechanistic studies proved the upregulation of anionic PS on the outer leaflet of the lipid bilayer of endothelial cell membranes by low-energy US and a consecutive higher potential of these preapoptotic endothelial cells to activate coagulation via tTF-NGR and coagulation factor X as being a basis for this synergistic activity.. Combining retargeted tTF to tumor vessels with proapoptotic stimuli for the tumor vascular endothelium increases the antitumor effects of tumor vascular infarction. Ultrasound treatment may thus be useful in this respect for regional tumor therapy.

Topics: Angiogenesis Inhibitors; Animals; Cell Line, Tumor; Disease Models, Animal; Endothelium, Vascular; Female; Fibrosarcoma; Flow Cytometry; Humans; Infarction; Mice; Mice, Nude; Microbubbles; Neovascularization, Pathologic; Thromboplastin; Ultrasonic Therapy

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

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

The major regulator of the neuroendocrine stress response in the brain is corticotropin releasing factor (CRF), whose transcription is controlled by CREB and its cofactors CRTC2/3 (TORC2/3). Phosphorylated CRTCs are sequestered in the cytoplasm, but rapidly dephosphorylated and translocated into the nucleus following a stressful stimulus. As the stress response is attenuated by oxytocin (OT), we tested whether OT interferes with CRTC translocation and, thereby, Crf expression. OT (1 nmol, i.c.v.) delayed the stress-induced increase of nuclear CRTC3 and Crf hnRNA levels in the paraventricular nucleus of male rats and mice, but did not affect either parameter in the absence of the stressor. The increase in Crf hnRNA levels at later time points was parallel to elevated nuclear CRTC2/3 levels. A direct effect of Thr(4) Gly(7)-OT (TGOT) on CRTC3 translocation and Crf expression was found in rat primary hypothalamic neurons, amygdaloid (Ar-5), hypothalamic (H32), and human neuroblastoma (Be(2)M17) cell lines. CRTC3, but not CRCT2, knockdown using siRNA in Be(2)M17 cells prevented the effect of TGOT on Crf hnRNA levels. Chromatin-immunoprecipitation demonstrated that TGOT reduced CRTC3, but not CRTC2, binding to the Crf promoter after 10 min of forskolin stimulation. Together, the results indicate that OT modulates CRTC3 translocation, the binding of CRTC3 to the Crf promoter and, ultimately, transcription of the Crf gene.. The neuropeptide oxytocin has been proposed to reduce hypothalamic-pituitary-adrenal (HPA) axis activation during stress. The underlying mechanisms are, however, elusive. In this study we show that activation of the oxytocin receptor in the paraventricular nucleus delays transcription of the gene encoding corticotropin releasing factor (Crf), the main regulator of the stress response. It does so by sequestering the coactivator of the transcription factor CREB, CRTC3, in the cytosol, resulting in reduced binding of CRTC3 to the Crf gene promoter and subsequent Crf gene expression. This novel oxytocin receptor-mediated intracellular mechanism might provide a basis for the treatment of exaggerated stress responses in the future.

Topics: Animals; Cells, Cultured; Colforsin; Corticotropin-Releasing Hormone; CREB-Binding Protein; Disease Models, Animal; Female; Gene Expression Regulation; Hypothalamus; Male; Mice; Mice, Inbred C57BL; Neurons; Oxytocics; Oxytocin; Protein Transport; Rats; Rats, Wistar; Receptors, Oxytocin; Signal Transduction; Stress, Psychological; Thromboplastin

Cancer is often associated with an increased risk of thrombotic events which are exacerbated by treatment with chemotherapeutics such as cyclosphosphamide (CP). Evidence suggests that thrombin can stimulate tumor progression via formation of fibrin and activation of protease-activated receptors (PARs) and platelets. We examined the effect of co-treatment with CP and dabigatran etexilate, a direct inhibitor of thrombin, using the murine orthotopic 4T1 tumor model. Mice receiving co-treatment with both low dose CP and dabigatran etexilate had significantly smaller mammary tumors and fewer lung metastases than mice treated with CP or dabigratran etexilate alone. Co-treatment with dabigatran etexilate and low dose CP also significantly decreased the number of arginase(+)Gr-1(+)CD11b(+) myeloid derived suppressor cells as well as levels of TGF-β in spleens from tumor bearing mice. 4T1 tumors express procoagulant tissue factor (TF) and spontaneously release TF(+) microparticles which are potent procoagulant factors that promote thrombin generation. Treatment with dabigatran etexilate alone prevented tumor-induced increases in circulating TF(+) microparticles and also decreased the numbers of tumor-induced activated platelets by 40%. These results show that co-treatment with dabigatran etexilate and CP synergistically inhibits growth and metastasis of mammary tumors, suggesting that oral administration of the thrombin inhibitor dabigatran etexilate may be beneficial in not only preventing thrombotic events in cancer patients but also in treating malignant tumors themselves.

Topics: Animals; Antineoplastic Agents, Alkylating; Antithrombins; Cell Line, Tumor; Cell-Derived Microparticles; Cyclophosphamide; Dabigatran; Disease Models, Animal; Disease Progression; Drug Synergism; Female; Mice; Myeloid Cells; Neoplasm Metastasis; Neoplasms; Platelet Activation; Thrombin; Thromboplastin

The aim of this study is to evaluate the effect of these new generation hemostatic agents on early-stage soft tissue healing of warfarin-treated rats by measuring the tissue factor (TF) activities. Rats in the warfarin group were treated intraperitonally with 0.1 mg/kg warfarin, and rats in the control group were treated with 1 mL/kg saline. All rats had 3 incisions on dorsal dermal tissue applied Celox, Ankaferd Blood Stopper (ABS), or no hemostatic agent. Six rats from each group were killed on day 4, and the other 6 were killed on day 8. Prothrombin time (PT) and TF activities were evaluated, respectively. Both the hemostatic agents positively affected the hemostasis. Warfarin treatment increased the PT levels as expected. Celox-treated dermal tissues had higher TF activity when compared to ABS-treated ones. The ABS affected the early-stage healing positively in clinical aspect, whereas Celox was more effective on hemostasis by means of increasing TF activities.

Topics: Animals; Biopolymers; Disease Models, Animal; Drug Interactions; Hemostasis; Male; Plant Extracts; Prothrombin Time; Rats; Rats, Wistar; Skin; Thromboplastin; Warfarin

The plasma zymogens factor XII (fXII) and factor XI (fXI) contribute to thrombosis in a variety of mouse models. These proteins serve a limited role in hemostasis, suggesting that antithrombotic therapies targeting them may be associated with low bleeding risks. Although there is substantial epidemiologic evidence supporting a role for fXI in human thrombosis, the situation is not as clear for fXII. We generated monoclonal antibodies (9A2 and 15H8) against the human fXII heavy chain that interfere with fXII conversion to the protease factor XIIa (fXIIa). The anti-fXII antibodies were tested in models in which anti-fXI antibodies are known to have antithrombotic effects. Both anti-fXII antibodies reduced fibrin formation in human blood perfused through collagen-coated tubes. fXII-deficient mice are resistant to ferric chloride-induced arterial thrombosis, and this resistance can be reversed by infusion of human fXII. 9A2 partially blocks, and 15H8 completely blocks, the prothrombotic effect of fXII in this model. 15H8 prolonged the activated partial thromboplastin time of baboon and human plasmas. 15H8 reduced fibrin formation in collagen-coated vascular grafts inserted into arteriovenous shunts in baboons, and reduced fibrin and platelet accumulation downstream of the graft. These findings support a role for fXII in thrombus formation in primates.

Topics: Animals; Antibodies, Monoclonal; Blood Coagulation; Disease Models, Animal; Factor XI; Factor XII; Factor XII Deficiency; Factor XIIa; Fibrin; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Papio; Recombinant Proteins; Thrombin; Thromboplastin; Thrombosis

Atherothrombosis is the main cause of myocardial infarction and ischemic stroke. Although the extrinsic (tissue factor-factor VIIa [FVIIa]) pathway is considered as a major trigger of coagulation in atherothrombosis, the role of the intrinsic coagulation pathway via coagulation FXII herein is unknown. Here, we studied the roles of the extrinsic and intrinsic coagulation pathways in thrombus formation on atherosclerotic plaques both in vivo and ex vivo.. Plaque rupture after ultrasound treatment evoked immediate formation of subocclusive thrombi in the carotid arteries of Apoe(-/-) mice, which became unstable in the presence of structurally different FXIIa inhibitors. In contrast, inhibition of FVIIa reduced thrombus size at a more initial stage without affecting embolization. Genetic deficiency in FXII (human and mouse) or FXI (mouse) reduced ex vivo whole-blood thrombus and fibrin formation on immobilized plaque homogenates. Localization studies by confocal microscopy indicated that FXIIa bound to thrombi and fibrin particularly in luminal-exposed thrombus areas.. The FVIIa- and FXIIa-triggered coagulation pathways have distinct but complementary roles in atherothrombus formation. The tissue factor-FVIIa pathway contributes to initial thrombus buildup, whereas FXIIa bound to thrombi ensures thrombus stability.

Topics: Animals; Aorta, Thoracic; Aortic Diseases; Apolipoproteins E; Atherosclerosis; Blood Coagulation; Blood Platelets; Carotid Arteries; Carotid Artery Diseases; Cholesterol, Dietary; Disease Models, Animal; Factor VIIa; Factor XI; Factor XII; Factor XII Deficiency; Factor XIIa; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Plaque, Atherosclerotic; Rupture, Spontaneous; Thromboplastin; Thrombosis; Time Factors

Heparanase is implicated in cell invasion, tumour metastasis and angiogenesis. It forms a complex and enhances the activity of the blood coagulation initiator - tissue factor (TF). We describe new peptides derived from the solvent accessible surface of TF pathway inhibitor 2 (TFPI-2) that inhibit the heparanase procoagulant activity. Peptides were evaluated in vitro by measuring activated coagulation factor X levels and co-immunoprecipitation. Heparanase protein and/or lipopolysaccharide (LPS) were injected intra-peritoneally and inhibitory peptides were injected subcutaneously in mouse models. Plasma was analysed by ELISA for thrombin-antithrombin complex (TAT), D-dimer as markers of coagulation activation, and interleukin 6 as marker of sepsis severity. Peptides 5, 6, 7, 21 and 22, at the length of 11-14 amino acids, inhibited heparanase procoagulant activity but did not affect TF activity. Injection of newly identified peptides 5, 6 and 7 significantly decreased or abolished TAT plasma levels when heparanase or LPS were pre-injected, and inhibited clot formation in an inferior vena cava thrombosis model. To conclude, the solvent accessible surface of TFPI-2 first Kunitz domain is involved in TF/heparanase complex inhibition. The newly identified peptides potentially attenuate activation of the coagulation system induced by heparanase or LPS without predisposing to significant bleeding tendency.

Topics: Animals; Blood Coagulation; Disease Models, Animal; Enzyme Activation; Factor X; Fibrin Fibrinogen Degradation Products; Glucuronidase; Glycoproteins; HEK293 Cells; Humans; Interleukin-6; Lipopolysaccharides; Mice; Mice, Inbred ICR; Mice, Knockout; Peptide Fragments; Thrombin; Thromboplastin; Thrombosis; Vena Cava, Inferior

Thrombus formation is determined by the balance between pro- thrombotic mediators and anti-thrombotic factors.High-density lipoprotein (HDL) from healthy subjects exerts anti-thrombotic properties. Whether this is also the case for HDL from patients with stable coronary heart disease (CHD) or acute coronary syndrome (ACS) is unknown.In human aortic endothelial cells in culture,HDL (50 µg/ml) from healthy subjects (HS) inhibited thrombin-induced tissue factor (TF) expression and activity, while HDL (50 µg/ml) from CHD and ACS patients did not. Similarly, only healthy HDL increased endothelial tissue factor pathway inhibitor (TFPI) expression and tissue plasminogen activator (tPA) release, while HDL from CHD and ACS patients had no effect. Healthy HDL inhibited thrombin-induced plasminogen activator inhibitor type 1 (PAI-1) expression, while HDL from ACS patients enhanced endothelial PAI-1 expression. Inhibition of nitric oxide (NO) formation with L-NAME (100 µmol/l) abolished the anti-thrombotic effects of healthy HDL on TF, TFPI, and tPA expression. The exogenous nitric oxide donor, DETANO, mimicked the effects of healthy HDL and counterbalanced the loss of anti-thrombotic effects of HDL from CHD and ACS patients in endothelial cells. In line with this observation, healthy HDL, in contrast to HDL from CHD and ACS patients, increased endothelial NO production. In the laser-injured carotid artery of the mouse, thrombus formation was delayed in animals treated with healthy HDL compared with mice treated with vehicle or HDL from patients with CHD or ACS. In conclusion, HDL from CHD and ACS patients loses the ability of healthy HDL to suppress TF and to increase TFPI and t-PA and instead enhances PAI-1 and arterial thrombus formation.

Topics: Acute Coronary Syndrome; Adult; Aged; Animals; Aorta; Blood Coagulation; Carotid Artery Injuries; Cells, Cultured; Coronary Disease; Disease Models, Animal; Endothelial Cells; Humans; Lipoproteins; Lipoproteins, HDL; Mice; Middle Aged; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroso Compounds; Plasminogen Activator Inhibitor 1; Platelet Aggregation; Thrombin; Thromboplastin; Tissue Plasminogen Activator

Choroidal neovascularization (CNV) occurs as a result of age-related macular degeneration (AMD) and causes severe vision loss among elderly patients. High expression of tissue factor (TF) was found in the retinas of AMD patients. In this study, we used anti-TF monoclonal antibody to test the effect of the TF blockage on experimental CNV induced by laser photocoagulation of retina in mice. Anti-TF monoclonal antibody or vehicle was administered intravitreally at day 1, 2 or 3 after laser application. We found that TF expression increased, and reached the peak at the 3rd week after the after laser application. Anti-TF monoclonal antibody can predominantly decrease the expression of TF, VEGF and F4/80, and reduced the area of CNV. Anti-TF monoclonal antibody also decreased incidence of CNV and leakage area. There were no pathological changes in the liver, heart, brain or kidney tissue. We conclude that TF plays an important role in CNV and anti-TF monoclonal antibody significantly decreases CNV in mouse model and anti-TF monoclonal antibody may have therapeutic potential in AMD.

Topics: Animals; Antibodies, Blocking; Antibodies, Monoclonal; Antigens, Differentiation; Blotting, Western; Capillary Permeability; Choroidal Neovascularization; Disease Models, Animal; Fluorescein Angiography; Immunohistochemistry; Immunotherapy; Intravitreal Injections; Laser Coagulation; Mice; Mice, Inbred C57BL; Real-Time Polymerase Chain Reaction; Thromboplastin; Vascular Endothelial Growth Factor A

Alternatively spliced tissue factor (asTF) is a novel isoform of full-length tissue factor, which exhibits angiogenic activity. Although asTF has been detected in human plaques, it is unknown whether its expression in atherosclerosis causes increased neovascularization and an advanced plaque phenotype.. Carotid (n=10) and coronary (n=8) specimens from patients with stable or unstable angina were classified as complicated or uncomplicated on the basis of plaque morphology. Analysis of asTF expression and cell type-specific expression revealed a strong expression and colocalization of asTF with macrophages and neovessels within complicated, but not uncomplicated, human plaques. Our results showed that the angiogenic activity of asTF is mediated via hypoxia-inducible factor-1α upregulation through integrins and activation of phosphatidylinositol-3-kinase/Akt and mitogen-activated protein kinase pathways. Hypoxia-inducible factor-1α upregulation by asTF also was associated with increased vascular endothelial growth factor expression in primary human endothelial cells, and vascular endothelial growth factor-Trap significantly reduced the angiogenic effect of asTF in vivo. Furthermore, asTF gene transfer significantly increased neointima formation and neovascularization after carotid wire injury in ApoE(-/-) mice.. The results of this study provide strong evidence that asTF promotes neointima formation and angiogenesis in an experimental model of accelerated atherosclerosis. Here, we demonstrate that the angiogenic effect of asTF is mediated via the activation of the hypoxia-inducible factor-1/vascular endothelial growth factor signaling. This mechanism may be relevant to neovascularization and the progression and associated complications of human atherosclerosis as suggested by the increased expression of asTF in complicated versus uncomplicated human carotid and coronary plaques.

Topics: Alternative Splicing; Animals; Apolipoproteins E; Carotid Arteries; Coronary Vessels; Disease Models, Animal; Endothelium, Vascular; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice; Mice, Inbred C57BL; Neointima; Neovascularization, Pathologic; Plaque, Atherosclerotic; Signal Transduction; Thromboplastin; Up-Regulation; Vascular Endothelial Growth Factor A

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

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

It is known that Toll-like receptor (TLR)-4 plays an important role in myocardial infarction and atherothrombosis. The role of TLR-4 in arterial thrombosis is undefined. Both TLR-4-deficient (TLR-4(-/-)) and wild-type (WT) mice were subjected to FeCl3 carotid artery injury, and the time required to form an occlusive thrombus was measured. The mean time to occlusion in TLR-4(-/-) mice was significantly greater than that in WT mice after injury (303 ± 32 vs. 165 ± 34 s, P < 0.05). Furthermore, when we used a WT or TLR-4(-/-)-derived platelet reinfusion in a platelet depletion/reinfusion procedure, there was no significant change in the occlusion time and tissue factor (TF) activity in injured arteries between WT mice and platelet-depleted WT mice. Similarly, no significant difference was observed between TLR-4(-/-) mice and platelet-depleted TLR-4(-/-) mice for the WT or TLR-4(-/-)-derived platelet reinfusion. However, TF expression and activity were significantly reduced in the vascular wall of TLR-4(-/-) mice compared with WT mice. In vivo, lipopolysaccharide accelerated the occlusion time in WT mice but not TLR-4(-/-) mice. In vitro, LPS-induced TF activity was reduced in endothelial cells of TLR-4(-/-) mice relative to WT mice. The data demonstrate that TLR-4 contributes to arterial thrombosis formation in vivo and causes increased TF expression and activity in vitro. The results further suggest that the stimulation is mainly derived by endothelial cells but is not due to platelet-derived TLR-4.

Topics: Animals; Blood Platelets; Carotid Artery Injuries; Disease Models, Animal; Endothelial Cells; Lipopolysaccharides; Male; Mice, Inbred C57BL; Mice, Knockout; Thromboplastin; Thrombosis; Toll-Like Receptor 4

To evaluate the effects of short-term intra-arterial delivery of paclitaxel on neointimal hyperplasia and the local thrombotic environment after angioplasty.. An experimental common carotid artery injury model was established in 60 rats, which were divided into experimental groups (40 rats) and controls (20 rats). Local intra-arterial administration of paclitaxel was applied at 2 doses (90 and 180 μg/30 μl), and the effects of short-term delivery of paclitaxel on neointimal hyperplasia and the expression of tissue factor (TF), plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (t-PA) were evaluated at days 15 and 30 by hematoxylin and eosin staining and immunohistochemistry.. At 15 and 30 days after injury, neointimal thickness and area, the ratio of intimal area to medial area and the stenotic rate were all significantly decreased in the group provided the high concentrations (180 μg/30 μl) of paclitaxel for 2 min or 10 min and in the group provided the low concentration (90 μg/30 μl) of paclitaxel for 10 min (p < 0.05). At 30 days after injury, there were no significant changes in TF expression among all experimental groups. PAI-1 expression increased in the neointima of the high concentration 10 min group (p < 0.05), while t-PA expression decreased in the neointima of the high concentration 2 min group (p < 0.05).. In the rat common carotid artery injury model, the short-term delivery of paclitaxel could effectively inhibit neointimal hyperplasia in the long term, with very little influence on the local expression of TF and PAI-1.

Topics: Angioplasty, Balloon; Animals; Biopsy, Needle; Carotid Artery Injuries; Carotid Artery, Common; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Hyperplasia; Immunohistochemistry; Infusions, Intra-Arterial; Male; Neointima; Paclitaxel; Random Allocation; Rats; Rats, Wistar; Risk Assessment; Thromboplastin; Thrombosis; Time Factors; Tissue Plasminogen Activator; Treatment Outcome

Thrombin is a multifunctional trypsin-like serine protease that plays key roles in coagulation and thrombogenesis. HY023016, a novel Dabigatran prodrug, is an oral direct thrombin inhibitor. The purpose of this study was to compare the anti-thrombotic activities and haemorrhagic effects of HY023016 with Dabigatran etexilate and tetramethylpyrazine in several animal thrombosis models.. To investigate drug exposure, liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to determine the pharmacokinetic profile of HY023016. After single intragastric administrations of HY023016, Dabigatran etexilate or tetramethylpyrazine, the anti-thrombotic activities were evaluated through rabbit jugular vein thrombosis model, rat inferior vena cava thrombosis model, ex vivo rabbit platelet aggregation assay, in vivo rabbit coagulation assay, and direct thrombin binding assay. Meanwhile, we evaluated the effect of HY023016 on expression of tissue factor (TF) by RT-PCR. Rabbit cuticle bleeding assay and mouse tail bleeding assay were applied to evaluate the effects of HY023016 on haemorrhage.. Pharmacokinetic parameters indicated that HY023016 can convert to Dabigatran and tetramethylpyrazine. Our studies showed that HY023016 was able to significantly inhibit thrombus formation in a dose-dependent manner in rabbit and rat models (P<0.05). Similarly, it was able to dose-dependently inhibit thrombin- or ADP-induced platelet aggregation, prolonging the activated partial thromboplastin time (APTT) and prothrombin time (PT), inhibiting the activity of thrombin and inhibiting thrombin- or ADP-induced expression of TF (P<0.05 or 0.01). Dabigatran etexilate was also able to dose-dependently and significantly inhibit thrombus formation (P<0.01) but was unable to affect ADP-induced platelet aggregation and expression of TF. In contrast, tetramethylpyrazine could only exhibit mild antithrombotic activity compared with HY023016 and Dabigatran etexilate (P<0.05). HY023016 could prolong bleeding time (P<0.001), but the prolongations were significantly less than Dabigatran etexilate (P<0.05).. HY023016 showed thrombosis-inhibition activities comparable to those of Dabigatran etexilate, but better than those of tetramethylpyrazine. The attendant bleeding risk of HY023016 was lower than Dabigatran etexilate in rabbits and mice.

Topics: Animals; Antithrombins; Benzimidazoles; beta-Alanine; Dabigatran; Disease Models, Animal; Mice; Prodrugs; Rabbits; Rats; RNA, Messenger; Thrombin; Thromboplastin; Thrombosis

Amla (Emblica officinalis Gaertn.) has been used for many centuries in traditional Indian Ayurvedic formulations for the prevention and treatment of many inflammatory diseases. The present study evaluated the anti-inflammatory and anticoagulant properties of amla fruit extract. The amla fruit extract potentially and significantly reduced lipopolysaccharide (LPS)-induced tissue factor expression and von Willebrand factor release in human umbilical vein endothelial cells (HUVEC) in vitro at clinically relevant concentrations (1-100 μg/ml). In a leucocyte adhesion model of inflammation, it also significantly decreased LPS-induced adhesion of human monocytic cells (THP-1) to the HUVEC, as well as reduced the expression of endothelial-leucocyte adhesion molecule-1 (E-selectin) in the target cells. In addition, the in vivo anti-inflammatory effects were evaluated in a LPS-induced endotoxaemia rat model. Oral administration of the amla fruit extract (50 mg/kg body weight) significantly decreased the concentrations of pro-inflammatory cytokines, TNF-α and IL-6 in serum. These results suggest that amla fruit extract may be an effective anticoagulant and anti-inflammatory agent.

Topics: Animals; Anti-Inflammatory Agents; Anticoagulants; Cell Adhesion; Cells, Cultured; Cytokines; Disease Models, Animal; E-Selectin; Endothelial Cells; Endotoxemia; Fruit; Human Umbilical Vein Endothelial Cells; Humans; Inflammation Mediators; Lipopolysaccharides; Male; Monocytes; Phyllanthus emblica; Phytotherapy; Plant Extracts; Rats; Rats, Wistar; Thromboplastin; von Willebrand Factor

Among circulating cell-derived microparticles, those derived from red cells (RMP) have been least well investigated. To exploit potential haemostatic benefit of RMP, we developed a method of producing them in quantity, and here report on their haemostatic properties. High-pressure extrusion of washed RBC was employed to generate RMP. RMP were identified and enumerated by flow cytometry. Their size distribution was assessed by Doppler electrophoretic light scattering analysis (DELSA). Interaction with platelets was studied by platelet aggregometry, and shear-dependent adhesion by Diamed IMPACT-R. Thrombin generation and tissue factor (TF) expression was also measured. The effect of RMP on blood samples of patients with bleeding disorders was investigated ex vivo by thromboelastography (TEG). Haemostatic efficacy in vivo was assessed by measuring reduction of blood loss and bleeding time in rats and rabbits. RMP have mean diameter of 0.45 µm and 50% of them exhibit annexin V binding, a proxy for procoagulant phospholipids (PL). No TF could be detected by flow cytometry. At saturating concentrations of MPs, RMP generated thrombin robustly but after longer delay compared to PMP and EMP. RMP enhanced platelet adhesion and aggregation induced by low-dose ADP or AA. In TEG study, RMP corrected or improved haemostatic defects in blood of patients with platelet and coagulation disorders. RMP reduced bleeding time and blood loss in thrombocytopenic rabbits (busulfan-treated) and in Plavix-treated rats. In conclusion, RMP has broad haemostatic activity, enhancing both primary (platelet) and secondary (coagulation) haemostasis, suggesting potential use as haemostatic agent for treatment of bleeding.

Topics: Adenosine Diphosphate; Animals; Bleeding Time; Blood Coagulation Disorders; Busulfan; Cell Separation; Cell-Derived Microparticles; Clopidogrel; Disease Models, Animal; Erythrocytes; Flow Cytometry; Hemostasis; Humans; Male; Platelet Aggregation; Rabbits; Rats; Rats, Sprague-Dawley; Thrombelastography; Thrombin; Thrombocytopenia; Thromboplastin; Ticlopidine

To determine the lung expression of tissue factor (TF) mRNA in hepatopulmonary syndrome (HPS) using a rat model system and to investigate the potential significance of its differential expression.. Forty male Sprague-Dawley rats were used to establish models of cirrhosis (n = 20) and HPS (n = 20). Blood gas analysis was used to investigate the effects of each model on pulmonary function. Effects on the expression of TF mRNA in lung were determined by qRT-PCR and on lung pathology by histological analysis.. The HPS rats showed significantly lower PaO2 than the cirrhosis rats (58.20 +/- 3.19 mmHg vs. 85.00 +/- 2.53 mmHg, P less than 0.05) but significantly higher TF mRNA expression in lung (0.77 +/- 0.22 vs. 0.33 +/- 0.14, P less than 0.05). TF mRNA expression was negatively correlated with the value of PaO2 (r = -0.565, P less than 0.05). The lungs of the cirrhosis rats showed widened alveolar intervals, diversified sizes of alveolar spaces, reduced lung capacity, inflammatory cell infiltration, and hyperemia in the pulmonary vessels. The lungs of the HPS rats showed all of the same changes but also with accumulated macrophages and micro-thrombosis in the pulmonary vessels. Among the HPS rats, those with micro-thrombosis in pulmonary vessels showed a greater increase in TF mRNA expression than those without (0.68 +/- 0.17 vs. 0.40 +/- 0.12, P less than 0.05).. The expression of TF mRNA in lung of hepatopulmonary syndrome model rats was elevated and might increase the incidence of thromboembolism in the lung.

Topics: Animals; Disease Models, Animal; Hepatopulmonary Syndrome; Lung; Male; Rats; Rats, Sprague-Dawley; RNA, Messenger; Thromboplastin

Radiosurgery for glioblastoma is limited to the development of resistance, allowing tumor cells to survive and initiate tumor recurrence. Based on our previous work that coadministration of tissue factor and lipopolysaccharide following radiosurgery selectively induced thrombosis in cerebral arteriovenous malformations, achieving thrombosis of 69% of the capillaries and 39% of medium sized vessels, we hypothesized that a rapid and selective shutdown of the capillaries in glioblastoma vasculature would decrease the delivery of oxygen and nutrients, reducing tumor growth, preventing intracranial hypertension, and improving life expectancy. Glioblastoma was formed by implantation of GL261 cells into C57Bl/6 mouse brain. Mice were intravenously injected tissue factor, lipopolysaccharide, a combination of both, or placebo 24 hours after radiosurgery. Control mice received both agents after sham irradiation. Coadministration of tissue factor and lipopolysaccharide led to the formation of thrombi in up to 87 ± 8% of the capillaries and 46 ± 4% of medium sized vessels within glioblastoma. The survival rate of mice in this group was 80% versus no survivor in placebo controls 30 days after irradiation. Animal body weight increased with time in this group (r = 0.88, P = 0.0001). Thus, radiosurgery enhanced treatment with tissue factor, and lipopolysaccharide selectively induces thrombosis in glioblastoma vasculature, improving life expectancy.

Topics: Animals; Combined Modality Therapy; Disease Models, Animal; Glioblastoma; Humans; Lipopolysaccharides; Mice; Neoplasm Recurrence, Local; Neoplasms, Experimental; Radiosurgery; Thromboplastin; Thrombosis

Idiopathic avascular necrosis (AVN) of bone causes significant morbidity in adults although the pathophysiology is unknown. The present treatment options include systemic biphosphonate therapy and local bone drilling decompression, ameliorating the healing process and their by render the weight bearing femur head less vulnerable to collapse. In the present study we demonstrate the involvement of heparanase in AVN and in the acceptable treatments.. 56 female rats were studied. In 8 control rats AVN was induced by ligamentum teres ligation of the right femur while the left femur remained intact. In the rest of the rats, in addition to right femur AVN, treatment was added by subcutaneous biphosphonate therapy, femoral head drilling or combination of the treatments. All rats were scarified after 6weeks. Immunostaining of the femur heads were performed to heparanase, tissue factor pathway inhibitor (TFPI), tissue factor (TF) and hematoxylin-eosin.. Staining of heparanase, TFPI and TF were most prominent in the bone-marrow tissue of the femur heads. Staining by hematoxylin-eosin revealed damaged femur heads with prominent heparanase and TFPI staining in the femur with AVN compared to the contra lateral side of the same rat. No difference was found in the TF staining between the two sides. In the drilling and / or biphosphonate therapy groups, in contrast to the control group, femur heads were preserved with no significant difference in heparanase and TFPI staining between the two sides.. Heparanase and TFPI are locally elevated in the process of AVN and are normalized by the acceptable treatments. Inhibition of heparanase by heparins can potentially improve the nowadays therapy modalities.

Topics: Alendronate; Animals; Bone Density Conservation Agents; Combined Modality Therapy; Decompression, Surgical; Disease Models, Animal; Female; Femur Head; Femur Head Necrosis; Glucuronidase; Hemostasis; Immunohistochemistry; Injections, Subcutaneous; Lipoproteins; Orthopedic Procedures; Rats; Rats, Sprague-Dawley; Staining and Labeling; Thromboplastin; Time Factors

Acyl-CoA:cholesterol acyltransferase (ACAT) converts cholesterol to cholesteryl esters in plaque foam cells. Complete deficiency of macrophage ACAT has been shown to increase atherosclerosis in hypercholesterolemic mice because of cytotoxicity from free cholesterol accumulation, whereas we previously showed that partial ACAT inhibition by Fujirebio compound F1394 decreased early atherosclerosis development. In this report, we tested F1394 effects on preestablished, advanced lesions of apolipoprotein-E-deficient mice.. Apolipoprotein-E-deficient mice on Western diet for 14 weeks developed advanced plaques, and were either euthanized (Baseline), or continued on Western diet with or without F1394 and euthanized after 14 more weeks. F1394 was not associated with systemic toxicity. Compared with the baseline group, lesion size progressed in both groups; however, F1394 significantly retarded plaque progression and reduced plaque macrophage, free and esterified cholesterol, and tissue factor contents compared with the untreated group. Apoptosis of plaque cells was not increased, consistent with the decrease in lesional free cholesterol. There was no increase in plaque necrosis and unimpaired efferocytosis (phagocytic clearance of apoptotic cells). The effects of F1394 were independent of changes in plasma cholesterol levels.. Partial ACAT inhibition by F1394 lowered plaque cholesterol content and had other antiatherogenic effects in advanced lesions in apolipoprotein-E-deficient mice without overt systemic or plaque toxicity, suggesting the continued potential of ACAT inhibition for the clinical treatment of atherosclerosis, in spite of recent trial data.

Topics: Acetyl-CoA C-Acyltransferase; Animals; Aorta; Aortic Diseases; Apolipoproteins E; Apoptosis; Atherosclerosis; Cholesterol; Cyclohexanes; Diet, Atherogenic; Dioxanes; Disease Models, Animal; Disease Progression; Enzyme Inhibitors; Foam Cells; Male; Mice; Mice, Knockout; Necrosis; Plaque, Atherosclerotic; Thromboplastin

Acute lung injury is a principal cause of morbidity and mortality in response to mustard gas (SM) inhalation. Obstructive, fibrin-containing airway casts have recently been reported in a rat inhalation model employing the SM analog 2-chloroethyl ethyl sulfide (CEES). The present study was designed to identify the mechanism(s) causing activation of the coagulation cascade after CEES-induced airway injury. Here we report that CEES inhalation elevates tissue factor (TF) activity and numbers of detached epithelial cells present in lavage fluid (BALF) from rats after exposure (18 h). In vitro studies using 16HBE cells, or with rat BALF, indicated that detached epithelial cells could convert factor X (FX) to the active form FXa when incubated with factor VII and could elicit rapid clotting of plasma. In addition, immunocytochemical analysis demonstrated elevated cell surface (TF) expression on CEES-exposed 16HBE cells as a function of time. However, total cell TF expression did not increase. Since membrane surfaces bearing TF are important determinants of clot initiation, anticoagulants directed against these entities were tested for ability to limit plasma clotting or FX activation capacity of BALF or culture media. Addition of tifacogin, a TF pathway inhibitor, effectively blocked either activity, demonstrating that the procoagulant actions of CEES were TF pathway dependent. Lactadherin, a protein capable of competing with clotting factors for phospholipid-binding sites, was partially effective in limiting these procoagulant actions. These findings indicate that TF pathway inhibition could be an effective strategy to prevent airway obstruction after SM or CEES inhalation.

Topics: Airway Obstruction; Animals; Antigens, Surface; Bronchi; Bronchoalveolar Lavage Fluid; Cell Line, Transformed; Chemical Warfare Agents; Disease Models, Animal; Epithelial Cells; Factor VII; Factor Xa; Humans; Inhalation Exposure; Male; Milk Proteins; Mustard Gas; Proteins; Rats; Rats, Sprague-Dawley; Thromboplastin; Time Factors

Acute lung injury is still a significant clinical problem with a high mortality rate and there are few effective therapies in clinic. Here, we studied the inhibitory effect of ruscogenin, an anti-inflammatory and anti-thrombotic natural product, on lipopolysaccharide (LPS)-induced acute lung injury in mice basing on our previous studies. The results showed that a single oral administration of ruscogenin significantly decreased lung wet to dry weight (W/D) ratio at doses of 0.3, 1.0 and 3.0 mg/kg 1 h prior to LPS challenge (30 mg/kg, intravenous injection). Histopathological changes such as pulmonary edema, coagulation and infiltration of inflammatory cells were also attenuated by ruscogenin. In addition, ruscogenin markedly decreased LPS-induced myeloperoxidase (MPO) activity and nitrate/nitrite content, and also downregulated expression of tissue factor (TF), inducible NO synthase (iNOS) and nuclear factor (NF)-κB p-p65 (Ser 536) in the lung tissue at three doses. Furthermore, ruscogenin reduced plasma TF procoagulant activity and nitrate/nitrite content in LPS-induced ALI mice. These findings confirmed that ruscogenin significantly attenuate LPS-induced acute lung injury via inhibiting expressions of TF and iNOS and NF-κB p65 activation, indicating it as a potential therapeutic agent for ALI or sepsis.

Topics: Acute Lung Injury; Animals; Anti-Inflammatory Agents; Disease Models, Animal; Lipopolysaccharides; Male; Mice; Mice, Inbred ICR; NF-kappa B; Nitrates; Nitric Oxide Synthase Type II; Nitrites; Peroxidase; Spirostans; Thromboplastin

The coagulation-inflammation cycle has been implicated as a critical component in malaria pathogenesis. Defibrotide (DF), a mixture of DNA aptamers, displays anticoagulant, anti-inflammatory, and endothelial cell (EC)-protective activities and has been successfully used to treat comatose children with veno-occlusive disease. DF was investigated here as a drug to treat cerebral malaria.. DF blocks tissue factor expression by ECs incubated with parasitized red blood cells and attenuates prothrombinase activity, platelet aggregation, and complement activation. In contrast, it does not affect nitric oxide bioavailability. We also demonstrated that Plasmodium falciparum glycosylphosphatidylinositol (Pf-GPI) induces tissue factor expression in ECs and cytokine production by dendritic cells. Notably, dendritic cells, known to modulate coagulation and inflammation systemically, were identified as a novel target for DF. Accordingly, DF inhibits Toll-like receptor ligand-dependent dendritic cells activation by a mechanism that is blocked by adenosine receptor antagonist (8-p-sulfophenyltheophylline) but not reproduced by synthetic poly-A, -C, -T, and -G. These results imply that aptameric sequences and adenosine receptor mediate dendritic cells responses to the drug. DF also prevents rosetting formation, red blood cells invasion by P. falciparum and abolishes oocysts development in Anopheles gambiae. In a murine model of cerebral malaria, DF affected parasitemia, decreased IFN-γ levels, and ameliorated clinical score (day 5) with a trend for increased survival.. Therapeutic use of DF in malaria is proposed.

Topics: Animals; Anti-Inflammatory Agents; Anticoagulants; Antimalarials; Blood Coagulation; Cells, Cultured; Complement Activation; Cytokines; Dendritic Cells; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelial Cells; Female; Glycosylphosphatidylinositols; Hemoglobins; Humans; Inflammation Mediators; Malaria, Cerebral; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Nitric Oxide; Plasmodium berghei; Plasmodium falciparum; Platelet Aggregation; Polydeoxyribonucleotides; Receptors, Purinergic P1; Severity of Illness Index; Thromboplastin; Time Factors

Tissue factor (TF) is a potent initiator of the extrinsic coagulation cascade. The role and source of TF in venous thrombotic disease is not clearly defined. Our study objective was to identify the contribution of myeloid cell TF to venous thrombogenesis in mice.. The mouse electrolytic inferior vena cava model was used to induce thrombosis. The following groups of mice were used (1) TF(flox/flox)LysMCre(+) mice that have reduced TF expression in myeloid cells, (2) TF(flox/flox)LysMCre(-) littermate controls, (3) Wild type mice given a monoclonal anti-mouse TF antibody (1H1) to inhibit TF activity, and (4) Wild type mice given rat IgG. Evaluations at baseline, day 2, and day 6 post thrombosis included thrombus weight, vein wall inflammatory cell migration, vein wall TF mRNA, and plasma D-dimer levels.. Inhibition of TF significantly decreased thrombus weight 2days post venous thrombosis. In contrast, TF(flox/flox)LysMCre(+) had no change in thrombus weight when compared to littermate controls. The absence of myeloid cell TF did not affect infiltration of neutrophils or monocytes into the vein wall. TF mRNA expression in the vein wall decreased at 2days but then returned to baseline levels by 6days post thrombosis. D-dimer levels peaked at 2days post thrombosis in mice with or without myeloid cell TF.. TF is important in the formation of venous thrombi in the macrovasculature. However, TF expression by myeloid cells does not significantly contribute to venous thrombogenesis in this model.

Topics: Animals; Disease Models, Animal; Fibrin Fibrinogen Degradation Products; Gene Expression Regulation; Male; Mice; Mice, Inbred C57BL; Myeloid Cells; Rats; RNA, Messenger; Thromboplastin; Vena Cava, Inferior; Venous Thrombosis

Sepsis, a leading cause of death worldwide, involves widespread activation of inflammation, massive activation of coagulation, and lymphocyte apoptosis. Calpains, calcium-activated cysteine proteases, have been shown to increase inflammatory reactions and lymphocyte apoptosis. Moreover, calpain plays an essential role in microparticle release.. We investigated the contribution of calpain in eliciting tissue damage during sepsis.. To test our hypothesis, we induced polymicrobial sepsis by cecal ligation and puncture in wild-type (WT) mice and transgenic mice expressing high levels of calpastatin, a calpain-specific inhibitor.. In WT mice, calpain activity increased transiently peaking at 6 hours after cecal ligation and puncture surgery. Calpastatin overexpression improved survival, organ dysfunction (including lung, kidney, and liver damage), and lymphocyte apoptosis. It decreased the sepsis-induced systemic proinflammatory response and disseminated intravascular coagulation, by reducing the number of procoagulant circulating microparticles and therefore delaying thrombin generation. The deleterious effect of microparticles in this model was confirmed by transferring microparticles from septic WT to septic transgenic mice, worsening their survival and coagulopathy.. These results demonstrate an important role of the calpain/calpastatin system in coagulation/inflammation pathways during sepsis, because calpain inhibition is associated with less severe disseminated intravascular coagulation and better overall outcomes in sepsis.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Cell-Derived Microparticles; Cytokines; Disease Models, Animal; Disseminated Intravascular Coagulation; Lymphocytes; Mice; Mice, Inbred C57BL; Mice, Transgenic; Multiple Organ Failure; NF-kappa B; Sepsis; Thromboplastin

Despite improvements in treatment, myocardial infarction (MI) remains an important cause of morbidity and mortality. Inflammation arising from ischaemic and reperfusion injury is a key mechanism which underpins myocardial damage and impairment of cardiac function. Early growth response-1 (Egr-1) is an early immediate gene and a master regulator that has been implicated in the pathogenesis of ischaemia-reperfusion (IR) injury. This study sought to examine the effect of selective inhibition of Egr-1 using catalytic deoxyribonucleic acid molecules (DNAzymes, DZs) delivered via the clinically relevant coronary route in a large animal model of myocardial IR. It was hypothesized that Egr-1 inhibition with intracoronary DZ would reduce infarction size by modulating its downstream effector molecules. Egr-1 DZs inhibited the adherence of THP-1 monocytes to IL-1β-activated endothelial cells in vitro and retained its catalytic activity up to 225 min after in vivo administration. In a porcine model of myocardial IR (45 min ischaemia/3 h reperfusion), DZ was taken up in the cytoplasm and nuclei of cardiomyocytes and endothelial cells in the myocardium after intracoronary delivery. Egr-1 DZs reduced infarct size and improved cardiac functional recovery following intracoronary delivery at the initiation of IR in this large animal model of MI. This was associated with inhibition of pro-inflammatory Egr-1 and ICAM-1 expression, and the reduced expression of TNF-α, PAI-1, TF, and myocardial MPO activity in tissue derived from the border zone of the infarct. Taken together, these data suggest that strategies targeting Egr-1 via the intracoronary route after IR injury in pigs have potential therapeutic implications in human MI.

Topics: Animals; Cell Adhesion; Cells, Cultured; Disease Models, Animal; DNA, Single-Stranded; Early Growth Response Protein 1; Endothelial Cells; Female; Genetic Therapy; Humans; Injections; Intercellular Adhesion Molecule-1; Interleukin-1beta; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Monocytes; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Peroxidase; Plasminogen Activator Inhibitor 1; Recovery of Function; Swine; Thromboplastin; Time Factors; Transfection; Tumor Necrosis Factor-alpha

Pharmaceutical grade heparins from porcine intestine and bovine lung consist mainly of repeating tri-sulfated units, of the disaccharide →4-α-IdoA2S-1→4-α-GlcNS6S-1→. Heparin preparations from bovine intestine, in contrast, are more heterogeneous. Nuclear magnetic resonance (NMR) and disaccharide analysis after heparinase digestions show that heparin from bovine intestine contains α-glucosamine with significant substitutive variations: 64% are 6-O-sulfated and N -sulfated, as in porcine intestinal heparin while 36% are 6-desulfated. Desulfated α-iduronic acid units are contained in slightly lower proportions in bovine than in porcine heparin. NMR data also indicate N-, 3- and 6-trisulfated α-glucosamine (lower proportions) and α-GlcNS-1→4-α-GlcA and α-IdoA2S-1→4-α-GlcNAc (higher amounts) in bovine than in porcine heparin. Porcine and bovine heparins can be fractionated by anion exchange chromatography into three fractions containing different substitutions on the α-glucosamine units. Each individual fraction shows close disaccharide composition and anticoagulant activity, regardless of their origin (bovine or porcine intestine). However, these two heparins differ markedly in the proportions of the three fractions. Interestingly, fractions with the typical heparin disaccharides of porcine intestine are present in bovine intestinal heparin. These fractions contain high in vitro anticoagulant activity, reduced antithrombotic effect and high bleeding tendency. These observations indicate that the prediction of haemostatic effects of heparin preparations cannot rely exclusively on structural analysis and anticoagulant assays in vitro . Minor structural components may account for variations on in vivo effects. In conclusion, we suggest that pharmaceutical grade bovine intestinal heparin, even after purification procedures, is not an equivalent drug to porcine intestinal heparin.

Topics: Animals; Anion Exchange Resins; Anticoagulants; Antithrombin Proteins; Blood Coagulation; Cattle; Chromatography, Ion Exchange; Disaccharides; Disease Models, Animal; Factor Xa; Factor Xa Inhibitors; Female; Fibrinolytic Agents; Glycosylation; Hemorrhage; Heparin; Heparin Antagonists; Heparin Lyase; Humans; Intestinal Mucosa; Magnetic Resonance Spectroscopy; Male; Molecular Structure; Partial Thromboplastin Time; Protamines; Prothrombin; Rats; Rats, Wistar; Structure-Activity Relationship; Sulfates; Swine; Thromboplastin; Venous Thrombosis

Heart failure (HF) has been recognized as a hypercoagulable state. However, the natural anticoagulation systems in the failing heart have not been studied. Recent experimental and clinical data have indicated that not only the thrombomodulin (TM)/protein C (PC) pathway but also the protein S (PS)/tissue factor pathway inhibitor (TFPI) system function as potent natural anticoagulants. To investigate the balance between procoagulant and anticoagulant activities in the failing heart, we measured the cardiac expression of tissue factor (TF), type 1 plasminogen activator inhibitor (PAI-1), TM, PC, PS, and TFPI by RT-PCR and/or Western blot analysis in male transgenic (TG) mice with heart-specific overexpression of TNF-α. Both procoagulant (TF and PAI-1) and anticoagulant (PS and TFPI) factors were upregulated in the myocardium of 24-wk-old TG (end-stage HF) but not in that of 4-wk-old TG (early decompensated HF) compared with the wild-type mice. Both factors were also upregulated in the infarcted myocardium at 3 days after coronary ligation in the wild-type mice. The expression of TM was downregulated in the TG heart, and PC was not detected in the hearts. The transcript levels of PS orphan receptors, Mer and Tyro3, but not Axl, were significantly upregulated in the TG heart. Double immunohistochemical staining revealed that myocardial infiltrating CD3-positive T cells may produce PS in the TG myocardium. In conclusion, the PS/TFPI was upregulated in the myocardium of a different etiological model of HF, thus suggesting a role for the PS/TFPI system in the protection of the failing heart under both inflammatory and hypercoagulable states.

Topics: Animals; Disease Models, Animal; Heart Failure; Lipoproteins; Male; Mice; Mice, Transgenic; Myocardium; Plasminogen Activator Inhibitor 1; Protein C; Protein S; RNA, Messenger; Thrombomodulin; Thromboplastin; Tumor Necrosis Factor-alpha; Up-Regulation

Although the lung expresses procoagulant proteins under inflammatory conditions, underlying mechanisms remain unclear. Here, we addressed lung endothelial expression of tissue factor (TF), which initiates the coagulation cascade and expression of which signifies development of a procoagulant phenotype in the vasculature. To establish the model of acid-induced acute lung injury (ALI), we intranasally instilled anesthetized mice with saline or acid. Then 2 h later, we isolated pulmonary vascular cells for flow cytometry and confocal microscopy to detect the leukocyte antigen, CD45 and the endothelial markers VE-cadherin and von Willebrand factor (vWf). Acid increased both the number of vWf-expressing cells as well as TF and P-selectin expressions on these cells. All of these effects were markedly inhibited by treating mice with antiplatelet serum, suggesting the involvement of platelets. The increased expressions of TF, vWf, and P-selectin in response to acid also occurred in platelets. Moreover, the effects were replicated in endothelial cells derived from isolated, blood-perfused lungs. However, the effect was inhibited completely in lungs perfused with platelet-depleted and, to a lesser extent, with leukocyte-depleted blood. Acid injury increased endothelial expressions of the platelet proteins, CD41 and CD42b, providing evidence that platelet proteins were transferred to the vascular surface. Reactive oxygen species (ROS) were implicated in these responses, in that the endothelial and platelet protein expressions were inhibited. We conclude that acid-induced ALI causes NOX2-mediated ROS generation that activates platelets, which then generate a procoagulant endothelial surface.

Topics: Acute Lung Injury; Animals; Antigens, CD; Blood Coagulation; Blood Platelets; Cadherins; Disease Models, Animal; Endothelial Cells; Endothelium, Vascular; Hydrochloric Acid; Leukocyte Common Antigens; Lung; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; NADPH Oxidase 2; NADPH Oxidases; P-Selectin; Platelet Activation; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIb-IX Complex; Platelet Membrane Glycoprotein IIb; Reactive Oxygen Species; Thromboplastin; von Willebrand Factor

Tissue factor (TF) is a significant risk factor for tumor growth and hepatic metastasis in patients with colorectal cancer (CRC). This study aimed to investigate whether hyperthermia has synergistic anti-tumor effects with TF knockdown in suppressing CRC progression and metastasis in vitro and in vivo.. Human colorectal cancer LOVO cells were treated by hyperthermia at 44°C for 2 hr or/and TF siRNA. Then the cells were subjected to colony formation assay. Apoptosis was analyzed by flow cytometry, confocal microscopy, and transmission electron microscopy. The cell migration and invasion abilities were analyzed by wound healing and matrigel assay. In addition, orthotopic nude mice model of CRC was established.. Hyperthermia synergized with TF knockdown to reduce colony formation ability, induce apoptosis, and suppress the migration and invasion of LOVO cells in vitro. Moreover, hyperthermia in combination with TF depletion inhibited the growth and hepatic metastasis of CRC in orthotopic nude mice model. Mechanistically, the synergistic effects were at least partly mediated by inducing JNK mediated apoptosis and suppressing matrix metalloproteinases (MMPs) mediated invasion.. Hyperthermia in combination with TF-targeted therapy could be a potential approach for CRC treatment.

Topics: Animals; Apoptosis; Cell Line, Tumor; Colorectal Neoplasms; Disease Models, Animal; Female; Hyperthermia, Induced; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; NF-kappa B; RNA, Small Interfering; Thromboplastin

Hemophilia is treated by IV replacement therapy with Factor VIII (FVIII) or Factor IX (FIX), either on demand to resolve bleeding, or as prophylaxis. Improved treatment may be provided by drugs designed for subcutaneous and less frequent administration with a reduced risk of inhibitor formation. Tissue factor pathway inhibitor (TFPI) down-regulates the initiation of coagulation by inhibition of Factor VIIa (FVIIa)/tissue factor/Factor Xa (FVIIa/TF/FXa). Blockage of TFPI inhibition may facilitate thrombin generation in a hemophilic setting. A high-affinity (K(D) = 25pM) mAb, mAb 2021, against TFPI was investigated. Binding of mAb 2021 to TFPI effectively prevented inhibition of FVIIa/TF/FXa and improved clot formation in hemophilia blood and plasma. The binding epitope on the Kunitz-type protease inhibitor domain 2 of TFPI was mapped by crystallography, and showed an extensive overlap with the FXa contact region highlighting a structural basis for its mechanism of action. In a rabbit hemophilia model, an intravenous or subcutaneous dose significantly reduced cuticle bleeding. mAb 2021 showed an effect comparable with that of rFVIIa. Cuticle bleeding in the model was reduced for at least 7 days by a single intravenous dose of mAb 2021. This study suggests that neutralization of TFPI by mAb 2021 may constitute a novel treatment option in hemophilia.

Topics: Animals; Antibodies, Blocking; Antibodies, Monoclonal, Humanized; Antibodies, Neutralizing; Bleeding Time; Blood Coagulation; Cross Reactions; Disease Models, Animal; Epitopes; Factor VIII; Factor Xa; Female; Fibrin; HEK293 Cells; Hemophilia A; Hemostasis; Human Umbilical Vein Endothelial Cells; Humans; Lipoproteins; Models, Molecular; Neutralization Tests; Protein Binding; Protein Structure, Tertiary; Rabbits; Species Specificity; Thromboplastin

Hypoxemia in the circulation can lead to venous thrombosis (VT) through tissue factor (TF) activation, but the mechanism of TF activation in hypoxia remains obscure. Ligands released from damaged tissues or cells due to hypoxia are identified by various pattern-recognition receptors (PRR), including Toll-like receptor3 (TLR3). In the present study, we investigated the mechanism of TF activation during acute hypoxia in a rat model. The expression of TLR3 and TF was analyzed by immunoblotting and RT-PCR. The TF activity was evaluated by two-stage chromogenic assay and fibrin deposition was detected by immunohistochemistry. The expression of TLR3, TF, and TF activity was increased significantly 6 h post acute hypoxia and then decreased gradually. The contribution of TLR3 in TF activation was investigated by poly I:C and TLR3 neutralizing antibody. We also found increased ERK phosphorylation both in acute hypoxia and poly I:C treatment. We further showed that the pre-treatment of TLR3 neutralizing antibody or ERK inhibitor (PD98059) 2 h prior to acute hypoxia or poly I:C treatment completely abrogated ERK phosphorylation and TF activation. The pre-treatment of TLR3 neutralizing antibody also inhibited fibrin deposition in lung vasculature. These data indicate that acute hypoxia induced TF activation is mediated through TLR3-ERK1/2 pathway.

Topics: Animals; Antibodies, Neutralizing; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Fibrin; Flavonoids; Gene Expression Regulation; Hypoxia; Male; MAP Kinase Signaling System; Phosphorylation; Poly I-C; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Thromboplastin; Toll-Like Receptor 3

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

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

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

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

Patients with diabetes mellitus have an increased risk of suffering atherothrombotic syndromes and are prone to clustering cardiovascular risk factors. However, despite their dysregulated glucose metabolism, intensive glycemic control has proven insufficient to reduce thrombotic complications. Therefore, we aimed to elucidate the determinants of thrombosis in a model of type 2 diabetes mellitus with cardiovascular risk factors clustering.. Intravital microscopy was used to analyze thrombosis in vivo in Zucker diabetic fatty rats (ZD) and lean normoglycemic controls. Bone marrow (BM) transplants were performed to test the contribution of each compartment (blood or vessel wall) to thrombogenicity. ZD showed significantly increased thrombosis compared with lean normoglycemic controls. BM transplants demonstrated the key contribution of the hematopoietic compartment to increased thrombogenicity. Indeed, lean normoglycemic controls transplanted with ZD-BM showed increased thrombosis with normal glucose levels, whereas ZD transplanted with lean normoglycemic controls-BM showed reduced thrombosis despite presenting hyperglycemia. Significant alterations in megakaryopoiesis and platelet-endoplasmic reticulum stress proteins, protein disulfide isomerase and 78-kDa glucose-regulated protein, were detected in ZD, and increased tissue factor procoagulant activity was detected in plasma and whole blood of ZD.. Our results indicate that diabetes mellitus with cardiovascular risk factor clustering favors BM production of hyperreactive platelets with altered protein disulfide isomerase and 78-kDa glucose-regulated protein expression that can contribute to increase thrombotic risk independently of blood glucose levels.

Topics: Animals; Blood Coagulation; Blood Coagulation Tests; Blood Glucose; Blood Platelets; Bone Marrow Cells; Bone Marrow Transplantation; Diabetes Complications; Diabetes Mellitus, Type 2; Disease Models, Animal; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Green Fluorescent Proteins; Heat-Shock Proteins; Platelet Activation; Platelet Function Tests; Protein Disulfide-Isomerases; Rats; Rats, Transgenic; Rats, Wistar; Rats, Zucker; Thromboplastin; Thrombopoiesis; Thrombosis; Time Factors

Elevated serum levels of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) correlate with an increased risk for atherothrombotic events and TNFα is known to induce prothrombotic molecules in endothelial cells. Based on the preexisting evidence for the impact of TNFα in the pathogenesis of autoimmune disorders and their known association with an acquired hypercoagulability, we investigated the effects of TNFα and the role of the TNF receptor subtypes TNFR1 and TNFR2 for arteriolar thrombosis in vivo.. Arteriolar thrombosis and platelet-rolling in vivo were investigated in wildtype, TNFR1-/-, TNFR2-/- and TNFR1-/R2-/- C57BL/6 mice using intravital microscopy in the dorsal skinfold chamber microcirculation model. In vitro, expression of prothrombotic molecules was assessed in human endothelial cells by real-time PCR and flow cytometry.. In wildtype mice, stimulation with TNFα significantly accelerated thrombotic vessel occlusion in vivo upon ferric chloride injury. Arteriolar thrombosis was much more pronounced in TNFR1-/- animals, where TNFα additionally led to increased platelet-endothelium-interaction. TNFα dependent prothrombotic effects were not observed in TNFR2-/- and TNFR1-/R2- mice. In vitro, stimulation of human platelet rich plasma with TNFα did not influence aggregation properties. In human endothelial cells, TNFα induced superoxide production, p-selectin, tissue factor and PAI-1, and suppressed thrombomodulin, resulting in an accelerated endothelial dependent blood clotting in vitro. Additionally, TNFα caused the release of soluble mediators by endothelial cells which induced prothrombotic and suppressed anticoagulant genes comparable to direct TNFα effects.. TNFα accelerates thrombus formation in an in vivo model of arteriolar thrombosis. Its prothrombotic effects in vivo require TNFR2 and are partly compensated by TNFR1. In vitro studies indicate endothelial mechanisms to be responsible for prothrombotic TNFα effects. Our results support a more selective therapeutic approach in anticytokine therapy favouring TNFR2 specific antagonists.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Endothelium, Vascular; Female; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microcirculation; P-Selectin; Plasminogen Activator Inhibitor 1; Receptors, Tumor Necrosis Factor, Type I; Receptors, Tumor Necrosis Factor, Type II; Skin; Superoxides; Thrombomodulin; Thromboplastin; Thrombosis; Tumor Necrosis Factor-alpha

A suitable animal model is required to investigate plaque biology. Here, we examined 6 rabbit models of plaque generated by balloon injury and sequential combinations of normal and high-cholesterol diets.. Fifty-eight male Japanese White rabbits were used. Lipid-rich macrophages accumulated in the center of the intima, and smooth muscle cells were located on the luminal side of the intima (similar to stable plaques in human coronary arteries) of a model in which balloon injury was followed by a normal diet for 4 weeks and then by a high-cholesterol diet for 4 weeks. Extending the high-cholesterol diet for a further 4 weeks increased accumulation of lipid-rich macrophages, diminished the amounts of elastic fibers and smooth muscle cells in the intima and caused the expression of matrix metalloproteinase-9 and tissue factor. All of these features are characteristic of unstable plaques. Moreover, quantitative analysis revealed that matrix metalloproteinase-9 expression and elastic-fiber content inversely correlated with statistical significance (R(2) = 0.52, p = 0.0003).. A high-cholesterol diet for 0 to 8 weeks after a normal diet for the first 4 weeks following balloon injury induced various arterial lesions resembling the diffuse intimal thickening, as well as stable and unstable plaques that accumulate in human coronary arteries. The present models might be useful for plaque studies.

Topics: Angioplasty, Balloon, Coronary; Animals; Cell Division; Cholesterol, Dietary; Coronary Artery Disease; Coronary Vessels; Disease Models, Animal; Elasticity; Foam Cells; Humans; Lipids; Liver; Macrophages; Male; Matrix Metalloproteinase 9; Muscle, Smooth, Vascular; Rabbits; Thromboplastin

The mammalian silent information regulator-two 1 (Sirt1) blunts the noxious effects of cardiovascular risk factors such as type 2 diabetes mellitus and obesity. Nevertheless, the role of Sirt1 in regulating the expression of tissue factor (TF), the key trigger of coagulation, and arterial thrombus formation remains unknown.. Human as well as mouse cell lines were used for in vitro experiments, and C57Bl/6 mice for in vivo procedures. Sirt1 inhibition by splitomicin or sirtinol enhanced cytokine-induced endothelial TF protein expression as well as surface activity, while TF pathway inhibitor protein expression did not change. Sirt1 inhibition further enhanced TF mRNA expression, TF promoter activity, and nuclear translocation as well as DNA binding of the p65 subunit of nuclear factor-kappa B (NFκB/p65). Sirt1 siRNA enhanced TF protein and mRNA expression, and this effect was reduced in NFκB/p65(-/-) mouse embryonic fibroblasts reconstituted with non-acetylatable Lys(310)-mutant NFκB/p65. Activation of the mitogen-activated protein kinases p38, c-Jun NH(2)-terminal kinase, and p44/42 (ERK) remained unaffected. In vivo, mice treated with the Sirt1 inhibitor splitomicin exhibited enhanced TF activity in the arterial vessel wall and accelerated carotid artery thrombus formation in a photochemical injury model.. We provide pharmacological and genetic evidence that Sirt1 inhibition enhances TF expression and activity by increasing NFκB/p65 activation in human endothelial cells. Furthermore, Sirt1 inhibition induces arterial thrombus formation in vivo. Hence, modulation of Sirt1 may offer novel therapeutic options for targeting thrombosis.

Topics: Animals; Benzamides; Binding Sites; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelial Cells; Enzyme Activators; Genes, Reporter; Histone Deacetylase Inhibitors; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitogen-Activated Protein Kinases; Naphthalenes; Naphthols; Promoter Regions, Genetic; Pyrones; Resveratrol; RNA Interference; RNA, Messenger; Sirtuin 1; Stilbenes; Thromboplastin; Thrombosis; Transcription Factor RelA; Transfection

High plasma levels of C-reactive protein (CRP) constitute a powerful predictive marker of cardiovascular events. Several lines of evidence suggest that CRP has prothrombogenic effects. However, whether CRP directly participates in the pathogenesis of thrombosis in vivo has not been fully clarified.. To test whether human CRP (hCRP) affects arterial thrombus formation after balloon injury of smooth muscle cell (SMC)-rich or macrophage-rich neointima.. We compared the susceptibility of transgenic (Tg) rabbits expressing hCRP (46.21 ± 13.85 mg L(-1), n = 22) and non-Tg rabbits to arterial thrombus formation after balloon injury of SMC-rich or macrophage-rich neointima.. Thrombus size on SMC-rich or macrophage-rich neointima was significantly increased, and was accompanied by an increase in fibrin content in hCRP-Tg rabbits, as compared with non-Tg rabbits. Thrombus size did not significantly differ between SMC-rich and macrophage-rich neointima in hCRP-Tg rabbits. Tissue factor (TF) mRNA expression and activity in these neointimal lesions were significantly increased in hCRP-Tg rabbits as compared with non-Tg rabbits. The degree of CRP deposition correlated with the elevated TF expression and thrombus size on injured neointima. In addition, hCRP isolated from hCRP-Tg rabbit plasma induced TF mRNA expression and activity in rabbit cultured vascular SMCs.. These results suggest that elevated plasma hCRP levels promote thrombus formation on injured SMC-rich neointima by enhancing TF expression, but have no additive effects in macrophage-rich neointima.

Topics: Animals; Animals, Genetically Modified; C-Reactive Protein; Catheterization; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Femoral Artery; Humans; Hyperlipidemias; Macrophages; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Rabbits; RNA, Messenger; Thromboplastin; Thrombosis; Time Factors; Tunica Intima; Up-Regulation; Vascular System Injuries

The objective of this study was to investigate the effects of irbesartan, carvedilol, and irbesartan plus carvedilol on the expression of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) mRNA and protein in rat myocardium after myocardial infarction (MI). MI was induced in male Wistar rats by ligation of the anterior descending branch of the left coronary artery. Irbesartan at 50 mg/kg/day, carvedilol at 1 mg/kg/day, irbesartan plus carvedilol, or placebo was administered intragastrically; expression of TF and TFPI mRNA and protein was determined by RT-PCR and Western blot analysis. The relative left ventricle weights were lower in all three treatment groups than in the placebo group, with the lowest relative weight in the irbesartan plus carvedilol group (P < 0.001). The size of the infarcted area was lower in the carvedilol and the combined groups than in the placebo group (P < 0.001). The levels of expression of TF and TFPI mRNA and protein were lower in the combined group than in the placebo group or the carvedilol group (P < 0.001). Treatment with irbesartan plus carvedilol reduced the expression of TF and TFPI mRNA and protein after MI in rats, and combined treatment with both agents had greater effects than the single agents alone. These findings suggest that the beneficial effects of these drugs may include anticoagulation and that combined therapy with both agents is an option that should be evaluated further.

Topics: Adrenergic Antagonists; Angiotensin II Type 1 Receptor Blockers; Animals; Biphenyl Compounds; Blood Coagulation; Blotting, Western; Carbazoles; Carvedilol; Disease Models, Animal; Down-Regulation; Drug Therapy, Combination; Irbesartan; Lipoproteins; Male; Myocardial Infarction; Myocardium; Propanolamines; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetrazoles; Thromboplastin

To investigate the therapeutic effect of ligustrazine on hepatic veno-occlusive disease (HVOD) induced by Gynura segetum and the possible mechanism of it.. Female Kunming mice (115) were randomly divided into four groups, gavaged with 30 g/kg per day Gynura segetum (group A), 30 g/kg per day Gynura segetum + 100 mg/kg per day ligustrazine (group B), 30 g/kg per day Gynura segetum + 200 mg/kg per day ligustrazine (group C) or 30 mL/kg per day phosphate-buffered saline (PBS) (group D). Thirty days later, all of the mice were killed. Blood samples and livers were harvested. Histological changes were evaluated by light microscopy. Liver function was measured, and the expression of tissue factor (TF), early growth response factor-1 (Egr-1) and nuclear factor-KBp65 (NF-KBp65) were determined by reverse transcription-polymerase chain reaction and Western blot.. A total of 24 mice in group A developed HVOD. Compared with the controls, they had increased liver ratio, serum total bilirubin (TBIL), direct bilirubin (DBIL), transaminase and decreased albumin (ALB) (P < 0.05). Administration of ligustrazine improved the clinical signs and biochemistry parameters in a dose-dependent manner. Compared with group A, the expression of TF, Egr-1 and NF-KB p65 decreased in groups B and C (P < 0.05).. Ligustrazine has a therapeutic effect on HVOD, improving clinical manifestations and liver function. The possible mechanism may be that ligustrazine could reduce the expression of TF by downregulating the expression of transcription factors: Egr-1 and NF-KB p65.

Topics: Animals; Asteraceae; Bilirubin; Biomarkers; Blotting, Western; Disease Models, Animal; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Early Growth Response Protein 1; Female; Gene Expression Regulation; Hepatic Veno-Occlusive Disease; Liver; Mice; Pyrazines; Reverse Transcriptase Polymerase Chain Reaction; Serum Albumin; Thromboplastin; Time Factors; Transaminases; Transcription Factor RelA

These studies were aimed at characterizing an animal model of inflammation-induced hepatotoxicity that would mimic features of idiosyncratic liver toxicity observed in humans. An attempt was made to identify oxidative damage and the involvement of coagulation system in liver after monocrotaline (MCT) administration under the modest inflammatory condition induced by lipopolysaccharide (LPS) exposure. Mice were given MCT (200 mg/kg) or an equivalent volume of sterile saline (Veh.) po followed 4 h later by ip injection of LPS (6 mg/kg) or vehicle. Mice co-treated with MCT and LPS showed increased plasma alanine aminotransferase (ALT), decrease in platelet number, and a reduction in hematocrit. Accumulation of oxidized low-density lipoprotein (ox-LDL) was remarkably higher in the liver sections of mice co-treated with MCT and LPS compared to those given MCT or LPS alone. A similar trend was observed in the expression of CXCL16 receptor in the same liver sections. Elevated expression of tissue factor (TF) and fibrinogen was also observed in the liver sections of MCT/LPS co-treated mice. The in vitro results showed that incubation of HepG2 cells with CXCL16 antibody strongly diminished uptake of ox-LDL. Expression of ox-LDL, CXCL16, and TF represents an early event in the onset of hepatotoxicity induced by MCT/LPS; thus, it may contribute to our understanding of idiosyncratic liver injury and points to potential targets for protection or intervention.

Topics: Alanine Transaminase; Animals; Cell Culture Techniques; Chemical and Drug Induced Liver Injury; Chemokine CXCL16; Chemokine CXCL6; Chemokines, CXC; Collagen; Data Interpretation, Statistical; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Hep G2 Cells; Humans; Lipopolysaccharides; Lipoproteins, LDL; Liver; Male; Mice; Mice, Inbred Strains; Monocrotaline; Oxidation-Reduction; Receptors, Scavenger; Thromboplastin

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

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

The microvasculature assumes an inflammatory and procoagulant state in a variety of different diseases, including sickle cell disease (SCD), which may contribute to the high incidence of ischemic stroke in these patients. This study provides evidence for accelerated thrombus formation in arterioles and venules in the cerebral vasculature of mice that express hemoglobin-S (β(s) mice). Enhanced microvascular thrombosis in β(s) mice was blunted by immunologic or genetic interventions that target tissue factor, endothelial protein C receptor, activated protein C, or thrombin. Platelets from β(s) mice also exhibited enhanced aggregation velocity after stimulation with thrombin but not ADP. Neutropenia also protected against the enhanced thrombosis response in β(s) mice. These results indicate that the cerebral microvasculature is rendered vulnerable to thrombus formation in β(s) mice via a neutrophil-dependent mechanism that is associated with an increased formation of and enhanced platelet sensitivity to thrombin.

Topics: Anemia, Sickle Cell; Animals; Blood Platelets; Bone Marrow Transplantation; Cerebral Arteries; Disease Models, Animal; Hemoglobin, Sickle; Intracranial Thrombosis; Male; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Mutant Strains; Microcirculation; Neutrophils; Platelet Aggregation; Protein C; Thrombin; Thromboplastin

Hemophilia A and B are caused by deficiencies in coagulation factor VIII (FVIII) and factor IX, respectively, resulting in deficient blood coagulation via the intrinsic pathway. The extrinsic coagulation pathway, mediated by factor VIIa and tissue factor (TF), remains intact but is negatively regulated by tissue factor pathway inhibitor (TFPI), which inhibits both factor VIIa and its product, factor Xa. This inhibition limits clot initiation via the extrinsic pathway, whereas factor deficiency in hemophilia limits clot propagation via the intrinsic pathway. ARC19499 is an aptamer that inhibits TFPI, thereby enabling clot initiation and propagation via the extrinsic pathway. The core aptamer binds tightly and specifically to TFPI. ARC19499 blocks TFPI inhibition of both factor Xa and the TF/factor VIIa complex. ARC19499 corrects thrombin generation in hemophilia A and B plasma and restores clotting in FVIII-neutralized whole blood. In the present study, using a monkey model of hemophilia, FVIII neutralization resulted in prolonged clotting times as measured by thromboelastography and prolonged saphenous-vein bleeding times, which are consistent with FVIII deficiency. ARC19499 restored thromboelastography clotting times to baseline levels and corrected bleeding times. These results demonstrate that ARC19499 inhibition of TFPI may be an effective alternative to current treatments of bleeding associated with hemophilia.

Topics: Animals; Aptamers, Nucleotide; Bleeding Time; Blood Coagulation; Disease Models, Animal; Factor VIIa; Factor VIII; Factor Xa; Hemophilia A; Hemophilia B; Hemostasis; Humans; In Vitro Techniques; Lipoproteins; Macaca fascicularis; Recombinant Proteins; Thrombin; Thromboplastin

Epidemiologic studies suggest that the incidence and severity of sepsis are ameliorated in patients on statins (3- hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) for cholesterol lowering indications. We sought to understand the mechanism underlying such protection and hypothesized that simvastatin would be protective in mice against acute infection with Staphylococcus aureus, the primary etiologic agent in sepsis. Mice were treated with simvastatin or buffer for two weeks and were subsequently challenged with S. aureus intratracheally or intravenously. Relative to buffer-treated mice, bacterial killing was enhanced 4-fold (p=0.02), systemic dissemination was reduced, and lethality was decreased (hazard ratio 8.8, 95% CI 2.5 to 31.3, p=0.001) in mice that were pretreated with simvastatin for two weeks. Systemic inflammatory response was abrogated and the local elaboration of inflammatory mediators was diminished. Serum concentrations of pro-fibrinolytic protein C were elevated (p=0.034), while the concentration of pro-coagulant tissue factor in bronchoalveolar lavage fluids was attenuated (reduced 25%), p=0.001, in simvastatin-treated mice. Taken together, these data indicate that extended treatment with simvastatin is protective during infection with S. aureus through enhanced bacterial clearance, anti-inflammatory, and anti-coagulant activities. These studies provide insights into the mechanism by which statins confer protection in acute infection, support the notion that statins may be effective adjuncts in the treatment of sepsis, and provide a rationale for randomized control trials in patients that are at a high risk for infection characterized by coagulopathy.

Topics: Animals; Bacterial Load; Bronchoalveolar Lavage Fluid; C-Reactive Protein; Cytokines; Disease Models, Animal; Gene Expression; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Mice; Peroxidase; Pneumonia, Staphylococcal; Protein C; Simvastatin; Thromboplastin

Fibrin deposition is integral to the pathogenesis of collagen-induced arthritis (CIA), an experimental model of rheumatoid arthritis (RA). Membrane-associated fibrinogen-like protein 2 (mFGL2), a novel inducible prothrombinase, generates fibrin by an alternate pathway and has been reported to be involved in the pathogenesis of a number of immune-mediated diseases. We hypothesized that expression of mFGL2 in inflamed synovium contributes to the fibrin deposition and subsequent inflammation in arthritis.. DBA/1 mice were immunized with 100 µg bovine collagen type II (CII) emulsified in complete Freund's adjuvant (CFA) followed by lipopolysaccharide (LPS) injection. Expression of mFGL2 prothrombinase in association with fibrin deposition was examined in mice with CIA and CD200-treated mice following induction of CIA. To directly assess the contribution of mFGL2, fgl2(-/-) mice were injected with antibody to CII (anti-CII).. Levels of fgl2 mRNA transcripts and mFGL2 protein were markedly up-regulated in joints of mice that developed CIA. Fibrin deposition was prominent within the synovial lining and articular joint space associated with expression of mFGL2. Inhibition of CIA by the immunosuppressant CD200 was associated with decreased expression of fgl2 mRNA and mFGL2 protein and absence of fibrin deposition. Following injection of anti-CII, all fgl2(+/+) mice developed severe arthritis with clinical and histological manifestations characteristic of RA, whereas fgl2(-/-) mice failed to develop any clinical manifestation or histological evidence of arthritis.. This study demonstrates that the prothrombinase activity of mFGL2 contributes to the pathogenesis of experimental arthritis. These studies may have therapeutic implications for patients with RA.

Topics: Animals; Antigens, CD; Arthritis, Experimental; Disease Models, Animal; Fibrin; Fibrinogen; Immunosuppressive Agents; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Knockout; Signal Transduction; Synovial Membrane; Thromboplastin; Up-Regulation

Antiphospholipid syndrome (APS) is characterized by thromboembolic phenomena and recurrent fetal loss associated with elevated circulating anti-phospholipid/beta2glycoprotein-I(β2GPI)-binding-antibodies(Abs). Individual APS patients harbor diverse clusters of circulating anti-β2GPI Abs, targeting different epitopes on the β2GPI molecule. Our novel approach was to construct a peptide composed of β2GPI-ECs-binding-site (phospholipids-membrane), named "EMBI". EMBI exert dual activities: a) At first EMBI prevented β2GPI ECs binding, thus reduced by 89% the binding of β2GPI/anti-β2GPI to the cells in comparison with 9.3% inhibition by EMBI scrambled form (scEMBI). b) Longer exposure of ECs to EMBI resulted in intracellular EMBI penetration which did not prevent β2GPI/anti-β2GPI binding to HUVEC. Surprisingly, β2GPI/anti-β2GPI did not activate ECs harboring EMBI, illustrated by prevention of E-selectin and tissue factor (TF) expression. The inhibition of TF mRNA transcription was illustrated by quantitative RT-PCR. EMBI decreased the expression of phosphorylated JNK1/2, p38, HSP27 and enhanced phosphorylation of glycogen synthase kinase-3β (pGSK3β). Knocking down the GSK3β expression by siRNA-GSK3β, reduced the TF expression by β2GPI/anti-β2GPI-exposed-HUVEC. In-vivo, EMBI significantly decreased the percentage of fetal loss in naïve mice infused with anti-β2GPI Abs, p<0.04. Thus, the dual activity of EMBI may introduce EMBI as a potential novel candidate peptide, to treat patients with APS.

Topics: Animals; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Disease Models, Animal; E-Selectin; Endothelial Cells; Enzyme Inhibitors; Female; Fetal Death; Gene Expression Regulation, Enzymologic; Glycogen Synthase Kinase 3; Humans; Male; Mice; Mice, Inbred BALB C; Peptides; Phospholipids; Phosphorylation; Protein Binding; Protein Conformation; RNA, Messenger; Thromboplastin

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

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

The role and underlying mechanisms of rosiglitazone, a peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonist, on myocardial infarction are poorly understood. We investigated the effects of this PPAR-γ agonist on the expression of tissue factor (TF), a primary molecule for thrombosis, and elucidated its underlying mechanisms. The PPAR-γ agonist inhibited TF expression in response to TNF-α in human umbilical vein endothelial cells, human monocytic leukemia cell line, and human umbilical arterial smooth muscle cells. The overexpression of TF was mediated by increased phosphorylation of mitogen-activated protein kinase (MAPK), which was blocked by the PPAR-γ agonist. The effective MAPK differed depending on each cell type. Luciferase and ChIP assays showed that transcription factor, activator protein-1 (AP-1), was a pivotal target of the PPAR-γ agonist to lower TF transcription. Intriguingly, two main drugs for drug-eluting stent, paclitaxel or rapamycin, significantly exaggerated thrombin-induced TF expression, which was also effectively blocked by the PPAR-γ agonist in all cell types. This PPAR-γ agonist did not impair TF pathway inhibitor (TFPI) in three cell types. In rat balloon injury model (Sprague-Dawley rats, n = 10/group) with continuous paclitaxel infusion, the PPAR-γ agonist attenuated TF expression by 70±5% (n = 4; P<0.0001) in injured vasculature. Taken together, rosiglitazone reduced TF expression in three critical cell types involved in vascular thrombus formation via MAPK and AP-1 inhibitions. Also, this PPAR-γ agonist reversed the paclitaxel-induced aggravation of TF expression, which suggests a possibility that the benefits might outweigh its risks in a group of patients with paclitaxel-eluting stent implanted.

Topics: Animals; Catheterization; Disease Models, Animal; Enzyme Activation; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; Infusions, Intra-Arterial; Lipoproteins; Mitogen-Activated Protein Kinases; Models, Biological; Monocytes; Muscle, Smooth, Vascular; Paclitaxel; Phosphorylation; PPAR gamma; Promoter Regions, Genetic; Rats; Rats, Sprague-Dawley; RNA, Messenger; Rosiglitazone; Sirolimus; Thiazolidinediones; Thromboplastin; Umbilical Arteries

To evaluate the effects of aging on venous thrombosis.. Anesthetized male mice (C57BL/6, n=125) underwent complete inferior vena cava occlusion to produce venous thrombosis. Experimental groups included 11-month-old mice (OLD), 2-month-old mice (YOUNG), and age-matched non-thrombosed controls. Mice were euthanized and the following parameters were evaluated two days post-thrombosis: thrombus mass (grams/cm), vein wall inflammatory cells (cells per 5 high powered fields), active plasma plasminogen activator inhibitor-1 (PAI-1, ng/mL), vein wall P-selectin protein determination by ELISA (pg/mL), circulating plasma microparticles (MPs, MPs/200microL), MP tissue factor (TF) activity (pM), and in vivo MP re-injection experiments.. Thrombosed OLD mice had greater thrombus mass than YOUNG mice (389+/-18 vs. 336+/-14 gx10(-4)/cm, P<.05). OLD mice had decreased vein wall monocyte, lymphocyte, and total inflammatory cell populations versus YOUNG mice (P<.05). Vein wall P-selectin levels were greater in OLD thrombosed mice versus YOUNG (7306+/-938 vs. 3805+/-745pg/mL, P<.05). Active plasma PAI-1 concentrations were increased in OLD mice versus YOUNG thrombosed animals (20+/-4 vs. 8+/-2ng/mL, P<.05). OLD mice had significantly higher circulating leukocyte-derived MPs versus YOUNG mice (5817+/-850 vs. 2563+/-283 MPs/200muL PPP, P<.01). OLD mice had plasma MPs with increased TF activity versus YOUNG animals post-thrombosis (34+/-4 vs. 24+/-2 pM, P<.05). Finally, YOUNG recipient animals, whether re-injected with OLD or YOUNG donor MPs, had a significant increase in thrombus mass versus OLD recipient animals (P<.01).. Aging influenced several circulating and vein wall factors that decreased thrombus resolution in older animals compared to younger ones in our mouse thrombosis model.

Topics: Aging; Animals; Disease Models, Animal; Male; Mice; Mice, Inbred C57BL; P-Selectin; Plasminogen Activator Inhibitor 1; Thromboplastin; Thrombosis; Venous Thrombosis

We investigated the neuroprotective effects of fasudil's active metabolite, hydroxyfasudil, a Rho-kinase inhibitor, in a rat stroke model in which endothelial damage and subsequent thrombotic occlusion were selectively induced in perforating arteries. By examining the effects on the endothelial damage/dysfunction, we thought to explore the mechanism of Rho-kinase inhibitors. Hydroxyfasudil (10mg/kg, i.p., once daily for 3 days) significantly improved neurological functions and reduced the size of the infarct area produced by internal carotid artery injection of sodium laurate in a rat cerebral microthrombosis model. Treatment with fasudil or hydroxyfasudil concentration-dependently inhibited tumor necrosis factor alpha-induced tissue factor expression on the surface of cultured human umbilical vein endothelial cells. They also inhibited thrombin-induced endothelial hyperpermeability. The present findings suggest that hydroxyfasudil is efficacious in preventing brain damage associated with cerebral ischemia, and is partially responsible for fasudil's cytoprotective potential. The results also suggest that the therapeutic benefits against ischemic injury of Rho-kinase inhibitors are attributed, at least in part, to activity upon endothelial damage/dysfunction.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Brain; Brain Ischemia; Capillary Permeability; Cells, Cultured; Disease Models, Animal; Endothelium; Enzyme Inhibitors; Humans; In Vitro Techniques; Male; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; rho-Associated Kinases; Stroke; Thromboplastin; Tumor Necrosis Factor-alpha; Umbilical Veins

The vascular pathobiology of sickle cell anemia involves inflammation, coagulation, vascular stasis, reperfusion injury, iron-based oxidative biochemistry, deficient nitric oxide (NO) bioavailability, and red cell sickling. These disparate pathobiologies intersect and overlap, so it is probable that multimodality therapy will be necessary for this disease. We have, therefore, tested a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), for efficacy in reducing endothelial activation. We found that pulmonary vascular endothelial VCAM-1 and tissue factor (TF) expression (both are indicators of endothelial activation) are powerfully and significantly inhibited by TSA. This is seen both with pretreatment before the inducing stress of hypoxia/reoxygenation (NY1DD sickle transgenic mouse), and upon longer-term therapy after endothelial activation has already occurred (hBERK1 sickle mouse at ambient air). In addition, TSA prevented vascular stasis in sickle mice, it exhibited activity as an iron chelator, and it induced expression of the antisickling hemoglobin, hemoglobin F. Notably, the TSA analog SAHA (suberoylanilide hydroxaminc acid) that is already approved for human clinical use exhibits the same spectrum of biologic effects as TSA. We suggest that SAHA possibly could provide true, multimodality, salubrious effects for prevention and treatment of the chronic vasculopathy of sickle cell anemia.

Topics: Anemia, Sickle Cell; Animals; beta-Thalassemia; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Enzyme Inhibitors; Fetal Hemoglobin; Hemoglobin A; Hemoglobin, Sickle; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Intercellular Adhesion Molecule-1; Iron Chelating Agents; Mice; Mice, Inbred C57BL; Mice, Transgenic; Pulmonary Veins; Regional Blood Flow; Thromboplastin; Vascular Cell Adhesion Molecule-1; Venules; Vorinostat

Tissue factor (TF) antagonists targeting the factor VII (FVII) binding domain have been shown to interrupt acute vascular thrombus formation without impairing haemostasis in non-human primates. In this study, we evaluate whether a human/mouse chimeric monoclonal antibody (ALT-836, formerly known as Sunol-cH36) blocking the factor X/factor IX (FX/FIX) binding site of tissue factor could achieve similar clinical benefits in an arterial thrombosis model induced by surgical endarterectomy in chimpanzees. In this model, sequential surgical endarterectomies on right and left superficial femoral arteries were performed 30 days apart in five chimpanzees. A bolus (1 mg/kg) of ALT-836 was injected intravenously immediately preceding the restoration of flow in the endarterectomised femoral artery. Pre-surgical labelling of autologous platelets using (111)In-Oxine and post-surgical gamma camera imaging of (111)In-platelet deposition at endarterectomy sites was performed. The manipulated arterial segments were harvested for patency analysis 30 days following surgery. The results indicate that ALT-836 was highly effective at reducing acute vascular thrombosis, with no significant variations in surgical blood loss and template-bleeding time in the treated group compared to the control animals. These data suggest that ALT-836 is an effective and safe antithrombotic agent in preventing TF-initiated vascular thrombogenesis without compromising haemostasis.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Binding Sites; Blood Coagulation; Blood Loss, Surgical; CHO Cells; Cricetinae; Cricetulus; Disease Models, Animal; Dose-Response Relationship, Drug; Endarterectomy; Factor IX; Factor VIIa; Factor X; Female; Femoral Artery; Fibrinolytic Agents; Hemorrhage; Humans; Injections, Intravenous; Mice; Mice, Inbred BALB C; Pan troglodytes; Radionuclide Imaging; Recombinant Fusion Proteins; Thromboplastin; Thrombosis

To evaluate tissue factor (TF) expression in vein grafts interposed in the arterial circulation of hypercholesterolemic rabbits. Veins implanted in the arterial circulation of normocholesterolemic rabbits respond by inflammation and infiltration by monocytes with transient TF expression. In a hypercholesterolemic milieu these monocytes may differentiate into macrophages capable of enhanced TF synthesis, which may facilitate hyperplasia and thrombosis.. Autologous jugular veins interposed in the carotid artery of hypercholesterolemic rabbits were harvested at 1, 2, 4, 6, and 8 weeks after surgery and examined for presence and localization of rabbit TF antigen. Protein extracted from vein segments was evaluated for procoagulant activity by bioassay and for TF protein content by western blotting.. Rabbit TF antigen was observed mostly in the subendothelium of vein grafts. Peak TF procoagulant activity observed at 1-2 weeks postsurgery (2.3+/-1.8 pg/mg, P<0.006) declined to 0.9+/-0.5, 0.2+/-0.1, and 0.15+/-0.06 pg/mg at 4, 6, and 8 weeks, respectively (P<0.03). Western blotting showed a time-dependent pattern for rabbit TF protein with prolonged expression peaking at 6 weeks.. Prolonged expression of biologically active rabbit TF and TF protein were shown within jugular vein grafts of hypercholesterolemic rabbits. This response, reported for the first time and attributed to increased cholesterol levels, may possibly contribute to enhanced hyperplasia. These results suggest that TF expression could serve as another mechanism underlying vein graft failure and that hypercholesterolemia in bypass patients should be treated aggressively beginning within the weeks after surgery.

Topics: Animals; Blood Coagulation; Blood Coagulation Tests; Blotting, Western; Carotid Artery, Common; Cholesterol; Disease Models, Animal; Graft Rejection; Hypercholesterolemia; Immunohistochemistry; Jugular Veins; Rabbits; Thromboplastin; Time Factors; Transplantation, Autologous

Early growth response gene-1 (Egr-1) regulates the expression of genes important to cardiovascular disease. Within atherosclerotic lesions, Egr-1 is expressed in smooth muscle cells, endothelial cells, and macrophages. Since macrophages play a pivotal role in atherosclerotic lesion initiation and progression, this study investigated the effects of Egr-1 deficiency within bone marrow-derived cells on the development of atherosclerosis in a hyperlipidaemic mouse model.. Bone marrow from Egr-1-deficient mice and wild-type controls was transplanted into lethally irradiated LDL receptor null mice. After 26 weeks on a high fat diet, atherosclerotic lesion size within the aortic sinus of recipients was evaluated. Mice receiving Egr-1-deficient bone marrow had significantly decreased lesion size compared with controls. Lesions of these mice contained fewer macrophages and had reduced expression of vascular cell adhesion molecule-1 (VCAM-1), tissue factor, as well as transforming growth factor receptor type II, which are target genes of Egr-1. These results were validated by in vitro analysis of Egr-1-deficient peritoneal macrophages which, after lipopolysaccharide stimulation, had decreased VCAM-1 and tissue factor mRNA expression compared with wild-type controls.. This study demonstrates that bone marrow-derived Egr-1 promotes macrophage accumulation, atherosclerotic lesion development, and lesion complexity.

Topics: Animals; Aorta, Thoracic; Atherosclerosis; Bone Marrow Cells; Bone Marrow Transplantation; Disease Models, Animal; Early Growth Response Protein 1; Female; Gene Expression Regulation; Hyperlipidemias; Lipids; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Protein Serine-Threonine Kinases; Radiation Chimera; Receptor, Transforming Growth Factor-beta Type II; Receptors, LDL; Receptors, Transforming Growth Factor beta; RNA, Messenger; Thromboplastin; Time Factors; Vascular Cell Adhesion Molecule-1; Whole-Body Irradiation

Acadesine, an adenosine-regulating agent and activator of AMP-activated protein kinase, has been shown to possess antiinflammatory activity. This study investigated whether and how acadesine inhibits tissue factor (TF) expression and thrombus formation.. Human umbilical vein endothelial cells and human peripheral blood monocytes were stimulated with lipopolysaccharide to induce TF expression. Pretreatment with acadesine dramatically suppressed the clotting activity and expression of TF (protein and mRNA). These inhibitory effects of acadesine were unchanged for endothelial cells treated with ZM241385 (a specific adenosine A(2A) receptor antagonist) or AMP-activated protein kinase inhibitor compound C, and in macrophages lacking adenosine A(2A) receptor or alpha1-AMP-activated protein kinase. In endothelial cells and macrophages, acadesine activated the phosphoinositide 3-kinase/Akt signaling pathway, reduced the activity of mitogen-activated protein kinases, and consequently suppressed TF expression by inhibiting the activator protein-1 and NF-kappaB pathways. In mice, acadesine suppressed lipopolysaccharide-mediated increases in blood coagulation, decreased TF expression in atherosclerotic lesions, and reduced deep vein thrombus formation.. Acadesine inhibits TF expression and thrombus formation by activating the phosphoinositide 3-kinase/Akt pathway. This novel finding implicates acadesine as a potentially useful treatment for many disorders associated with thrombotic pathology, such as angina pain, deep vein thrombosis, and sepsis.

Topics: Adenosine A2 Receptor Antagonists; Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; Apolipoproteins E; Atherosclerosis; Blood Coagulation; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelial Cells; Enzyme Activation; Fibrinolytic Agents; Humans; Lipopolysaccharides; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Monocytes; NF-kappa B; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrazoles; Pyrimidines; Receptor, Adenosine A2A; Ribonucleosides; RNA, Messenger; Sepsis; Signal Transduction; Thromboplastin; Transcription Factor AP-1; Triazines; Triazoles; Up-Regulation; Venous Thrombosis

Our studies and those of many others have implicated hepatocyte necrosis and apoptosis mediated by fibrinogen-like protein-2 (fgl2) prothrombinase and tumor necrosis factor receptor (TNFR) in the development of fulminant viral hepatitis, a disease with a mortality rate greater than 80% in cases lacking immediate organ transplantation. This study was designed to explore the efficacy of dual short hairpin RNA (shRNA) interference with fgl2 and TNFR1 in the treatment of murine hepatitis virus strain 3 (MHV-3)-induced fulminant hepatitis in mice. Plasmids p-mfgl2shRNA and p-mTNFR1shRNA, complementary to the sequences for mfgl2 and mTNFR1, were constructed. Plasmids pEGFP-mfgl2 and pEGFP-mTNFR1 expressing mfgl2-EGFP (enhanced green fluorescent protein) and mTNFR1-EGFP fusion proteins were also constructed to screen the inhibitory effect of p-mfgl2shRNA and p-mTNFR1shRNA on mfgl2 and mTNFR1 expression. Cotransfection of individual shRNA plasmids and pcDNA3.0-mfgl2 and pcDNA3.0-mTNFR1 expression constructs into Chinese hamster ovary (CHO) cells significantly inhibited mfgl2 and mTNFR1 gene expression, as evidenced by fluorescence microscopy, reverse transcription-polymerase chain reaction, and Western blotting. In vivo hydrodynamic delivery of dual-interference shRNA plasmids for mfgl2 and mTNFR1 significantly decreased mfgl2 and mTNFR1 expression; markedly ameliorated fibrin deposition, hepatocyte necrosis, and apoptosis; and prolonged survival against fulminant viral hepatitis induced by MHV-3 in BALB/cJ mice compared with mfgl2 or TNFR1 single-gene interference. These results indicate that in vivo interference with genes for more than one key target provides superior treatment efficacy compared with single-gene interference.

Topics: Animals; Apoptosis; Blotting, Western; CHO Cells; Cricetinae; Cricetulus; Disease Models, Animal; Female; Fibrinogen; Gene Expression; Hydrodynamics; Liver Failure, Acute; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Murine hepatitis virus; Receptors, Tumor Necrosis Factor, Type I; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Thromboplastin; Transcription, Genetic

Increased phosphatidic acid (PA) and phospholipase D (PLD) activity are frequently observed in various disease states including cancers, diabetes, sepsis, and thrombosis. Previously, PA has been regarded as just a precursor for lysophosphatidic acid (LPA) and diacylglycerol (DAG). However, increasing evidence has suggested independent biological activities of PA itself. In the present study, we demonstrated that PA can enhance thrombogenic activities in human erythrocytes through phosphatidylserine (PS) exposure in a Ca(2+)-dependent manner. In freshly isolated human erythrocytes, treatment of PA or PLD induced PS exposure. PA-induced PS exposure was not attenuated by inhibitors of phospholipase A(2) or phosphatidate phosphatase, which converts PA to LPA or DAG. An intracellular Ca(2+) increase and the resultant activation of Ca(2+)-dependent PKC-alpha appeared to underlie the PA-induced PS exposure through the activation of scramblase. A marginal decrease in flippase activity was also noted, contributing further to the maintenance of exposed PS on the outer membrane. PA-treated erythrocytes showed strong thrombogenic activities, as demonstrated by increased thrombin generation, endothelial cell adhesion, and erythrocyte aggregation. Importantly, these procoagulant activations by PA were confirmed in a rat in vivo venous thrombosis model, where PA significantly enhanced thrombus formation. In conclusion, these results suggest that PA can induce thrombogenic activities in erythrocytes through PS exposure, which can increase thrombus formation and ultimately contribute to the development of cardiovascular diseases.

Topics: Animals; Blood Coagulation; Calcium; Cell Adhesion; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Enzyme Activation; Enzyme Inhibitors; Erythrocyte Aggregation; Erythrocyte Membrane; Erythrocytes; Humans; Male; Phosphatidate Phosphatase; Phosphatidic Acids; Phosphatidylserines; Phospholipase A2 Inhibitors; Phospholipase D; Phospholipases A2; Phospholipid Transfer Proteins; Protein Kinase C-alpha; Rats; Rats, Sprague-Dawley; Thrombin; Thromboplastin; Thrombosis; Time Factors

Canine models have been good predictors of efficacy of hemophilia treatments, including recombinant human coagulation factor (F)VIIa (hFVIIa). However, canine FVIIa and tissue factor (TF) have remained incompletely characterized..  To explore canine-human cross-species FVIIa-TF compatibility in order to strengthen the predictive value of canine models in research on FVIIa and TF.. Canine FVIIa (cFVIIa) and canine TF((1-217)) [cTF((1-217))] were produced by recombinant techniques, and canine-human cross-species FVIIa-TF interactions were characterized in vitro..  Recombinant cFVIIa and soluble cTF((1-217)) were produced and purified to homogeneity. hFVIIa and cFVIIa bound with comparably high affinities to cTF((1-217)) (K(D)=6.0±0.7 nm and K(D)=6.0±0.3 nm, respectively) and to cell surface-expressed cTF (K(D)=8.4±0.4 nm and K(D)=7.2±1.2 nm, for (125) I-labeled hFVIIa and cFVII, respectively). In contrast, cFVIIa bound to human TF (hTF) with decreased affinity, both in solution and on cell surfaces. The decreased binding resulted in reduced activity of cFVIIa in functional assays with hTF((1-209)) . In direct comparison, cFVIIa was more active than hFVIIa, both in the absence and the presence of cognate TF.. The present finding that hFVIIa binds to cTF essentially as it does to hTF substantiates the hypothesis that human FVIIa-TF biology can be reliably recapitulated in canine models on administration of hFVIIa to dogs.

Topics: Animals; Blood Coagulation; Cell Membrane; Cloning, Molecular; Disease Models, Animal; Dogs; Factor VII; Factor VIIa; Fibroblasts; Humans; Kinetics; Protein Binding; Recombinant Proteins; Species Specificity; Thromboplastin

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

Factor (F) VIIa in association with tissue factor (TF) is the primary in vivo initiator of blood coagulation and activates FX and FIX to generate thrombin, which plays a key role in the pathogenesis of thrombosis. We evaluated the enzyme kinetics, antithrombotic and antihaemostatic properties of BMS-593214, an active-site, direct FVIIa inhibitor. Studies were conducted in enzymatic assays, and in anesthetised rabbit models of electrically-induced carotid arterial thrombosis (AT), thread-induced vena cava venous thrombosis (VT) and cuticle bleeding time (BT). Antithrombotic efficacy of BMS-593214 given intravenously was evaluated for both the prevention and treatment of AT and VT. BMS-593214 displayed direct, competitive inhibition of human FVIIa in the hydrolysis of a tripeptide substrate with Ki of 5 nM. However, it acted as a noncompetitive inhibitor of the activation of the physiological substrate FX by TF/VIIa with Ki of 9.3 nM. BMS-593214 showed selectivity for FVIIa and exhibited species differences in TF-FVIIa-dependent anticoagulation with similar potency in human and rabbit plasma. BMS-593214 was efficacious in the prevention and treatment models of AT and VT with ED50 values of 1.1 to 3.1 mg/kg. Furthermore, BMS-593214 exhibited a wide therapeutic window with respect to BT. These results suggest that inhibition of FVIIa with small-molecule active-site inhibitors represents a promising antithrombotic approach for the development of new therapies for the prevention and treatment of AT and VT.

Topics: Animals; Benzoates; Bleeding Time; Blood Coagulation; Carotid Artery Thrombosis; Catalytic Domain; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Factor VIIa; Factor Xa; Fibrinolytic Agents; Hemostasis; Hemostatics; Heterocyclic Compounds, 4 or More Rings; Humans; Injections, Intravenous; Kinetics; Male; Rabbits; Recombinant Proteins; Thromboplastin; Venous Thrombosis

Validation of animal models of disseminated intravascular coagulation (DIC) to human DIC is crucial in order to translate findings in research models to treatment modalities for DIC in humans. ISTH classifications of overt and non-overt human DIC have proven to have a high diagnostic accuracy, and we have previously established a rabbit model of non-overt DIC based on the ISTH classification of non-overt DIC. In this rabbit model, we used purified rabbit brain thromboplastin to induce DIC and test applicability of ISTH classifications of overt human DIC. Cardiovascular and haematological parameters from rabbits, either saline-injected or administered a 2.5 mg thromboplastin/kg bolus and a 15 minutes 1.25 mg thromboplastin/kg infusion, were determined at four time points over a 90 minute period. All groups of rabbits were scored at each time point according to the ISTH classifications of overt DIC. Despite the fact that injection of purified thromboplastin resulted in decreased platelet count, increased prothrombin time, activated partial thromboplastin time, level of thrombin-antithrombin complexes and fibrin degradation products, and pulmonary micro-thrombosis, none of the rabbits were diagnosed as having overt DIC according to ISTH classification. We conclude that purified thromboplastin causes haemostatic abnormalities in the rabbit but this experimental model was not diagnosed as overt DIC.

Topics: Animals; Disease Models, Animal; Disseminated Intravascular Coagulation; Female; Fibrin Fibrinogen Degradation Products; Fibrinogen; Hemostasis; Humans; Partial Thromboplastin Time; Platelet Count; Prothrombin Time; Rabbits; Thrombelastography; Thromboplastin

Thrombin amplifies the blood coagulation via factor V (FV)-mediated positive feedback loop. We hypothesised that factor Xa (FXa) inhibitors would interfere more gradually with this feedback activation loop than thrombin inhibitors, thereby achieving a better balance between haemostasis and prevention of thrombosis. In this study, we compared the effects of TAK-442, a novel FXa inhibitor, versus ximelagatran, a thrombin inhibitor, on FV-mediated positive feedback, venous thrombosis and bleeding. In normal plasma, TAK-442 delayed the onset of tissue factor-induced thrombin generation and prolonged prothrombin time (PT) with more gradual concentration-response curve than melagatran, the active form of ximelagatran. The effect of melagatran on the onset of thrombin generation decreased in an FVa-concentration-dependent manner in FV-deficient plasma supplemented with FVa. Furthermore, in FV-deficient plasma, the PT-prolonging potency of melagatran was markedly increased with a change in its concentration-response curve from steep to gradual. In the rat venous thrombosis model, TAK-442 (10 mg/kg, p.o.) prevented thrombus formation by 55% with 1.2 times prolongation of PT; a similar effect was observed in ximelagatran-treated (3 mg/kg, p.o.) rats. TAK-442 at 100 mg/kg prolonged PT by only 2.1 times with no change in bleeding time (BT), whereas ximelagatran at 10 mg/kg prolonged PT by 3.9 times and significantly increased BT. These results suggest that the differential effects of the two agents on FV-mediated amplification of thrombin generation may underlie the observation of a wider therapeutic window for TAK-442 than for ximelagatran.

Topics: Administration, Oral; Animals; Anticoagulants; Antithrombins; Azetidines; Benzylamines; Blood Coagulation; Disease Models, Animal; Dose-Response Relationship, Drug; Factor V; Factor Xa; Factor Xa Inhibitors; Feedback, Physiological; Hemorrhage; Humans; Male; Phospholipids; Prothrombin Time; Pyrimidinones; Rats; Rats, Sprague-Dawley; Risk Assessment; Sulfones; Thrombin; Thromboplastin; Venous Thrombosis

To assess the effects of aging on arterial thrombus formation by comparing 2-year-old with 11-week-old C57Bl6 mice.. Aging is a major risk factor for cardiovascular disease. In humans, assessing the direct effects of aging on vascular homeostasis is difficult because it occurs in the presence of other risk factors. Arterial thrombosis is the critical event in cardiovascular diseases; however, it is not known whether aging per se promotes its occurrence. Mice represent an interesting system to address this issue because they age without spontaneously developing other risk factors. Organ chamber experiments confirmed the advanced level of aging of old mice. As previously shown, old mice exhibited endothelial dysfunction; however, arterial thrombosis induced by photochemical injury was unchanged. Arterial tissue factor expression and activity; expressions of tissue factor pathway inhibitor, thrombomodulin, and plasminogen activator inhibitor 1; prothrombin time; partial thromboplastin time; thrombin-antithrombin complex; and platelet activation were comparable in both groups.. Although these results cannot be directly extrapolated to humans, this study contributes novel important information on the direct effect of aging on arterial thrombosis and underscores the importance of controlling modifiable risk factors in aged individuals.

Topics: Aging; Animals; Aorta, Thoracic; Aortic Diseases; Base Sequence; Blood Coagulation; Blood Platelets; Carotid Artery Thrombosis; Disease Models, Animal; DNA Primers; Endothelium, Vascular; Humans; Inflammation Mediators; Male; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Risk Factors; RNA, Messenger; Thromboplastin; Vasodilation

In a rat model of branch retinal vein occlusion (BRVO), changes in gene expression of factors implicated in the development of retinal edema and alterations in the properties of Müller cells were determined.. In adult Long-Evans rats, BRVO was induced by laser photocoagulation of retinal veins; untreated eyes served as controls. The mRNA levels of after factors were determined with real-time RT-PCR in the neural retina and retinal pigment epithelium after 1 and 3 days of BRVO: VEGF-A, pigment epithelium-derived factor (PEDF), tissue factor, prothrombin, the potassium channel Kir4.1, and aquaporins 1 and 4. Potassium currents were recorded in isolated Müller cells, and cellular swelling was assessed in retinal slices.. In the neural retina, the expression of VEGF was upregulated within 1 day of BRVO and returned to the control level after 3 days. PEDF was upregulated in the neuroretina and retinal pigment epithelium after 3 days of BRVO. Prothrombin, Kir4.1, and both aquaporins were downregulated in the neuroretina. After BRVO, Müller cells displayed a decrease in their potassium currents and an altered distribution of Kir4.1 protein, an increase in the size of their somata, and cellular swelling under hypoosmotic stress that was not observed in control tissues.. BRVO results in a rapid transient increase in the expression of VEGF and a delayed increase in the expression of PEDF. The downregulation of Kir4.1 and aquaporins, the mislocation of Kir4.1 protein, and the osmotic swelling of Müller cells may contribute to the development of edema and neuronal degeneration.

Topics: Animals; Aquaporin 1; Aquaporin 4; Disease Models, Animal; Electrophysiology; Eye Proteins; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Macular Edema; Membrane Potentials; Nerve Growth Factors; Potassium Channels, Inwardly Rectifying; Prothrombin; Rats; Rats, Long-Evans; Retina; Retinal Degeneration; Retinal Neurons; Retinal Vein Occlusion; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serpins; Thromboplastin; Vascular Endothelial Growth Factor A; Vimentin

Ischemia is the defined pathway leading to steroid-associated osteonecrosis (ON). Early detection of ischemic condition may help predict later ON occurrence. Bone marrow perfusion function evaluation by perfusion magnetic resonance imaging (MRI) may be a unique modality for this application. Twenty-five adult male New Zealand white rabbits were used in this study. Lipopolysaccharide (LPS) and methylprednisolone (MPS) were administrated for ON induction based on a published protocol. T1-weighted and fat suppression T2-weighted MR imaging (conventional MRI) were performed for ON lesion detection based on the abnormal signal in the proximal femora at week 0 as the baseline (before LPS injection), and week 1 and week 2 after MPS injection. At the same time, the blood perfusion function in the proximal femora was measured by perfusion MRI. Maximum enhancement (ME)--an index of MRI perfusion function was analyzed. After MRI scanning, the proximal femora were prepared histopathologically for ON lesion analysis. The rabbit with bilateral histopathological ON lesions was defined as an ON+ rabbit and included in the ON+ group evaluated at week 1 and week 2, respectively, and the rabbit without ON lesions in bilateral femora was classified into the ON- group. For the underlying mechanism of perfusion change, the extravascular marrow fat cells were measured and the intravascular endothelium inflammation injury indicator of tissue factor (TF) expression and thrombus formation were detected. In ON+ group, ME in perfusion MRI showed a significant decrease at week 1 and week 2 as compared with the baseline (p < 0.01). There was a more than 50% decrease in ME at week 1 in ON+ group; whereas there were no detectable ON lesions by conventional MRI at week 1, though 93% (14/15) rabbits could be detected at week 2 in ON+ group. In ON- group, ME showed a slight decrease at week 1 (less than 30%), and nearly recovered to normal at week 2 as compared with the baseline. Histological results showed a much larger average marrow fat area and more severe marrow blood sinusoids compression from surrounding crowded fat cells, and stronger positive TF expression in marrow endothelium and more thrombus formation in ON+ rabbits than ON- rabbits. This study demonstrated that functional perfusion MRI could predict development of steroid-associated ON. Our experimental data suggested that perfusion MRI might be a sensitive noninvasive modality for monitoring steroid-associated ON in patients

Topics: Adipose Tissue; Animals; Bone Marrow; Disease Models, Animal; Endothelium, Vascular; Glucocorticoids; Ischemia; Lipopolysaccharides; Magnetic Resonance Imaging; Male; Methylprednisolone; Osteonecrosis; Predictive Value of Tests; Rabbits; Sensitivity and Specificity; Thromboplastin

Pregnancy loss and intrauterine growth restriction (IUGR) are serious pregnancy complications, and the triggers and mediators of placental and fetal damage are not completely understood. Using a mouse model of recurrent spontaneous miscarriages (DBA/2-mated CBA/J mice) that shares features with human recurrent miscarriage and fetal growth restriction, we identified tissue factor (TF) as an essential participating factor in placental and fetal injury. We have previously shown that C5a releases antiangiogenic molecule sFlt-1 in monocytes that causes defective placental development and fetal death in DBA/2-mated CBA/J mice. In this study, we found that TF not only activates the coagulation pathway, but it also mediates sFlt-1 release in monocytes causing defective placental development and fetal death. Blockade of TF with a monoclonal antibody inhibited sFlt-1 release, prevented the pathological activation of the coagulation pathway, restored placental blood flow, prevented placental oxidative stress, and rescued pregnancies. We also demonstrated that pravastatin, by down-regulating TF expression on monocytes and trophoblasts, prevented placental damage and protected pregnancies in DBA/2-mated CBA/J mice. These studies indicate that TF is an important mediator in fetal death and growth restriction and that statins may be a good treatment for women with recurrent miscarriages and IUGR.

Topics: Abortion, Spontaneous; Animals; Animals, Newborn; Anticoagulants; Antithrombin III; Cells, Cultured; Disease Models, Animal; Female; Humans; Infant, Newborn; Male; Mice; Monocytes; Nitrogen Oxides; Oxidative Stress; Placenta; Pravastatin; Pregnancy; Protein Binding; Thrombin; Thromboplastin; Trophoblasts; Vascular Endothelial Growth Factor Receptor-1

Although stasis is important in the pathogenesis of deep vein thrombosis (DVT), how it contributes to thrombogenesis is largely unknown. To gain mechanistic insight, we used a rat model of inferior vena cava (IVC) ligation.. Rats were subjected to IVC ligation for 15 to 60 minutes. Ligation resulted in rapid IVC dilatation and by 60 minutes, thrombi were detected in all rats. Small thrombi were detected in the IVC of most rats after 15 minutes of ligation. Thrombi were rich in fibrin, contained aggregated platelets as well as trapped leukocytes and red cells, and most originated at sites of localized endothelial denudation. Immunohistochemical analysis revealed tissue factor (TF)-expressing leukocytes within the thrombi and adherent to the vessel wall. Despite a largely intact vessel wall, endothelial cells also stained for TF. The expression of TF colocalized with that of protein disulfide isomerase (PDI), an enzyme implicated in TF decryption.. These findings suggest that the rapid development of DVT after IVC ligation reflects a combination of stasis-induced vein wall injury and enhanced TF expression in endothelial cells and leukocytes. Because TF expression occurs so soon after ligation, new synthesis is unlikely. Instead, stasis-induced venous dilatation with or without exposure of subendothelial TF, may be responsible for vessel wall TF expression. Colocalization of TF and PDI raises the possibility that PDI-mediated TF decryption plays a role in the pathogenesis of DVT.

Topics: Animals; Cell Adhesion; Dilatation, Pathologic; Disease Models, Animal; Endothelial Cells; Leukocytes; Ligation; P-Selectin; Protein Disulfide-Isomerases; Rats; Rats, Sprague-Dawley; Thromboplastin; Time Factors; Up-Regulation; Vena Cava, Inferior; Venous Thrombosis

Microparticles (MP) are lipid vesicles from platelets, leukocytes and endothelial cells that are involved in early thrombogenesis. We evaluated a detailed time-course analysis of MPs on thrombogenesis and the associated tissue factor (TF) activity in wild-type, in gene-deleted for E- and P-selectins and with high levels of P-selectin expression after the initiation of venous thrombosis in mice. Inferior vena cava (IVC) ligation was performed on C57BL/6 mice (n = 191, 59 = wild-type [WT], 55 = gene-deleted for E- and P - selectins [knock-outs, EPKO] and 77 = elevated levels of soluble P-selectin, named Delta Cytoplasmic Tail (DeltaCT). Animals were euthanised at various time points to assess MP production, origin and thrombus weight. MPs were re-injected into separate mice at concentrations of 80,000 and 160,000 units, as well as from different ages. In addition, MPs from thrombosed animals were pooled and TF activity quantitated using a chromogenic assay. Thrombus weight correlated negatively with MPs derived from leukocytes, and positively with MPs derived from platelets for WT animals (p < 0.05), while MPs from platelets presented a positive correlation to thrombus weight in the WT and EPKO groups (p < 0.01). Total MPs correlated negatively with thrombus weight in the DeltaCT group (p < 0.05). MP re-injections led to greater thrombus weight, while older MP reinjections tended to form larger thrombus than younger. Finally, TF bearing MPs showed a significant correlation to MP concentrations (R = 0.99). In conclusion, MPs appear to be an important element in venous thrombogenesis.

Topics: Animals; Blood Coagulation; Blood Platelets; Cell-Derived Microparticles; Disease Models, Animal; E-Selectin; Leukocytes; Ligation; Mice; Mice, Inbred C57BL; Mice, Knockout; P-Selectin; Thromboplastin; Time Factors; Vena Cava, Inferior; Venous Thrombosis

Validation of animal models of disseminated intravascular coagulation (DIC) to human DIC is crucial in order to translate findings in research models to treatment modalities for DIC in humans. ISTH classifications of overt and non-overt human DIC have proven to have a high diagnostic accuracy, but the scoring systems have rarely been applied to animal models of DIC. In this study, we use rabbit brain thromboplastin (thromboplastin) to induce DIC in a rabbit model and test the applicability of the ISTH criteria for standardized diagnosis of DIC. Cardiovascular and haematological parameters from rabbits, either saline-injected or administered 0.625, 1.25, 2.5 or 5 mg thromboplastin/kg as a single bolus, were collected at four timepoints over a 90 minute period. All groups of rabbits were scored at each time point according to the ISTH diagnostic criteria for non-overt DIC. Injection of 5 mg thromboplastin/kg was lethal. For the remaining groups, a dose dependent decrease in blood pressure, platelet count and fibrinogen level together with a dose dependent increase in prothrombin time, activated partial thromboplastin time, level of thrombin-antithrombin complexes, fibrin degradation products and number of thrombi in lung vasculature was seen. The administration of a bolus of 1.25 - 2.5 mg thromboplastin/kg to rabbits induced a reproducible dose dependent model of non-overt DIC according to the ISTH diagnostic criteria. We conclude that the non-overt ISTH score can be applied to evaluate severity and progression of DIC in a standardized manner in this thromboplastin induced rabbit model.

Topics: Animals; Blood Pressure; Disease Models, Animal; Disseminated Intravascular Coagulation; Dose-Response Relationship, Drug; Female; Fibrinogen; Platelet Count; Prothrombin Time; Rabbits; Thromboplastin; Time Factors

Using different mouse monoclonal and human antiphospholipid (aPL) antibodies, we developed a new animal model of renal injury that shares many features with thrombotic microangiopathy (TMA). We found that more than 1 mechanism/signaling pathway is involved in glomerular injury induced by aPL antibodies in this model. Both complement-dependent and complement-independent pathways were identified that lead to glomerular endothelial cell damage and renal function impairment. We also found that C5a-C5aR interaction is a crucial step for the activation of the coagulation cascade and glomerular injury induced by complement-activating antibodies. In addition, our studies demonstrated complement-independent mechanisms in which reactivity with beta(2) glycoprotein I (beta2GPI) plays an important role in aPL-induced glomerular damage and renal failure. Independently of the mechanism responsible for aPL-induced TMA, mice that express low levels of tissue factor (TF) were protected from glomerular injury. That genetic reduction of TF prevents renal injury induced by different aPL antibodies indicates that TF is a common mediator of glomerular damage and a possible target for selective pharmacologic intervention. Treatment with pravastatin, which down-regulates glomerular TF synthesis, prevents aPL-induced TMA in this mouse model, thus emphasizing that targeting TF might be a good therapeutic intervention in patients with TMA.

Topics: Adult; Animals; Antibodies, Antiphospholipid; Antibodies, Monoclonal; Antigen-Antibody Reactions; Disease Models, Animal; Female; Humans; Kidney Glomerulus; Male; Mice; Mice, Inbred C57BL; Microvessels; Middle Aged; Renal Insufficiency; Thromboplastin; Thrombosis

Topics: Animals; Disease Models, Animal; Drugs, Chinese Herbal; Male; Random Allocation; Rats; Rats, Wistar; Sepsis; Thromboplastin

To determine if neurally adjusted ventilatory assist (NAVA) that delivers pressure in proportion to diaphragm electrical activity is as protective to acutely injured lungs (ALI) and non-pulmonary organs as volume controlled (VC), low tidal volume (Vt), high positive end-expiratory pressure (PEEP) ventilation.. Prospective, randomized, laboratory animal study.. Twenty-seven male New Zealand white rabbits.. Anesthetized rabbits with hydrochloric acid-induced ALI were randomized (n = 9 per group) to 5.5 h NAVA (non-paralyzed), VC (paralyzed; Vt 6-ml/kg), or VC (paralyzed; Vt 15-ml/kg). PEEP was adjusted to hemodynamic goals in NAVA and VC6-ml/kg, and was 1 cmH2O in VC15-ml/kg.. PaO2/FiO2; lung wet-to-dry ratio; lung histology; interleukin-8 (IL-8) concentrations in broncho-alveolar-lavage (BAL) fluid, plasma, and non-pulmonary organs; plasminogen activator inhibitor type-1 and tissue factor in BAL fluid and plasma; non-pulmonary organ apoptosis rate; creatinine clearance; echocardiography. PEEP was similar in NAVA and VC6-ml/kg. During NAVA, Vt was lower (3.1 +/- 0.9 ml/kg), whereas PaO2/ FiO2, respiratory rate, and PaCO2 were higher compared to VC6-ml/kg (p<0.05 for all). Variables assessing ventilator-induced lung injury (VILI), IL-8 levels, non-pulmonary organ apoptosis rate, and kidney as well as cardiac performance were similar in NAVA compared to VC6-ml/kg. VILI and non-pulmonary organ dysfunction was attenuated in both groups compared to VC15-ml/kg.. In anesthetized rabbits with early experimental ALI, NAVA is as effective as VC6-ml/kg in preventing VILI, in attenuating excessive systemic and remote organ inflammation, and in preserving cardiac and kidney function.

Topics: Acute Lung Injury; Analysis of Variance; Animals; Bronchoalveolar Lavage Fluid; Diaphragm; Disease Models, Animal; Electrophysiological Phenomena; Feedback, Physiological; Interleukin-8; Male; Multiple Organ Failure; Plasminogen Activator Inhibitor 1; Positive-Pressure Respiration; Prospective Studies; Rabbits; Random Allocation; Respiration, Artificial; Statistics, Nonparametric; Thromboplastin; Tidal Volume; Ventilator-Induced Lung Injury

To study the pathogenetic role of tissue factor (TF) in endothelial-injury in GVHD.. Gene and protein expressions of TF in the organs of allogenic hematopoietic stem cell transplantation (allo-HSCT) and autologous HSCT (auto-HSCT) mice were determined by real-time PCR and Western blot. The effect of allogeneic T lymphocytes on the expression of TF and other cytokines and activation of MAPKs in human umbilical vein endothelial cells (HUVECs) was detected by flow cytometry, real-time PCR or Western blot. The influence of TF antibodies (SB203580 and SP600125) on allogeneic T lymphocytes-induced cytokines expression was also tested.. (1) TF gene and protein expression in the liver, skin, small intestine and stomach of allo-HSCT mice was significantly elevated about 15.1+/-2.1, 5.5+/-1.4, 9.7+/-2.3, 14.2+/-2.9 folds and 13.5+/-2.7, 6.2+/-0.9, 7.9+/-1.6, 15.3+/-3.2 folds respectively compared with that of auto-HSCT mice. (2) Allogeneic CD4+ CD8+ T lymphocytes significantly enhanced TF, VCAM-1, TNF-alpha, IFN-gamma and IL-6 expression in TNF-alpha prestimulated HUVECs. (3) Allogeneic T lymphocytes enhanced p38MAPK and JNK phosphorylation in HUVECs, but did not affect ERK phosphorylation. p38 MAPK JNK inhibitors SB203580 and SP600125 reduced allogeneic T lymphocytes-induced TF expression in HUVECs. (4) SB203580 and SP600125 down-regulated allogeneic T lymphocytes-induced VCAM-1, TNF-alpha, IFN-gamma, IL-6 expression in HUVECs.. TF mediates vascular endothelial-injury and activation in GVHD via phosphorylation of p38MAPK and JNK.

Topics: Animals; Anthracenes; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Endothelium; Endothelium, Vascular; Graft vs Host Disease; Hematopoietic Stem Cell Transplantation; Humans; Imidazoles; Interferon-gamma; Interleukin-6; Mice; Mitogen-Activated Protein Kinases; Pyridines; T-Lymphocytes; Thromboplastin; Tumor Necrosis Factor-alpha

Induction of tumor vasculature occlusion by targeting a thrombogen to newly formed blood vessels in tumor tissues represents an intriguing approach to the eradication of primary solid tumors. In the current study, we construct and express a fusion protein containing vascular endothelial growth factor (VEGF) and tissue factor (TF) to explore whether this fusion protein has the capability of inhibiting tumor growth in a colon carcinoma model. The murine cDNA of VEGF A and TF were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), and then cloned into prokaryotic expression plasmid pQE30 with a linker. The expression product recombinant VEGF-TF (rVEGF-TF) was purified and proved to have comparable enzyme activity to a commercial TF and the capability of specific binding to tumor vessels. Significant decrease of tumor growth was found in the mice administered with rVEGF-TF on Day 6 after initiated rVEGF-TF treatment (P<0.05), and the tumor masses in 2 of 10 mice were almost disappeared on Day 14 after the first treatment. In addition, valid thrombogenesis and tumor necrosis were observed in the tumor tissues injected with rVEGF-TF. Our results demonstrate that occlusion of tumor vasculature with rVEGF-TF is potentially an effective approach for cancer therapy.

Topics: Animals; Cell Line, Tumor; Cloning, Molecular; Colonic Neoplasms; Disease Models, Animal; Disease Progression; Gene Expression; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Plasmids; Recombinant Fusion Proteins; Thromboplastin; Thrombosis; Vascular Endothelial Growth Factor A

Women with antiphospholipid syndrome (APS), a condition characterized by the presence of antiphospholipid antibodies (aPL), often suffer pregnancy-related complications, including miscarriage. We have previously shown that C5a induction of tissue factor (TF) expression in neutrophils contributes to respiratory burst, trophoblast injury, and pregnancy loss in mice treated with aPL. Here we analyzed how TF contributes to neutrophil activation and trophoblast injury in this model. Neutrophils from aPL-treated mice expressed protease-activated receptor 2 (PAR2), and stimulation of this receptor led to neutrophil activation, trophoblast injury, and fetal death. An antibody specific for human TF that has little impact on coagulation, but potently inhibits TF/Factor VIIa (FVIIa) signaling through PAR2, inhibited aPL-induced neutrophil activation in mice that expressed human TF. Genetic deletion of the TF cytoplasmic domain, which allows interaction between TF and PAR2, reduced aPL-induced neutrophil activation in aPL-treated mice. Par2-/- mice treated with aPL exhibited reduced neutrophil activation and normal pregnancies, which indicates that PAR2 plays an important role in the pathogenesis of aPL-induced fetal injury. We also demonstrated that simvastatin and pravastatin decreased TF and PAR2 expression on neutrophils and prevented pregnancy loss. Our results suggest that TF/FVIIa/PAR2 signaling mediates neutrophil activation and fetal death in APS and that statins may be a good treatment for women with aPL-induced pregnancy complications.

Topics: Animals; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Disease Models, Animal; Factor VIIa; Female; Fetal Death; Gene Deletion; Gene Expression Regulation; Humans; Immunoglobulin G; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophil Activation; Neutrophils; Phagocytosis; Pregnancy; Reactive Oxygen Species; Receptor, PAR-1; Receptor, PAR-2; Respiratory Burst; Signal Transduction; Simvastatin; Thromboplastin

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

The induction of heme oxygenase-1 (HO-1) may protect against tissue injury. The present study examines the induction of HO-1 in a murine model of venous thrombosis and explores the downstream consequences of this induction. In a model of stasis-induced thrombosis created by ligation of the inferior vena cava, HO-1 expression is markedly induced. Such expression occurs primarily in smooth muscle cells in the venous wall and in leukocytes infiltrating the venous wall and clot. To determine the significance of HO-1 induction in venous thrombosis, this model was imposed in HO-1(+/+) and HO-1(-/-) mice. The initial clot size did not differ in either group by day 2, but was significantly larger in HO-1(-/-) mice by day 10, where an exaggerated inflammatory response in the venous wall was also observed. Following ligation of the inferior vena cava, HO-1(-/-) mice exhibited increased nuclear factor kappaB activation and markedly increased up-regulation of tissue factor, selectins, inflammatory cytokines, and matrix metalloproteinase-9, the latter incriminated in both clot lysis and vascular injury. We conclude that HO-1 deficiency impairs thrombus resolution and exaggerates the inflammatory response to thrombus formation. These findings offer insight into recent observations that polymorphisms in the HO-1 gene may increase the risk for recurrent venous thrombosis and dysfunction of hemodialysis arteriovenous fistulas, the latter caused, in part, by thrombosis.

Topics: Animals; Chemokine CCL2; Chemokine CXCL1; Chemokine CXCL12; Disease Models, Animal; Enzyme Induction; Female; Heme Oxygenase-1; Humans; Interleukin-6; Male; Matrix Metalloproteinase 9; Mice; Mice, Knockout; NF-kappa B; Thromboplastin; Thrombosis; Vena Cava, Inferior; Venous Thrombosis

Healing of skin wounds is delayed in hemophilia B (HB) mice. HB mice do not bleed excessively at wounding, yet rebleed hours to days later. Tissue factor (TF) expression is up-regulated by inflammatory cytokines and has been linked to angiogenesis. We hypothesized that impaired thrombin generation in HB leads to impaired TF expression following injury. Punch biopsies were placed on wild-type (WT) and HB mice. Tissues from wound sites were immunostained for TF. Blood vessels are normally surrounded by a coat of pericytes expressing TF. Surprisingly, within a day after wounding TF disappeared from around nearby vessels; returning after 8 days in WT and 10 days in HB mice. The granulation tissue filling the wound during healing also lacked TF around angiogenic vessels. Thus, perivascular TF expression is down-regulated during wound healing. This may prevent thrombosis of neovessels during angiogenesis but renders hemophiliacs vulnerable to hemorrhage during healing.

Topics: Animals; Biopsy; Disease Models, Animal; Down-Regulation; Factor X Deficiency; Gene Expression Regulation; Hemophilia B; Humans; Mice; Mice, Knockout; Skin; Thromboplastin; Wound Healing; Wounds and Injuries

To investigate the effect of the combined treatment of photodynamic therapy and specific VEGF165 inhibition with pegaptanib sodium (Macugen; Eyetech Pharmaceuticals, Lexington, MA) on ocular neovascularization.. Photodynamic therapy's (PDT's) effects on the integrity of pegaptanib sodium were analyzed by HPLC, a VEGF165-binding assay, and a VEGF165-induced tissue factor gene expression assay. The effects of mono- or combined treatment on vessel growth and regression were determined in a murine corneal neovascularization model. The effects of combined treatment on vessel growth were also determined in a murine choroidal neovascularization model.. PDT did not affect the chemical composition of pegaptanib sodium nor the efficacy of pegaptanib sodium in the inhibition of VEGF165 binding to Flt-1 and VEGF165-induced gene expression. In an animal model of effects on existing ocular neovascular lesions (corneal neovascularization), PDT monotherapy yielded an initial regression of these vessels, but there followed a rapid regrowth. In contrast, pegaptanib sodium monotherapy yielded little regression but potently abrogated further vessel growth. The combination of pegaptanib sodium and PDT resulted in the regression of the neovascular lesions, as observed with PDT alone, but also prevented significant vessel regrowth, leading to a significantly greater reduction in lesion size than did each monotherapy. In addition, there was a significantly greater effect of the combination of pegaptanib sodium and PDT on lesion size in choroidal neovascularization than with each monotherapy. Pretreatment with pegaptanib sodium appeared to decrease the efficacy of PDT-induced vessel regression in corneal neovascularization, and as such the enhanced efficacy over monotherapy when the agents were delivered simultaneously was not observed.. Although the combined simultaneous treatment of ocular neovascularization with PDT and pegaptanib sodium may provide a more effective approach for the regression and overall treatment of CNV associated with AMD, the order of addition of these treatments may play a role in achieving optimal efficacy.

Topics: Animals; Aptamers, Nucleotide; Choroidal Neovascularization; Chromatography, High Pressure Liquid; Cornea; Corneal Neovascularization; Disease Models, Animal; Drug Therapy, Combination; Male; Mice; Mice, Inbred C57BL; Photochemotherapy; Photosensitizing Agents; Porphyrins; RNA, Messenger; Thromboplastin; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Verteporfin

Recent studies have suggested a link between inhaled particulate matter (PM) exposure and atherogenesis. We investigated tissue factor (TF) expression with ambient fine particulate matter (diameter < 2.5 microm, PM(2.5)) exposure and in response to in vitro exposure to fine and ultrafine PM in cultured human bronchial epithelial cells, vascular smooth muscle cells (hSMCs), and monocytes. ApoE-/- mice, fed with normal chow (NC) or high-fat chow (HFC), were exposed to concentrated PM(2.5) or filtered air (FA) for 6 mo (6 h/day, 5 day/wk, n = 28). Following in vivo ultrasound bio-microscopy (UBM) assessment of plaque area, macrophage infiltration (CD68) and TF expression in the aorta were quantified. Cultured cells were incubated with size-fractionated PM from cascade impactors, or with standard reference PM material (SRM, number 1649a) and assayed for TF protein, mRNA, and activity. UBM-derived plaque areas were 7 +/- 1% larger in the PM(2.5)-HFC than the FA-HFC group (p = .04), but not significantly different between the PM(2.5)-NC and FA-NC groups (p = .07). Immunohistochemistry revealed increased TF (15 +/- 3% vs. 8 +/- 2%, p < .01) and macrophage infiltration (19 +/- 2% vs. 14 +/- 3%, p < .01) in the plaques of PM(2.5)-HFC compared with FA-HFC groups. Impactor-collected PM(2.5) and ultrafine particles consistently increased TF protein in bronchial epithelial cells, monocytes, and hSMCs. TF mRNA expression increased rapidly (within 1 h) in response to SRM PM. We conclude that in vivo and in vitro exposure to ambient air PM(2.5) induces TF expression.

Topics: Air Pollution; Animals; Apolipoproteins E; Atherosclerosis; Bronchi; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Environmental Exposure; Epithelial Cells; Gene Expression Regulation; Gene Silencing; Humans; Image Processing, Computer-Assisted; Inhalation Exposure; Male; Mice; Mice, Knockout; Monocytes; Muscle, Smooth, Vascular; Particulate Matter; RNA, Messenger; Thromboplastin; Ultrasonography

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

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

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

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

Postoperative bleeding and adhesion formation remain the two major problems after endoscopic sinus surgery (ESS). This study investigates the effect on adhesion formation and wound healing in a sheep model of chronic sinusitis of three topical agents: recombinant tissue factor (rTF, Dade Innovin, Marburg, Germany), poly-ethylene glycol (SprayGel, Confluent Surgical, Waltham, MA), and a novel chitosan-dextran derivative gel (CD, Department of Chemistry, University of Otago, Dunedin, New Zealand).. Twenty sheep with chronic sinusitis underwent ESS with standardized mucosal injuries created on the lateral nasal wall and the ethmoid region. Injured areas were divided into four groups, and one of the three agents or control (no treatment) was randomly applied. The presence and severity of adhesions were noted and the healing was evaluated by taking brushings for ciliary beat frequency and biopsies of the injured regions at day 28, 56, 84, and 112 post initial surgery. The biopsy specimens were assessed for re-epithelialisation using light microscopy and scanning electron microscopy for reciliation. The cytobrush specimens assessed cilial function by measuring ciliary beat frequency.. CD significantly decreased lateral nasal wall and ethmoidal adhesions compared to tissue factor at all time points (5% vs. 25%, and 0 vs. 50%, respectively). There was a noticeable trend toward decreased adhesions on the lateral nasal wall and ethmoids in the SprayGel group (10% and 14%) and the CD group (10% and 0%) compared to controls (15% and 40%). The CD group had a significantly greater percentage of re-epithelialisation at day 28 and day 84 compared to the rTF group (70% vs. 33%, P < .001; 84.5% vs. 61%, P < 0.05). At day 28, the CD group was significantly more ciliated than control (62% vs. 31%, P < .01) and than rTF (62% vs. 23%, P < .001). This difference between CD and rTF reciliation remained significant at day 56 (67% vs. 40%, P < .05). In addition, the mean cilial grade for CD at day 112 was significantly better than control (1.9 vs. 2.7, P < .05).. In the sheep model of chronic sinusitis, CD significantly improves microscopic wound healing and reduces adhesion formation after ESS.

Topics: Animals; Biocompatible Materials; Chitosan; Chronic Disease; Dextrans; Disease Models, Animal; Endoscopy; Epithelium; Gels; Mucous Membrane; Polyethylene Glycols; Postoperative Complications; Random Allocation; Recombinant Proteins; Sheep; Sinusitis; Thromboplastin; Tissue Adhesions; Wound Healing

Therapeutic use of unfractionated heparin and low molecular weight heparins (LMWHs) is limited by hemorrhagic adverse effects. We compared the antithrombotic effect of LMW fucoidan (LMWF) and LMWH in an experimental model.. Thrombosis was induced in femoral arteries of male New Zealand White rabbits by in situ induction of endothelial apoptosis with staurosporine (10(-5)M for 30 min). Starting the day before apoptosis induction, the animals received subcutaneous LMWF (15 mg/kg), LMWH (enoxaparin 2.5 mg/kg) or saline solution (control group) twice a day for 4 days.. The degrees of apoptosis and endothelial denudation were similar in the 3 groups. The thrombotic score was significantly lower in the LMWF group than in the LMWH and control groups (p = 0.01). Tissue factor expression was significantly lower in the LMWF group than in the control and LMWH groups (p = 0.01). The plasma concentration of tissue factor pathway inhibitor was significantly increased after LMWF injection (137 +/- 28 vs. 102 +/- 17; p = 0.01), whereas no change was observed after LMWH treatment. LMWF did not prolong the bleeding time or decrease platelet aggregation.. LMWF appeared to be more effective than LMWH for preventing arterial thrombosis in this experimental model. LMWF also had a lower hemorrhagic risk than LMWH.

Topics: Animals; Apoptosis; Blood Coagulation; Disease Models, Animal; Dose-Response Relationship, Drug; Femoral Artery; Fibrinolytic Agents; Hemorrhage; Heparin, Low-Molecular-Weight; Lipoproteins; Male; Platelet Aggregation; Polysaccharides; Rabbits; Staurosporine; Thromboplastin; Thrombosis

Plasma-derived human antithrombin (pAT) is used for the treatments of disseminated intravascular coagulation (DIC) and hereditary antithrombin deficiencies. We expressed recombinant human antithrombin (rAT) in Chinese hamster ovary (CHO) cells. The purified rAT is composed of 55% alpha-isoform and 45% beta-isoform. The structure of the N-linked oligosaccharides of rAT is the same biantennary complex type as previously found in pAT with less sialylated on the non-reducing ends. Most of the oligosaccharides of rAT are fucosylated at the reducing ends of N-acetylglucosamine, while those of pAT are not fucosylated. Despite of the difference in sialylation and fucosylation of the oligosaccharide units, rAT and pAT showed indistinguishable heparin cofactor and progressive activities, and they bound to thrombin in a one-to-one stoichiometric manner. In lipopolysaccharide (LPS)-induced and thromboplastin-induced DIC rat models, rAT reduced fibrinogen and platelet consumption to a similar extent with pAT. In LPS-induced DIC model, both ATs similarly restrained the increase of alanine aminotransferase and aspartate aminotransferase activities. Finally, pharmacokinetic analysis showed that both ATs had similar half-lives in the circulation of normal rats. Together, the present study demonstrated that rAT prepared in CHO cells has potential for a substitute of pAT in therapeutic use.

Topics: Alanine Transaminase; Animals; Antithrombin III Deficiency; Antithrombins; Aspartate Aminotransferases; CHO Cells; Cricetinae; Cricetulus; Disease Models, Animal; Disseminated Intravascular Coagulation; Fibrinogen; Glycosylation; Humans; Lipopolysaccharides; Male; Oligosaccharides; Protein Isoforms; Rats; Rats, Wistar; Recombinant Proteins; Stereoisomerism; Thromboplastin; Time Factors

Platelet endothelial cell adhesion molecule-1 (PECAM-1) (CD31) is known to inhibit platelet function and thrombus formation. The mechanisms involved in PECAM-1's roles as a modulator of hemostasis are still not completely understood. We examined the role of PECAM-1 as a regulator of tissue factor (TF) expression, a known important inducer of thrombosis. Wildtype and CD31KO mice underwent transient (30 min) renal ischemia followed by 24 h re-perfusion and their kidneys assessed for apoptosis, fibrin formation, and tissue factor expression. CD31KO mice exhibited increased tubular epithelial and endothelial apoptosis, increased fibrin deposition, and tissue factor expression. Human umbilical vein endothelial cells (HUVEC) transfected with antisense (AS) PECAM-1 oligonucleotides to downregulate PECAM-1 expression, exhibited greater induction of TF mRNA and protein expression as well as increased expression and nuclear localization of the transcription factor Egr-1 compared to scrambled AS PECAM-1 (Scr)-treated HUVEC following thrombin stimulation. TF induction was found to be mediated through thrombin receptor PAR-1 and the Galphai/o subunit of G-protein, confirmed by PAR-1 antagonist and pertussis toxin inhibition respectively. Thrombin-mediated TF induction was dependent on Rho Kinase activity, phosphorylation of p38(MAPK) and p85 & Akt dephosphorylation. The inverse correlation of PI3K-Akt phosphorylation with p38 (MAPK) phosphorylation was confirmed by pharmacological inhibition. These studies suggest that PECAM-1 is involved in regulating a signaling pathway, affecting PI3K and Akt activation, p38 (MAPK) phosphorylation, which in turn, affects Egr-1 expression and nuclear translocation, ultimately affecting TF expression. These findings provide new insights into the action of PECAM-1 as a modulator of thrombosis.

Topics: Active Transport, Cell Nucleus; Animals; Apoptosis; Blood Coagulation; Cells, Cultured; Disease Models, Animal; Down-Regulation; Early Growth Response Protein 1; Endothelial Cells; Fibrin; Humans; Kidney; Male; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Mice, Knockout; Oligodeoxyribonucleotides, Antisense; Platelet Endothelial Cell Adhesion Molecule-1; Receptor, PAR-1; Reperfusion Injury; RNA, Messenger; Thrombin; Thromboplastin; Thrombosis

Blockade of the thrombin receptors protease-activated receptor (PAR)1 and PAR4 with pepducins, cell-penetrating lipopeptides based on the third intracellular loop of PAR1 and PAR4, effectively inhibits platelet aggregation. We have previously shown that PAR1 pepducin also exerts an anticoagulant activity by partial inhibition of the thrombin plus collagen-induced externalization of phosphatidylserine (PS) at the platelet plasma membrane.. In the present study we examined the effects of PAR1 and PAR4 pepducins on tissue factor (TF)-initiated thrombin generation in platelet-rich plasma (PRP) and the interaction between PAR4 pepducin-loaded mouse platelets and a growing thrombus to confirm the relevance of the in vitro data.. Localization of pepducins at the inner leaflet of the plasma membrane was confirmed with a fluorescence resonance energy transfer assay. Both the PAR1 pepducin, P1pal12, and the PAR4 pepducin, P4pal10, inhibited TF-initiated thrombin generation in PRP. Concentrations of P1pal12 and P4pal10, which blocked the thrombin-induced influx of extracellular calcium ions and inhibited platelet aggregation, reduced the rate of thrombin generation during the propagation phase by 38% and 36%, respectively. Whether this anticoagulant effect is relevant in inhibiting in vivo arterial thrombin growth is uncertain because P4pal10 prevented the incorporation of platelets in a growing thrombus.. Our findings suggest that in spite of their potential anticoagulant activities the in vivo antithrombotic effect of intracellular PAR pepducins is mainly based on inhibiting platelet-platelet interactions.

Topics: Animals; Anticoagulants; Blood Platelets; Carotid Artery, Common; Cell Membrane; Cell Membrane Permeability; Disease Models, Animal; Dose-Response Relationship, Drug; Fibrinolytic Agents; Flow Cytometry; Fluorescence Resonance Energy Transfer; Humans; In Vitro Techniques; Lipoproteins; Male; Mice; Microscopy, Video; Platelet Aggregation; Platelet Aggregation Inhibitors; Receptor, PAR-1; Receptors, Proteinase-Activated; Receptors, Thrombin; Thrombin; Thromboplastin; Thrombosis; Time Factors

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

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

Current anticoagulant therapies for the prevention and treatment of thromboembolic disorders have many drawbacks: vitamin K antagonists interact with food and drugs and require frequent laboratory monitoring, and heparins require parenteral administration. Oral rivaroxaban (BAY 597939) is a new, highly selective and potent direct factor-Xa (FXa) inhibitor with a predictable pharmacodynamic and pharmacokinetic profile and could therefore be an attractive antithrombotic drug. It was the objective of this study to investigate the antithrombotic efficacy of oral rivaroxaban in two rabbit models of experimental venous thrombosis. In the venous stasis (prevention) model, animals were randomized to receive oral rivaroxaban 0.3, 1.0, 3.0 or 10.0 mg/kg or vehicle control. Thrombosis was induced by jugular vein stasis and injection of thromboplastin into the ear vein. In the venous thrombosis (treatment) model, intravenous (1.0 and 3.0 mg/kg) and oral (3.0 mg/kg) rivaroxaban was compared with intravenous nadroparin (40 U bolus and 20 U/h), fondaparinux (42 microg/kg) and vehicle control. Thrombus growth was assessed by measuring the accretion of radiolabelled fibrinogen into preformed clots in the jugular veins. Bleeding was assessed using an ear bleeding model. In the prevention model, rivaroxaban reduced thrombus formation dose-dependently (calculated ED(50) 1.3 mg/kg). In the treatment model, oral rivaroxaban (3.0 mg/kg) reduced thrombus growth to a similar extent to intravenous rivaroxaban (1.0 mg/kg), nadroparin and fondaparinux. Oral rivaroxaban did not prolong bleeding time. In conclusion, the orally available selective, direct FXa inhibitor rivaroxaban is effective in the prevention and treatment of venous thrombosis in two well-established models of experimental thrombosis.

Topics: Administration, Oral; Animals; Anticoagulants; Blood Coagulation Tests; Disease Models, Animal; Dose-Response Relationship, Drug; Factor Xa Inhibitors; Fibrinolytic Agents; Fondaparinux; Hemorrhage; Humans; Infusions, Intravenous; Injections, Intravenous; Jugular Veins; Ligation; Morpholines; Nadroparin; Polysaccharides; Rabbits; Random Allocation; Rivaroxaban; Thiophenes; Thromboplastin; Venous Thrombosis

Annexin V (AV), a protein with anticoagulant activity, exerts antithrombotic activity by binding to phosphatidylserine (PS), inhibiting activation of serine proteases important in blood coagulation. The potential use of this protein as an anticoagulant is limited as it rapidly passes from the blood into the kidneys due to its relatively small size (36 kDa). We used recombinant DNA technology to produce a homodimer of human AV (DAV, 73 kDa), which exceeds the renal filtration threshold, and has a 6.5-hour half-life in the rat circulation. Human red blood cells with externalized PS were used to show that DAV had a higher affinity for PS-exposing cells than AV. DAV labeling sensitively identifies PS-exposing cells, was found to be a potent inhibitor of the activity of the prothombinase complexes and inhibits the ability of secretory phospholipaseA(2) to hydrolyze phospholipids of PS-exposing cells, reducing the formation of mediators of blood coagulation and reperfusion injury. DAV exerts dose-dependent antithrombotic activity in rat veins. This combination of activities suggests that DAV is a valuable probe to measure PS exposure and may be efficacious as a novel drug in a wide range of clinical situations.

Topics: Anemia, Sickle Cell; Animals; Annexin A5; Anticoagulants; Blood Coagulation; Cell Membrane; Dimerization; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Erythrocytes; Factor Va; Fibrinolytic Agents; Humans; Male; Mice; Phosphatidylserines; Phospholipases A; Rats; Rats, Wistar; Recombinant Proteins; Thrombin; Thromboplastin; Venous Thrombosis

Inflammation contributes importantly to all stages of atherosclerosis, including the onset of acute thrombotic complications. In clinical trials, statins are beneficial in the primary and secondary prevention of coronary heart disease. Moreover, statins have been shown to possess several pleiotropic properties independent of cholesterol lowering in experimental settings. Based on these premises, we investigated the anti-inflammatory and anti-atherothrombotic properties of rosuvastatin in vivo, testing its effect on cholesterol and monocyte accumulation, and on adhesion molecules and tissue factor (TF) expression. ApoE-deficient female mice were fed a cholesterol-rich diet containing rosuvastatin (0, 1, 2 or 10 mg kg(-1)d(-1)) for 12 weeks. Treatment with rosuvastatin did not significantly affect either body weight gain or plasma total cholesterol (C) and triglyceride levels. However, rosuvastatin treatment dose-dependently reduced ICAM-1 expression in the aortic valves (V) (up to 40% inhibition, p<0.05) and in the proximal segment of the ascending aorta (AA) (-50%, p<0.001). Similarly, rosuvastatin inhibited VCAM-1 expression in the V (-40%) and in the AA (-35%, p<0.05). Moreover, there was a reduced accumulation of macrophages in the V in a dose-dependent and statistically significant manner (-45%, p<0.01). These anti-inflammatory effects were reflected in a reduction of cholesterol deposition in the entire aorta, both in the free and in the esterified form. Finally, the expression of tissue factor, the most potent pro-thrombogenic agent, was consistently reduced in AA by rosuvastatin treatment (-71%, p<0.001). Altogether, these data demonstrate that rosuvastatin has anti-inflammatory and anti-atherothrombotic activities in apoE-deficient mice that could translate in a beneficial effect on atherogenesis.

Topics: Animals; Anti-Inflammatory Agents; Aorta; Aortic Valve; Apolipoproteins E; Atherosclerosis; Cardiovascular Agents; Cholesterol; Cholesterol, Dietary; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Fluorobenzenes; Intercellular Adhesion Molecule-1; Macrophages; Mice; Mice, Knockout; Pyrimidines; Rosuvastatin Calcium; Sulfonamides; Thromboplastin; Time Factors; Vascular Cell Adhesion Molecule-1

Tissue factor (TF) initiates coagulation and indirectly triggers thrombin-dependent protease activated receptor (PAR) signaling. The TF-VIIa complex also directly cleaves PAR2 and promotes angiogenesis in vitro in TF cytoplasmic domain-deleted (TF(deltaCT)) mice. Here we address the effect of PAR1 and PAR2 deficiency on angiogenesis in vivo.. In hypoxia-driven angiogenesis of oxygen induced retinopathy (OIR), wild-type, PAR1-/-, PAR2-/-, and TF(deltaCT) mice showed a comparable regression of the superficial vascular plexus during the initial exposure of mice to hyperoxia. However, TF(deltaCT) mice revascularized areas of central vaso-obliteration significantly faster than wild-type animals. Pharmacological inhibition of the TF-VIIa complex, but not of Xa, and blockade of tyrosine kinase receptor pathways with Gleevec reversed accelerated angiogenesis of TF(deltaCT) mice to revascularization rates observed in wild-type mice. Genetic deletion of PAR2, but not of PAR1, abolished enhanced revascularization of TF(deltaCT) mice. PAR1 knock-out animals were indistinguishable from wild-type mice in the model of retinal neoangiogenesis and angiogenesis-dependent subcutaneous tumor growth was unaltered in PAR1- and PAR2-deficient animals.. Loss of the TF cytoplasmic domain results in accelerated hypoxia-induced angiogenesis mediated by TF-VIIa signaling. PAR2 signaling is sufficient for this proangiogenic effect without apparent contributions of mouse host cell PAR1.

Topics: Animals; Benzamides; Blood Coagulation Factor Inhibitors; Cell Line, Tumor; Disease Models, Animal; Factor VIIa; Hyperoxia; Hypoxia; Imatinib Mesylate; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Neoplasms, Experimental; Neovascularization, Pathologic; Oxygen; Piperazines; Protein Kinase Inhibitors; Pyrimidines; Receptor, PAR-1; Receptor, PAR-2; Retinal Neovascularization; Retinal Vessels; Signal Transduction; Thromboplastin; Time Factors

Inhaled ultrafine particles trigger peripheral thrombotic complications.. We have analyzed the systemic prothrombotic risk following lung inflammation induced by pulmonary carbon nanotubes (CNTs).. Intratracheal instillation in Swiss mice of 200 and 400 microg of multiwall ground CNTs triggered substantial lung neutrophil, but not macrophage influx, 24 h later. The detection of circulating platelet-leukocyte conjugates exclusively 6 h after CNT instillation pointed to early but transient activation of circulating platelets. At 24 h, elevated plasma procoagulant microvesicular tissue factor activity was found in CNT-exposed but not in saline-exposed mice. However, at 24 h, both the tail and jugular vein bleeding times were prolonged in CNT-exposed but not in saline-exposed mice, arguing against strong CNT-induced platelet activation at this point. Nevertheless, at 24 h, enhanced peripheral thrombogenicity was detected in CNT-exposed but not in saline-exposed mice, via quantitative photochemically induced carotid artery thrombosis measurements. P-selectin neutralization abrogated platelet-leukocyte conjugate formation and microvesicular tissue factor generation, and abolished the CNT-induced thrombogenicity amplification. In contrast, the weak vascular injury-triggered thrombus formation in saline-treated mice was not affected by P-selectin neutralization at 24 h.. The mild CNT-induced lung inflammation translates via rapid but mild and transient activation of platelets into P-selectin-mediated systemic inflammation. Leukocyte activation leads to tissue factor release, in turn eliciting inflammation-induced procoagulant activity and an associated prothrombotic risk.

Topics: Animals; Blood Platelets; Disease Models, Animal; Female; Granulocytes; Leukocytes; Male; Mice; Nanotubes, Carbon; P-Selectin; Platelet Activation; Pneumonia; Thromboplastin; Thrombosis

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

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

Severe pulmonary arterial hypertension (PAH) occurs in idiopathic form and in association with diverse diseases. The pathological hallmarks are distal smooth muscle hypertrophy, obliteration of small pulmonary arteriole lumens, and disorganized cellular proliferation in plexiform lesions. In situ thrombosis is also observed. A detailed understanding of the disease progression has been hampered by the absence of an animal model bearing all the pathological features of human disease. To create a model with these characteristics, we gave young (200-g) rats monocrotaline 1 wk following left pneumonectomy; controls with vehicle treatment or sham operation were also studied. In experimental rats, pulmonary arteries had distal smooth muscle hypertrophy and proliferative perivascular lesions. The lesions had a plexiform appearance, occurred early in disease development, and were composed of cells expressing endothelial antigens. Three-dimensional microangiography revealed severe vascular pruning and disorganized vascular networks. We found that expression of tissue factor (TF), the membrane glycoprotein that initiates coagulation, facilitates angiogenesis, and mediates arterial injury in the systemic circulation, was increased in the pulmonary arterioles and plexiform-like lesions of the rats. TF was also heavily expressed in the vessels and plexiform lesions of humans with pulmonary arterial hypertension. We conclude that plexiform-like lesions can be reproduced in rats, and this model will facilitate experiments to address controversies about the role of these lesions in PAH. Increased TF expression may contribute to the prothrombotic diathesis and vascular cell proliferation typical of human disease.

Topics: Angiography; Animals; Cell Proliferation; Disease Models, Animal; Endothelial Cells; Humans; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Male; Monocrotaline; Pneumonectomy; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Thromboplastin; Vascular Endothelial Growth Factor Receptor-2; von Willebrand Factor

Topics: Animals; Disease Models, Animal; Humans; Hypertension, Pulmonary; Monocrotaline; Pneumonectomy; Pulmonary Artery; Radiography; Rats; Thromboplastin; Vascular Endothelial Growth Factor Receptor-2

Topics: Aldosterone; Animals; Blood Coagulation; Blood Pressure; Carboxypeptidase B2; Disease Models, Animal; Eplerenone; Infusions, Intravenous; Male; Mineralocorticoid Receptor Antagonists; Plasminogen Activator Inhibitor 1; Rats; Rats, Wistar; Receptors, Mineralocorticoid; Spironolactone; Thromboplastin; Thrombosis; Tissue Plasminogen Activator

Radiosurgery for arteriovenous malformations is limited to small lesions and may take 3 years to produce total occlusion. It has recently been shown that coadministration of low-dose lipopolysaccharide (LPS) and soluble tissue factor (sTF) selectively induces thrombosis in murine tumor models, attributable perhaps to the prothrombotic phenotype of tumor vasculature. Radiosurgery may induce changes in endothelial prothrombotic molecules similar to those found in tumors. This study aimed to determine if a similar strategy could be used to stimulate thrombus formation in an animal arteriovenous malformation model.. Seventeen rats underwent creation of a carotid-to-jugular anastomosis. Animals were intravenously injected with sTF, low-dose LPS, a combination of both, or placebo 24 hours after stereotactic irradiation of the anastomosis. Control animals received both agents after sham irradiation.. Coadministration of sTF and LPS led to the formation of thrombi in up to 69% of small vessels and 39% of medium-sized vessels within the target region. The irradiated vasculature demonstrated intermediate rates of thrombosis after treatment with either sTF or LPS alone as did vessels within the fistula in the control group. Logistic regression analysis demonstrated significant associations between development of thrombi and treatment with radiation, sTF, or LPS (P < 0.005). There was no evidence of systemic thrombus formation or toxicity in any group.. Treatment with sTF and LPS selectively induces thrombosis of irradiated vessels in a rat model of arteriovenous malformation. Stimulation of thrombosis may improve the efficacy of radiosurgery, increasing the treatable lesion size and reducing latency.

Topics: Animals; Arteriovenous Malformations; Disease Models, Animal; Drug Administration Schedule; Lipopolysaccharides; Male; Radiosurgery; Rats; Rats, Sprague-Dawley; Thromboplastin; Thrombosis

To investigate the characteristics of the thrombus at different time points after thrombosis of the intracranial venous sinus, we have developed a new reversible superior sagittal sinus (SSS) model in rats. In this new model, thrombosis was induced by slow injections of the thrombogenic agent into the SSS using a microcatheter. The success of SSS thrombosis was confirmed by magnetic resonance images (MRI), magnetic resonance venographs (MRV), and electron microscopy. T2-weighted MRI and MRV were performed every week for 4 weeks to investigate the process of SSS occlusion. We also examined thrombus formation and the surrounding tissue pathology, as well as endothelial cell injury following SSS occlusion. SSS occlusion occurred at the beginning of the injection of the partial thromboplastin time reagent, and the occluded SSS reopened at the beginning of the second week. MRI images revealed that T2 signals were detected in the parieto-occipital lobes 24 h after SSS thrombosis and disappeared at the end of week two. During week two, the rate of thrombus organization was evident and increased significantly in week three. Thrombus calcification was detected in week three and increased significantly in week four. Electron microscopy examination showed the damaged endothelial cell detected at week three following SSS thrombosis. All of these findings suggest that this reversible SSS thrombosis model is feasible and reproducible. The occlusion state can be maintained for at least 4 weeks, providing an opportunity to study the mechanisms of SSS thrombosis.

Topics: Animals; Cerebrovascular Circulation; Disease Models, Animal; Endothelial Cells; Magnetic Resonance Imaging; Male; Rats; Rats, Sprague-Dawley; Sagittal Sinus Thrombosis; Superior Sagittal Sinus; Thromboplastin; Thrombosis

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

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

Diabetes mellitus leads to thiamine deficiency and multiple organ damage including diabetic neuropathy. This study was designed to examine the effect of benfotiamine, a lipophilic derivative of thiamine, on streptozotocin (STZ)-induced cerebral oxidative stress. Adult male FVB mice were made diabetic with a single injection of STZ (200 mg/kg, i.p.). Fourteen days later, control and diabetic (fasting blood glucose >13.9 mM) mice received benfotiamine (100 mg/kg/day, i.p.) for 14 days. Oxidative stress and protein damage were evaluated by glutathione/glutathione disulfide (GSH/GSSG) assay and protein carbonyl formation, respectively. Pro-oxidative or pro-inflammatory factors including advanced glycation end-product (AGE), tissue factor and tumor necrosis factor-alpha (TNF-alpha) were evaluated by immunoblot analysis. Four weeks STZ treatment led to hyperglycemia, enhanced cerebral oxidative stress (reduced GSH/GSSG ratio), elevated TNF-alpha and AGE levels without changes in protein carbonyl or tissue factor. Benfotiamine alleviated diabetes-induced cerebral oxidative stress without affecting levels of AGE, protein carbonyl, tissue factor and TNF-alpha. Collectively, our results indicated benfotiamine may antagonize diabetes-induced cerebral oxidative stress through a mechanism unrelated to AGE, tissue factor and TNF-alpha.

Topics: Adjuvants, Immunologic; Analysis of Variance; Animals; Blood Glucose; Diabetes Mellitus, Experimental; Disease Models, Animal; Glutathione; Glutathione Disulfide; Glycation End Products, Advanced; Male; Mice; Oxidative Stress; Protein Carbonylation; Thiamine; Thromboplastin; Tumor Necrosis Factor-alpha

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

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

CD39 (nucleoside triphosphate diphosphohydrolase [NTPDase-1]) expressed on the luminal surface of endothelial cells rapidly metabolizes extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to adenosine monophosphate (AMP), and abrogates platelet reactivity. Optimization of CD39 enzymatic activity appears dependent upon the expression of both transmembrane domains within plasma membranes. Thus, motivation exists to examine therapeutic antiplatelet formulations that consist of liposomal CD39.. Full-length human CD39 was produced by using a yeast expression system, purified, and reconstituted within lipid vesicles. The catalytic efficiency (kcat/Km) of CD39-mediated phosphohydrolysis of ADP and ATP was determined both for detergent-solubilized and protein-reconstituted CD39 within lipid membranes. The capacity of CD39-containing lipid vesicles to inhibit platelet activation induced by ADP, collagen, or thrombin was determined in vitro by platelet aggregometry. A murine model of thromboplastin-induced thromboembolism was used to determine the effectiveness of intravenous liposomal CD39 in limiting platelet consumption and mortality.. Reconstitution of human CD39 in lipid vesicles was associated with a decrease in Km of nearly an order of magnitude over the detergent-solubilized form. There was a concomitant increase in both ADPase and ATPase catalytic efficiencies (kcat/Km ADPase: sol CD39: 2.7 x 10(6) vs liposomal CD39: 1.4 x 10(7) min/ M; kcat/Km ATPase: sol CD39: 7.2 x 10(6) vs liposomal CD39: 2.0 x10(7) min/M). Furthermore, CD39 lipid vesicles effectively inhibited platelet aggregation when activated by ADP, collagen, or thrombin, and also promoted platelet disaggregation (60.4% +/- 6.1%). Treatment with CD39 lipid vesicles preserved platelet counts after thromboplastin injection (pretreatment, 906.8 +/- 42.9 platelets/microm3; empty vesicles, 278.6 +/- 34.8 platelets/microm3; CD39 vesicles, 563.6 +/- 42.2 platelets/microm3; n = 10 mice/test group; P < .0001). In parallel survival studies, liposomal CD39 reduced mortality from 73% to 33% (P < or = .05; n = 12 mice/experimental test group, n = 15 mice/control test group).. Incorporation of solubilized CD39 into a lipid bilayer restores enzyme activity and optimizes thromboregulatory potential. Treatment with CD39 in liposomal formulations decreased mortality in a murine model of thromboplastin-induced thromboembolism by limiting intravascular platelet aggregation and thrombosis.

Topics: Adenosine Diphosphate; Animals; Antigens, CD; Apyrase; Biopsy, Needle; Blotting, Western; Disease Models, Animal; Humans; Immunohistochemistry; Injections, Intravenous; Liposomes; Mice; Mice, Inbred Strains; Platelet Activation; Platelet Aggregation Inhibitors; Platelet Count; Pulmonary Embolism; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Thromboplastin; Time Factors

The tissue factor (TF)-factor VIIa (FVIIa) complex not only is essential for activation of blood coagulation but also affect the inflammatory response during sepsis. The objective of this study was to determine the role of TF-FVIIa in pneumonia caused by Streptococcus pneumoniae, the most important causative organism in community-acquired pneumonia and a major cause of sepsis.. A controlled, in vivo laboratory study.. Research laboratory of a health sciences university.. Patients with unilateral community-acquired pneumonia and female BALB/c mice.. Bilateral bronchoalveolar lavage was performed in patients with community-acquired pneumonia. In mice, pneumonia was induced by intranasal inoculation with S. pneumoniae with or without concurrent inhibition of TF-FVIIa by subcutaneous injections of recombinant nematode anticoagulant protein (rNAPc2).. Patients with unilateral community-acquired pneumonia demonstrated elevated concentrations of FVIIa, soluble TF, and thrombin-antithrombin complexes in bronchoalveolar lavage fluid obtained from the infected site compared with the uninfected site. Mice with S. pneumoniae pneumonia displayed increased TF expression and fibrin deposits in lungs together with elevated thrombin-antithrombin complex levels in bronchoalveolar lavage fluid; inhibition of TF-FVIIa by rNAPc2 attenuated the procoagulant response in the lung but did not affect host defense, as reflected by an unaltered outgrowth of pneumococci and an unchanged survival.. These data suggest that TF-FVIIa activity contributes to activation of coagulation in the lung during pneumococcal pneumonia but does not play an important role in the antibacterial host defense in this murine model.

Topics: Adult; Animals; Antithrombin III; Blood Coagulation; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Factor VIIa; Female; Helminth Proteins; Humans; Injections, Subcutaneous; Male; Mice; Mice, Inbred BALB C; Middle Aged; Peptide Hydrolases; Pneumonia, Pneumococcal; Streptococcus pneumoniae; Thromboplastin

Substantial progress has been made in understanding the contribution of alterations in coagulation and fibrinolysis to the pathogenesis of acute lung injury (ALI). Findings from mouse, rat, baboon, and human studies indicate that alterations in coagulation and fibrinolysis may be of major pathogenetic importance in ALI and other inflammatory conditions in the lung including pneumonia, sepsis, and ventilator-induced lung injury. Therapies targeted at both activation of coagulation through the extrinsic coagulation cascade and modulation of coagulation through the protein C system have the potential to favorably impact clinical ALI/acute respiratory distress syndrome.

Topics: Animals; Blood Coagulation; Disease Models, Animal; Fibrinolysis; Humans; Models, Immunological; Papio; Pneumonia; Protein C; Receptors, Proteinase-Activated; Respiration, Artificial; Respiratory Distress Syndrome; Sepsis; Thrombomodulin; Thromboplastin

The coagulation trigger tissue factor has been implicated in tumor growth, angiogenesis, and metastasis. In this study, we explore the effects of ex vivo and in vivo delivery of short interfering RNA (siRNA) targeting tissue factor on B16 melanoma colonization of the lung in a murine model for metastasis. The purposes of this work are to establish a noncytotoxic in vivo model for investigation of tissue factor function and provide preclinical assessment of the therapeutic potential of tissue factor siRNA for prevention of metastasis.. C57BL/6 mice were evaluated for pulmonary metastases following tail vein injection of B16 cells transfected with either active or inactive siRNA. Mice receiving cells transfected with active siRNA had significantly lower numbers of pulmonary tumors compared with mice injected with control cells (transfected with inactive siRNA). The average time point at which the mice started to exhibit tumor-associated stress was also increased significantly from 22 days for the control group to 27 days for the experimental group (P = 0.01). In a therapeutically more relevant model, where the siRNA was delivered i.p. and the cells (untransfected) by tail vein injection, an inhibitory effect on metastasis was observed when the siRNA treatment was initiated either before or at the time of cell injection.. The results suggest that tissue factor has a crucial function in promoting lung tumor metastasis of blood-borne tumor cells in the early stages of the tumor take process and further suggest that treatment with tissue factor siRNA may become a viable clinical strategy for prevention of tumor metastasis.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Drug Delivery Systems; Female; Injections, Intravenous; Injections, Subcutaneous; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; RNA, Small Interfering; Thromboplastin

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

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

Diagnosis of cerebral sinus vein thrombosis is still a challenge for imaging. MRI and MRA play a major role in sinus imaging. For further development of MR techniques, MR-compatible animal models are required. The aim of this study was to develop an animal model for sinus thrombosis and additional cortical vein thrombosis with a clot of human blood for MR imaging studies.. A combined surgical and interventional approach was carried out in 13 pigs. After minimal invasive surgical access to the anterior superior sagittal sinus and cortical vein, thrombosis with human blood was induced using an interventional catheter approach. MR imaging was performed prior to and after thrombus induction.. Sinus thrombosis was induced in 12 of 13 animals. Three animals suffered acute subdural haemorrhage; one of these animals died during the intervention, and one died after thrombus induction. MR imaging of the thrombosed sinus could easily be performed without significant artefacts in 11 of 13 animals.. This new model of sinus and cortical vein thrombosis with a clot of human blood allows artefact-free imaging studies on MR.

Topics: Animals; Balloon Occlusion; Blood Transfusion; Disease Models, Animal; Hemostatics; Humans; Intracranial Thrombosis; Magnetic Resonance Imaging; Sinus Thrombosis, Intracranial; Swine; Thromboplastin; Venous Thrombosis

Subacute stent thrombosis is a major clinical concern, and the search for new molecules to cover stents remains important. Dimethyl sulfoxide (DMSO) is used for preservation of hematopoietic progenitor cells and is infused into patients undergoing bone marrow transplantation. Despite its intravenous application, the impact of DMSO on vascular cells has not been assessed.. In human endothelial cells, monocytes, and vascular smooth muscle cells (VSMC), DMSO inhibited tissue factor (TF) expression and activity in response to tumor necrosis factor-alpha or thrombin in a concentration-dependent manner. DMSO did not exert any toxic effects as assessed by phase-contrast microscopy, trypan blue exclusion, and lactate dehydrogenase release. Real-time polymerase chain reaction revealed that inhibition of TF expression occurred at the mRNA level. This effect was mediated by reduced activation of the mitogen-activated protein kinases c-Jun terminal NH2 kinase (51+/-6%; P=0.0005) and p38 (50+/-3%; P<0.0001) but not p44/42 (P=NS). In contrast to TF, DMSO did not affect expression of TF pathway inhibitor or plasminogen activator inhibitor-1. In vivo, DMSO treatment suppressed TF activity (41%; P<0.002) and prevented thrombotic occlusion in a mouse carotid artery photochemical injury model. DMSO also inhibited VSMC proliferation (70%; P=0.005) and migration (77%; P=0.0001) in a concentration-dependent manner; moreover, it prevented rapamycin and paclitaxel-induced upregulation of TF expression.. DMSO suppresses TF expression and activity, as well as thrombus formation; in addition, it inhibits VSMC proliferation and migration. Given its routine use in modern clinical practice, we propose DMSO as a novel strategy for coating drug-eluting stents and treating acute coronary syndromes.

Topics: Animals; Carotid Artery Thrombosis; Cell Movement; Cell Proliferation; Cells, Cultured; Dimethyl Sulfoxide; Disease Models, Animal; Gene Expression Regulation; Humans; Mice; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Thromboplastin; Thrombosis

Phosphatidylserine (PtdSer) is exposed on the external leaflet of the plasma membrane during apoptosis. The protein annexin A5 (anxA5) shows high affinity for PtdSer. When anxA5 binds to the PtdSer-expressing membranes during apoptosis, it crystallizes as an extended two-dimensional network and activates thereby a novel portal of cell entry that results in the internalization of the PtdSer-expressing membrane patches. This novel pathway of cell entry is potentially involved in the regulation of the surface expression of membrane receptors. In this study we report the regulation of surface expression of the initiator of blood coagulation tissue factor (TF) by this novel pathway of cell entry. AnxA5 induces the internalization of tissue factor expressed on the surface of apoptotic THP-1 macrophages. This down-regulation depends on the abilities of anxA5 to bind to PtdSer and to form a two-dimensional crystal at the membrane. We furthermore show that THP-1 cells produce and externalize anxA5 that cause the internalization of TF in an autocrine type of mechanism. We extended our in vitro work to the in vivo situation and show in a mouse model that anxA5 causes the down-regulation of TF expression by smooth muscle cells of the media of the carotid artery that was mechanically injured. In conclusion, anxA5 down-regulates surface-expressed TF by activating the novel portal of cell entry. This mechanism may be part of a more general autocrine function of anxA5 to regulate the plasma membrane receptor repertoir under stress conditions associated with the surface expression of PtdSer.

Topics: Animals; Annexin A5; Apoptosis; Carotid Artery Injuries; Cell Line; Cell Membrane; Disease Models, Animal; Down-Regulation; Endocytosis; Etoposide; Factor VII; Humans; Macrophages; Male; Mice; Phosphatidylserines; Receptors, Cell Surface; Signal Transduction; Tetradecanoylphorbol Acetate; Thromboplastin

Photochemically induced thrombosis (a thrombin-dependent process) was measured in rats treated with moderate doses of anticoagulants, but which appeared to be unchanged. We considered the possibility that platelet-inhibiting agents, which also indirectly inhibit coagulation, would act as more potent antithrombotic agents. Inhibitors used as such were prostaglandin E1 (PGE1), which elevates cyclic AMP levels, and the P2Y12 ADP-receptor antagonist, AR-C69931MX. Effects of these agents were investigated in an ex vivo model system, in which whole blood under coagulant conditions was perfused over fibrinogen at defined wall shear rate. Perfusion of blood (rat or human) in the presence of tissue factor resulted in deposition of activated platelets and subsequent aggregate formation, along with exposure of procoagulant phosphatidylserine (PS) on the platelet surface and formation of fibrin fibers. In the presence of PGE1 aggregation was completely inhibited, but platelet adhesion and PS exposure were only party reduced, while fibrin formation was hardly affected. Treatment with AR-C69931MX caused similar, but less complete effects. These results indicate that in tissue factor-triggered blood under conditions of flow: (i) the platelet procoagulant response is independent of aggregate formation; (ii) the platelet-inhibiting effect of PGE1 and AR-C69931MX is sufficient to suppress aggregation, but not platelet adhesion and coagulation. These platelet inhibitors thus maintain their aggregation-inhibiting effect at sites of thrombin formation.

Topics: Adenosine Monophosphate; Alprostadil; Animals; Blood Coagulation; Disease Models, Animal; Drug Interactions; Fibrin; Fibrinogen; Fibrinolytic Agents; Male; Perfusion; Phosphatidylserines; Platelet Activation; Platelet Aggregation; Rats; Rats, Wistar; Thrombin; Thromboplastin

BAY 59-7939 is an oral, direct Factor Xa (FXa) inhibitor in development for the prevention and treatment of arterial and venous thrombosis. BAY 59-7939 competitively inhibits human FXa (K(i) 0.4 nm) with > 10 000-fold greater selectivity than for other serine proteases; it also inhibited prothrombinase activity (IC(50) 2.1 nm). BAY 59-7939 inhibited endogenous FXa more potently in human and rabbit plasma (IC(50) 21 nm) than rat plasma (IC(50) 290 nm). It demonstrated anticoagulant effects in human plasma, doubling prothrombin time (PT) and activated partial thromboplastin time at 0.23 and 0.69 microm, respectively. In vivo, BAY 59-7939 reduced venous thrombosis (fibrin-rich, platelet-poor thrombi) dose dependently (ED(50) 0.1 mg kg(-1) i.v.) in a rat venous stasis model. BAY 59-7939 reduced arterial (fibrin- and platelet-rich) thrombus formation in an arteriovenous (AV) shunt in rats (ED(50) 5.0 mg kg(-1) p.o.) and rabbits (ED(50) 0.6 mg kg(-1) p.o.). Slight inhibition of FXa (32% at ED(50)) reduced thrombus formation in the venous model; to affect arterial thrombosis in the rat and rabbit, stronger inhibition of FXa (74%, 92% at ED(50)) was required. Calculated plasma levels in rabbits at the ED(50) were 14-fold lower than in the rat AV shunt model, correlating with the 14-fold lower IC(50) of FXa inhibition in rabbit compared with rat plasma; this may suggest a correlation between FXa inhibition and antithrombotic activity. Bleeding times in rats and rabbits were not significantly affected at antithrombotic doses (3 mg kg(-1) p.o., AV shunt). Based on these results, BAY 59-7939 was selected for clinical development.

Topics: Animals; Blood Coagulation Tests; Disease Models, Animal; Dose-Response Relationship, Drug; Factor Xa Inhibitors; Fibrinolytic Agents; Humans; Inhibitory Concentration 50; Morpholines; Rabbits; Rats; Rivaroxaban; Serine Proteinase Inhibitors; Species Specificity; Thiophenes; Thromboplastin; Venous Thrombosis

Topics: Animals; Blood Coagulation Tests; Chlorides; Disease Models, Animal; Female; Ferric Compounds; Hemostasis; Male; Nadroparin; Rats; Rats, Wistar; Regression Analysis; Sensitivity and Specificity; Thromboplastin; Thrombosis; Venous Thrombosis

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

We compared the antithrombotic properties of a factor Xa inhibitor (DX-9065a) with those of a thrombin inhibitor (melagatran) in a rat disseminated intravascular coagulation model and a rat venous thrombosis model. Rat disseminated intravascular coagulation and venous thrombosis models were produced by injection of tissue factor and platinum wire placement, respectively. DX-9065a exerted antithrombotic effects dose dependently in both models. Melagatran was also effective in the venous thrombosis model, whereas it showed an aggravation in the disseminated intravascular coagulation model at low but not high doses. In the in vitro study, DX-9065a decreased the C(max) of the thrombin generation curve in plasma irrespective of whether protein C was present or not. However, melagatran increased the C(max) at low concentrations when protein C was present. This increase was not detected in protein C-deficient plasma. These results suggest that, unlike DX-9065a, melagatran in low doses aggravates disseminated intravascular coagulation by increasing thrombin generation, which may be partly due to suppression of negative feedback by activated protein C.

Topics: Animals; Azetidines; Benzylamines; Blood Coagulation; Disease Models, Animal; Dose-Response Relationship, Drug; Factor Xa Inhibitors; Fibrinolytic Agents; Glycine; Male; Naphthalenes; Propionates; Rats; Rats, Wistar; Thrombin; Thromboplastin; Thrombosis

Inflammation and platelet activation are critical phenomena in the setting of acute coronary syndromes. Platelets may contribute to increase ischemic injury by enhancing the inflammatory response of leukocytes and endothelial myocardial cells. Pharmacological inhibition of platelet activation prevents ischemic complications in patients with coronary diseases. Agents directed against the integrin glycoprotein IIb/IIIa (GP IIb/IIIa) receptor not only inhibit platelet aggregation but also have been demonstrated to limit the inflammatory response in acute coronary syndromes. The question then raised is if the inhibition of platelet activation by other mechanisms than the blockade of GP IIb/IIIa may also exert anti-inflammatory effects. The aim of the present study was to analyze if clopidogrel may exert anti-inflammatory effects during the acute phase of myocardial infarction. A ligature was placed around the left anterior descending coronary artery of New Zealand White rabbits. After 15 min of ischemia, the myocardium was reperfused and the ischemic coronary artery was isolated 24 h after the ischemia. A group of ischemic rabbits was given a single oral dose of clopidogrel (20 mg kg(-1)) just after the arterial occlusion and the animal was recovered. Sham-operated animals served as control. P-selectin expression was significantly increased in infarcted rabbits with respect to control rabbits. Clopidogrel administration reduced P-selectin expression with respect to untreated infarcted rabbits. CD40 ligand and tissue factor expression was increased in the ischemic coronary artery and reduced after clopidogrel administration. Clopidogrel also protected endothelial nitric oxide synthase protein expression in the ischemic coronary artery, a protein that has been found downregulated under inflammatory conditions. In conclusion, inhibition of platelet activation by clopidogrel exerted anti-inflammatory effects on the ischemic coronary artery.

Topics: Animals; Anti-Inflammatory Agents; CD40 Ligand; Clopidogrel; Coronary Vessels; Disease Models, Animal; Male; Myocardial Ischemia; P-Selectin; Platelet Aggregation Inhibitors; Rabbits; Thromboplastin; Ticlopidine

To observe the expressions of early growth response factor-1 (Egr-1) and tissue factor (TF) in rats with cerulein-induced acute pancreatitis and to explore its significance.. A large dose of cerulein was used to create the experimental acute pancreatitis model in rats. The changes of Egr-1 mRNA and protein in rats were observed during 30 min to 4 h after the treatment and immunohistochemical method was used to observe the localized expression of Egr-1 in tissues. In addition to the mRNA expression of Egr-1 target gene, TF was also observed. A blank control group, and a bombesin-administered group were used for comparison.. After the stimulation of a large dose of cerulein, the rats showed typical inflammatory changes of acute pancreatitis. Thirty minutes after the stimulation, the mRNA expression of Egr-1 in the pancreatic tissue reached its peak and then declined, while the expression of Egr-1 protein reached its peak 2 h after the stimulation. Histologically, 2 h after the stimulation, almost all pancreatic acinar cells had the expression of Egr-1 protein, which was focused in the nuclei. The mRNA expression of TF occurred 1 h after the stimulation and gradually increased within 4 h. However, a large dose of bombesin only stimulated the pancreatic tissue to produce a little mRNA expression of Egr-1 and no mRNA expression of Egr-1 protein and TF.. Egr-1 as a pro-inflammatory transcription factor may play an important role in the pathogenesis of acute pancreatitis by modulating the expression of TF.

Topics: Acute Disease; Animals; Ceruletide; Disease Models, Animal; DNA-Binding Proteins; Early Growth Response Protein 1; Gene Expression; Immediate-Early Proteins; Male; Pancreatitis; Rats; Rats, Wistar; Thromboplastin; Transcription Factors

Endothelium plays a critical role in the pathobiology of sepsis by integrating systemic host responses and local rheological stimuli. We studied the differential expression and activation of tissue factor (TF)-dependent coagulation on linear versus branched arterial segments in a baboon sepsis model. Animals were injected intravenously with lethal doses of Escherichia coli or saline and sacrificed after 2 to 8 hours. Whole-mount arterial segments were stained for TF, TF-pathway inhibitor (TFPI), factor VII (FVII), and markers for endothelial cells (ECs), leukocytes, and platelets, followed by confocal microscopy and image analysis. In septic animals, TF localized preferentially at branches, EC surface, leukocytes, and platelet aggregates and accumulated in large amounts in the subendothelial space. FVII strongly co-localized with TF on ECs and leukocytes but less so with subendothelial TF. TFPI co-localized with TF and FVII on endothelium and leukocytes but not in the subendothelial space. Focal TF increases correlated with fibrin deposition and increased endothelial permeability to plasma proteins. Biochemical analysis confirmed that aortas of septic baboons expressed more TF mRNA and protein than controls. Branched segments contained higher TF protein levels and coagulant activity than equivalent linear areas. These data suggest that site-dependent endothelial heterogeneity and rheological factors contribute to focal procoagulant responses to E. coli.

Topics: Animals; Aorta; Arteries; Biomarkers; Blood Coagulation; Blood Platelets; Disease Models, Animal; Endothelial Cells; Escherichia coli Infections; Factor VII; Image Processing, Computer-Assisted; Leukocytes; Lipopolysaccharides; Lipoproteins; Microscopy, Confocal; Models, Biological; Papio; RNA, Messenger; Shock, Septic; Thromboplastin; Up-Regulation

NO-releasing statins are new chemical entities, combining HMG-CoA reductase inhibition and slow NO release, that possess stronger anti-inflammatory and antiproliferative activities than the native statins.. We evaluated the antithrombotic effects of nitropravastatin (NCX-6550) by assessing its activity on platelet activation and tissue factor (TF) expression by mononuclear cells in vitro and in vivo.. In vitro, NCX-6550 inhibited (1) U46619- and collagen-induced platelet aggregation in buffer and plasma; (2) collagen-induced P-selectin expression in whole blood and (3) platelet adhesion to collagen-coated coverslips under high shear stress. These effects were displayed at concentrations of NCX-6550 ranging from 25 to 100 mum, and were totally reverted by the guanylylcyclase inhibitor ODQ (10 microm). Equimolar concentrations of pravastatin had no influence on these parameters of platelet function. LPS- and PMA-induced TF expression by blood mononuclear cells was also inhibited by NCX-6550 (IC50 13 microm), but not by pravastatin, as assessed by functional and immunological assays and by real-time PCR. In a mouse model of platelet pulmonary thromboembolism, induced by the i.v. injection of collagen plus epinephrine, pretreatment with NCX-6550 (24-48 mg kg(-1)) significantly reduced platelet consumption, lung vessel occlusion and mortality. Moreover, nitropravastatin markedly inhibited the generation of procoagulant activity by spleen mononuclear cells and peritoneal macrophages in mice treated with LPS. In these in vivo models too, pravastatin failed to affect platelet activation and monocyte/macrophage procoagulant activity.. Our results show that nitropravastatin exerts strong antithrombotic effects in vitro and in vivo, and may represent an interesting antiatherothrombotic agent for testing in acute coronary syndromes.

Topics: Animals; Blood Platelets; Disease Models, Animal; Dose-Response Relationship, Drug; Humans; In Vitro Techniques; Inhibitory Concentration 50; Leukocytes, Mononuclear; Macrophages, Peritoneal; Male; Mice; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitrites; Nitro Compounds; P-Selectin; Platelet Adhesiveness; Platelet Aggregation; Pravastatin; Pulmonary Embolism; RNA, Messenger; Spleen; Thromboplastin

Topics: Animals; Disease Models, Animal; Humans; Hypertension, Pulmonary; Rats; Rats, Sprague-Dawley; Thromboplastin; Tunica Intima

To evaluate the expression of fibrinogen-like protein 2 (fgl2) and its correlation with disease progression in both mice and patients with severe viral hepatitis.. Balb/cJ or A/J mice were infected intraperitoneally (ip) with 100 PFU of murine hepatitis virus type 3 (MHV-3), liver and serum were harvested at 24, 48, and 72 h post infection for further use. Liver tissues were obtained from 23 patients with severe acute chronic (AOC) hepatitis B and 13 patients with mild chronic hepatitis B. Fourteen patients with mild chronic hepatitis B with cirrhosis and 4 liver donors served as normal controls. In addition, peripheral blood mononuclear cells (PBMC) were isolated from 30 patients (unpaired) with severe AOC hepatitis B and 10 healthy volunteers as controls. Procoagulant activity representing functional prothrombinase activity in PBMC and white blood cells was also assayed. A polyclonal antibody against fgl2 was used to detect the expression of both mouse and human fgl2 protein in liver samples as well as in PBMC by immunohistochemistry staining in a separate set of studies. Alanine aminotransferase (ALT) and total bilirubin (TBil) in serum were measured to assess the severity of liver injury.. Histological changes were found in liver sections 12-24 h post MHV-3 infection in Balb/cJ mice. In association with changes in liver histology, marked elevations in serum ALT and TBil were observed. Mouse fgl2 (mfgl2) protein was detected in the endothelium of intrahepatic veins and hepatic sinusoids within the liver 24 h after MHV-3 infection. Liver tissues from the patients with severe AOC hepatitis B had classical pathological features of acute necroinflammation. Human fgl2 (hfgl2) was detected in 21 of 23 patients (91.30%) with severe AOC hepatitis B, while only 1 of 13 patients (7.69%) with mild chronic hepatitis B and cirrhosis had hfgl2 mRNA or protein expression. Twenty-eight of thirty patients (93.33%) with severe AOC hepatitis B and 1 of 10 with mild chronic hepatitis B had detectable hfgl2 expression in PBMC. No hfgl2 expression was found either in the liver tissue or in the PBMC from normal donors. There was a positive correlation between hfgl2 expression and the severity of the liver disease as indicated by the levels of TBil. PCA significantly increased in PBMC in patients with severe AOC hepatitis B.. The molecular and cellular results reported here in both mice and patients with severe viral hepatitis suggest that virus-induced hfgl2 prothrombinase/fibroleukin expression and the coagulation activity associated with the encoded fgl2 protein play a pivotal role in initiating severe hepatitis. The measurement of hfgl2/fibroleukin expression in PBMC may serve as a useful marker to monitor the severity of AOC hepatitis B and a target for therapeutic intervention.

Topics: Animals; Disease Models, Animal; Disease Progression; Female; Fibrinogen; Hepatitis B; Hepatitis, Viral, Animal; Humans; Liver; Mice; Mice, Inbred BALB C; Murine hepatitis virus; Thromboplastin

While the adenosine 5'-diphosphate (ADP) pathway is known to enhance thrombus formation by recruiting platelets and leukocytes to the primary layer of collagen-adhering platelets, its role for the initiation of coagulation has not been revealed. Ex vivo inhibition of the P2Y12 ADP receptor by clopidogrel administration diminished the rapid exposure of tissue factor (TF), the major initiator of coagulation, in conjugates of platelets with leukocytes established by the contact of whole blood with fibrillar collagen. Under in vitro conditions, the P2Y12 and P2Y1 ADP receptors were both found to be implicated in the exposure of TF in collagen-activated whole blood. Immunoelectron-microscopy revealed that collagen elicited the release of TF from its storage pools within the platelets. Functional activation of the intravascular TF was reduced by inhibition of the ADP receptors, partially due to the disruption of the platelet-neutrophil adhesions. Injection of collagen into the venous system of mice increased the number of thrombin-antithrombin complexes, indicative for the formation of thrombin in vivo. In P2Y1-deficient mice, the ability of collagen to enhance the generation of thrombin was impaired. In conclusion, the platelet ADP pathway supports the initiation of intravascular coagulation, which is likely to contribute to the concomitant formation of fibrin at the site of the growing thrombus.

Topics: Adult; Animals; Blood Coagulation; Blood Platelets; Clopidogrel; Collagen; Disease Models, Animal; Disseminated Intravascular Coagulation; Factor Xa; Humans; Leukocytes; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Immunoelectron; Platelet Adhesiveness; Platelet Aggregation Inhibitors; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Receptors, Purinergic P2Y1; Reference Values; Thrombin; Thromboplastin; Thrombosis; Ticlopidine

Sepsis is associated with a systemic activation of coagulation and an excessive inflammatory response. Anticoagulants have been shown to inhibit both coagulation and inflammation in sepsis. In this study, we used both genetic and pharmacologic approaches to analyze the role of tissue factor and protease-activated receptors in coagulation and inflammation in a mouse endotoxemia model. We used mice expressing low levels of the procoagulant molecule, tissue factor (TF), to analyze the effects of TF deficiency either in all tissues or selectively in hematopoietic cells. Low TF mice had reduced coagulation, inflammation, and mortality compared with control mice. Similarly, a deficiency of TF expression by hematopoietic cells reduced lipopolysaccharide (LPS)-induced coagulation, inflammation, and mortality. Inhibition of the down-stream coagulation protease, thrombin, reduced fibrin deposition and prolonged survival without affecting inflammation. Deficiency of either protease activated receptor-1 (PAR-1) or protease activated receptor-2 (PAR-2) alone did not affect inflammation or survival. However, a combination of thrombin inhibition and PAR-2 deficiency reduced inflammation and mortality. These data demonstrate that hematopoietic cells are the major pathologic site of TF expression during endotoxemia and suggest that multiple protease-activated receptors mediate crosstalk between coagulation and inflammation.

Topics: Adaptor Proteins, Signal Transducing; Animals; Blood Coagulation; Carrier Proteins; Cell Adhesion Molecules; Cell Cycle Proteins; Disease Models, Animal; Endotoxemia; Fibrin; Hematopoiesis; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Receptor Cross-Talk; Receptor, PAR-1; Receptor, PAR-2; Receptors, Proteinase-Activated; Survival Rate; Thromboplastin

Tissue factor (TF) expressed in arterial atherosclerotic plaque plays a key role in activating the extrinsic coagulation pathway and triggering acute coronary syndromes. In this study, we developed and characterized a TF-factor (F)VIIa-mediated thrombosis model in rabbits. Balloon catheter-induced endothelial denudation in the femoral artery and a 4-week high cholesterol diet produced a localized atherosclerotic plaque at the injured site. High levels of TF mRNA and TF protein antigen (152 +/- 25 vs. 49 +/- 12 pg mg-1 protein in normal vessels) were detected in these atherosclerotic plaques. Plasma FVII coagulant activity (FVII:C) was significantly increased in the hypercholesterolemic rabbits (36 +/- 1 s) compared with the normal rabbits (44 +/- 1 s, P < 0.0001). Plaque rupture was induced by balloon angioplasty, which resulted in thrombus formation in the injured vessel segment after a brief period of stasis. FVIIai, a specific TF-FVIIa inhibitor, was administered intravenously to rabbits before plaque rupture at 0.3 and 1.0 mg kg-1. FVIIai dose-dependently reduced thrombus mass (14.7 +/- 2.5 and 5.9 +/- 2.2 mg, respectively, vs. 21.6 +/- 1.9 mg in the control group). PD198961, a novel factor Xa inhibitor, and argatroban, a thrombin inhibitor, also dose-dependently inhibited thrombosis. These results indicate that thrombus formation in this model is initiated by the activation of TF-FVIIa pathway, which is attributed to TF expression in the atherosclerotic plaque and enhanced plasma FVII coagulant activity. This model may be useful for evaluating in vivo efficacy of new antithrombotic drugs, particularly TF-FVIIa inhibitors.

Topics: Animals; Arteriosclerosis; Disease Models, Animal; Factor VIIa; Fibrinolytic Agents; Gene Expression; Hypercholesterolemia; Lipids; Rabbits; RNA, Messenger; Thromboplastin; Thrombosis

Abnormal tissue factor (TF) expression has been demonstrated on blood monocytes and circulating endothelial cells in humans with sickle cell anemia. We have now studied sickle transgenic mice to help define the biology of endothelial TF expression in sickle disease. Using immunostaining of tissue sections, we find that this is confined almost exclusively to the pulmonary veins. About 15% and 13% of these exhibit TF-positive endothelium in the wild-type normal mouse and the normal human hemoglobin (HbA)-expressing control transgenic mouse, respectively. The mild sickle mouse is indistinguishable from normal (approximately 14% positive), but TF expression is significantly elevated in the moderate and severe mouse models of sickle disease (approximately 29% and approximately 41% positive, respectively). Exposure of the mild sickle mouse to hypoxia for 3 hours, followed by reoxygenation, converted its TF expression phenotype to that of the severe sickle mouse (approximately 36% positive). Pretreatment with lovastatin eliminated excessive expression of TF in the posthypoxic mild sickle mouse (approximately 16% positive) and in the more severe mouse at ambient air (approximately 21% positive). In addition to identifying tissue expression of endothelial TF in the sickle lung, these studies implicate reperfusion injury physiology in its expression and suggest a rationale for use of statins in sickle disease.

Topics: Anemia, Sickle Cell; Animals; Disease Models, Animal; Endothelium, Vascular; Humans; Lovastatin; Mice; Mice, Inbred C57BL; Mice, Transgenic; Thromboplastin

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

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

Plaque disruption does not always result in complete thrombotic occlusion. The mechanism of arterial thrombus propagation remains unclear.. We studied how vascular wall thrombogenicity and blood flow reduction affect thrombus propagation using a rabbit model of single and repeated balloon injury. After balloon injury of the normal femoral artery, the blood flow was reduced to 50%, 25%, or 10% (n=5). Small mural thrombi composed of aggregated platelets were produced, but no occlusive thrombi developed in any flow reduction. Three weeks after the first balloon injury, neointima with tissue factor expression and increased procoagulant activity was developed. Balloon injury of the neointima with the same blood flow reduction (n=5) induced fibrin-rich thrombus formation. Additionally, injury with flow reduced to 25% and 10% promoted thrombus propagation resulting in vessel occlusion within 160+/-18 and 71+/-17 seconds, respectively. An injection of anti-von Willebrand factor (vWF) monoclonal antibody (AJW200; 1.0 mg/kg) prevented occlusive thrombus formation.. Increased vascular wall thrombogenicity together with a substantial blood flow reduction is crucial for occlusive thrombus formation, and vWF plays an important role in thrombus propagation. Reduced blood flow at plaque disruption sites might contribute to thrombus propagation leading to acute coronary syndromes.

Topics: Animals; Arterial Occlusive Diseases; Catheterization; Connective Tissue; Constriction, Pathologic; Disease Models, Animal; Femoral Artery; Male; Microscopy, Electron, Transmission; Neovascularization, Pathologic; Rabbits; Regional Blood Flow; Thromboplastin; Thrombosis; Tunica Intima

Administration of antithrombin III-enriched plasma to rabbits with acute Masugi nephritis inhibited prothrombinase formation and increased the release of component C3 from the kidneys. This treatment had a cytoprotective effect and was probably followed by dissociation of antigen--antibody complexes.

Topics: Animals; Antithrombin III; Complement C3; Disease Models, Animal; Hemostasis; Heparin; Kidney; Nephritis; Protein Precursors; Rabbits; Thromboplastin

In septic shock, tissue factor (TF) activates blood coagulation, and cytokines and chemokines orchestrate an inflammatory response. In this study, the role of Egr-1 in lipopolysaccharide (LPS) induction of TF and inflammatory mediators in vivo was evaluated using Egr-1(+/+) and Egr-1(-/-) mice. Administration of LPS transiently increased the steady-state levels of Egr-1 mRNA in the kidneys and lungs of Egr-1(+/+) mice with maximal induction at one hour. Egr-1 was expressed in epithelial cells in the kidneys and lungs in untreated and LPS-treated mice. LPS induction of monocyte chemoattractant protein mRNA in the kidneys and lungs of Egr-1(-/-) mice was not affected at 3 hours, but its expression was significantly reduced at 8 hours compared with the expression observed in Egr-1(+/+) mice. Similarly, LPS induction of TF mRNA expression in the kidneys and lungs at 8 hours was reduced in Egr-1(-/-) mice. However, Egr-1 deficiency did not affect plasma levels of tumor necrosis factor alpha in endotoxemic mice. Moreover, Egr-1(+/+) and Egr-1(-/-) mice exhibited similar survival times in a model of acute endotoxemia. These data indicate that Egr-1 does not contribute to the early inflammatory response in the kidneys and lungs or the early systemic inflammatory response in endotoxemic mice. However, Egr-1 does contribute to the sustained expression of inflammatory mediators and to the maximal expression of TF at 8 hours in the kidneys and lungs.

Topics: Animals; Blotting, Northern; Disease Models, Animal; DNA-Binding Proteins; Early Growth Response Protein 1; Endotoxemia; Immediate-Early Proteins; Intercellular Adhesion Molecule-1; Kidney; Lipopolysaccharides; Mice; Mice, Knockout; Plasminogen Activator Inhibitor 1; RNA, Messenger; Thromboplastin; Transcription Factors; Transcription, Genetic; Zinc Fingers

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

Age-related macular degeneration (AMD) is the leading cause of blindness after age 55 in the industrialized world. Severe loss of central vision frequently occurs with the exudative (wet) form of AMD, as a result of the formation of a pathological choroidal neovasculature (CNV) that damages the macular region of the retina. We tested the effect of an immunotherapy procedure, which had been shown to destroy the pathological neovasculature in solid tumors, on the formation of laser-induced CNV in a mouse model simulating exudative AMD in humans. The procedure involves administering an Icon molecule that binds with high affinity and specificity to tissue factor (TF), resulting in the activation of a potent cytolytic immune response against cells expressing TF. The Icon binds selectively to TF on the vascular endothelium of a CNV in the mouse and pig models and also on the CNV of patients with exudative AMD. Here we show that the Icon dramatically reduces the frequency of CNV formation in the mouse model. After laser treatment to induce CNV formation, the mice were injected either with an adenoviral vector encoding the Icon, resulting in synthesis of the Icon by vector-infected mouse cells, or with the Icon protein. The route of injection was i.v. or intraocular. The efficacy of the Icon in preventing formation of laser-induced CNV depends on binding selectively to the CNV. Because the Icon binds selectively to the CNV in exudative AMD as well as to laser-induced CNV, the Icon might also be efficacious for treating patients with exudative AMD.

Topics: Adenoviridae; Animals; Cells, Cultured; Choroid; Disease Models, Animal; DNA, Complementary; Gene Library; Genetic Vectors; Humans; Immunotherapy; Lasers; Liver; Macular Degeneration; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Neovascularization, Pathologic; Protein Binding; Retina; Swine; Thromboplastin

Clinical and experimental evidence suggests that extravascular fibrin deposition in arthritic joints is prominent and deleterious. The aim of this study was to investigate the contributions of tissue factor (TF) and its inhibitor, TF pathway inhibitor (TFPI), in arthritis.. Synovial tissue specimens obtained from 10 patients with rheumatoid arthritis (RA) and 12 patients with osteoarthritis (OA) were scored histologically for inflammation and fibrin content. TF and TFPI levels were assayed at antigenic and functional levels. TF messenger RNA (mRNA) levels were determined using RNase protection assays. The effect of TF inhibition in murine antigen-induced arthritis (AIA) was assessed by administering systemically active site-blocked activated factor VIIa (FVIIai).. Functional TF activity was significantly increased in synovial membranes from RA patients compared with those from OA patients. In contrast, no difference in TF mRNA and TF antigenic levels was observed between these 2 groups. This discrepancy can be accounted for by TFPI, because we observed a negative correlation between TF activity and TFPI activity. There was a significant difference between the RA and OA groups in terms of synovial inflammation, with more inflammation observed in the RA group. Most importantly, TF activity was associated with fibrin (P = 0.024) and with histologic inflammation (P = 0.03) scores. In AIA, inhibition of TF-induced coagulation by FVIIai led, on day 9 of arthritis, to decreased synovial thickness and decreased articular cartilage damage, although only the latter difference between controls and treated mice reached significance (P < 0.04). Finally, in FVIIai-treated mice, there was a strong negative association between the prothrombin time and intraarticular fibrin deposition.. Our results show that TF expression in arthritic synovial tissue favors extravascular coagulation and may play a role in inflammation in RA. In this context, TF inhibitors may be of therapeutic value.

Topics: Aged; Animals; Arthritis, Experimental; Arthritis, Rheumatoid; Dansyl Compounds; Disease Models, Animal; Factor VIIa; Female; Fibrin; Fibrinolytic Agents; Hindlimb; Humans; Immunohistochemistry; Lipoproteins; Male; Mice; Mice, Inbred C57BL; Middle Aged; Osteoarthritis; Radionuclide Imaging; RNA, Messenger; Synovitis; Thromboplastin

Tissue factor (TF) is a small-molecular-weight glycoprotein that initiates the extrinsic coagulation pathway but may have important noncoagulation vascular functions as well. Tissue factor pathway inhibitor (TFPI) is a major physiological inhibitor of TF-initiated coagulation. Enhancement of vascular TFPI either by overexpression using gene transfer or delivery of protein to the vessel has been shown to reduce neointimal formation. However, the inherent role of TFPI in this process has not been defined. To do so, we utilized a murine model of vascular remodeling using flow cessation in mice, which are heterozygous for a genetic deletion of the first Kunitz domain of TFPI or wild type littermates. The heterozygotic mice had 50% of wild type TFPI activity in plasma as well as vascular homogenates. To study the effect of TFPI deficiency on neointimal formation, age matched TFPI(K1)+/- and wildtype littermates underwent unilateral common carotid artery ligation. Mice were sacrificed at 4 weeks and the ligated carotid arteries were analyzed. There was a significantly greater neointima to media ratio and less luminal area in the TFPI(K1)+/- mice compared to their TFPI(K1)+/+ littermates. The proliferative index of intimal cells in TFPI(K1)+/- mice at 1 week was significantly higher compared to TFPI(K1)+/+ mice. We conclude that TFPI deficiency enhances neointimal formation and proliferation associated with flow cessation. This suggests that TFPI may regulate vascular remodeling primarily through modulation of neointimal formation.

Topics: Animals; Blood Vessels; Carotid Arteries; Cell Division; Coagulants; Disease Models, Animal; Gene Deletion; Gene Transfer Techniques; Heterozygote; Homozygote; Lipoproteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Protein Structure, Tertiary; Thromboplastin

Tissue factor (TF) and lipopolysaccharide (LPS) are frequently used to induce disseminated intravascular coagulation (DIC) in experimental animal models. Although the pathophysiology of DIC may differ depending on which agent is used for induction, previous studies on models of DIC have not distinguished which DIC-inducing agent was used. In the present paper, we evaluate the characteristics of TF-induced and LPS-induced DIC using two types of DIC models, with special reference to selected hemostatic parameters and pathological findings within the kidney. Male Wistar rats were administered TF (3.75 U/kg; sustained infusion for 4 h) or LPS (30 mg/kg; sustained infusion for 4 h) via the tail vein, and blood sampling was performed at 0, 1, 3, 4, 5, 7, 9, 11, and 28 h. Judging from changes in the levels of thrombin-antithrombin complex, fibrinogen levels, and platelet counts, it is reasonable to conclude that the severity of both types of experimental DIC is similar with regard to hemostatic activation and consumption coagulopathy. A marked elevation in the level of D-dimer was noted without any organ dysfunction or much fibrin deposition in the kidney upon administration of TF. However, a markedly prolonged period of elevation in plasminogen activator inhibitor activity, a prolonged depression in antithrombin III activity, severe organ failure, and a markedly prolonged period of fibrin deposition in the kidney was observed following LPS administration. A modest number of the rats from the TF-induced DIC model died during the experimental period, whereas a large number of rats died during LPS-induced DIC, especially after 9 h. Since the time course of the pathophysiology differed remarkably among the TF-induced and LPS-induced DIC models in rats, we recommend that TF-induced and LPS-induced DIC be approached as distinct models in order to determine the implications of their experimental and clinical use.

Topics: Animals; Biomarkers; Blood Coagulation Factor Inhibitors; Blood Coagulation Factors; Disease Models, Animal; Disease Progression; Disseminated Intravascular Coagulation; Hemostasis; Kidney; Kinetics; Lipopolysaccharides; Male; Rats; Rats, Wistar; Thromboplastin

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

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

Iron overload has been implicated in the pathogenesis of ischemic cardiovascular events. However, the effects of iron excess on vascular function and the thrombotic response to vascular injury are not well understood.. We examined the effects of chronic iron dextran administration (15 mg over 6 weeks) on thrombosis, systemic and vascular oxidative stress, and endothelium-dependent vascular reactivity in mice. Thrombus generation after photochemical carotid artery injury was accelerated in iron-loaded mice (mean time to occlusive thrombosis, 20.4+/-8.5 minutes; n=10) compared with control mice (54.5+/-35.5 minutes, n=10, P=0.009). Iron loading had no effect on plasma clotting, vessel wall tissue factor activity, or ADP-induced platelet aggregation. Acute administration of dl-cysteine, a reactive oxygen species scavenger, completely abrogated the effects of iron loading on thrombus formation, suggesting that iron accelerated thrombosis through a pro-oxidant mechanism. Iron loading enhanced both systemic and vascular reactive oxygen species production. Endothelium-dependent vasorelaxation was impaired in iron-loaded mice, indicating reduced NO bioavailability.. Moderate iron loading markedly accelerates thrombus formation after arterial injury, increases vascular oxidative stress, and impairs vasoreactivity. Iron-induced vascular dysfunction may contribute to the increased incidence of ischemic cardiovascular events that have been associated with chronic iron overload.

Topics: Adenosine Diphosphate; Animals; Blood Coagulation; Carotid Arteries; Carotid Artery Thrombosis; Cysteine; Disease Models, Animal; Disease Progression; Endothelium, Vascular; Free Radical Scavengers; Iron; Iron Overload; Iron-Dextran Complex; Male; Mice; Mice, Inbred C57BL; Nitric Oxide; Oxidative Stress; Platelet Aggregation; Reactive Oxygen Species; Thromboplastin; Time; Vascular Patency; Vasodilation; Vasodilator Agents

Recent studies suggest that the beneficial effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins) in reducing cardiovascular events may in part, be independent of their capacity to lower plasma lipids. To test this hypothesis, simvastatin (50 mg/kg/d) was administered to 30-week-old apolipoprotein E deficient mice (apo E-/-) for 12, 18 and 24 weeks. In contrast to other experimental models and humans, simvastatin treatment increases plasma cholesterol levels in apo E-/- mice. Quantitative real-time polymerase chain reaction was used to quantify expression of tissue factor (TF) and monocyte chemoattractant protein-1 (MCP-1) in the aorta of each mouse. Expression of TF was reduced to 34, 24, and 13% of control levels at 12, 18 and 24 weeks, respectively, of simvastatin administration. Advanced lesions in the innominate arteries of the simvastatin treated mice had reduced levels of TF, fewer macrophages and reduced expression of early growth response-1 (Egr-1). In vitro studies in mouse macrophages demonstrated decreased lipopolysaccharide induced binding of nuclear proteins to the Egr-1 consensus DNA sequence following pretreatment with simvastatin. RNA levels for MCP-1 were reduced to 30% of control values following 24 weeks of simvastatin treatment. In conclusion, these data suggest that chronic administration of simvastatin to older apo E-/- mice can inhibit the expression of pro-thrombotic/pro-inflammatory genes within established atherosclerotic lesions via mechanisms that are independent of reductions in plasma lipids.

Topics: Animals; Apolipoproteins E; Arteriosclerosis; Base Sequence; Chemokine CCL2; Cholesterol; Culture Techniques; Disease Models, Animal; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Immunohistochemistry; Male; Mice; Mice, Inbred Strains; Molecular Sequence Data; Probability; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Severity of Illness Index; Simvastatin; Thromboplastin

Tissue factor (TF) is normally expressed at low levels in the media of blood vessels, but it is readily induced after vessel injury. It is not known whether vascular damage per se or thrombus formation is responsible for this phenomenon.. Cyclic flow variations (CFVs), attributable to recurrent thrombus formation, were induced in stenotic rabbit carotid arteries with endothelial injury. CFVs were observed for 30 minutes and 2, 4, and 8 hours in different groups of animals. Another group of rabbits pretreated with hirudin before inducing arterial damage to inhibit thrombus formation was observed for 8 hours. Arterial sections were immunostained for TF. Undamaged arteries served as controls. In additional rabbits, in situ hybridization experiments were performed. No TF expression was observed in the media of control vessels, whereas a progressive increase in TF mRNA and protein expression was observed in carotid arteries as CFVs progressed. No increase in TF expression was observed in animals pretreated with hirudin. In vitro experiments demonstrated that TF mRNA is induced in smooth muscle cells stimulated with activated platelets as well as with some platelet-derived mediators.. This phenomenon may contribute to sustain intravascular thrombus formation after the initial thrombogenic stimulus.

Topics: Animals; Blood Platelets; Carotid Arteries; Carotid Artery Thrombosis; Carotid Stenosis; Disease Models, Animal; In Situ Hybridization; Monocytes; Muscle, Smooth, Vascular; Neutrophils; Platelet Activating Factor; Platelet-Derived Growth Factor; Rabbits; Recurrence; Regional Blood Flow; RNA, Messenger; Thromboplastin; Tunica Intima; Tunica Media

Previous studies in experimental models revealed a role for the P2Y1 platelet ADP receptor in systemic vascular thromboembolism models. In the present work, we used models of localized arterial and venous thrombosis to assess the role of the P2Y1 receptor in these processes. Arterial thrombosis was induced in one mesenteric arteriole of a mouse using FeCl3, while venous thrombosis was studied in a Wessler model adapted to rats. P2Y1-deficient mice and mice treated with the P2Y1 antagonist MRS2179 displayed significantly less arterial thrombosis than their respective controls. Combination of P2Y1 deficiency with P2Y12 inhibition led to a significant additive effect. Venous thrombosis was slightly but significantly inhibited in MRS2179-treated rats. These results demonstrate a role for the P2Y1 receptor in both arterial and venous thrombosis, further establishing this receptor as a potential target for antithrombotic drugs.

Topics: Adenosine Diphosphate; Animals; Clopidogrel; Disease Models, Animal; Drug Interactions; Mesenteric Arteries; Mice; Mice, Mutant Strains; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Receptors, Purinergic P2Y1; Thromboplastin; Thrombosis; Ticlopidine; Vena Cava, Inferior

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

Infection with the Ebola virus induces overexpression of the procoagulant tissue factor in primate monocytes and macrophages, suggesting that inhibition of the tissue-factor pathway could ameliorate the effects of Ebola haemorrhagic fever. Here, we tested the notion that blockade of fVIIa/tissue factor is beneficial after infection with Ebola virus.. We used a rhesus macaque model of Ebola haemorrhagic fever, which produces near 100% mortality. We administered recombinant nematode anticoagulant protein c2 (rNAPc2), a potent inhibitor of tissue factor-initiated blood coagulation, to the macaques either 10 min (n=6) or 24 h (n=3) after a high-dose lethal injection of Ebola virus. Three animals served as untreated Ebola virus-positive controls. Historical controls were also used in some analyses.. Both treatment regimens prolonged survival time, with a 33% survival rate in each treatment group. Survivors are still alive and healthy after 9 months. All but one of the 17 controls died. The mean survival for the six rNAPc2-treated macaques that died was 11.7 days compared with 8.3 days for untreated controls (p=0.0184). rNAPc2 attenuated the coagulation response as evidenced by modulation of various important coagulation factors, including plasma D dimers, which were reduced in nearly all treated animals; less prominent fibrin deposits and intravascular thromboemboli were observed in tissues of some animals that succumbed to Ebola virus. Furthermore, rNAPc2 attenuated the proinflammatory response with lower plasma concentrations of interleukin 6 and monocyte chemoattractant protein-1 (MCP-1) noted in the treated than in the untreated macaques.. Post-exposure protection with rNAPc2 against Ebola virus in primates provides a new foundation for therapeutic regimens that target the disease process rather than viral replication.

Topics: Animals; Blood Coagulation; Disease Models, Animal; Ebolavirus; Factor VIIa; Helminth Proteins; Hemorrhagic Fever, Ebola; Macaca mulatta; Recombinant Proteins; Survival Rate; Thromboplastin; Virus Replication

Peroral administration of peptide Pro-Gly-Pro to 10-11-month-old rats with modeled prethrombotic state normalized functions of the anticoagulation system and produced a potent antiplatelet effect. Peroral administration of Pro-Gly peptide before provocation of thrombin generation and thrombus formation prevented death of animals from thrombosis. Experiments on rats with venous thrombosis induced by stasis and administration of thrombin showed that pretreatment with Pro-Gly peptide decreased the weight of formed thrombi.

Topics: Administration, Oral; Animals; Animals, Outbred Strains; Anticoagulants; Blood Platelets; Disease Models, Animal; Fibrinolysis; Hemostatics; Injections, Intraperitoneal; Male; Oligopeptides; Platelet Aggregation; Platelet Aggregation Inhibitors; Rats; Survival Rate; Thrombophlebitis; Thromboplastin; Thrombosis; Time Factors

Pharmacological treatment of deep vein thrombosis (DVT) in the future may target inhibitors of specific procoagulant proteins. This study used a non-human primate model to test the effect of PHA-798, a specific inhibitor of the tissue factor/Factor VIIa complex (TF/VIIa), on venous thrombus formation.. PHA inhibits the TF/VIIa complex with an IC(50) of 13.5 nM (K(i) 9 nM) and is more than 2000-fold selective for the TF/VIIa complex with respect to IC(50)s for factor Xa and thrombin. In the model, a thrombogenic surface was introduced into the vena cava of a primate, and the amount of thrombus accumulated after 30 min was determined.. PHA-798 reduced thrombus formation on the thrombogenic surface in a dose-dependent manner (56+/-1.9% and 85+/-0.3% inhibition with 100 and 200 microg/kg/min PHA-798, respectively) indicating that the model is sensitive to TF/VIIa inhibition. Treatment with 1 mg/kg intravenous (IV) acetyl salicylic acid (ASA) resulted in only a slight (4-12%), non-significant inhibition of thrombus formation. However, the combination of 100 microg/kg/min PHA-798 and 1 mg/kg ASA resulted in an 89% inhibition of thrombus formation. Additionally, while ASA alone increased bleeding time (BT) from 3.3 min at baseline to 4.6 min following treatment, addition of PHA-798 (100 microg/kg/min) to ASA did not significantly increase the BT further (4.7 min).. The results of this study indicate that inhibition of TF/VIIa may be safe and effective for the prevention of the proprogation of venous thrombosis and that the combination of ASA and PHA may provide increased efficacy with little change in safety.

Topics: Animals; Aspirin; Bleeding Time; Body Weight; Disease Models, Animal; Factor VIIa; Macaca fascicularis; Male; Platelet Aggregation Inhibitors; Thromboplastin; Thrombosis

Systemic activation of coagulation leading to disseminated intra-vascular coagulation (DIC) is an important feature in patients with severe sepsis. Tissue factor has been shown to play a primary role in this pathological response, as revealed by the use of specific inhibitors and antagonists of the tissue factor/factor VIIa pathway. This class of agents has been demonstrated to attenuate the coagulation response in human volunteers with induced low-grade endotoxemia and to reduce mortality in primate models of Gram-negative sepsis. The efficacy of these agents in attenuating the activation of coagulation and formation of microvascular thrombosis in sepsis may depend on the mechanism of inhibition. Here we demonstrate the efficacy of recombinant nematode anticoagulant protein c2 (rNAPc2) that specifically inhibits the tissue factor/factor VIIa complex by a novel mechanism, in a model of endotoxin-induced coagulation activation in chimpanzees. Administration of a low dose of Gram-negative endotoxin induced marked increases of thrombin generation as measured by plasma levels of prothrombin activation fragment F(1+2) and thrombin-antithrombin complexes, which were completely blocked by rNAPc2. In chimpanzees receiving rNAPc2 alone, there was a significant reduction in the activation of factor X but not factor IX, compared to animals receiving placebo. In contrast to the effect of rNAPc2 on thrombin generation, there was no effect of this inhibitor on the well known enhanced systemic fibrinolytic response induced by endotoxin. In conclusion, the recombinant peptide rNAPc2 is an effective inhibitor of tissue factor-driven thrombin generation during low grade endotoxemia. These results suggest that rNAPc2 may be a promising therapeutic option to inhibit coagulation activation in patients with sepsis.

Topics: Animals; Blood Coagulation; Blood Coagulation Factors; Disease Models, Animal; Disseminated Intravascular Coagulation; Endotoxemia; Endotoxins; Factor VIIa; Helminth Proteins; Pan troglodytes; Protein Binding; Recombinant Proteins; Thrombin; Thromboplastin

We have investigated the role of two vasoactive substances, nitric oxide (NO)and endothelin (ET), in the pathophysiology of disseminated intravascular coagulation (DIC), using two types of DIC models. Experimental DIC was induced by sustained infusion of 0.1, 1, 10, or 50 mg/kg lipopolysaccharide (LPS), or 3.75 U/kg thromboplastin (TF), for 4 h via the rat tail vein. Plasma levels of both NOX (metabolites of NO) and ET were significantly increased following infusion of 0.1 mg/kg or greater of LPS in the LPS-induced DIC rat model. In contrast, although a marked increase in the plasma levels of NOX was observed, only a slight increase in plasma ET levels was seen in the TF-induced DIC rat model. No significant differences in the plasma levels of platelets or thrombin-ATIII complex were observed among the TF-induced and LPS (50 mg/dl)-induced DIC models. However, plasma NOX levels rose significantly higher in the TF-induced model, relative to the LPS-induced model (p <0.01). Conversely, plasma ET levels were significantly greater after LPS-induction, compared to TF-induction, of DIC (p <0.01). Vasoconstriction, as well as depressed fibrinolytic activity, may be additional factors leading to severe organ dysfunction in the LPS-induced DIC rat model. Moreover, vasodilatation, as well as enhanced fibrinolytic activity, may help to prevent rats from severe organ dysfunction in the TF-induced DIC model. Our results suggest that modulator of vasoactive substances should be examined in the treatment of DIC.

Topics: Animals; Antithrombin III; Disease Models, Animal; Disseminated Intravascular Coagulation; Endothelins; Hemostasis; Lipopolysaccharides; Male; Nitric Oxide; Peptide Hydrolases; Platelet Count; Rats; Rats, Wistar; Thromboplastin; Up-Regulation; Vasoconstriction; Vasodilation

Tissue factor and lipopolysaccharide frequently have been used to induce disseminated intravascular coagulation in experimental animal models. Although the pathophysiology of disseminated intravascular coagulation may differ according to the agents used to induce it, these previous models have not distinguished between the use of different disseminated intravascular coagulation-inducing agents. In this study, we attempted to evaluate the characteristic features of these agents in two types of disseminated intravascular coagulation models, with special reference to selected hemostatic parameters and pathologic findings in the kidney.. Prospective, comparative, experimental study.. Laboratory at a university hospital.. Twenty-seven male Wistar rats, age 6-7 wks, weighing 160-170 g.. Three groups of animals were studied: a control group (n = 8) receiving physiologic saline, a tissue factor-treated group (n = 11) receiving tissue factor 3.75 units/kg, and a lipopolysaccharide-treated group (n = 8) receiving lipopolysaccharide 30 mg/kg; each group sustained infusion for 4 hrs via the tail vein.. The degree of hemostatic activation in both types of experimental disseminated intravascular coagulation was identical, based on the results of thrombin-antithrombin III complex levels. Markedly elevated D-dimer concentrations were observed without organ dysfunction or fibrin deposition in the kidney on administration of tissue factor, whereas markedly elevated plasminogen activator inhibitor activity, decreased antithrombin III activity, severe organ failure, and marked fibrin deposition in the kidney were observed for lipopolysaccharide administration.. Because pathophysiology differed remarkably between the tissue factor- and lipopolysaccharide-induced disseminated intravascular coagulation models in rats, we recommend that they be assessed carefully as distinct entities to determine implications of their experimental and clinical use.

Topics: Animals; Antithrombin III; Disease Models, Animal; Disseminated Intravascular Coagulation; Fibrin; Fibrin Fibrinogen Degradation Products; Hemostasis; Kidney; Lipopolysaccharides; Male; Peptide Hydrolases; Prospective Studies; Rats; Rats, Wistar; Thromboplastin

Tissue factor (TF) and its specific inhibitor TF pathway inhibitor (TFPI) are produced by vascular smooth muscle cells (SMCs) in vitro and are increased in vivo in atherosclerotic compared to normal vessels. Besides local regulation of the hemostatic balance, this may be related to non-hemostatic TF/protease dependent functions such as SMC proliferation, adhesion and migration. The aim of the study was to compare the expression of both proteins between the contractile (normal adult) and synthetic (neo-intimal) SMC phenotypes. Primary cultures of SMCs isolated from rat thoracic aorta before and 10 days after balloon injury displayed stable characteristics of the contractile and synthetic phenotype, respectively. Synthetic SMCs expressed more TF mRNA than contractile SMCs, but released excess TF in the conditioned medium, so that the cell-associated TF activity measured by a factor Xa generating assay remained similar in the two subtypes. Accordingly, cell surface thrombogenicity measured under blood flow conditions was also similar. The production and release of functional TFPI was enhanced by a factor 3 to 6 (p < 0.01) in synthetic SMCs. A difference in the quantitative expression of TF and TFPI is a new distinctive feature of SMC phenotypes. Matrix-associated TFPI derived from synthetic SMCs may serve as an anchorage for their migration and regulate protease-activated processes during neo-intima formation.

Topics: Animals; Aorta; Cell Culture Techniques; Disease Models, Animal; Fibrin; Lipoproteins; Male; Muscle, Smooth, Vascular; Perfusion; Phenotype; Rats; Rats, Wistar; Thromboplastin; Thrombosis; Up-Regulation

ADP plays a key role in hemostasis, acting through 2 platelet receptors: the P2Y(1) receptor and an unidentified P2 receptor, called P2cyc, coupled to adenylyl cyclase inhibition, which is the target of the antiplatelet drug clopidogrel. We showed that the P2Y(1) receptor is an essential cofactor in thrombotic states induced by intravenous infusion of collagen and epinephrine. The aim of the present study was to assess the role of this receptor in thrombin-dependent tissue factor-induced thromboembolism.. Human thromboplastin was injected intravenously into wild-type or P2Y(1)-deficient mice, and the effects on platelet count and mortality were determined and plasma thrombin-antithrombin III (TAT) complexes were quantified. P2Y(1)-deficient mice were resistant to the thromboembolism induced by injection of thromboplastin. Whereas the platelet count decreased sharply in wild-type mice, there was no significant drop in platelets in P2Y(1)-knockout mice. The platelet consumption in wild-type mice was probably due to thrombin generation, because it was abolished by hirudin. Thromboplastin also led to a rise in TAT complexes in plasma, again reflecting thrombin formation. This effect, however, was less important in P2Y(1)-knockout mice than in wild-type mice, indicating that less thrombin was generated in the absence of P2Y(1). Similar results were obtained after intravenous administration of N:(6)-methyl-2'-deoxyadenosine-3':5'-bisphosphate, a selective antagonist of the P2Y(1) receptor, to wild-type mice.. Our results demonstrate a role of the P2Y(1) receptor in thrombotic states involving thrombin generation and provide further evidence for the potential relevance of this receptor as a target for antithrombotic drugs.

Topics: Acute Disease; Adenosine Diphosphate; Animals; Antithrombin III; Disease Models, Animal; Fibrinolytic Agents; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Peptide Hydrolases; Platelet Count; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Receptors, Purinergic P2Y1; Thrombin; Thromboembolism; Thromboplastin

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

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

Topics: Angioplasty, Balloon, Coronary; Animals; Arteriosclerosis; Blood Coagulation; Constriction, Pathologic; Disease Models, Animal; Disease Progression; Humans; Rats; Thromboplastin; Thrombosis; Tunica Intima

1Alpha,25-dihydroxyvitamin D3 (active form of vitamin D3; vitamin D3) has been reported to induce the upregulation of thrombomodulin and downregulation of tissue factor (TF) on monocytes. The possibility exists that vitamin D3 prevents the development of disseminated intravascular coagulation (DIC). In particular, monocyte TF production plays an important role in the pathophysiology of DIC in septic patients. We have attempted to determine whether vitamin D3 is effective against DIC in a rat model induced by lipopolysaccharides (LPS) (30 mg/kg, 4 h) or TF (3.75 U/kg, 4 h) using selective hemostatic parameters, markers of organ dysfunction and pathological findings (assessment of glomelular fibrin deposition). Vitamin D3 was administered orally each day at a dose of 2.0 mg/kg/day for 3 days, or low molecular weight heparin (LMWH 200 u/kg; i.v.) was given 10 min before the injection of TF or LPS in each treatment group. Vitamin D3 was effective against DIC in the rat model induced by LPS only, whereas LMWH was effective against DIC in both rat models induced by either TF or LPS. The anti-DIC effect of vitamin D3 was equal to (or more potent than) that of LMWH. The results suggested that vitamin D3 was useful for the treatment of LPS-induced DIC, and that the assessment of a drug's efficacy should be done carefully given the markedly different results obtained according to the agents used to induce DIC.

Topics: Administration, Oral; Animals; Anticoagulants; Cholecalciferol; Coagulants; Disease Models, Animal; Disseminated Intravascular Coagulation; Fibrin; Heparin; Kidney Diseases; Kidney Glomerulus; Lipopolysaccharides; Male; Rats; Rats, Wistar; Sepsis; Thromboplastin; Thrombosis

Although hyperhomocysteinemia (HHcy) is a well-known risk factor for the development of cardiovascular disease, the underlying molecular mechanisms are not fully elucidated. Here we show that induction of HHcy in apoE-null mice by a diet enriched in methionine but depleted in folate and vitamins B6 and B12 increased atherosclerotic lesion area and complexity, and enhanced expression of receptor for advanced glycation end products (RAGE), VCAM-1, tissue factor, and MMP-9 in the vasculature. These homocysteine-mediated (HC-mediated) effects were significantly suppressed, in parallel with decreased levels of plasma HC, upon dietary supplementation with folate and vitamins B6/B12. These findings implicate HHcy in atherosclerotic plaque progression and stability, and they suggest that dietary enrichment in vitamins essential for the metabolism of HC may impart protective effects in the vasculature.

Topics: Animals; Apolipoproteins E; Arteriosclerosis; Cells, Cultured; Diet; Disease Models, Animal; Folic Acid; Humans; Hyperhomocysteinemia; Immunohistochemistry; Male; Matrix Metalloproteinase 9; Methionine; Mice; Mice, Inbred C57BL; Mice, Knockout; Pyridoxine; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Thromboplastin; Vascular Cell Adhesion Molecule-1; Vasculitis; Vitamin B 12

The substrate recognition region of tissue factor contains two residues, Lys165 and Lys166, which are important for macromolecular substrate activation by the tissue factor:factor VIIa complex. Replacement of these two residues with alanine in a soluble version of human tissue factor resulted in a mutant, hTFAA, which can bind factor VIIa but forms an enzymatically inactive complex. We found that hTFAA inhibits the activity of guinea pig factor VIIa, allowing us to evaluate hTFAA's effects on thrombosis and hemostasis in a guinea pig model of recurrent arterial thrombosis. In addition to heparin, the effects of hTFAA were compared to active site inhibited factor IXa (F.IXai) and factor Xa (F.Xai). We found that hTFAA, F.IXai and F.Xai were potent antithrombotics and may possess a decreased risk of hemorrhage when compared to unfractionated heparin. When administered at a dose that inhibited thrombosis by about 90%, hTFAA neither affected cuticle bleeding nor the activated partial thromboplastin time, and had only a modest effect on the prothrombin time. At equi-efficacious doses, F.IXai, F.Xai and heparin prolonged bleeding times by 20% (p >0.5), 50% (p <0.05) and 100% (p <0.01), respectively. In summary, our study demonstrates that, unlike heparin, specific inhibitors of factors VIIa, IXa and Xa can produce antithrombotic effects without or with only minimally disturbing normal hemostasis. The results further suggest that factor VIIa and factor IXa are especially promising targets for antithrombotic drug development.

Topics: Amino Acid Substitution; Animals; Arterial Occlusive Diseases; Bleeding Time; Carotid Artery Thrombosis; Catalytic Domain; Disease Models, Animal; Factor IXa; Factor VIIa; Factor Xa; Factor Xa Inhibitors; Fibrinolytic Agents; Guinea Pigs; Heparin; Humans; Solubility; Thromboplastin; Thrombosis

All-trans retinoic acid (ATRA) has been introduced to the management of acute promyelocytic leukemia (APL) as a differentiation treatment. This drug not only causes complete remission, but also improves disseminated intravascular coagulation (DIC) without adding anticoagulants in APL. We have attempted to determine whether ATRA is effective against DIC in rat models induced by tissue factor (TF) or lipopolysaccharide (LPS), because the anticoagulant effect of ATRA has been considered to induce thrombomodulin upregulation and TF downregulation on endothelial cells as well as on APL cells. In male Wistar rats, DIC was induced by a 4-h infusion of thromboplastin (3.75 U/kg) or lipopolysaccharide (30 mg/kg). The rats were given ATRA orally each day at a dose of 100 mg/kg per day for 1 week before the injection of TF or LPS in ATRA treatment groups, or given low molecular weight heparin (LMWH) 10 min before the injection of TF or LPS (200 U/kg, bolus intravenously) in LMWH treatment groups. No significant changes in hemostatic parameters or markers of organ dysfunction were caused by the ATRA administration, while DIC was significantly improved by LMWH in the TF-induced model. DIC was significantly improved by both ATRA and LMWH in the LPS-induced model. These findings suggested that ATRA was useful for treating DIC only in the LPS-induced model, and that drug efficacy should be carefully assessed because the agents used to induce DIC considerably influenced the outcome.

Topics: Animals; Disease Models, Animal; Disseminated Intravascular Coagulation; Keratolytic Agents; Lipopolysaccharides; Male; Rats; Rats, Wistar; Thromboplastin; Tretinoin

Danaparoid and heparin, on the basis of anti-activated factor X (anti-FXa) activity, were equipotent in accelerating the rate of interaction of FXa and antithrombin III. In rat tissue factor-induced disseminated intravascular coagulation (DIC) models, an intravenous administration of danaparoid inhibited the decrease in plasma fibrinogen and platelet counts and the increase in serum fibrinogen degradation products. Expressed on the basis of anti-FXa activity, these effects were comparable with those of dalteparin and heparin. In rat mesenteric small artery and vein, less bleeding was observed after intravenous administration of danaparoid than after dalteparin or heparin. Danaparoid did not affect adenosine diphosphate- or collagen-induced platelet aggregation, and showed weaker inhibitory effects on aggregation induced by thrombin, or collagen + thrombin, than did dalteparin or heparin. These findings suggest that danaparoid may be useful for the prevention of DIC and has less tendency to cause bleeding than dalteparin or heparin, probably as a result of its weaker ability to inhibit platelet aggregation.

Topics: Animals; Anticoagulants; Antithrombin III; Bleeding Time; Chondroitin Sulfates; Dalteparin; Dermatan Sulfate; Disease Models, Animal; Disseminated Intravascular Coagulation; Drug Combinations; Drug Evaluation, Preclinical; Enzyme Inhibitors; Factor Xa; Factor Xa Inhibitors; Heparin; Heparitin Sulfate; Kinetics; Male; Platelet Aggregation; Rats; Rats, Wistar; Risk Assessment; Thromboplastin

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

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

Tissue factor (TF) is a transmembrane glycoprotein that complexes with factor VIIa to initiate blood coagulation. We previously reported that expression of high levels of TF in a human melanoma cell line promotes metastasis. Both the cytoplasmic domain of TF and its extracellular domain complexed with factor VIIa are required for the metastatic effect. To further explore the mechanism of TF-mediated metastasis, we investigated the possibility that a protease-activated receptor (PAR) might play a role. For this purpose, we first determined the expression levels of the known PARs (PAR1-4) in a human melanoma cell line, SIT1, that has low endogenous levels of TF and low metastatic potential. We found negligible levels of all of the known PARs and transfection of this cell line with human TF cDNA did not alter expression of the known PARs. To study the possible role of PAR1 in TF-mediated metastasis, we prepared a panel of transfected cell lines with varying levels of TF and PAR1. Our studies show that TF promotes metastasis by a pathway that does not involve high expression of known PARs by tumor cells. In addition, while overexpression of PAR1 is insufficient to induce metastasis in cells with low TF expression, it enhances the metastatic potential of cells with high TF expression, indicating a possible synergy between TF and PAR1 in promoting metastasis.

Topics: Animals; Disease Models, Animal; Drug Synergism; Female; Humans; Lung Neoplasms; Melanoma; Mice; Mice, SCID; Neoplasm Metastasis; Receptor, PAR-1; Receptors, Thrombin; Thromboplastin; Transfection; Tumor Cells, Cultured

Post-thrombotic inflammation probably contributes to chronic venous insufficiency, and little effective treatment exists. IL-10 is an anti-inflammatory cytokine that previously has been shown to decrease perithrombotic inflammation and thrombosis. We investigated in a rat model whether local expression of viral IL-10 (vIL-10) in a segment of vein that undergoes thrombosis would confer an anti-inflammatory effect and how this effect might be mediated. Rats underwent inferior vena cava isolation, cannulation, and instillation of saline or adenovirus encoding either beta-galactosidase or vIL-10. Two days after transfection, thrombosis was induced, 2 days after this the rats underwent gadolinium (Gd)-enhanced magnetic resonance venography exam, and the vein segments were harvested. Tissue transfection was confirmed by either RT-PCR of vIL-10 or positive 5-bromo-4-chloro-3-indolyl beta-d-galactopyranoside (X-Gal) staining. vIL-10 significantly decreased both leukocyte vein wall extravasation and area of Gd enhancement compared with those in controls, suggesting decreased inflammation. Immunohistochemistry demonstrated decreased endothelial border staining of P- and E-selectin, while ELISA of vein tissue homogenates revealed significantly decreased P- and E-selectin and ICAM-1 levels in the vIL-10 group compared with those in controls. Importantly, native cellular IL-10 was not significantly different between the groups. However, neither clot weight nor coagulation indexes, including tissue factor activity, tissue factor Ag, or von Willebrand factor levels, were significantly affected by local vIL-10 expression. These data suggest that local transfection of vIL-10 decreases venous thrombosis-associated inflammation and cell adhesion molecule expression, but does not directly affect local procoagulant activity.

Topics: Animals; Blood Coagulation Tests; Cell Adhesion Molecules; Cytokines; Disease Models, Animal; Epoprostenol; Gene Transfer Techniques; Herpesvirus 4, Human; Interleukin-10; Male; Rats; Rats, Sprague-Dawley; Thromboplastin; Transfection; Vena Cava, Inferior; Venous Thrombosis; Viral Proteins

Thrombin plays a central role in the genesis of thrombotic events and is the most potent known platelet agonist. This enzyme activates platelets by cleaving G-protein coupled protease activated receptors (PARs) and by binding to glycoprotein (GP) Ib. Thrombin also cleaves platelet GPV to liberate a soluble 69 kDa fragment (GPVf1), leaving a 20 kDa fragment (GPVf2) attached to the membrane. The aim of this study was to assess the value of GPV as an in vivo marker of the activation of platelets by thrombin. Newly developed monoclonal and polyclonal antibodies recognizing rat GPVf1 and GPVf2 respectively were used to detect soluble GPV by ELISA and the new NH2-terminus exposed by thrombin using flow cytometry. These assays were employed in a rat thrombosis model designed to trigger thrombin formation in vivo. When thromboplastin (4.8 ml/kg/h) was infused for 30 min, thrombin generation was reflected by a rapid increase in thrombin-antithrombin (TAT) complexes in plasma and by the appearance of GPVf2 at the surface of circulating platelets. Simultaneously, GPVf1 disappeared from the surface of platelets and accumulated as a soluble fragment in plasma, where it was detected by GPV ELISA. These effects were inhibited by pretreatment of the rats with hirudin. Levels of plasma PF4 also increased in this model, but unlike GPV levels which returned slowly (> 2 hours) to baseline, PF4 had a very short half-life. In conclusion, GPV is cleaved by thrombin in vivo, circulates and is a reliable in vivo marker of the activation of platelets by thrombin. Monitoring of GPV levels in rats should be useful to evaluate the effects of antithrombotic and antiplatelet drugs, while further studies will be required to confirm the potential interest of GPV as a marker of thrombotic states in humans.

Topics: Animals; Antibodies, Monoclonal; Antithrombin III; Biomarkers; Blood Platelets; Coagulants; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Epitopes; Flow Cytometry; Pancreatic Elastase; Peptide Hydrolases; Platelet Activation; Platelet Factor 4; Platelet Glycoprotein GPIb-IX Complex; Platelet Membrane Glycoproteins; Rats; Receptors, Cell Surface; Sensitivity and Specificity; Solubility; Thrombin; Thromboplastin; Thrombosis

Topics: Angiotensin II; Angiotensin Receptor Antagonists; Animals; Animals, Genetically Modified; Coronary Disease; Disease Models, Animal; Humans; Hypertension; Mice; Rats; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Thromboplastin; Thrombosis

Tissue factor pathway inhibitor (TFPI) is the sole known inhibitor of the extrinsic coagulation pathway of physiological importance; however, its role in modulating thrombosis in vivo is still unclear.. Intravascular thrombosis was initiated by placing an external constrictor around endothelially injured rabbit carotid arteries (n=10). Carotid blood flow velocity was measured by a Doppler flow probe. After placement of the constrictor, cyclic flow reductions (CFRs), due to recurrent thrombosis, developed at the site of stenosis. Transstenotic TFPI plasma activity was measured in blood samples before induction of CFRs and after 30, 60, and 180 minutes of CFRs. TFPI plasma activity distal to the site of thrombosis was significantly lower than the corresponding proximal values at 30, 60, and 180 minutes of CFRs. In addition, a progressive decrease in TFPI plasma activity was observed in both the proximal and the distal samples, indicating consumption of TFPI during thrombus formation. In 10 additional rabbits, CFRs were abolished by administration of aspirin (10 mg/kg). In the animals in which aspirin abolished CFRs, endogenous TFPI was depleted by a bolus of a polyclonal antibody against rabbit TFPI, and the effects on restoration of CFRs were monitored. In 5 of 6 animals in which aspirin abolished CFRs, depletion of endogenous TFPI activity caused full restoration of CFRs.. The data of the present study support the involvement of endogenous TFPI in the process of thrombus formation in vivo and its active role in modulating arterial thrombosis.

Topics: Animals; Antibodies; Aspirin; Blood Coagulation; Blood Flow Velocity; Carotid Artery Injuries; Carotid Stenosis; Disease Models, Animal; Endothelium, Vascular; Female; Male; Platelet Aggregation Inhibitors; Rabbits; Thromboplastin; Thrombosis

Hemostasis factors may influence the pathophysiology of stroke. The role of brain hemostasis in ischemic hypertensive brain injury is not known. We studied ischemic injury in spontaneously hypertensive rats in relation to cerebrovascular fibrin deposition and activity of different hemostasis factors in brain microcirculation. In spontaneously hypertensive rats subjected to transient middle cerebral artery occlusion versus normotensive Wistar-Kyoto (W-K) rats, infarct and edema volumes were increased by 6.1-fold (P < 0.001) and 5.8-fold (P < 0.001), respectively, the cerebral blood flow (CBF) reduced during middle cerebral artery occlusion (MCAO) by 55% (P < 0.01), motor neurologic score increased by 6.9-fold (P < 0.01), and cerebrovascular fibrin deposition increased by 6.8-fold (P < 0.01). Under basal conditions, brain capillary protein C activation and tissue plasminogen activator activity were reduced in spontaneously hypertensive rats compared with Wistar-Kyoto rats by 11.8-fold (P < 0.001) and 5.1-fold (P < 0.001), respectively, and the plasminogen activator inhibitor-1 antigen and tissue factor activity were increased by 154-fold (P < 0.00001) and 74% (P < 0.01), respectively. We suggest that hypertension reduces antithrombotic mechanisms in brain microcirculation, which may enhance cerebrovascular fibrin deposition and microvascular obstructions during transient focal cerebral ischemia, which results in greater neuronal injury.

Topics: Animals; Blood Gas Analysis; Brain Ischemia; Capillaries; Cerebrovascular Circulation; Disease Models, Animal; Endothelium, Vascular; Fibrin; Fibrinolysis; Gene Expression; Hemostasis; Hypertension; Intracranial Thrombosis; Male; Microscopy, Electron; Neurologic Examination; Plasminogen Activator Inhibitor 1; Protein C; Rats; Rats, Inbred SHR; Rats, Inbred WKY; RNA, Messenger; Stroke; Thromboplastin

We studied the antithrombotic activity of a mixed micellar formulation containing 14 mg/ml argatroban administered by the subcutaneous (s.c.) route in rat and rabbit models of venous thrombosis. The effects on bleeding time in the rat tail transection bleeding time test were also studied. In a tissue factor-dependent arterio-venous shunt model, argatroban treatment led to dose-dependent reduction in thrombus weight with an estimated ID50 of 1.8 mg/kg s.c. In the same model, heparin had an estimated ID50 of 179 IU/kg. The antithrombotic activity of argatroban was accompanied by increases in the thrombin and ecarin clotting times but not the aPTT, whereas heparin increased the thrombin time and aPTT but not the ecarin clotting times. Argatroban also inhibited thrombus formation in a rabbit model of thromboplastin + stasis induced thrombosis in the rabbit jugular vein with an estimated ID50 of 1 mg/kg s.c. When tested in the rat tail transection bleeding time test, the mixed micellar formulation of argatroban caused significant increases in the bleeding time as from 8 mg/kg s.c., while heparin significantly increased the bleeding time at 800 U/kg. Mixed micellar argatroban appears to have a superior safety margin to heparin in terms of antithrombotic efficacy and bleeding risk. Thus, a mixed micellar formulation of argatroban, which markedly enhances its solubility, could be useful as a potential antithrombotic agent for subcutaneous administration.

Topics: Animals; Anticoagulants; Antithrombins; Arginine; Arteriovenous Shunt, Surgical; Bleeding Time; Blood Coagulation Tests; Carotid Arteries; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Compounding; Drug Evaluation, Preclinical; Hemostatics; Heparin; Injections, Subcutaneous; Male; Micelles; Pipecolic Acids; Rabbits; Rats; Sulfonamides; Thromboplastin; Venous Thrombosis

Statins are effective in prevention of end-organ damage; however, the benefits cannot be fully explained on the basis of cholesterol reduction. We used an angiotensin II (Ang II)-dependent model to test the hypothesis that cerivastatin prevents leukocyte adhesion and infiltration, induction of inducible nitric oxide synthase (iNOS), and ameliorates end-organ damage.. We analyzed intracellular targets, such as mitogen-activated protein kinase and transcription factor (nuclear factor-kappaB and activator protein-1) activation. We used immunohistochemistry, immunocytochemistry, electrophoretic mobility shift assays, and enzyme-linked immunosorbent assay techniques. We treated rats transgenic for human renin and angiotensinogen (dTGR) chronically from week 4 to 7 with cerivastatin (0.5 mg/kg by gavage).. Untreated dTGR developed hypertension, cardiac hypertrophy, and renal damage, with a 100-fold increased albuminuria and focal cortical necrosis. dTGR mortality at the age of seven weeks was 45%. Immunohistochemistry showed increased iNOS expression in the endothelium and media of small vessels, infiltrating cells, afferent arterioles, and glomeruli of dTGR, which was greater in cortex than medulla. Phosphorylated extracellular signal regulated kinase (p-ERK) was increased in dTGR; nuclear factor-kappaB and activator protein-1 were both activated. Cerivastatin decreased systolic blood pressure compared with untreated dTGR (147 +/- 14 vs. 201 +/- 6 mm Hg, P < 0.001). Albuminuria was reduced by 60% (P = 0.001), and creatinine was lowered (0.45 +/- 0.01 vs. 0.68 +/- 0.05 mg/dL, P = 0. 003); however, cholesterol was not reduced. Intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression was diminished, while neutrophil and monocyte infiltration in the kidney was markedly reduced. ERK phosphorylation and transcription factor activation were reduced. In addition, in vitro incubation of vascular smooth muscle cells with cerivastatin (0.5 micromol/L) almost completely prevented the Ang II-induced ERK phosphorylation.. Cerivastatin reduced inflammation, cell proliferation, and iNOS induction, which led to a reduction in cellular damage. Our findings suggest that 3-hydroxy-3-methylglutaryl coenzyme (HMG-CoA) reductase inhibition ameliorates Ang II-induced end-organ damage. We suggest that these effects were independent of cholesterol.

Topics: Albuminuria; Angiotensin II; Angiotensinogen; Animals; Animals, Genetically Modified; Blood Pressure; Cell Division; Cholesterol; Creatinine; Disease Models, Animal; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Intercellular Adhesion Molecule-1; Kidney; Kidney Failure, Chronic; Leukocytes; Male; Mitogen-Activated Protein Kinases; NF-kappa B; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Organ Size; Phosphorylation; Plasminogen Activators; Pyridines; Rats; Rats, Sprague-Dawley; Renin; Thromboplastin; Transcription Factor AP-1; Urea; Vascular Cell Adhesion Molecule-1; Vasoconstrictor Agents

To investigate whether impaired endothelial function was related to alteration of nitric oxide (NO) formation during endotoxic shock, we studied the effects of supplementation of L-arginine (L-Arg), D-arginine (D-Arg), and N(G)-nitro-L-arginine methyl ester (L-NAME), on endothelial function and structure in a rabbit model. Endotoxic shock was induced by a single lipopolysaccharide bolus (0.5 mg/kg i.v., Escherichia coli endotoxin). Coagulation factors and expression of monocyte tissue factor were determined by functional assays. Endothelium-dependent vascular relaxation was assessed by in vitro vascular reactivity. Immunohistochemical staining (CD31) was performed to assess damaged endothelial cell surface of the abdominal aorta. These parameters were studied 5 days after the onset of endotoxic shock and were compared under three conditions: in absence of treatment, with L-Arg or D-Arg supplementation, or with L-NAME. Both L-Arg and D-Arg significantly improved endothelium-dependent relaxation and endothelial morphological injury. L-NAME did not alter endothelial histological injury induced by lipopolysaccharide. These data indicate that arginine supplementation nonspecifically prevents endothelial dysfunction and histological injury in rabbit endotoxic shock. Moreover, L-Arg has no effect on coagulation activation and expression of monocyte tissue factor induced by endotoxic shock.

Topics: Acetylcholine; Animals; Arginine; Blood Gas Analysis; Calcimycin; Disease Models, Animal; Endothelium, Vascular; Enzyme Inhibitors; Hemostasis; Ionophores; Lipopolysaccharides; Male; Monocytes; NG-Nitroarginine Methyl Ester; Nitroprusside; Phenylephrine; Rabbits; Shock, Septic; Thromboplastin; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents; Weight Loss

Automated human plasma, continuous monitoring of the formation and inactivation of thrombin during the coagulation process provides an adequate way to detect hypo- and hypercoagulant conditions. Here, we describe an analogous procedure to determine the endogenous thrombin potential (ETP), i. e. the free thrombin concentration-time integral, of coagulating rat plasma. When activated with tissue factor, the ETP of plasma from Wistar rats was comparable to the ETP of human plasma, in spite of a relatively short half-life time of free thrombin in rat plasma. The ETP was highly sensitive to heparin as well as to administration of vitamin K antagonist or feeding of the animals with a vitamin K-deficient diet. In plasma that was activated under sub-optimal conditions (reduced levels of tissue factor or vitamin K-dependent coagulation factors), the ETP increased with the rate of thrombin formation in the first minutes of the coagulation process. Since both parameters are dependent of the prothrombin concentration, it appears that this level plays an important role in determining both the initial and total activity of the coagulation system. Thus, automated measurement of free thrombin during the coagulation process of rat plasma allows a detailed analysis of hypocoagulability in this animal model.

Topics: Animals; Blood Coagulation Disorders; Blood Coagulation Tests; Disease Models, Animal; Electronic Data Processing; Humans; Kinetics; Male; Rats; Rats, Wistar; Sensitivity and Specificity; Thrombin; Thromboplastin; Warfarin

In order to investigate the leukemogenic potential of PLZF-RARA fusion protein in vivo, hCG-PLZF-RARA transgenic mice were generated, in which PIZF-RARA fusion gene was driven by hCG promoter to express in myeloid cells of mice.. Molecular cloning technology was used to construct hCG-PLZF-RARA gene. The genotype and phenotype of the hCG-PLZF-RARA transgenic mice were analyzed by PCR, RT-PCR, immunofluorescence, morphology of bone marrow (BM) cells, pathology and retinoic acid differentiation assays.. Six hCG-PLZF-RARA transgenic mice developed leukemia resembling human chronic myeloid leukemia. TF(tissue factor) was not expressed in BM cells of normal mice nor in mice without the expressed transgene, but it was expressed in mice expessing the transgene.. PLZF-RARA fusion protein plays a crucial role in leukemogenesis. TF is up-regulated by PLZF-RARA fusion gene.

Topics: Animals; Cell Transformation, Neoplastic; Chorionic Gonadotropin; Disease Models, Animal; Humans; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Mice, Transgenic; Neoplasm Proteins; Oncogene Proteins, Fusion; Recombinant Fusion Proteins; Thromboplastin; Up-Regulation

Tissue factor (TF) is the major activator of the coagulation protease cascade and contributes to lethality in sepsis. Despite several studies analyzing TF expression in animal models of endotoxemia, there remains debate about the cell types that are induced to express TF in different tissues. In this study, we performed a detailed analysis of the induction of TF mRNA and protein expression in two rabbit models of endotoxemia to better understand the cell types that may contribute to local fibrin deposition and disseminated intravascular coagulation. Northern blot analysis demonstrated that lipopolysaccharide (LPS) increased TF expression in the brain, lung, and kidney. In situ hybridization showed that TF mRNA expression was increased in cells identified morphologically as epithelial cells in the lung and as astrocytes in the brain. In the kidney, in situ hybridization experiments and immunohistochemical analysis showed that TF mRNA and protein expression was increased in renal glomeruli and induced in tubular epithelium. Dual staining for TF and vWF failed to demonstrate TF expression in endothelial cells in LPS-treated animals. These results demonstrate that TF expression is induced in many different cell types in LPS-treated rabbits, which may contribute to local fibrin deposition and tissue injury during endotoxemia.

Topics: Animals; Brain; Disease Models, Animal; Endotoxemia; Gene Expression; Immunohistochemistry; In Situ Hybridization; Kidney; Lipopolysaccharides; Lung; Rabbits; RNA, Messenger; Thromboplastin; Tissue Distribution

Topics: Animals; Disease Models, Animal; Disseminated Intravascular Coagulation; Humans; Lipoproteins; Papio; Thromboplastin

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

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

The intimal thickening that follows vascular injury is inhibited by periprocedural tissue factor pathway inhibitor (TFPI) treatment in animal models. TFPI is a multivalent Kunitz-type protease inhibitor that inhibits factor Xa via its second Kunitz domain and the factor VIIa/tissue factor (TF) complex via its first Kunitz domain. The basic C-terminus of TFPI is required for the binding of TFPI to cell surfaces and cell-bound TFPI mediates the internalization and degradation of factor X and the down regulation of surface factor VIIa/TF activity. The C-terminus of TFPI is also required for its reported direct inhibition of smooth muscle cell proliferation in vitro. To examine the structural requirements for the inhibition of neointimal formation by TFPI, several TFPI-related proteins were tested in the rat carotid angioplasty model: 1) XK(1), a hybrid protein containing the N-terminal portion of factor X and the first Kunitz domain of TFPI that directly inhibits factor VIIa/TF; 2) TFPI(WT), the full-length TFPI molecule that inhibits factor Xa and factor VIIa/TF and binds cell surfaces; 3) TFPI(K36I), an altered form of TFPI that inhibits factor Xa, but not factor VIIa/TF, and binds cell surfaces; 4) TFPI(13-161), a truncated form of TFPI that inhibits factor VIIa/TF but interacts with factor Xa poorly and does not bind to cell surfaces. Seven day infusions of XK(1), TFPI(WT), and high levels of TFPI(K36I) begun the day before balloon-induced vascular injury produced a significant reduction in the intimal hyperplasia measured 28 days after angioplasty. The infusion of high concentrations of TFPI(13-161) was ineffective in this model. These in vivo results directly mirror the ability of each TFPI-related protein to inhibit tissue thromboplastin-induced coagulation in rat plasma: XK(1) approximately TFPI(WT)>TFPI(K36I)>>TFPI(13-161). The studies confirm the important role of TF-mediated coagulation in the smooth muscle proliferation and neointimal thickening that follows vascular injury and suggest that the anticoagulant effect alone of TFPI and TFPI-related proteins is sufficient to explain their therapeutic action.

Topics: Angioplasty, Balloon; Animals; Carotid Artery Injuries; Carotid Artery, Common; Cricetinae; Disease Models, Animal; Factor VIIa; Female; Fibrinolytic Agents; Kidney; Lipoproteins; Peptide Fragments; Rats; Rats, Sprague-Dawley; Reagent Kits, Diagnostic; Recombinant Proteins; Thromboplastin; Tunica Intima

Induction of tissue factor (TF) activity by endotoxin and cytokines is an important mechanism for initiation of disseminated intravascular coagulation (DIC) seen in patients with gram-negative sepsis. Based on data from an in vitro study in which dithiocarbamates abrogated endothelial cell TF activity by inhibition of the NF-kappaB pathway, we investigated whether dithiocarbamates had in vivo activity in an animal model of DIC. Dithiocarbamates ameliorated the adverse clinical and histological effects of endotoxin-induced DIC, including morbidity, hypofibrinogenemia, and target organ damage, especially in the liver and kidney, even when given up to 1 hour after administration of endotoxin. This pilot study confirms the key role of the nuclear factor-kappa beta (NF-kappaB) pathway in induction of TF activity in initiating sepsis-associated DIC and suggests that dithiocarbamates may be useful in treatment of DIC associated with excessive TF expression because of gram-negative sepsis. Additional studies of dithiocarbamates in DIC models are warranted.

Topics: Animals; Disease Models, Animal; Disseminated Intravascular Coagulation; Endotoxins; Female; Fibrinogen; NF-kappa B; Rabbits; Thiocarbamates; Thromboplastin; Transcription, Genetic

We studied the use of the Ecarin Clotting Time (ECT) as a predictive assay of the antithrombotic effects of argatroban in a new tissue factor-dependent model of venous thrombosis and a model of arterial thrombosis in the rat. Heparin was used as a reference anticoagulant. Infusions of argatroban dose-dependently increased the ECT across the range of doses required for antithrombotic activity in models of venous and arterial thrombosis (1.25-40 microg/kg/min). The TT was only useful as a marker in the case of venous thrombosis, since, in the arterial thrombosis model, the clotting times were >200 s in the majority of animals receiving antithrombotic doses. The aPTT is not sufficiently sensitive to be predictive of an antithrombotic effect in the venous model, and shows only modest increases in the arterial thrombosis model. Heparin did not significantly increase the ECT at antithrombotic doses in the venous thrombosis model, and only increased the ECT by 53% at 40 microg/kg/min in the arterial model, despite a marked antithrombotic effect. Both the TT and aPTT were dose-dependently increased by heparin at doses active in the venous model, whereas both parameters were >200 s at doses active in the arterial thrombosis model. Thus, the ECT provides a predictive marker for the antithrombotic activity of argatroban in both venous and arterial thrombosis, at least in the rat.

Topics: Animals; Antithrombins; Arginine; Arteriovenous Shunt, Surgical; Blood Coagulation Tests; Disease Models, Animal; Male; Pipecolic Acids; Rats; Rats, Inbred Strains; Reference Values; Sulfonamides; Thrombophlebitis; Thromboplastin; Thrombosis; Treatment Outcome

To study whether locally administered recombinant inactivated human coagulation factor VIIa (FFR-rFVIIa) would reduce the thrombus formation and improve patency in an experimental venous thrombosis model without inducing systemic changes in the coagulation.. Experimental double-dummy randomised study.. In 20 healthy New Zealand White rabbits both jugular veins were exposed under general anaesthesia.. The thrombi were induced in a 10 mm long jugular vein segment with a combination of chemical destruction of the intima and a restriction of the bloodflow. Each segment was treated with either FFR-rFVIIa or placebo injected directly into the vein.. 1.5 mg topically applied FFR-rFVIIa significantly reduced the thrombus weight (p < 0.001). The 30 and the 120 min patency tests were significantly improved (p < 0.05 and p < 0.001, respectively) Plasma analyses (APTT, dilute-TF time, FVII protein) were evaluated as baseline, 3 min after declamping and at sacrifice. No prolongation of the clotting times were seen. FFR-rFVIIa protein was detected in minute amounts (ng/ml); however, this was not enough to prolong the dilute-TF time.. Local application of recombinant active-site inhibited human FVIIa reduced both thrombus weight and improved patency significantly in an experimental venous thrombosis model without affecting the systemic clotting times.

Topics: Anesthesia, General; Animals; Blood Coagulation; Constriction, Pathologic; Disease Models, Animal; Double-Blind Method; Factor VIIa; Humans; Injections, Intravenous; Jugular Veins; Partial Thromboplastin Time; Placebos; Rabbits; Random Allocation; Recombinant Proteins; Regional Blood Flow; Thromboplastin; Thrombosis; Tunica Intima; Vascular Patency

Tissue factor (TF) is a membranous protein normally present on the surface of the fibroblasts and smooth muscle cells of vessels. TF is an initiation factor for blood coagulation, and its expression is induced on macrophages and endothelial cells during the inflammatory or immune response. We studied the significance of TF expression in warm ischemic-reperfusion injury of the liver using a rat model.. Following laparotomy of Lewis rats, the branches of the hepatic artery and portal vein leading to the median, left, and caudate lobes of the liver were clamped for 2 hr. The liver was reperfused after 120 min of ischemia. Rats were killed at 0, 1, 3, 5, 8, and 12 hr after reperfusion, and liver tissues were harvested. TF activity was measured by the chromophilic substrate S-2222. TF expression was studied by immunohistochemical staining with the monoclonal antibody HTF-K108.. TF activity in the blood showed a peak at 3 hr after reperfusion (8.9+/-0.5 U/L), then decreased and returned to the normal level by 12 hr (0.9+/-0.3 U/L). TF activity in ischemic liver tissue increased gradually over 12 hr after reperfusion (1223+/-275 U/g dry weight before ischemia and 2545+/-284 U/g weight at 12 hr after reperfusion). Histologically spotty necroses were observed in the liver tissue 5 hr after reperfusion. The necrotic area extended and encompassed almost all of the ischemic liver by 12 hr after reperfusion. Histochemically, TF staining was negative on the hepatocytes and slightly positive on sinusoid cells of the normal liver. On the other hand, TF was strongly stained, especially on the hypertrophic monocytic cells accumulating at the site of the necrosis, but staining was not evident on the necrotic hepatocytes. A slight degree of TF staining was observed on the alveolar epithelium of the lung, irrespective of liver ischemia and reperfusion.. These results demonstrate that TF plays an important role in the development of the hepatic ischemic-reperfusion injury, and the subsequent microcirculatory incompetence might cause the formation of microthrombus and the development of necrosis.

Topics: Alanine Transaminase; Animals; Disease Models, Animal; Hyaluronic Acid; Immunohistochemistry; Ischemia; Liver; Lung; Male; Rats; Rats, Inbred Lew; Reperfusion Injury; Temperature; Thromboplastin; Tumor Necrosis Factor-alpha

The anticoagulant and antithrombotic effects of YM-75466 (N-[4-[(1-acetimidoyl-4-piperidyl)oxy]phenyl]-N-[(7-amidino-2-naph thyl)methyl]sulfamoyl acetic acid monomethanesulfonate), a novel orally-active factor Xa (FXa) inhibitor, and warfarin were compared in mice. Both agents were orally administered in all studies. In ex vivo studies, the peak effects of YM-75466 occurred 1 hr after administration while the peak of warfarin activity occurred 18 hr after administration. At each peak, both YM-75466 and warfarin prolonged coagulation time dose-dependently. The dose response curve of warfarin for prothrombin time was steeper than that of YM-75466. In a thromboplastin-induced thromboembolism model, administration of 30 mg/kg YM-75466 or 3 mg/kg warfarin significantly improved the lethality ratio. In blood loss studies, YM-75466 did not increase blood loss from the tail even at 30 mg/kg, while warfarin markedly increased blood loss at 3 mg/kg. Agents that interfere with warfarin action did not interfere with YM-75466 action. In conclusion, this study shows that YM-75466 has advantages over warfarin: i) rapid onset of anticoagulant activity, ii) wide therapeutic range, iii) little effect on bleeding and iv) lack of drug interaction with agents that interfere with warfarin. These results suggest that YM-75466 may be promising as a novel oral anticoagulant agent.

Topics: Administration, Oral; Analgesics, Non-Narcotic; Animals; Anti-Bacterial Agents; Anticoagulants; Anticonvulsants; Antifibrinolytic Agents; Blood Coagulation; Carbamazepine; Cimetidine; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Erythromycin; Factor Xa Inhibitors; Fibrinolytic Agents; Hemorrhage; Male; Mice; Mice, Inbred ICR; Partial Thromboplastin Time; Phenytoin; Piperidines; Prothrombin Time; Rifampin; Sulfonamides; Thromboembolism; Thromboplastin; Vitamin K 1; Warfarin

Recently, we demonstrated that neutrophils express tissue factor (TF) in a model of acute obstructive cholangitis (AOC). However, the regulation of TF expression was not clear. In this study, we clarified the role of platelet-activating factor (PAF) in TF expression in neutrophils.. In a model of AOC, intravenous PAF antagonist, (SM-12502, 200 mg/kg) was administered 5 min before sepsis was induced. Normal saline was given as a control. Coagulation parameters and TF activity were monitored for 6 h. Thereafter, the liver was harvested for histological examination.. The percentage of neutrophils which stained positive for TF was significantly reduced by SM-12502 (74.9 +/- 19.3% vs 96.3 +/- 2.8%) (P < 0.01). The number of leukocytes infiltrating the liver was also significantly reduced. Coagulation abnormalities, TF activity, and focal necrosis of the hepatocytes were reduced by SM-12502.. SM-12502 inhibits TF expression in neutrophils which have infiltrated the liver sinusoids, reducing the subsequent infiltration of leukocytes. These results suggest that PAF plays an important role in the expression of TF in neutrophils in vivo.

Topics: Animals; Aspartate Aminotransferases; Blood Coagulation; Cholangitis; Disease Models, Animal; Leukocytes; Liver; Male; Neutrophils; Platelet Activating Factor; Rabbits; Thiazoles; Thiazolidines; Thromboplastin

Inhibition of the tissue factor/factor VIIa (TF/F.VIIa) complex attenuates thrombosis in different animal models of arterial thrombosis. However, it remains unclear to what extent the antithrombotic effects are associated with changes in hemostatic functions and how this compares with inhibition of thrombin, an enzyme acting at a later stage in the coagulation cascade. The antithrombotic and the antihemostatic effects of a monoclonal anti-TF antibody (AP-1) were compared in a model of arterial thrombosis to those of a direct thrombin inhibitor (napsagatran) and heparin. In anesthetized rabbits transient arterial thrombi were induced by mechanical damage to the subendothelium of a moderately stenosed carotid artery. Recurrent formation and dislodgement of thrombi resulted in cyclic flow variations (CFVs) which were monitored over 2 hours. Rabbits received intravenously either a placebo (control), a monoclonal anti-rabbit TF antibody (AP-1, 0.05 mg/kg as an i.v. bolus repeated every 15 min, a specific low molecular weight thrombin inhibitor (napsagatran, 3 microg/kg/min) or heparin (3 and 13 microg/kg/min). The effect of the inhibitors on the hemostatic system was studied in a separate set of rabbits by measuring template bleeding times (BT) in the ear arterioles, marginal ear vein and the nail cuticle of the foreleg. AP-1 and napsagatran showed a similar antithrombotic activity (78% and 80% abolition of the CFVs, respectively), whereas either low or high dose heparin was poorly effective (43% and 40% inhibition of CFVs, respectively). At these antithrombotic doses and even at 4-fold higher dosage, AP-1 did not significantly alter the BT, whereas napsagatran and heparin prolonged the ear vessels and cuticle BT in a dose-dependent manner. These results suggest that in contrast to direct thrombin inhibition, the blockade of the TF/F. VIIa function did not result in a concomitant prolongation of the bleeding time. Thus, dissociation of antithrombotic and antihemostatic effects indicates that inhibition of the coagulation system at its initial stage represents a promising approach for the development of new anticoagulants.

Topics: Animals; Antibodies, Monoclonal; Anticoagulants; Antithrombins; Bleeding Time; Blood Coagulation; Disease Models, Animal; Hemodynamics; Heparin; Naphthalenes; Piperidines; Rabbits; Thrombin; Thromboplastin; Thrombosis

Acute obstructive cholangitis (AOC) is one of the most fatal outcomes in sepsis, and frequently complicates disseminated intravascular coagulation (DIC). Recently we found that the plasma tissue factor (TF) level increased and changed in parallel with plasma markers of DIC in patients with AOC. To elucidate the role of TF in the pathogenesis of coagulopathy in AOC, we investigated the plasma levels of TF and its localization by immunohistochemical staining in rabbit models of AOC. Plasma TF activity significantly increased 3 h after the insult (0.63 +/- 0.1¿9 U/ml; p < 0.01) compared with that beforehand (0.05 +/- 0.02 U/ml), then reached a maximum level at 6 h (0.94 +/- 0.16 U/ml). The fluctuations in plasma TF activity correlated with those of the coagulation parameters including platelet count, fibrinogen, prothrombin time, and antithrombin III activity. Immunohistochemically, enhanced expression of TF was mainly detected in macrophages and neutrophils that had infiltrated into the liver sinusoids and around the bile duct, but not in the sinusoidal endothelial cells. A double immunofluorescence study revealed the concomitant presence of TF and fibrin at sites where macrophages and neutrophils had conglomerated. However, we could not detect an apparent change in TF expression in the lung or kidney. These data suggest that macrophages and neutrophils infiltrating into the liver sinusoids and around the bile duct play a pivotal role in TF expression, leading to coagulopathy in the acute phase of obstructive cholangitis in rabbits.

Topics: Acute Disease; Animals; Blood Coagulation; Cell Movement; Cholangitis; Disease Models, Animal; Female; Immunohistochemistry; Liver; Macrophages; Neutrophils; Rabbits; Thromboplastin

Rethrombosis limits the efficacy of coronary thrombolysis and may result from surface-associated thrombin, de-novo prothrombin activation, or both. This study was designed to determine the relative roles of thrombin, factor Xa, and the complex of tissue factor and factor VIIa in the procoagulant activity on injured arteries with evolving thrombi.. Extensive vascular injury and platelet-rich thrombi were induced in the abdominal aorta of 25 anesthetized rabbits by applying anodal current through a transluminal electrode for 3 h. Injured vessel segments were excised and placed in a chamber permitting perfusion over the luminal surface and associated thrombus.. Vessel segments perfused with recalcified, citrated human plasma induced marked increases in the concentration of fibrinopeptide A, a marker of thrombin-induced fibrin formation, in the effluent plasma after 10 min (4636 +/- 1894% of fibrinopeptide A in the nonperfused plasma, n = 5). Perfusion with plasma depleted of vitamin K-dependent coagulation factors prevented the increase in fibrinopeptide A (122 +/- 30%, n = 4), indicating the lack of preformed functional thrombin. Furthermore, appearance of fibrinopeptide A was attenuated by perfusion with plasma containing 0.1 mumol/l recombinant tick anticoagulant peptide, a specific inhibitor of factor Xa (594 +/- 320%, n = 3), and by preincubation of vessel segments with a monoclonal antibody to rabbit tissue factor (438 +/- 220%, n = 3).. Procoagulant activity on injured vessels and associated thrombi is mediated by factor Xa, a product of the functional initiation of coagulation by factor VIIa associated with tissue factor. Accordingly, inhibition of tissue factor-mediated coagulation may be effective for attenuation of active thrombogenesis on injured vessels and during thrombolysis.

Topics: Animals; Aorta, Abdominal; Blood Coagulation; Coronary Thrombosis; Disease Models, Animal; Factor VIIa; Factor Xa; Fibrinopeptide A; Humans; Microscopy, Electron, Scanning; Prothrombin; Rabbits; Thrombin; Thrombolytic Therapy; Thromboplastin; Thrombosis

We investigated the protective effects of DX-9065a, an orally active, newly synthesized, and specific inhibitor of factor Xa, against experimental disseminated intravascular coagulation (DIC) in rats. Experimental DIC was induced by a 4 hour sustained infusion of thromboplastin at a dose of 2.5 mg/kg. The rats were orally administered DX-9065a at 10, 30, 100 mg/kg 30 minutes before thromboplastin injection. In this DIC model, DX-9065a showed a protective effect against DIC, at all doses and in all parameters, including fibrin(ogen) degradation products, fibrinogen level, thrombin-antithrombin III complex level, prothrombin time (PT), activated partial thromboplastin time (APTT), platelet count, and the number of renal glomeruli with fibrin thrombi. When DX-9065a was orally administered at 100 mg/kg without thromboplastin, no significant changes were seen in hemostatic parameters except PT and APTT, and no fibrin thrombi or abnormal bleeding were seen in renal specimens. These findings suggested that the new oral anti-Xa drug, DX-9065a, has a protective effect against thromboplastin-induced DIC model with little risk of bleeding.

Topics: Administration, Oral; Animals; Anticoagulants; Disease Models, Animal; Disseminated Intravascular Coagulation; Factor X; Factor Xa Inhibitors; Male; Naphthalenes; Propionates; Rats; Rats, Wistar; Thromboplastin

Tissue factor pathway inhibitor (TFPI) plays a key role in modulating tissue factor-dependent blood coagulation. This study was done to determine not only the inhibitory effects of recombinant human TFPI (rTFPI) on thrombus formation in rat models with disseminated intravascular coagulation (DIC), but also to identify the distribution of exogenous TFPI in vivo. Disseminated intravascular coagulation was induced by administering a priming dose of carrageenan 10 mg/kg body weight and was followed 24 hours later by a provocative dose of lipopolysaccharide (LPS) 500 mg/kg body weight. The rTFPI was administered intravenously at a dose of either 1 or 4 mg/kg body weight immediately after LPS treatment. Exogenous rTFPI at a dose of 4 mg/kg significantly inhibited the consumption of fibrinogen, platelets and factor VIIa (P < .05) and also reduced the number of fibrin thrombi formed in the liver, lungs, kidneys, and spleen (P < .05), whereas rTFPI at a dose of 1 mg/kg had no significant inhibitory effect on these DIC parameters. Recombinant human rTFPI activity was rapidly cleared from the plasma; however, a significant amount of the inhibitor was still present in tissues even 3 to 6 hours after intravenous administration. Exogenous TFPI was mainly identified in Kupffer cells, macrophages, and on the microvascular endothelial lining of different organs. In the kidney, rTFPI was identified on both the abluminal surface of the renal tubules and the luminal surface of the proximal convoluted tubules. No rTFPI, however, was detected in the hepatocytes. Tissue factor was mainly expressed by monocytes/macrophages. These findings suggest that TFPI plays an important role in modulating TF-dependent thrombogenesis. The elucidation of the rTFPI distribution and interactions in vivo might thus provide valuable insight into its inhibitory mechanisms as well as its therapeutic implications in DIC.

Topics: Animals; Anticoagulants; Carrageenan; Disease Models, Animal; Disseminated Intravascular Coagulation; Fibrin; Humans; Immunohistochemistry; Lipoproteins; Male; Rats; Rats, Wistar; Recombinant Proteins; Shock, Septic; Survival Rate; Thromboplastin; Thrombosis; Tissue Distribution

Bleomycin-induced lung injury is an established murine model of human pulmonary fibrosis. Although procoagulant molecules (e.g., tissue factor [TF]) and fibrinolytic components (e.g., urokinase [u-PA] and type 1 plasminogen activator inhibitor [PAI-1]) have been detected in alveolar fluid from injured lungs, the origin of these molecules remains unknown. We therefore examined the expression of procoagulant and fibrinolytic components in relation to the distribution of parenchymal fibrin in bleomycin-injured lungs. Extravascular fibrin localized to the alveolar and extracellular matrix in injured lung tissue. Injured lung tissue extracts contained elevated levels of PAI-1 activity and decreased levels of u-PA activity. Whole lung PAI-1 and TF mRNAs were dramatically induced by lung injury. In situ hybridization of injured lungs revealed that PAI-1, u-PA, and TF mRNAs were induced within the fibrin-rich fibroproliferative lesions, primarily in fibroblast-like and macrophagelike cells, respectively, while TF mRNA was also induced in perilesional alveolar cells. Taken together, these observations suggest that the induction of PAI-1 and TF gene expression plays and important role in the formation and persistence of extracellular fibrin in bleomycin injured murine lungs.

Topics: Animals; Bleomycin; Disease Models, Animal; Female; Fibrin; Fibrinolysis; Gene Expression; In Situ Hybridization; Lung; Mice; Mice, Inbred C57BL; Plasminogen Activator Inhibitor 1; Pulmonary Fibrosis; RNA, Messenger; Thromboplastin; Time Factors; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator

An exposure of blood to tissue factor (TF) activates the coagulation system by the extrinsic pathway and may cause clot formation. Recombinant TF (r-TF) has been produced and subsequently reconstituted into phospholipid vesicles. The aim of these studies was to elucidate the in vitro procoagulant effects and the in vivo thrombogenicity of r-TF using a rabbit jugular vein stasis thrombosis model. The in vitro studies exhibited a clear concentration-dependent decrease in the clotting time when rabbit brain thromboplastin was replaced by r-TF in the prothrombin time assay. The in vivo studies revealed a dose-dependent thrombogenicity between 1.6 ng/kg and 50 ng/kg. Electron microscope scanning of the surface of representative clots revealed fibrin-rich structures of heterogeneous density. In comparison, thrombi obtained when FEIBA was utilized as the thrombogenic agent were more homogeneous. The injection of r-TF caused a slight transient drop in blood pressure with little or no effects on the pulse rate, complete blood count (CBC) profile, clotting and amidolytic assays when compared to sham control animals. In contrast, the whole blood clotting parameters (activated clotting time and thrombelastograph) were prolonged dose-dependently after r-TF injection. The antithrombotic activity of heparin was assessed in this model and compared to the antithrombotic activity when FEIBA is used as the thrombogenic agent. The apparent ED50 of heparin was found to be 4 times higher in the r-TF system. In control studies, no thrombogenic effects were observed by the phospholipid vesicles alone nor by r-TF not embedded in phospholipid vesicles. These data demonstrate that lipidated r-TF is a potent thrombogenic challenge that activates the hemostatic system by the extrinsic pathway.

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Hemostasis; Heparin; Male; Rabbits; Recombinant Proteins; Thromboplastin; Thrombosis

Several studies have established a link between blood coagulation and cancer, and more specifically between tissue factor (TF), a transmembrane protein involved in initiating blood coagulation, and tumor metastasis. In the study reported here, a murine model of human melanoma metastasis was used for two experiments. (i) The first experiment was designed to test the effect of varying the level of TF expression in human melanoma cells on their metastatic potential. Two matched sets of cloned human melanoma lines, one expressing a high level and the other a low level of the normal human TF molecule, were generated by retroviral-mediated transfections of a nonmetastatic parental line. The metastatic potential of the two sets of transfected lines was compared by injecting the tumor cells into the tail vein of severe combined immunodeficiency (SCID) mice and later examining the lungs and other tissues for tumor development. Metastatic tumors were detected in 86% of the mice injected with the high-TF lines and in 5% of the mice injected with the low-TF lines, indicating that a high TF level promotes metastasis of human melanoma in the SCID mouse model. This TF effect on metastasis occurs with i.v.-injected melanoma cells but does not occur with primary tumors formed from s.c.-injected melanoma cells, suggesting that TF acts at a late stage of metastasis, after tumor cells have escaped from the primary site and entered the blood. (ii) The second experiment was designed to analyze the mechanism by which TF promotes melanoma metastasis. The procedure involved testing the effect on metastasis of mutations in either the extracellular or cytoplasmic domains of the transmembrane TF molecule. The extracellular mutations introduced two amino acid substitutions that inhibited initiation by TF of the blood-coagulation cascade; the cytoplasmic mutation deleted most of the cytoplasmic domain without impairing the coagulation function of TF. Several human melanoma lines expressing high levels of either of the two mutant TF molecules were generated by retroviral-mediated transfection of the corresponding TF cDNA into the nonmetastatic parental melanoma line, and the metastatic potential of each transfected line was tested in the SCID mouse model. Metastases occurred in most mice injected with the melanoma lines expressing the extracellular TF mutant but were not detected in most mice injected with the melanoma lines expressing the cytoplasmic TF mutant. Results with the extrac

Topics: Animals; Blood Coagulation; Disease Models, Animal; Female; Humans; Melanoma; Mice; Mice, SCID; Mutation; Thromboplastin; Transfection; Tumor Cells, Cultured

Tissue factor (TF)/FVIIa initiates coagulation by activating factor IX (FIX) and factor X (FX). Tissue factor pathway inhibitor (TFPI)-FXa complexes form and inhibit TF/FVIIa. Blocking of TFPI may facilitate haemostasis initiated by FVIIa/TF thereby compensating for impaired FIX/FVIII-dependent coagulation. This hypothesis was tested in a study using rabbits made temporarily haemophilic by the injection of antibodies against FVIII. These rabbits were given i.v. injections of anti-TFPI IgG antibodies and 40 min later bleeding was initiated by cutting the nail including the apex of the cuticle. Injection of anti-TFPI IgG shortened the bleeding time significantly from 26 min to 11 min (normal mean bleeding time in non-haemophilia rabbits: 5 min). Treatment with anti-TFPI IgG also resulted in a shortening of the haemophilic aPTT to a level slightly longer than the normal aPTT. In addition, the prolonged dilute TF clotting time was shortened by the anti-TFPI IgG treatment. Thus, both bleeding and coagulation parameters indicated that blocking of TFPI may be potentially haemostatic in haemophilia.

Topics: Animals; Antibodies; Antibody Specificity; Bleeding Time; Blood Coagulation; Disease Models, Animal; Factor VIII; Female; Hemophilia A; Immunoglobulin G; Lipoproteins; Partial Thromboplastin Time; Rabbits; Thromboplastin

Thrombus formation and the sequential expression of tissue factor (TF), interleukin-1 beta (IL-1 beta) and interleukin-1 receptor antagonist (IL-1 ra) in several organs were examined immunohistochemically and morphometrically in a novel model of disseminated intravascular coagulation (DIC) developed by modifying the generalized Shwartzman reaction (GSR) in rabbits. The new model [carrageenan (CA)-lipopolysaccharide (LPS)] was induced by the administration of a priming dose of intraperitoneal CA, 10 mg/kg, followed 24 h later by a provocative dose of LPS 25 micrograms/kg, while GSR was induced by the intravenous injection of two doses of LPS 25 micrograms/kg. CA was detected predominantly within macrophages in the spleen and liver. Fibrin thrombi were formed as early as 1 h after the second LPS treatment in all examined organs reaching a peak at 3-9 h and their prevalence was higher in the CA-LPS group (p < 0.05). The sequential expressions of TF and IL-1 beta correlated well with each other in both groups reaching a peak at 3-9 h with the CA-LPS group showing a more pronounced expression than the GSR group. Macrophages in the liver, spleen and lungs, and Bowman's epithelial cells expressed both proteins, while IL-1 beta was also expressed by endothelial and epithelial cells. IL-1 ra was expressed by the same cells expressing IL-1 beta, however, its expression continued to increase gradually over 24 h. The mortality rate was lower (p < 0.05) and neutrophilic sequestration less prominent in the CA-LPS group than in the GSR group. These findings indicate that CA efficiently replaced the priming LPS treatment and the consequently enhanced production of IL-1 beta may have resulted in the upregulation of TF expression leading to the high level of thrombi in this new model which may provide a tool for further studies on the role of cytokines in DIC.

Topics: Animals; Carrageenan; Disease Models, Animal; Disseminated Intravascular Coagulation; Female; Immunohistochemistry; Injections, Intraperitoneal; Injections, Intravenous; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Kidney; Lipopolysaccharides; Liver; Lung; Rabbits; Shwartzman Phenomenon; Sialoglycoproteins; Spleen; Thromboplastin; Thrombosis; Time Factors; Tissue Distribution

Endothelins (ET) are the most important vasoconstrictors known, and administration results in contraction of vascular strips in man and experimental animals in vitro. We examined the effects of ET-1 on thrombus formation in rabbits. We used vasoconstrictor and thrombus forming agents and we selected an animal model, the vena jugularis thrombus model. In addition, intravascular endothelium was examined ultrastructurally. The ET-1 level is known to be high in patients with hypertension; if these patients also have atherosclerosis, then intravascular thrombus formation may increase. In the vena jugularis thrombus model, thromboplastin and ET-1 act synergistically to increase intravascular thrombus formation. On injection of ET-1 dose dependent vasoconstriction was shown in the vessel wall. Although similar maximal contraction is achieved, a decrease in vessel diameter is associated with increased potency of ET-1 and thromboplastin. The results suggest that ET-1 may regulate vascular tone through constriction of vessels.

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Synergism; Endothelins; Jugular Veins; Male; Rabbits; Thromboembolism; Thromboplastin

Alveolar and interstitial fibrin deposition is a prominent pathologic feature in many acute lung injury syndromes. Previous studies have suggested that ischemic lung preservation has a stimulatory effect on donor alveolar macrophages (Mphis) during transplantation. An animal model of lung preservation was developed to examine the hypothesis that ischemia enhances Mphi procoagulant activity (PCA) as a potential mechanism contributing to lung reperfusion injury. Histologic examination of ischemic lungs reperfused ex vivo revealed evidence of alveolar fibrin deposition. Mphis lavaged from lungs stored for at least 8 h at 21 degrees C exhibited increased PCA. The use of factor-deficient human plasma characterized this Mphi procoagulant as tissue factor (TF). Since increased PCA correlated with decreased airspace pO2 at the end of preservation, the effect of various O2 concentrations on PCA induction in vivo and in vitro was examined. Lung inflation during ischemia with decreasing O2 concentrations confirmed that hypoxia was associated with a rise in Mphi PCA in situ. However, in vitro exposure of Mphis to hypoxia did not increase Mphi PCA, suggesting that hypoxia alone was not responsible for induction of this procoagulant effect. Northern blot analysis demonstrated an increase in TF mRNA levels from in situ but not in vitro Mphis, thereby confirming transcriptional TF induction in this group. In addition, enhanced PCA was observed when Mphis were suspended in the bronchoalveolar lavage supernatant from the ischemic lungs stored at 21 degrees C. This suggests that in situ lung ischemia and hypoxia may produce soluble factors that either directly or indirectly stimulate Mphi TF expression. These factors may contribute to Mphi-mediated ischemic lung injury.

Topics: Animals; Blotting, Northern; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Fibrin; Hypoxia; Ischemia; Macrophages, Alveolar; Male; Microscopy, Electron, Scanning; Pulmonary Alveoli; Rats; Rats, Wistar; Reperfusion Injury; RNA, Messenger; Thromboplastin; Tubulin

The aims of the present investigation were to develop a new venous thrombosis animal model with low flow conditions in the venous blood stream and then evaluate this model for testing new anticoagulants. In this model, the vena cava of rats was narrowed with a Doppler flow probe, blood flow velocity continuously recorded and thrombus formation initiated by thromboplastin infusion. Sixty-five minutes following thromboplastin infusion the animals were sacrificed and the following parameters measured: thrombus wet weight, fibrinopeptide A (FpA), activated partial thromboplastin time and platelet number. The new model was evaluated with aspirin, a PGI2 mimetic, heparin and a soluble thrombomodulin analogue. Without thromboplastin infusion no thrombus formation or reduction of blood flow was observed. Controls receiving thromboplastin infusion developed a thrombus, blood flow was arrested, platelet number decreased and FpA was elevated. In contrast, animals pretreated with anticoagulants maintained a residual flow, while thrombus weight, thrombocytopenia and FpA elevation were reduced. The antiplatelet agents were not effective. This study demonstrates that, under low flow conditions, only a combination of blood flow reduction with a hypercoagulable state results in venous thrombus formation. This improved model of venous thrombosis more closely resembles the clinical situation and is applicable for testing anticoagulants.

Topics: Animals; Anticoagulants; Aspirin; Blood Flow Velocity; Constriction; Disease Models, Animal; Epoprostenol; Heparin; Male; Platelet Aggregation Inhibitors; Prostaglandins, Synthetic; Rats; Rats, Wistar; Recombinant Proteins; Thrombomodulin; Thrombophlebitis; Thromboplastin; Vena Cava, Inferior

This study demonstrated that intravenous infusion of recombinant human soluble thrombomodulin (rhs-TM) could inhibit disseminated intravascular coagulation (DIC) caused by 4 hr infusion of tissue factor (TF) in rats. Extended infusion of TF reduced fibrinogen and platelet counts and elevated serum FDP level. Pretreatment and coinfusion of rhs-TM could block changes of these DIC-parameters without prolongation of APTT. Heparin, which is a potent anti-DIC drug, could also inhibit these changes with extra prolongation of APTT and PT. Thus, these results suggest thrombomodulin prevent DIC less bleeding tendency than heparin.

Topics: Animals; Disease Models, Animal; Disseminated Intravascular Coagulation; Enzyme-Linked Immunosorbent Assay; Female; Fibronectins; Heparin; Infusions, Intravenous; Partial Thromboplastin Time; Platelet Count; Prothrombin Time; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Thrombomodulin; Thromboplastin

The possible involvement of platelet activating factor (PAF) in the pathogenesis of endotoxin-induced disseminated intravascular coagulation (DIC) was investigated in rats using a novel potent PAF antagonist, TCV-309. TCV-309 (> 1 mg/kg, i.v.) showed beneficial effects in rats with experimental DIC induced by a 4-hour sustained infusion of endotoxin (1 mg/kg) in a dose-dependent manner. TCV-309 (1 mg/kg) significantly ameliorated the decrease in platelet count and plasma fibrinogen, the prolongation of prothrombin time (PT) and activated partial thromboplastin time (APTT) and the increase in fibrin and fibrinogen degradation products (FDP) and inhibited glomerular fibrin deposition. Furthermore, plasma tissue factor (TF) activity was greatly increased in the DIC rats, and this was also significantly decreased by TCV-309 (1 mg/kg). TCV-309 (1 mg/kg) did not affect these parameters in normal rats. A 4-hour sustained infusion of PAF (60 micrograms/kg) caused mild but significant changes in some DIC parameters such as PT, fibrinogen and FDP concentration and increased the plasma TF activity. TCV-309 (1 mg/kg) inhibited all these PAF-induced changes. TCV-309 (0.1 mM) itself had no direct in vitro effects on the blood coagulation system including TF activity. These results strongly suggest that PAF plays a role in the pathogenesis of endotoxin-induced DIC via the generation of TF. Prophylactic use of PAF antagonists may therefore be useful for the treatment of DIC with sepsis.

Topics: Animals; Disease Models, Animal; Disseminated Intravascular Coagulation; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Isoquinolines; Male; Platelet Activating Factor; Pyridinium Compounds; Rats; Rats, Sprague-Dawley; Tetrahydroisoquinolines; Thromboplastin

Microvascular perfusion defects occur after occlusion and reperfusion of the middle cerebral artery in examples of focal cerebral ischemia. In addition to cellular (eg, polymorphonuclear leukocyte) contributors to the focal "no-reflow" phenomenon, activation of coagulation may also play a role. We have tested a potential role of tissue factor-mediated coagulation in the microvascular perfusion defects seen after focal cerebral ischemia-reperfusion in a baboon model of reversible middle cerebral artery occlusion with the murine anti-tissue factor monoclonal antibody TF9-6B4. Tissue factor is the principal resident procoagulant substance in cerebral tissues and has a distinct perivascular distribution.. Microvascular patency in the basal ganglia after 3-hour middle cerebral artery occlusion and 1-hour reperfusion was quantified by computerized video imaging of carbon-tracer perfused tissues. Animals were randomized to receive intravenous TF9-6B4 (10 mg/kg) 10 minutes before middle cerebral artery occlusion (n = 6) or no treatment (n = 6) in an open study.. In the control animals, a significant decrease in patency was confirmed in microvessels less than 30 microns in diameter. Infusion of TF9-6B4 before middle cerebral artery occlusion produced a stable maximal level of circulating antibody within 10 minutes, which lasted the duration of ischemia and reperfusion. An increase in reflow in microvessels of all size classes occurred after TF9-6B4 infusion, which was significant in those 7.5 to 30 microns (P = .038) and 30 to 50 microns (P = .013) in diameter.. These results indicate that tissue factor-mediated events may also contribute to no-reflow in noncapillary microvessels after focal cerebral ischemia.

Topics: Animals; Antibodies, Monoclonal; Blood Coagulation Disorders; Brain Ischemia; Cerebral Arteries; Disease Models, Animal; Male; Microcirculation; Papio; Reperfusion Injury; Thromboplastin

Previous studies on recombinant human soluble thrombomodulin (rsTM) from Chinese hamster ovary cells revealed that rsTM was expressed as two proteins that differed functionally in vitro due to the presence (rsTM beta) or absence (rsTM alpha) of chondroitin-4-sulfate. The current study evaluates the in vivo behavior of rsTM in rats and in a rat model of tissue factor-induced disseminated intravascular coagulation (DIC). rsTM beta was more potent than rsTM alpha for prolongation of the activated partial thromboplastin time (APTT) and their in vivo half-lives determined by ELISA were 20 min for rsTM beta and 5.0 h for rsTM alpha. Injection of a tissue factor suspension (5 mg/kg) resulted in DIC as judged by decreased platelet counts and fibrinogen concentrations, prolonged APTT, and increased fibrin and fibrinogen degradation products (FDP) levels. A bolus injection of either rsTM (0.2 mg/kg) 1 min before induction of DIC essentially neutralized effects on platelets, fibrinogen, and FDP levels, and had only a moderate effect on APTT prolongation. The dose of anticoagulant to inhibit the drop in platelet counts by 50% (ED50) was 0.2 mg/kg rsTM alpha, 0.07 mg/kg rsTM beta, and 7 U/kg heparin. The effect of increasing concentrations of rsTM and heparin on bleeding times were compared in experiments involving incision of the rat tail. Doubling of the bleeding times occurred at 5 mg/kg rsTM alpha, 3 mg/kg rsTM beta or 90 U/kg heparin. These values represent a 25-fold increase over the ED50 for rsTM alpha, 43-fold for rsTM beta and 13-fold for heparin.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Disease Models, Animal; Disseminated Intravascular Coagulation; Enzyme-Linked Immunosorbent Assay; Fibrinogen; Fibrinolysis; Glycosaminoglycans; Hemorrhage; Heparin; Male; Platelet Count; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, Thrombin; Recombinant Proteins; Solubility; Thromboplastin

Venous thrombosis was induced by ligature of the inferior vena cava in rats whose blood was made hypercoagulable by intravenous (IV) administration of tissue thromboplastin. From a dose-response showing that the administration of increasing doses of tissue thromboplastin resulted in a subsequent progressive increase of thrombus weight, two concentrations of tissue thromboplastin were chosen: a high dose (550 microL/kg, IV) where thrombus formation was optimal and a concentration (7 microL/kg, IV) where tissue thromboplastin-hypercoagulability was intermediate. In both experimental conditions, leukopenia provoked by a myelotoxic drug did not influence the development of venous thrombosis. However, after thrombocytopenia induced by an antiplatelet antiserum, a dramatic decrease in thrombus formation was observed in animals that had been pre-challenged with the lower dose of tissue thromboplastin, whereas decrease in platelet count did not affect venous thrombosis under high thrombogenic challenge. When administered orally 2 hours before thrombosis induction, the ticlopidine analogue clopidogrel showed dose-dependent inhibition of thrombus formation in animals that were pre-challenged with a low dose of tissue thromboplastin (ED50 = 7.9 +/- 1.5 mg/kg, orally) but remained ineffective against high tissue thromboplastin-induced venous thrombosis. We further determined the effect of heparin and hirudin, and showed that both of these drugs exhibited a more potent antithrombotic activity after injection of the lower dose of tissue thromboplastin than after injection of a high dose of tissue thromboplastin. Therefore, using our model of stasis and hypercoagulability, platelet activation played a major role in the development of venous thrombosis when the thrombogenitic stimulus was mild.

Topics: Animals; Blood Platelets; Clopidogrel; Disease Models, Animal; Heparin; Hirudins; Leukopenia; Male; Mechlorethamine; Partial Thromboplastin Time; Platelet Aggregation Inhibitors; Rabbits; Rats; Rats, Sprague-Dawley; Thrombocytopenia; Thrombophlebitis; Thromboplastin; Ticlopidine

The main objective of these experiments was to develop and characterize a new experimental model of venous thrombosis, and determine whether a combination of vascular wall damage (crushing with hemostat clamps) and prolonged stasis produced more reproducible clots than prolonged stasis per se. Rabbits were laparotomized, and a segment of the vena cava was dissected free and looped with two silk ligatures, 2.5 cm apart. The proximal tie was ligated 5 min after release of the clamps; the distal tie applied shortly thereafter, trapping a volume of blood in the isolated segment. At 2 hr after ligation, the isolated venous sac was excised and examined for the presence of a clot. Large, well-formed clots, which could be readily transferred and weighed, were invariably observed in the "clamp" and "no clamp" groups, the latter being a sham control. Mean clot weights did not differ in the two groups (23.1 +/- 1.6 versus 30.8 +/- 5.4 mg dry weight, clamped versus no clamp, respectively, p greater than 0.05). However, the precision of the method was improved significantly (p less than 0.005) by clamping as determined by homogeneity of variance testing. Time-course studies showed that a considerable lag period (about 60 min) preceded development of a detectable clot, and that the thrombus evolved rapidly during the interval of 60-90 min postligation. The location of the small clots at 60 min in clamped segments, as well as the failure of prolonged (120 min) stasis without caval isolation to cause substantial thrombi, strongly suggests that clot formation attributed to "stasis per se" is in fact due to focal vascular lesions created at the tie-down points. The present study is also the first report of blockade of localized venous thrombosis by recombinant tissue factor pathway inhibitor (rTFPI). When given as an i.v. bolus 20 min prior to ligation, rTFPI at 400 and 800 micrograms/kg completely blocked formation of the thrombus or greatly reduced its size in five of the six animals tested.

Topics: Animals; Disease Models, Animal; Lipoproteins; Male; Rabbits; Regional Blood Flow; Thrombophlebitis; Thromboplastin; Venae Cavae

It was shown in experiments on white rats that combined administration of nucleotide antiaggregants NAD+ and AMP, and NAD+ decomposition inhibitor--nicotinamide, decreased the increment of procoagulant (thromboplastic) activity of membrane particles in the ischemized kidneys and reduced thromboplastic activity of membrane particles in intact kidneys. When nucleotide antiaggregants were added to rabbit citrate plasma, the inhibition by membrane activation of recalcified plasma coagulation was more pronounced with the use of tissue thromboplastin of the kidneys and brain--proteophospholipid liposomes, than with the use of phospholipid liposomes obtained from thromboplastin. The data presented have evidenced an indirect anticoagulant effect of nucleotide aggregants at the level of tissue coagulation factors, and their direct anticoagulant action at the level of membrane activation of plasma factors.

Topics: Adenosine Monophosphate; Animals; Blood Coagulation; Disease Models, Animal; Ischemia; Kidney; Male; NAD; Platelet Aggregation Inhibitors; Rabbits; Rats; Thromboplastin

A study was made of thromboplastin, antiheparin and antithrombin activity of the brain, heart and liver tissues of rats in experimental chronic alcoholization. Appreciable changes in the tissue factors of coagulation according to the dyscoagulemia type depending on the duration and gravity of alcoholic intoxication may aggravate hemostatic disorders.

Topics: Alcoholism; Animals; Antithrombin III; Blood Coagulation; Brain; Disease Models, Animal; Ethanol; Liver; Myocardium; Platelet Factor 4; Rats; Thromboplastin

Coagulopathy results from many diverse events, including several neurogenic causes. Using a rabbit model, we produced coagulopathy by injecting autologous spinal cord and extracted thromboplastin intravenously. Serial coagulation panels were performed to evaluate the activation of the thrombotic and fibrinolytic pathways. Group 1 animals (n = 4) received intravenous injections of homogenized spinal cord tissue. Coagulopathy was not produced with 36 mg of homogenized spinal cord tissue, but 50 mg or more resulted in death. Group 2 animals (n = 12) received intravenous injections of extracted rabbit cord thromboplastin, which contained approximately 60% activity of a commercially purified rabbit brain thromboplastin. Five animals receiving 2.5 to 5.5 mg of thromboplastin per kilogram of body weight survived with evidence of coagulopathy. Seven animals receiving 2.5 to 100 mg of thromboplastin per kilogram of body weight died. Group 3 (4 control animals) received normal saline injections without changes in clinical or laboratory status. The thrombotic pathway was activated in all animals as evidenced by decreased platelet counts and fibrinogen levels. Activation of the fibrinolytic system was demonstrated by increased concentrations of protamine sulfate and abnormal euglobulin clot lysis times. The most sensitive parameters were the platelet count, protamine sulfate concentration, and white cell count (margination), which became abnormal within 15 minutes after the injections and returned to normal within 1 hour.

Topics: Animals; Blood Coagulation Disorders; Disease Models, Animal; Injections, Spinal; Platelet Count; Rabbits; Sodium Chloride; Spinal Cord; Thromboplastin

The bleeding disorder of hemophilia A currently treated by replacement therapy of the missing coagulation factor, factor VIII, is frequently complicated by the development of neutralizing antibodies. The therapeutic potential of attenuated forms of the lipid-associated glycoprotein tissue factor, a known initiator of coagulation, was investigated as a factor VIII-by-passing activity. The protein moiety of tissue factor (Apo-TF) was partially purified and exhibited minimal procoagulant activity before relipidation in vitro. In pilot studies, Apo-TF injection into rabbits previously anticoagulated with an antibody to factor VIII was found to have a procoagulant effect. The efficacy of the material was further demonstrated when injection of Apo-TF in hemophilic dogs resulted in a normalization of the cuticle bleeding time. Little or no change in the blood parameters associated with disseminated intravascular coagulation was observed at lower doses, although mild to moderate effects were seen at higher doses. These data suggest a novel role for Apo-TF preparations as a potential therapeutic agent for hemophiliacs with antibodies to factor VIII once the potential thrombogenicity of such materials is evaluated.

Topics: Animals; Blood Coagulation Tests; Cattle; Disease Models, Animal; Dogs; Factor IXa; Factor VIII; Hemophilia A; Phospholipids; Rabbits; Serine Endopeptidases; Thromboplastin

A synthetic pentasaccharide, representing the critical sequence required in heparin for binding to antithrombin III (AT III), produces strong anti-factor Xa activity in vitro in the presence of AT III and is devoid of any activity directed towards thrombin. This pentasaccharide provides a unique tool to study the question of whether an agent capable of inhibiting factor Xa but devoid of anti-factor IIa activity in vitro, has the capacity to produce an antithrombotic effect in vivo. We have previously demonstrated in a rabbit stasis thrombosis model using a human serum challenge, a significant antithrombotic effect of the pentasaccharide. This finding and discrepancies with some earlier reports on the antithrombotic actions of other oligosaccharide fragments, led us to extend these studies. Four modifications of the stasis thrombosis model were developed using the following thrombogenic challenges selected for their specified induction sites of thrombosis, thromboplastin, an activated prothrombin complex concentrate, a non-activated prothrombin complex concentrate administered simultaneously with Russell's viper venom, and factor Xa. Dose-dependent antithrombotic responses were obtained in all four systems with ED50 values between 25-43 ug/kg for pentasaccharide as compared to 16-47 ug/kg for heparin. Complete inhibition of induced thrombosis was obtained in all four systems for pentasaccharide. Ex vivo analysis revealed expected anti-factor Xa levels but no anti-factor IIa activity. It is concluded that an oligosaccharide with high anti-factor Xa activity and devoid of anti-factor IIa activity is capable of inhibiting thrombosis induced in rabbit stasis models, but that higher dosages than heparin are required for this effect.

Topics: Animals; Antithrombin III; Disease Models, Animal; Dose-Response Relationship, Drug; Factor X; Factor Xa; Heparin; Male; Oligosaccharides; Prothrombin; Rabbits; Thromboplastin; Thrombosis

Rats were subjected to gram-negative septicemia induced by cecal perforation or were sham-operated. Thromboplastin values increased in blood monocytes (40-fold), peritoneal macrophages (115-fold) pleural macrophages (5-fold), splenic macrophages (3-fold), and lung alveolar macrophages (1.4-fold) in septic animals as compared to controls. In septic animals disseminated intravascular coagulation was evidenced by a significant (p less than 0.05) fall in fibrinogen, factor VII, X and platelets. A simultaneous and significant (p less than 0.05) decrease in thromboplastin content of tissue-specimens from lung and spleen was observed in rats with septicemia, whereas increased thromboplastin values were demonstrated in tissue-samples from cecum - the infectious focus. This might reflect mobilization of mononuclear phagocytes in favour of the site of infection.

Topics: Animals; Blood Coagulation Factors; Cell Movement; Disease Models, Animal; Disseminated Intravascular Coagulation; Gram-Negative Bacteria; Macrophages; Male; Monocytes; Peritoneal Cavity; Pleura; Pulmonary Alveoli; Rats; Rats, Inbred Strains; Sepsis; Spleen; Thromboplastin; Tissue Distribution

Yucatan miniature swine were the experimental model used to examine the effect of ischemia-injury on post-ischemic monocyte (MO) and immune function. Monocyte plasminogen activator (PA) was depressed while MO tissue factor activity was increased. The ability of porcine monocytes to generate a primary in vitro antibody forming cell (AFC) response to sheep red blood cells (SRBC) also was depressed by ischemic injury. The mechanism by which ischemic injury modulated immunosuppression appeared to be through generation of immunosuppressive serum substances.

Topics: Animals; Antibody Formation; Disease Models, Animal; Extremities; Female; Immune Tolerance; Immunity, Cellular; Ischemia; Lymphocytes; Monocytes; Plasminogen Activators; Swine; Swine, Miniature; Thromboplastin; Wounds and Injuries

The original stasis thrombosis model of Wessler has been modified. Numerous thrombogenic agents were evaluated for their pathophysiologic effects and were classified in terms of stasis clot in the jugular vein. Alterations produced in coagulation parameters, such as the PT, APTT, thrombin time, activated recalcification time (Hemachron), and thrombelastographic pattern were recorded. Since the pathophysiologic activation of the hemostatic system varies considerably in different diseases, a proper animal model along with a proper type of thrombogenic trigger should be carefully selected to produce pathogenesis and to study the therapeutic responses of heparin and its derivatives. In the modified stasis thrombosis model, besides monitoring the formation of the jugular vein stasis clot, it is proposed that the following tests may be useful to establish hypercoagulable states: Functional levels of various coagulation factors, platelet counts, fibrinogen levels, and whole blood activated clotting times. The nature of activation processes in each thrombogenic challenge should be carefully analyzed in terms of pathways involved; for example, the administration of heterologous serum (such as human, monkey) to rabbits produces anaphylactoid reactions, including hemolysis, thrombocytopenia, clinical chemistry abnormalities (enzymes), and many problems that may involve the complement and immune systems. All previous data obtained using heterologous sera as a thrombogenic trigger are of questionable value as to the efficacy of some of the antithrombotic agents tested against it. In addition to the species and the thrombogenic challenge, the following factors may contribute significantly to the pathophysiologic response and its alteration by various agents: (1) Composition of the thrombogenic agent; (2) effect of preparatory drugs, such as anesthetics, on the hemostatic parameters; (3) alterations on injection time, volume, osmolarity, and temperature; (4) variations in the circulation time of the thrombogenic agent and stasis time of the ligated jugular vein stasis segment; and (5) blood sample collection and handling. Since the kallikrein-kinin cascade is closely associated with the coagulation and the fibrinolytic network, a systemic monitoring of blood pressure may provide information on the effect of thrombogenic agents on hemodynamics.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Blood; Blood Coagulation Factors; Disease Models, Animal; Dogs; Endopeptidases; Haplorhini; Hemostasis; Heparin; Humans; Jugular Veins; Male; Oligosaccharides; Prothrombin Time; Rabbits; Serine Endopeptidases; Thromboplastin; Thrombosis; Viper Venoms

A telescoped model of nephrotoxic nephritis in the rabbit, using guinea pig antiglomerular basement membrane IgG in rabbits preimmunized with guinea pig IgG, reproducibly induced crescentic nephritis. Procoagulant activity (PCA) was measured in sieve-isolated glomeruli that had been either sonicated or cultured for 48 hours. In both sonicated and cultured glomeruli PCA peaked on days 5 and 6. The time course for appearance of PCA corresponded precisely with the appearance of proteinaceous material containing fibrin in Bowman's space as measured by a light microscopic histologic scoring system and confirmed by immunofluorescence and electron microscopy. Glomerular PCA returned to baseline by days 9 and 10 in spite of progression of glomerular injury. PCA also appeared in urine. Urine PCA peaked on day 8 and persisted through day 12 when glomerular PCA had returned to baseline. Glomerular and urine PCA were characterized using human coagulation factor-deficient plasmas and antithromboplastin IgG. Both glomerular PCA and urine PCA were inhibited by antithromboplastin IgG, showing that thromboplastin (tissue factor) contributed to PCA. The PCA in glomerular sonicates was dependent on factor X, but independent of factor VII or Hageman factor, suggesting that factor VII was present. Following glomerular culture for 48 hours the PCA had changed and in some cases was dependent on Hageman factor, factor IX, and factor VII for full PCA expression. Urine PCA was uniformly Hageman factor dependent and sometimes independent of factors VII and X. No active thrombin was present. The forms of glomerular and urine PCA were, therefore, complex. They seemed to be primarily driven by thromboplastin but also appeared to require the presence of the intrinsic coagulation pathway for full expression of PCA.

Topics: Animals; Basement Membrane; Blood Coagulation Factors; Disease Models, Animal; Fibrin; Glomerulonephritis; Immunoglobulin G; Kidney Glomerulus; Macrophages; Rabbits; Thromboplastin; Time Factors

Topics: Animals; Antithrombins; Blood Coagulation; Blood Coagulation Factors; Body Weight; Castration; Disease Models, Animal; Dose-Response Relationship, Drug; Ethinyl Estradiol; Female; Fibrinogen; Fibrinolysis; Hysterectomy; Organ Size; Rats; Thromboplastin; Uterus

Topics: 5-Hydroxytryptophan; Abruptio Placentae; Animals; Blood Coagulation Factors; Desoxycorticosterone; Disease Models, Animal; Disseminated Intravascular Coagulation; Female; Fibrinogen; Fibrinolysis; Humans; Hypertension; Platelet Adhesiveness; Pre-Eclampsia; Pregnancy; Pregnancy Complications, Hematologic; Pulmonary Circulation; Rabbits; Rats; Thrombocytopenia; Thromboplastin

Topics: Animals; Blood Platelets; Depression, Chemical; Disease Models, Animal; Disseminated Intravascular Coagulation; Dogs; Factor V; Factor VII; Factor VIII; Factor XI; Factor XII; Factor XIII; Fibrinogen; Fibrinolysis; Prothrombin Time; Stimulation, Chemical; Thromboplastin

Topics: Animals; Disease Models, Animal; Disseminated Intravascular Coagulation; Female; Fibrin; Fibrinogen; Humans; Placenta; Pre-Eclampsia; Pregnancy; Rabbits; Thromboplastin

Topics: Animals; Catheterization; Diatrizoate; Disease Models, Animal; Dogs; Electrocardiography; Fibrinolysin; Fibrinolysis; Fluoroscopy; Pulmonary Artery; Pulmonary Embolism; Thromboplastin

Topics: Animals; Blood Coagulation; Coronary Disease; Disease Models, Animal; Dogs; Fibrinogen; Fibrinolysis; Heparin; Methods; Prothrombin Time; Thrombelastography; Thromboplastin; Time Factors

Topics: Abruptio Placentae; Animals; Cardiac Output; Disease Models, Animal; Dye Dilution Technique; Embolism, Amniotic Fluid; Female; Fibrin; Humans; Latex; Male; Meconium; Methods; Microspheres; Placenta; Pregnancy; Rabbits; Radioisotopes; Retinal Vessels; Thrombin; Thromboplastin; Time Factors; Tissue Extracts; Xenon

Topics: Animals; Antithrombins; Blood Coagulation; Blood Coagulation Tests; Disease Models, Animal; Dogs; Fibrin; Fibrinogen; Fibrinolysis; Humans; Lethal Dose 50; Methods; Placental Extracts; Shock, Hemorrhagic; Tachyphylaxis; Thromboplastin