thromboplastin and Venous-Thrombosis

Spontaneous venous thrombosis is often the first clinical sign of cancer, and it is linked to a worsened survival rate. Traditionally, tumor-cell induced platelet activation has been the main actor studied in cancer-associated-thrombosis. However, platelet involvement alone does not seem to be sufficient to explain this heightened pro-thrombotic state. Neutrophils are emerging as key players in both thrombus generation and cancer progression. Neutrophils can impact thrombosis through the release of pro-inflammatory cytokines and expression of molecules like P-selectin and Tissue Factor (TF) on their membrane and on neutrophil-derived microvesicles. Their role in cancer progression is evidenced by the fact that patients with high blood-neutrophil counts have a worsened prognosis. Tumors can attract neutrophils to the cancer site via pro-inflammatory cytokine secretions and induce a switch to pro-tumoral (or N2) neutrophils, which support metastatic spread and have an immunosuppressive role. They can also expel their nuclear contents to entrap pathogens forming Neutrophil Extracellular Traps (NETs) and can also capture coagulation factors, enhancing the thrombus formation. These NETs are also known to have pro-tumoral effects by supporting the metastatic process. Here, we strived to do a comprehensive literature review of the role of neutrophils as drivers of both cancer-associated thrombosis (CAT) and cancer progression.

Topics: Blood Platelets; Extracellular Traps; Humans; Neoplasms; Neutrophils; P-Selectin; Platelet Activation; Thromboplastin; Thrombosis; Venous Thrombosis

Venous thrombosis is a common complication in cancer patients, and some cancer chemotherapies are associated with an increased risk of venous thromboembolism. The regulatory mechanisms that control thrombus formation and subsequent resolution in patients with cancer, however, are incompletely understood, and novel treatments for cancer-associated thrombosis may arise from a better understanding of such mechanisms. In this chapter, pathways that regulate cancer-associated thrombus formation are outlined, and the effects of anti-angiogenic cancer chemotherapies on venous thrombus resolution are highlighted. Potentially pro-thrombotic effects of anti-angiogenic agents are important considerations when managing the complications of venous thrombosis in cancer patients.

Topics: Angiogenesis Inhibitors; Disease Management; Humans; Neoplasms; Neovascularization, Pathologic; Odds Ratio; Plasminogen Activator Inhibitor 1; Risk; Thromboplastin; Tumor Necrosis Factor-alpha; Venous Thromboembolism; Venous Thrombosis

Present review highlights some new aspects of the role of individual components of blood coagulation process and proposes a modified concept of hemocoagulation cascade. The role of FXII in the initiation of the so-called intrinsic coagulation system is currently questioned. Its role has been recently demonstrated mainly in the thrombus propagation and final stabilization together with factors XI and XIII. The edited concept underlines the common part of the tissue factor (TF) in the initiation of both the intrinsic and extrinsic pathways of the coagulation system and therefore may make it not improperly be called the TF coagulation pathway. The search for new antithrombotic agents shows that the level of the coagulation system blockade depends on which step in the coagulation cascade is targeted and results in different degrees of the antithrombotic efficiency and the risk of bleeding complications.

Topics: Blood Coagulation; Fibrinolytic Agents; Humans; Terminology as Topic; Thromboplastin; Venous Thrombosis

Microparticles (MP) are small extracellular vesicles (30-1,000 nm) that are released from activated cells or platelets. Exposure of negatively charged phospholipids and tissue factor (TF) renders MP procoagulant. Normal plasma levels of intravascular TF-exposing MP (TFMP) are low, but their number may rise in pathological conditions, including cancer and infectious disease. Emerging evidence indicates an important role for these circulating TFMP in the pathogenesis of thrombotic complications such as venous thromboembolism and disseminated intravascular coagulation, whereas their contribution to arterial thrombosis is less studied. Despite serious limitations of the currently available assays for measuring TFMP levels or the procoagulant activity associated with TFMP with respect to sensitivity and specificity, the scientific interest in TFMP is rapidly growing because their application as prognostic biomarkers for thrombotic complications is promising. Future advances in detection methods will likely provide more insight into TFMP and eventually improve their clinical utility.

Topics: Animals; Cell-Derived Microparticles; Disseminated Intravascular Coagulation; Humans; Thromboplastin; Venous Thrombosis

Patients with cancer have an increased risk for venous thrombosis. Interestingly, different cancer types have different rates of thrombosis, with pancreatic cancer having one of the highest rates. However, the mechanisms responsible for the increase in venous thrombosis in patients with cancer are not understood. Tissue factor (TF) is a transmembrane receptor and primary initiator of blood coagulation. Tumor cells express TF and spontaneously release TF-positive microparticles (MPs) into the blood. MPs are small membrane vesicles that are highly procoagulant. It has been proposed that these circulating tumor-derived, TF-positive MPs may explain the increased rates of venous thrombosis seen in patients with cancer. In animal models, increased levels of tumor-derived, TF-positive MPs are associated with activation of coagulation. Moreover, these MPs bind to sites of vascular injury and enhance thrombosis. We and others have found that patients with cancer have elevated levels of circulating TF-positive MPs. These MPs are derived from tumors because they express tumor markers and are decreased by tumor resection. Importantly, several studies have shown that increased levels of TF-positive MPs correlate with venous thrombosis in patients with cancer. Taken together, these results suggest that TF-positive MPs may be a useful biomarker to identify patients with cancer who are at high risk for thrombosis.

Topics: Animals; Cell-Derived Microparticles; Humans; Models, Biological; Neoplasms; Risk Factors; Thromboplastin; Venous Thromboembolism; Venous Thrombosis

Microvesicles (MVs) are small (0.1-1 µm) membrane vesicles released from activated cells. The surface of MVs can be highly procoagulant due to the presence of the procoagulant protein tissue factor (TF) and of negatively charged phospholipids, such as phosphatidylserine. This review focuses on the potential utility of plasma MVs as a biomarker of venous thrombosis. The majority of studies have not found a clear relationship between venous thrombosis and phosphatidylserine-positive MVs or platelet MVs. Conversely, TF-positive MVs are increased in patients with venous thromboembolism. Studies with pancreatic cancer patients suggest that elevated levels of TF-positive MVs may be predictive of the development of venous thrombosis and patient survival. However, further studies are needed to determine if TF-positive MVs are a good biomarker for venous thrombosis in cancer and other diseases.

Topics: Biomarkers; Cell-Derived Microparticles; Humans; Risk Factors; Thromboplastin; Venous Thrombosis

Cancer is characterized by bidirectional interrelations between tumour progression, coagulation activation, and inflammation. Tissue factor (TF), the principal initiator of the coagulation protease cascade, is centrally positioned in this complex triangular network due to its pleiotropic effects in haemostasis, angiogenesis, and haematogenous metastasis. While formation of macroscopic thrombi is the correlate of cancer-associated venous thromboembolism (VTE), a major healthcare burden in clinical haematology and oncology, microvascular thrombosis appears to be critically important to blood-borne tumour cell dissemination. In this regard, expression of TF in malignant tissues as well as shedding of TF-bearing microparticles into the circulation are thought to be regulated by defined genetic events relevant to pathological cancer progression, thus directly linking Trousseau's syndrome to molecular tumourigenesis. Because pharmacological inhibition of the TF pathway in selective tumour types and patient subgroups would be in line with the modern concept of individualized, targeted anti-cancer therapy, this review will focus on the role of TF in tumour biology and cancer-associated VTE.

Topics: Animals; Cell Transformation, Neoplastic; Hemostasis; Humans; Models, Biological; Neoplasms; Thromboplastin; Venous Thrombosis

Topics: Female; Gene Frequency; Genetic Predisposition to Disease; Genetic Variation; Humans; Incidence; Male; Middle Aged; Phenotype; Regression Analysis; Risk Assessment; Risk Factors; Thromboplastin; Venous Thrombosis

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

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

In 1856 Virchow proposed a triad of causes for venous thrombosis, postulating that stasis, changes in the vessel wall or changes in the blood could lead to thrombosis. We now know that abnormally high levels of some coagulation factors and defects in the natural anticoagulants contribute to thrombotic risk. Among these, factor V Leiden, which renders factor Va resistant to activated protein C, is the most prevalent with approximately 5% of the Caucasian population having this genetic alteration. These genetically controlled variants in coagulation factors work in concert with other risk factors, such as oral contraceptive use, to dramatically increase thrombotic risk. While these abnormalities in the blood coagulation proteins are associated with thrombotic disease propensity, they are less frequent contributors to thrombosis than age or cancer. Cancer increases thrombotic risk by producing tissue factor to initiate coagulation, by shedding procoagulant lipid microparticles or by impairing blood flow. Age is the strongest risk factor for thrombosis. Among possible reasons are fragility of the vessels potentially contributing to stasis, increased coagulation factor levels, impaired function of the venous valves, decreases in the efficacy of natural anticoagulants associated with the vessel wall, increased risk of immobilization and increased risk of severe infection.

Topics: Aging; Blood Coagulation; Factor V; Humans; Neoplasms; Protein C; Thromboplastin; 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

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

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

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

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

Topics: Angiogenesis Inhibitors; Animals; Anticoagulants; Guideline Adherence; Heparin, Low-Molecular-Weight; Humans; Membrane Glycoproteins; Neoplasms; P-Selectin; Practice Guidelines as Topic; Pulmonary Embolism; Risk Assessment; Thromboplastin; Venous Thrombosis

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

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

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

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

Angiogenesis is essential for tumor growth and metastasis. This is firmly established in solid tumors, but accumulating evidence suggests that this is also an important event in hematological neoplasias. Angiogenesis is therefore a putative target for therapy. The potential application of different angiogenesis inhibitors is currently under intense clinical investigation, and we will here review a number of these trials. The association between cancer and thromboembolic disease is even better documented, and again, this is not limited to solid tumors. It appears that many patients with hematological malignancies have a dysfunctional hemostatic system, with increased risk of thromboembolism. Furthermore, effective antithrombotic therapy seems to reduce the risk of cancer progression and even prolongs overall survival. In this review we will thus discuss the mechanisms involved in the regulation of angiogenesis and hemostasis and present evidence for a shared biology. A number of factors regulating the hemostatic system also have pro- or anti-angiogenic properties. Tissue factor (TF) and TF pathway inhibitor (TFPI) seem to play a central role, and there are several lines of evidence suggesting a close cooperation between TF/TFPI and pro-angiogenic factors like members of the vascular endothelial growth factor family. A better understanding of this shared biology may reveal new targets, and will probably increase the safety of targeting the blood supply.

Topics: Angiogenesis Inhibitors; Animals; Endothelial Cells; Hematologic Neoplasms; Hemostasis; Humans; Lipoproteins; Matrix Metalloproteinase Inhibitors; Neovascularization, Pathologic; Neovascularization, Physiologic; Thromboplastin; Venous Thrombosis

Anticoagulant therapy is effective at preventing the development of venous thromboembolism in high-risk patients, and reduces morbidity and mortality in individuals with established thromboembolic disease. Vitamin K antagonists and heparins are currently the most commonly used anticoagulant drugs, but they have practical limitations. Therefore, new antithrombotic agents with predictable dose-responses (thereby decreasing the need for monitoring without compromising efficacy or safety), ideally available in an oral formulation and with a rapidly reversible anticoagulant effect, are needed. New drugs fulfilling some of the above criteria have been developed and have proven to be effective agents for the treatment and prevention of venous thromboembolism.

Topics: Animals; Anticoagulants; Azetidines; Benzylamines; Blood Coagulation; Factor VII; Factor Xa Inhibitors; Fondaparinux; Helminth Proteins; Humans; Oligosaccharides; Polysaccharides; Recombinant Proteins; Thrombin; Thromboembolism; Thrombomodulin; Thromboplastin; Treatment Outcome; Venous Thrombosis

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

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

The role of the haemostatic system in colorectal cancer (CRC) is reviewed. Correlations between the activation of the haemostatic system and overall survival have been suggested. Experimental studies indicate that the haemostatic system plays a key role in growth, invasion and dissemination of tumour cells, and in tumour related angiogenesis. Additional activation by the surgical trauma and postoperative infections are discussed. Finally, anti-cancer modalities directed against regulation of the haemostatic system in CRC are considered.

Topics: Animals; Blood Coagulation; Chemotherapy, Adjuvant; Colorectal Neoplasms; Fibrinolysis; Hemostasis; Humans; Infections; Peptide Hydrolases; Postoperative Complications; Thromboplastin; Venous Thrombosis

Originally isolated from a haematophagous hookworm, recombinant nematode anticoagulant protein c2 (rNAPc2) is an 85-amino acid protein with potent anticoagulant properties. Unlike conventional anticoagulants that attenuate blood coagulation via inhibition of thrombin or activated factor X (FXa) at the downstream portion of the cascade, rNAPc2 is a potent inhibitor of the activated factor VII/tissue factor complex (FVIIa/TF), the key physiological initiator of blood coagulation. Its mechanism of action requires prerequisite binding to circulating FXa or zymogen factor X (FX) to form a binary complex prior to its interaction and inhibition of membrane-bound FVIIa/TF. The binding of rNAPc2 to FX results in an elimination half-life of longer than 50 h following either subcutaneous or intravenous administration. Recombinant NAPc2, like other inhibitors of FVIIa/TF including tissue factor pathway inhibitor (TFPI) and active site-blocked FVIIa (ASIS, FFR-rFVIIa or FVIIai), may have a promising role in the prevention and treatment of venous and arterial thrombosis, as well as potential efficacy in the management of disseminated intravascular coagulopathies because of their potent and selective inhibition of FVIIa/TF.

Topics: Anticoagulants; Blood Coagulation; Factor VIIa; Factor X; Factor Xa; Helminth Proteins; Humans; Recombinant Proteins; Thromboplastin; Thrombosis; Venous Thrombosis

Topics: Anticoagulants; Heparin, Low-Molecular-Weight; Home Care Services, Hospital-Based; Humans; Randomized Controlled Trials as Topic; Risk Factors; Thromboembolism; Thromboplastin; Venous Thrombosis

Topics: Arteriosclerosis; Blood Coagulation Factors; Blood Platelets; Endothelium, Vascular; Factor VIII; Fibrinogen; Hemostasis; Hepatocytes; Humans; Interleukin-6; Leukocytes; Models, Biological; Receptors, Interleukin-6; Thromboplastin; Thrombosis; Venous Thrombosis; von Willebrand Factor

Post-surgical deep vein thrombosis (DVT) is often underdiagnosed by clinical assessment alone. Subclinical DVT is a major source of pulmonary embolism, which is an important cause of death, particularly following total hip replacement surgery. Results from pathophysiological studies and recently conducted, prospective double-blind venographic studies in Europe and North America suggest that, in patients undergoing total hip replacement surgery, thromboprophylaxis with a low-molecular-weight heparin should be continued for at least 5 weeks post-operatively to minimize this serious complication.

Topics: Anticoagulants; Arthroplasty, Replacement, Hip; Double-Blind Method; Europe; Heparin, Low-Molecular-Weight; Humans; Leg; Multicenter Studies as Topic; North America; Outpatients; Phlebography; Postoperative Complications; Postoperative Period; Prevalence; Prospective Studies; Pulmonary Embolism; Randomized Controlled Trials as Topic; Regional Blood Flow; Risk Factors; Thromboembolism; Thromboplastin; Venous Thrombosis; Warfarin

Total joint arthroplasty (TJA) of the hip or knee (THA, TKA) has become an increasingly common procedure. While TJA is a successful treatment for individuals experiencing degenerative joint diseases, it is well known that one of the most common perioperative complications of TJA is deep venous thrombosis (DVT). To profile tissue factor (TF), microparticle-tissue factor (MP-TF), thrombin-activatable fibrinolysis inhibitor (TAFI), and fibrinogen levels in patients undergoing TJA to determine potential preexisting Hemostatic dysregulation. De-identified blood samples were obtained from patients undergoing TJA 1 day pre- and 1 day postprocedure. Plasma samples were analyzed using enzyme-linked immunosorbent assay kits for fibrinogen, TAFI, TF, and MP-TF; fibrinogen levels were also assessed using a clot-based activity assay. In comparison with healthy controls, there were significant increases of fibrinogen and MP-TF levels, while there were significant decreases in TF and TAFI levels in the preoperative and postoperative patients. Comparing the pre versus postoperative patients, no significant differences were found; interestingly, however, surgical intervention exacerbated the changes found in the preoperative samples compared to the controls. The results of this study confirm that patients undergoing TJA have preexisting alterations in the fibrinolytic system. Surgical intervention tended to exacerbate these changes. The alterations observed in this study may provide insight as to why TJA is associated with higher rates of DVT and thromboembolism.

Topics: Aged; Aged, 80 and over; Arthroplasty, Replacement, Hip; Biomarkers; Carboxypeptidase B2; Female; Fibrinogen; Humans; Male; Middle Aged; Thromboplastin; Venous Thrombosis

Multiple myeloma (MM) is associated with an increased risk of venous thromboembolic (VTE) complications. Aim of this study was to measure microparticle-associated tissue factor (MP-TF) activity in patients with newly diagnosed MM before and after chemotherapy and to investigate whether MP-TF activity is associated with VTE. MP-TF activity was assessed in 122 newly diagnosed MM patients who were eligible for combination chemotherapy. MP-TF activity levels (17.6 fM Xa/min [8.6-33.2] (median [IQR]) were higher in untreated MM patients compared to normal healthy volunteers (4.1 fM Xa/min [2.3-6.6], p <0.001). MP-TF activity prior to the start of treatment was not different between patients who developed a VTE during follow-up (n=15) and those who did not (n=107). In 75 patients in whom plasma was obtained before and after chemotherapy, MP-TF activity decreased significantly (from 17.4 [10.2-32.8] to 12.0 [7.0-18.5] fM Xa/min, P=0.006). MP-TF activity remained, however, elevated in patients who developed VTE (15.1 [10.3-25.2]), in contrast to patients not developing VTE (11.4 [7.0-25.2], P<0.001). In conclusion, MP-TF activity is increased in patients with MM. Whether MP-TF activity has a pathogenetic role in VTE in MM patients remains to be established in future studies.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Boronic Acids; Bortezomib; Case-Control Studies; Cell-Derived Microparticles; Dexamethasone; Doxorubicin; Female; Humans; Male; Middle Aged; Multiple Myeloma; Pyrazines; Thalidomide; Thromboplastin; Venous Thrombosis; Vincristine

BACKGROUND. Venous thromboembolism is known to be an important social and health care problem because of its high incidence among patients who undergo surgery. For instance, 20-30% of patients develop this problem after general surgical operations, while 5.5% of patients have this complication when laparoscopic fundoplications are performed without any prophylaxis. The aim of our study was to evaluate the hypocoagulation effect of the following treatments during and after laparoscopic fundoplication: a) intermittent pneumatic compression (IPC) and b) combination of low-molecular-weight heparin (LMWH) and IPC. MATERIAL AND METHODS. The study was performed on 20 consecutive patients who were randomized into two groups. The first group received IPC during operation, the second group received IPC during operation and LMWH before operation. Plasma prothrombin fragment F1+2 (F1+2), thrombin-antithrombin complex (TAT) - markers of thrombogenesis - and plasma free tissue factor pathway inhibitor (fTFPI) - a marker of hypocoagulation effect - were measured 1 h before, during, and after the laparoscopic operation. RESULTS. In the IPC group, plasma F1+2 and TAT levels increased significantly during and after laparoscopic gastrofundoplication. In the IPC+LMWH group, F1+2 and plasma TAT levels did not change during or after the operation. fTFPI levels significantly increased during and after the operation in the IPC+LMWH group; however, fTFPI levels did not change during or after the laparoscopic operation in the IPC group. CONCLUSIONS. A combination of low-molecular-weight heparin and intermittent pneumatic compression during laparoscopic fundoplication caused hypocoagulation effect in the patients, which was not observed in the patients who were treated with intermittent pneumatic compression alone.

Topics: Anticoagulants; Blood Coagulation; Blood Coagulation Factors; Coagulants; Enoxaparin; Female; Fibrinolytic Agents; Fundoplication; Humans; Informed Consent; Intermittent Pneumatic Compression Devices; Intraoperative Care; Laparoscopy; Male; Postoperative Care; Postoperative Complications; Prospective Studies; Statistics, Nonparametric; Thromboplastin; Venous Thrombosis

With the best prophylactics now available, venous thromboembolism after total knee replacement remains substantial (25% to 27%). Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent inhibitor of factor VIIa/tissue factor complex that has the potential to reduce this risk. The present study was performed to determine an efficacious and safe dose of rNAPc2 for prevention of venous thromboembolism after elective, unilateral total knee replacement.. This open-label, sequential dose-ranging study was conducted in 11 centers in Canada, Europe, and the United States. Five regimens were tested. Injections were administered subcutaneously on the day of surgery (day 1) and days 3, 5, and optionally, day 7. Primary efficacy outcome was a composite of overall deep vein thrombosis based on mandatory unilateral venography (day 7+/-2) and confirmed symptomatic venous thromboembolism recorded

Topics: Aged; Anticoagulants; Arthroplasty, Replacement, Knee; Canada; Dose-Response Relationship, Drug; Europe; Factor VIIa; Female; Helminth Proteins; Hemorrhage; Humans; Injections, Subcutaneous; Logistic Models; Male; Odds Ratio; Postoperative Complications; Risk Assessment; Survival Rate; Thromboplastin; United States; Venous Thrombosis

Deep venous thrombosis (DVT) is one of the major health problems worldwide. Apolipoprotein L domain containing 1 (APOLD1) was reported to be downregulated in DVT. The present study intended to investigate whether APOLD1 affects thrombus formation in a rat model of DVT.. The rat model of DVT was established by inferior vena cava (IVC) stenosis. At 6 hr, 12 hr, 24 hr, and 48 hr after IVC stenosis, the gross IVC with thrombus was dissected and observed. Then, the rats were preinjected with the lentiviral overexpression vector, APOLD1-LVs, 1 hr before IVC stenosis, to evaluate the influence of APOLD1 on thrombosis in rats. The serum levels of D-dimer and TAT as well as the content of TF in IVC tissues were detected by enzyme-linked immunosorbent assay (ELISA).. IVC stenosis resulted in thrombus formation in rats, increased serum levels of D-dimer and TAT, and decreased APOLD1 expression. APOLD1 overexpression inhibited in vivo thrombosis, reduced serum levels of D-dimer, and downregulated tissue factor (TF) activity and level. APOLD1 overexpression also increased p-PI3K and p-Akt protein levels.. APOLD1 suppresses thrombus formation in a rat model of DVT via downregulating TF expression by activating the PI3K/Akt pathway.

Topics: Animals; Apolipoprotein L1; Constriction, Pathologic; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Thromboplastin; Thrombosis; Treatment Outcome; Vena Cava, Inferior; Venous Thrombosis

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

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

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

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

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

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

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

Venous thromboembolism (VTE) is a common and severe complication of joint arthroplasty. Microparticles (MPs) containing phosphatidylserine (PS) and tissue factor (TF) can trigger coagulation in VTE. This study aims to measure and compare MP levels in joint arthroplasty patients with and without VTE.. This prospective cohort study enrolled 181 patients who underwent joint arthroplasty. Ultrasound examination was used to diagnose VTE on preoperative day 0 and postoperative day 6. MPs were analysed using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and flow cytometry. The levels of platelet-derived microparticles (PMPs), endothelial cell-derived microparticles (EMPs), granulocyte-derived microparticles (GMPs), red cell-derived microparticles (RMPs), monocyte-derived microparticles (MMPs), Annexin V. The levels of AV. AV

Topics: Annexin A5; Arthroplasty; Biomarkers; Cell-Derived Microparticles; Humans; Phosphatidylserines; Prospective Studies; Thromboplastin; Venous Thromboembolism; Venous Thrombosis

Krüppel-like factors (KLFs) play essential roles in multiple biological functions, including maintaining vascular homeostasis. KLF11, a causative gene for maturity-onset diabetes of the young type 7, inhibits endothelial activation and protects against stroke. However, the role of KLF11 in venous thrombosis remains to be explored. Utilizing stasis-induced murine deep vein thrombosis (DVT) model and cultured endothelial cells (ECs), we identified an increase of KLF11 expression under prothrombotic conditions both in vivo and in vitro. The expression change of thrombosis-related genes was determined by utilizing gain- and loss-of-function approaches to alter KLF11 expression in ECs. Among these genes, KLF11 significantly downregulated tumor necrosis factor-α (TNF-α)-induced tissue factor (

Topics: Animals; Apoptosis Regulatory Proteins; Endothelial Cells; Humans; Mice; Mice, Knockout; Repressor Proteins; Thromboplastin; Thrombosis; Transcription Factors; Tumor Necrosis Factor-alpha; Venous Thrombosis

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

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

CD248 is a pro-inflammatory, transmembrane glycoprotein expressed by vascular smooth muscle cells (VSMC), monocytes/macrophages, and other cells of mesenchymal origin. Its distribution and properties are reminiscent of those of the initiator of coagulation, tissue factor (TF).. We examined whether CD248 also participates in thrombosis.. We evaluated the role of CD248 in coagulation using mouse models of vascular injury, and by assessing its functional interaction with the TF-factor VIIa (FVIIa)-factor X (FX) complex.. The time to ferric chloride-induced occlusion of the carotid artery in CD248 knockout (KO) mice was significantly longer than in wild-type (WT) mice. In an inferior vena cava (IVC) stenosis model of thrombosis, lack of CD248 conferred relative resistance to thrombus formation compared to WT mice. Levels of circulating cells and coagulation factors, prothrombin time, activated partial thromboplastin time, and tail bleeding times were similar in both groups. Proximity ligation assays revealed that TF and CD248 are <40 nm apart, suggesting a potential functional relationship. Expression of CD248 by murine and human VSMCs, and by a monocytic cell line, significantly augmented TF-FVIIa-mediated activation of FX, which was not due to differential expression or encryption of TF, altered exposure of phosphatidylserine or differences in tissue factor pathway inhibitor expression. Rather, conformation-specific antibodies showed that CD248 induces allosteric changes in the TF-FVIIa-FX complex that facilitates FX activation by TF-FVIIa.. CD248 is a newly uncovered protein partner and potential therapeutic target in the TF-FVIIa-FX macromolecular complex that modulates coagulation.

Topics: Animals; Antigens, CD; Antigens, Neoplasm; Factor VIIa; Humans; Mice; Mice, Knockout; Prothrombin Time; Thromboplastin; Venous Thrombosis

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

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

Bleeding disorders and thrombotic complications are major causes of morbidity and mortality with many cases being unexplained. Thrombus formation involves aberrant expression and activation of tissue factor (TF) in vascular endothelial and smooth muscle cells. Here, we sought to identify factors that modulate

Topics: Animals; Genetic Variation; Humans; LIM-Homeodomain Proteins; Mice; Muscle Proteins; Thromboplastin; Transcription Factors; Venous Thrombosis

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

Topics: Biomarkers; Cell-Derived Microparticles; Humans; Neoplasms; Postthrombotic Syndrome; Risk Factors; Thromboplastin; Venous Thrombosis

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

To explore the change of coagulation function and associated potential mechanisms and the relationship between coagulation disorders and deep venous thrombosis (DVT) after subarachnoid hemorrhage (SAH) within 3 days of onset.. A total of 150 patients with SAH within 3 days of onset and 100 healthy individuals were recruited. Thrombelastography analysis and traditional laboratory tests were performed. Tissue factor (TF), tissue factor pathway inhibitor (TFPI) and activated protein C (APC) were tested by using enzyme-linked immunoassay kits. Extremities of patients with SAH were scanned by Doppler ultrasonography. Subgroup analysis was performed in patients with SAH based on the presence or lack of DVT.. Compared with control groups, R (an indicator of coagulation factor function in thrombelastography) was significantly decreased (4.32 ± 0.99 minutes vs. 6.00 ± 0.75 minutes; P < 0.001), especially in patients with DVT. TF was significantly increased (20.84 ± 4.15 pg/mL vs. 5.24 ± 1.86 pg/mL; P < 0.001). TFPI was decreased (50.42 ± 5.81 ng/mL vs. 64.10 ± 6.04 ng/mL). APC had no apparent changes. R was negatively correlated with TF (r = 0.358; P < 0.05) and positively correlated with TFPI (r = 0.325; P < 0.05) and APC (r = 0.162; P < 0.05). TF, TFPI, and APC had the same variation characteristics in the DVT subgroup compared with the no DVT subgroup. DVT was associated with R through association analysis (r = 0.369; P < 0.05). The R cutoff value for estimating the presence of DVT was 3.65 minutes.. Coagulation factor hyperfunction may be mainly accompanied within 3 days of SAH onset and may induce DVT. This situation may be associated with TF-TFPI-APC imbalance. R = 3.65 minutes was a potential intervention point to prevent the risk of DVT in this population.

Topics: Coagulation Protein Disorders; Enzyme-Linked Immunosorbent Assay; Female; Humans; Lipoproteins; Male; Middle Aged; Prospective Studies; Protein C; ROC Curve; Subarachnoid Hemorrhage; Thrombelastography; Thromboplastin; Time Factors; Venous Thrombosis

Cancer patients with Tissue Factor (TF)-bearing MPs have been presented association with increased risk of venous thromboembolism (VTE), but results of these studies have not been consistent. We aimed to conduct a meta-analysis to assess the relationship between TF-bearing MPs and risk of VTE in patients with cancer. PubMed, Web of Science and EMBASE Databases were systematically retrieved up to1

Topics: Cell-Derived Microparticles; Humans; Neoplasms; Risk Assessment; Thromboplastin; Venous Thrombosis

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

The objective of this study was to measure the role of platelets and red blood cells on thrombus propagation in an in vitro model of venous valvular stasis.. A microfluidic model with dimensional similarity to human venous valves consists of a sinus distal to a sudden expansion, where for sufficiently high Reynolds numbers, 2 countercurrent vortices arise because of flow separation. The primary vortex is defined by the points of flow separation and reattachment. A secondary vortex forms in the deepest recess of the valve pocket characterized by low shear rates. An initial fibrin gel formed within the secondary vortex of a tissue factor-coated valve sinus. Platelets accumulated at the interface of the fibrin gel and the primary vortex. Red blood cells at physiological hematocrits were necessary to provide an adequate flux of platelets to support thrombus growth out of the valve sinus. A subpopulation of platelets that adhered to fibrin expose phosphatidylserine. Platelet-dependent thrombus growth was attenuated by inhibition of glycoprotein VI with a blocking Fab fragment or D-dimer.. A 3-step process regulated by hemodynamics was necessary for robust thrombus propagation: First, immobilized tissue factor initiates coagulation and fibrin deposition within a low flow niche defined by a secondary vortex in the pocket of a model venous valve. Second, a primary vortex delivers platelets to the fibrin interface in a red blood cell-dependent manner. Third, platelets adhere to fibrin, activate through glycoprotein VI, express phosphatidylserine, and subsequently promote thrombus growth beyond the valve sinus and into the bulk flow.

Topics: Blood Coagulation; Blood Flow Velocity; Blood Platelets; Erythrocytes; Fibrin; Hematocrit; Hemodynamics; Humans; Lab-On-A-Chip Devices; Microfluidic Analytical Techniques; Phosphatidylserines; Platelet Membrane Glycoproteins; Signal Transduction; Stress, Mechanical; Thromboplastin; Venous Thrombosis; Venous Valves

The complement and coagulation cascades interact at multiple levels in thrombosis and inflammatory diseases. In venous thrombosis, complement factor 3 (C3) is crucial for platelet and tissue factor (TF) procoagulant activation dependent on protein disulfide isomerase (PDI). Furthermore, C5 selectively contributes to the exposure of leukocyte procoagulant phosphatidylserine (PS), which is a prerequisite for rapid activation of monocyte TF and fibrin formation in thrombosis. Here, we show that monoclonal cofactor-independent antiphospholipid antibodies (aPLs) rapidly activate TF on myelomonocytic cells. TF activation is blocked by PDI inhibitor and an anti-TF antibody interfering with PDI binding to TF, and requires C3 but unexpectedly not C5. Other prothrombotic, complement-fixing antibodies, for example, antithymocyte globulin, typically induce TF activation dependent on C5b-7-mediated PS exposure on the outer membrane of monocytes. We show that aPLs directly induce procoagulant PS exposure independent of C5. Accordingly, mice deficient in C3, but not mice deficient in C5, are protected from in vivo thrombus formation induced by cofactor-independent aPLs. Only immunoglobulin G (IgG) fractions with cofactor-independent anticardiolipin reactivity from patients with antiphospholipid syndrome (APS) induce complement-independent monocyte PS exposure and PDI-dependent TF activation. Neither a human monoclonal aPL directed against β2-glycoprotein I (β2GPI) nor patient IgG with selective reactivity to β2GPI rapidly activated monocyte TF. These results indicate that inhibitors of PDI and TF, but not necessarily clinically available drugs targeting C5, have therapeutic benefit in preventing thrombosis associated with APS caused by pathogenic aPLs primarily reactive with lipid, independent of β2GPI.

Topics: Animals; Antibodies, Antiphospholipid; Blood Coagulation; Complement C3; Complement C5; Mice; Mice, Knockout; Monocytes; Thromboplastin; Venous Thrombosis

Deep vein thrombosis (DVT) is multifactorial disorder and well known to cause substantial morbidity and mortality. There is sparse data in the Asian population, particularly India regarding association of tissue factor (TF) gene single nucleotide polymorphisms (SNPs) with plasma TF levels in DVT. So, we analyzed the distribution of SNPs (603A>G and 5466A>G) in India, to evaluate their effect on TF levels in DVT patients. Plasma level and SNPs (603A>G and 5466A>G) of TF gene were screened in subjects (100 DVT patients and 100 controls). Patients had significantly higher TF levels than controls (patients: 84.95 ± 17.16 pg/ml, controls: 70.55 ± 15.87 pg/ml, p < 0.001). G allele of 603A>G polymorphism was significantly higher in patients than controls (patients: 40.5% controls: 27.5%, p = 0.004). Subjects with AG and GG genotype had significantly higher TF levels than AA genotype (p = 0.001). After multiple logistic regression analysis, risk of DVT was increased 1.398 fold (95% CI 0.738-2.651) and 4.41 fold (95% CI 1.404-13.884) with AG and GG genotype respectively. Allelic and genotypic frequencies of 5466A>G polymorphism was neither associated with TF levels nor with DVT. We found high TF level in patients with TF 603A>G polymorphism, which is an important predisposing factor in increasing risk of DVT in young Indians. Furthermore, GG genotype of 603A>G polymorphism augments the risk of thrombosis by 4.4 fold, thus highlighting the significance of this polymorphism in the development of DVT. So, we suggest that inclusion of 603A>G polymorphism in prothrombotic work-up may be helpful in making the treatment strategy in DVT patients.

Topics: Case-Control Studies; Genetic Predisposition to Disease; Genotype; Humans; India; Polymorphism, Single Nucleotide; Risk; Thromboplastin; Venous Thrombosis

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

Topics: Actin-Related Protein 2; Alleles; Animals; Factor V; Female; Gene Frequency; Genetic Predisposition to Disease; Genotype; Heterozygote; Humans; Lipoproteins; Male; Mice; Mutation; Phenotype; Polymorphism, Single Nucleotide; Risk Factors; Sequence Analysis, DNA; Thromboplastin; Thrombosis; Venous Thrombosis

Ovarian clear cell carcinoma (OCCC) and high grade serous ovarian cancer (HGSOC) are associated with the highest risk of VTE among patients with epithelial ovarian cancer (EOC). Tissue factor (TF) is a transmembrane glycoprotein which can trigger thrombosis. We sought to evaluate if there is an association between VTE and tumor expression of tissue factor (TF), plasma TF, and microvesicle TF (MV TF) activity in this high-risk population.. We performed a case-control study of OCCC and HGSOC patients with and without VTE. 105 patients who underwent surgery at a tertiary care center between January 1995 and October 2013 were included. Plasma TF was measured with an enzyme-linked immunosorbent assay. A TF-dependent Factor Xa generation assay was used to measure MV TF activity. Immunohistochemical (IHC) analysis was performed to evaluate tumor expression of TF.. 35 women with OCCC or HGSOC diagnosed with VTE within 9months of surgery were included in the case group. Those with VTE had a worse OS, p<0.0001, with a greater than three-fold increase in risk of death, HR 3.33 (CI 1.75-6.35). There was no significant difference in median plasma TF level or MV TF activity level between patients with and without VTE. OCCC patients had greater expression of TF in their tumors than patients with HGSOC, p<0.0001.. TFMV activity and plasma TF level were not predictive of VTE in this patient population. Given the extensive expression of TF in OCCC tumors, it is unlikely IHC expression will be useful in risk stratification for VTE in this population.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Ovarian Epithelial; Case-Control Studies; Enzyme Multiplied Immunoassay Technique; Female; Humans; Immunohistochemistry; Middle Aged; Neoplasm Staging; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Survival Analysis; Thromboplastin; Venous Thrombosis

Non-muscular myosin heavy chain IIA (NMMHC IIA) plays a key role in tissue factor expression and venous thrombosis. Natural products might inhibit thrombosis through effects on NMMHC IIA. Here, we have shown that a natural saponin, D39, from Liriope muscari exerted anti-thrombotic activity in vivo, by targeting NMMHC IIA.. Expression and activity of tissue factor in endothelial cells were analysed in vitro by Western blot and simplified chromogenic assays. Interactions between D39 and NMMHC IIA were assessed by serial affinity chromatography and molecular docking analysis. D39-dependent interactions between NMMHC IIA and TNF receptor 2 (TNFR2) were measured by immunofluorescence, co-immunoprecipitation and proximity ligation assays. Anti-thrombotic activity of D39 in vivo was evaluated with a model of inferior vena cava ligation injury in mice.. D39 inhibited tissue factor expression and procoagulant activities in HUVECs and decreased thrombus weight in inferior vena cava-ligated mice dose-dependently. Serial affinity chromatography and molecular docking analysis suggested that D39 bound to NMMHC IIA. In HEK293T cells, D39 inhibited tissue factor expression evoked by NMMHC IIA overexpression. This effect was blocked by NMMHC IIA knockdown in HUVECs. D39 inhibited dissociation of NMMHC IIA from TNFR2, which subsequently modulated the Akt/GSK3β-NF-κB signalling pathways.. D39 inhibited tissue factor expression and thrombus formation by modulating the Akt/GSK3β and NF-κB signalling pathways through NMMHC IIA. We identified a new natural product that targeted NMMHC IIA, as a potential treatment for thrombotic disorders and other vasculopathies.

Topics: Animals; Cells, Cultured; Fibrinolytic Agents; Glycogen Synthase Kinase 3 beta; HEK293 Cells; Human Umbilical Vein Endothelial Cells; Humans; Male; Mice, Inbred C57BL; Molecular Docking Simulation; Myosin Heavy Chains; Proto-Oncogene Proteins c-akt; Receptors, Tumor Necrosis Factor, Type II; Saponins; Thromboplastin; Transcription Factor RelA; Tumor Necrosis Factor-alpha; Venous Thrombosis

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

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

Topics: Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Humans; Leukocytes; Thromboplastin; Thrombosis; Venous Thrombosis

Danhong huayu koufuye (DHK) has traditionally been used clinically for a long time in China. This study was to evaluate the effect of DHK in treating deep vein thrombosis (DVT) in rats and explore its possible mechanism.. Forty-eight Sprague-Dawley rats were divided into four groups, performed with incomplete inferior vena cava ligation to induce DVT, and orally administered with DHK (3.20 mg/kg/d), warfarin (2.00 mg/kg/d), or vehicle for 7 days. The involved inferior vena cava and thrombi were collected and measured in size. The tissue specimens were performed for routine histopathologic evaluation and immunohistochemical staining with tissue factor and matrix metalloproteinases-9. Blood samples were collected for detecting coagulation function, blood cell count, and the levels of interleukin-6 and tumor necrosis factor-α.. The treatment of DHK markedly reduced the size of thrombi by 49.26%, and the vein wall thickness by 47.86%. The recanalization rate was significant higher in the DHK-treated group than the vehicle-treated group (26.34 ± 6.53% versus 15.91 ± 3.93%, P < 0.01). Comparing to vehicle control, DHK significantly reduced neutrophils (P < 0.05) and lymphocytes (P < 0.05), serum tumor necrosis factor-α level (4.90 ± 1.14 pg/mL versus 6.60 ± 1.62 pg/mL, P < 0.01), and the expression of matrix metalloproteinases-9 and tissue factor (P < 0.05) in thrombi.. DHK effectively prevented DVT through anti-inflammatory action in rats.

Topics: Animals; Blood Cell Count; Blood Coagulation; Drug Evaluation, Preclinical; Drugs, Chinese Herbal; Interleukin-6; Male; Matrix Metalloproteinase 9; Random Allocation; Rats, Sprague-Dawley; Thromboplastin; Tumor Necrosis Factor-alpha; Vena Cava, Inferior; Venous Thrombosis

Topics: Bacteria; Endotoxemia; Endotoxins; Factor VIII; Gene Expression Regulation; Humans; Liver; Liver Cirrhosis; Nitric Oxide; Portal Vein; RNA, Messenger; Signal Transduction; Thromboplastin; Venous Thrombosis; von Willebrand Factor

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

Essentials Activated protein C (APC) resistance is a prevalent risk factor for venous thrombosis. A novel missense mutation (Ala512Val - FVBonn ) was characterized in vitro and in silico. FVBonn is a new cause of APC resistance and venous thrombosis. FVBonn expresses additionally enhanced procoagulant activity in the absence of APC.. Background Activated protein C (APC) resistance is a prevalent risk factor for venous thrombosis. This phenotype is most commonly associated with the factor V Arg506Gln mutation (FV Leiden), which impairs the APC-mediated inactivation of both activated FV (FVa) and activated FVIII (FVIIIa). Objectives Here, we report the identification and characterization of a novel FV mutation (Ala512Val, FVBonn ) in six patients with APC resistance and venous thrombosis or recurrent abortions. Methods FVBonn was expressed in a recombinant system and compared with recombinant wild-type (WT) FV and FV Leiden in several functional assays. Results FVBonn conferred APC resistance to FV-depleted plasma, both in the activated partial thromboplastin time (APTT)-based test (APC sensitivity ratio [APCsr] of 1.98 for FVBonn versus 4.31 for WT FV and 1.59 for FV Leiden) and in the thrombin generation-based test (normalized APCsr of 5.41 for FVBonn versus 1.00 for WT FV and 8.99 for FV Leiden). The APC-mediated inactivation of FVaBonn was slower than that of WT FVa (mainly because of delayed cleavage at Arg506), but was greatly stimulated by protein S. The APC cofactor activity of FVBonn in FVIIIa inactivation was ~ 24% lower than that of WT FV. In line with these findings, an in silico analysis showed that the Ala512Val mutation is located in the same loop as the Arg506 APC cleavage site and might hamper its interaction with APC. Moreover, FVBonn was more procoagulant than WT FV and FV Leiden in the absence of APC, because of an increased activation rate and, possibly, an enhanced interaction with activated FX. Conclusions FVBonn induces hypercoagulability via a combination of increased activation/procoagulant activity, decreased susceptibility to APC-mediated inactivation, and slightly reduced APC cofactor activity.

Topics: Abortion, Habitual; Activated Protein C Resistance; Adult; Aged; Blood Coagulation; Blood Coagulation Tests; Catalysis; Coagulants; Cohort Studies; Factor V; Factor Va; Factor VIIIa; Female; Humans; Male; Mutation; Mutation, Missense; Partial Thromboplastin Time; Pregnancy; Protein C; Thrombin; Thromboplastin; Venous Thrombosis; Young Adult

Thalidomide (Thal) treatment of patients with multiple myeloma (MM) is associated with vascular thrombosis, but the underlying mechanism is unknown.. To evaluate the hypothesis that Thal, dexamethasone (Dex) and MM serum perturb the hemostatic balance on human umbilical vein endothelial cells (HUVECs).. Drugs with or without the serum of MM patients or healthy controls were incubated with HUVECs. Analyses of phosphatidylserine (PS), tissue factor (TF), endothelial protein C receptor (EPCR) and thrombomodulin (TM) were performed using flow cytometry. The production of thrombin and activated protein C (APC) were measured by chromogenic assay. The roles of IL-6 and TNFα in regulating these indicators were also investigated.. We found that Thal or Dex alone could not increase TF and PS expression in HUVECs. However, when pretreated with MM serum, their expression was significantly increased by either Thal or Dex. Concurrent changes were also detected in thrombin generation. In contrast, Thal and Dex had a direct inhibitory effect on the expression of EPCR and TM, and this inhibitory effect was especially significant when MM serum was added. The generation of APC paralleled the expression of EPCR and TM. All of the above outcomes were reversed to a variable extent by anti-IL-6R and anti-TNFα antibodies.. These findings suggest Thal may act as a procoagulant by altering the balance between procoagulant and anticoagulant proteins on the surface of HUVECs, thereby contributing to thrombogenesis. MM serum plays a synergistic role in this process.

Topics: Adult; Angiogenesis Inhibitors; Antigens, CD; Coagulants; Dexamethasone; Endothelial Cells; Endothelial Protein C Receptor; Female; Hemostasis; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-6; Male; Middle Aged; Multiple Myeloma; Phosphatidylserines; Receptors, Cell Surface; Thalidomide; Thrombomodulin; Thromboplastin; Tumor Necrosis Factor-alpha; Venous Thrombosis

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

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

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

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

Topics: Animals; Carcinoma, Pancreatic Ductal; Cell-Derived Microparticles; Male; Pancreatic Neoplasms; Thromboplastin; Venous Thrombosis

Several studies highlight the role of inflammatory markers in thrombosis as well as in cancer. However, their combined role in cancer-associated deep vein thrombosis (DVT) and the molecular mechanisms, involved in its pathophysiology, needs further investigations. In the present study, C-reactive protein, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1β), matrix metalloproteases-9 (MMP-9), vascular endothelial growth factor (VEGF), tissue factor (TF), fibrinogen and soluble P-selectin, were analyzed in plasma and in monocyte samples from 385 cancer patients, of whom 64 were concomitantly affected by DVT (+). All these markers were higher in cancer patients DVT+ than in those DVT-. Accordingly, significantly higher NF-kB activity was observed in cancer patients DVT+ than DVT-. Significant correlation between data obtained in plasma and monocyte samples was observed. NF-kB inhibition was associated with decreased levels of all molecules in both cancer DVT+ and DVT-. To further demonstrate the involvement of NF-kB activation by the above mentioned molecules, we treated monocyte derived from healthy donors with a pool of sera from cancer patients with and without DVT. These set of experiments further suggest the significant role played by some molecules, regulated by NF-kB, and detected in cancer patients with DVT. Our data support the notion that NF-kB may be considered as a therapeutic target for cancer patients, especially those complicated by DVT. Treatment with NF-kB inhibitors may represent a possible strategy to prevent or reduce the risk of DVT in cancer patients.

Topics: Aged; Biomarkers; C-Reactive Protein; Female; Fibrinogen; Humans; Interleukin-1beta; Interleukin-6; Male; Matrix Metalloproteinase 9; Middle Aged; Monocytes; Neoplasms; NF-kappa B; Smoking; Thromboplastin; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; Venous Thrombosis

Recent studies have shown an association between thrombosis and factor VII (FVII), tissue factor (TF), and angiotensin-converting enzyme (ACE). This suggests that individuals with FVII-402 G/A, FVII-401 G/T, TF+5466 A/G, and ACE-287 insertion/deletion (I/D) polymorphisms present an increased risk of venous thrombosis, heart disease, and ischemic stroke compared with controls. In this study, we investigated the frequencies of these polymorphisms and their association with arterial and venous thrombosis. For the FVII-402 G/A polymorphism, there were 57.3% heterozygote (HT) genotypes and 8.3% homozygote (HM) genotypes in the patients, and 45.2% HT genotypes and 15.4% HM genotypes in the controls. For the FVII-401 G/T polymorphism, there were 37.5% HT genotypes and 3.1% HM genotypes in the patients, and 32.7% HT genotypes and 4.8% HM genotypes in the controls. The polymorphism TF+5466 A/G was not found in any of the samples analyzed. For the ACE-287 I/D polymorphism, there were 43 (40.6%) HT genotypes and 63 (59.4%) HM genotypes in the controls and 28 (45.2%) HT genotypes and 34 (54.8%) HM genotypes in the patients. No significant difference was observed by comparing patients and controls. In this study, no association was found between the presence of the evaluated polymorphisms and the occurrence of thrombotic events.

Topics: Adolescent; Adult; Alleles; Case-Control Studies; Child; Factor VII; Female; Gene Frequency; Genetic Association Studies; Genetic Predisposition to Disease; Genotype; Humans; Male; Middle Aged; Peptidyl-Dipeptidase A; Polymorphism, Genetic; Polymorphism, Single Nucleotide; Risk Factors; Thromboplastin; Thrombosis; Venous Thrombosis; Young Adult

Topics: Animals; Humans; Thromboplastin; Venous Thrombosis

Bradykinin has been shown to exert a variety of protective effects against vascular injury, and to reduce the levels of several factors involved in the coagulation cascade. A key determinant of thrombin generation is tissue factor (TF). However, whether bradykinin can regulate TF expression remains to be investigated.. To study the effect of bradykinin on TF expression, we used Lipopolysaccharides (LPS) to induce TF expression in human umbilical vein endothelial cells and monocytes. Transcript levels were determined by RT-PCR, protein abundance by Western blotting. In the in vivo study, bradykinin and equal saline were intraperitoneally injected into mice for three days ahead of inferior cava vein ligation that we took to induce thrombus formation, after which bradykinin and saline were injected for another two days. Eventually, the mice were sacrificed and tissues were harvested for tests.. Exogenous bradykinin markedly inhibited TF expression in mRNA and protein level induced by LPS in a dose-dependent manner. Moreover, the NO synthase antagonist L-NAME and PI3K inhibitor LY294002 dramatically abolished the inhibitory effects of bradykinin on tissue factor expression. PI3K/Akt signaling pathway activation induced by bradykinin administration reduced the activity of GSK-3β and MAPK, and reduced NF-x03BA;B level in the nucleus, thereby inhibiting TF expression. Consistent with this, intraperitoneal injection of C57/BL6 mice with bradykinin also inhibited the thrombus formation induced by ligation of inferior vena cava.. Bradykinin suppressed TF protein expression in human umbilical vein endothelial cells and monocytes in vitro; in line with this, it inhibits thrombus formation induced by ligation of inferior vena cava in vivo.

Topics: Animals; Bradykinin; Chromones; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Human Umbilical Vein Endothelial Cells; Humans; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Monocytes; Morpholines; NF-kappa B; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type III; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Receptor, Bradykinin B2; RNA, Messenger; Signal Transduction; Thromboplastin; Venous Thrombosis

Topics: Female; Genetic Variation; Hemostasis; Humans; Male; Plasma; Thromboplastin; Venous Thrombosis

Topics: Female; Genetic Variation; Hemostasis; Humans; Male; Plasma; Thromboplastin; Venous Thrombosis

Topics: Female; Genetic Variation; Hemostasis; Humans; Male; Plasma; Thromboplastin; Venous Thrombosis

C-reactive protein (CRP) promotes tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) expression in vitro, and an elevated plasma CRP concentration is associated with an increased risk of vein graft (VG) thrombosis after coronary artery bypass surgery. However, little is known about the effects of CRP on VG TF and PAI-1 expression in vivo, or on VG thrombosis.. We studied transgenic (Tg) mice expressing human CRP in a VG model to explore in vivo cause-and-effect relationships between CRP and TF, PAI-1, and VG thrombosis.. Vein segments from wild-type (WT) and CRP-Tg donors were transplanted into carotid arteries of WT and CRP-Tg recipients. VGs were analyzed 1-4 weeks later.. Human CRP accumulated in VGs during the first 4 weeks after surgery, but appeared to originate exclusively from systemic sources, rather than local production. Human CRP significantly increased TF gene expression, protein concentration and activity in VGs. Human CRP also increased PAI-1 concentrations in VGs, although only in vascular endothelial cells. Human CRP stimulated macrophage migration, invasion into VGs, and TF expression. Fibrin deposition was significantly greater in VGs of CRP-Tg mice than in WT controls.. CRP accumulates in VGs early after surgery, originating from systemic sources rather than local synthesis. Human CRP promotes TF and PAI-1 expression in VGs, although with different expression patterns. Human CRP stimulates macrophage invasion and fibrin deposition within VGs. These results suggest that CRP induces pathologic changes in VGs that contribute to early VG occlusion.

Topics: Animals; C-Reactive Protein; Cell Movement; Chlorides; Coronary Artery Bypass; Ferric Compounds; Fibrin; Humans; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Plasminogen Activator Inhibitor 1; Recombinant Proteins; Thromboplastin; Transgenes; Veins; Venous Thrombosis

Tissue factor (TF) is the main in-vivo initiator of blood coagulation. Microparticles (MPs) are small procoagulant membrane vesicles. Elevated TF-bearing MPs have been found in different prothrombotic conditions and MP-associated TF activity may contribute to the pathogenesis of unprovoked deep vein thrombosis (DVT).. To determine MP-TF activity levels at diagnosis of DVT and at four additional time points during the course of one year in a well-defined group of patients with unprovoked DVT of the lower limb.. In this study, 41 patients with acute unilateral symptomatic and unprovoked DVT of the lower limb were included and followed for 1 year. Venous blood samples for determination of MP-TF activity were drawn at diagnosis of acute DVT, and 1-, 3-, 6-, and 12 months later. In addition, 10 young and healthy control subjects were included.. The median MP-TF activity was 0.06 pg/mL (25th-75th percentile: 0.0-0.53) in patients with acute DVT and 0.18 pg/mL (0.07-0.33) in healthy controls, and did not differ significantly (p=0.35). No significant changes in MP-TF activity were found in the follow-up measurements. MP-TF activity did also not differ significantly between patients with proximal- or distal DVT and between those with- or without residual DVT after 6 months.. MP-TF activity is low at the acute event in patients with unprovoked DVT of the lower limb and remains unchanged during the course of the disease. Our data do not support the hypothesis that TF-bearing MPs play a determining role in the pathogenesis of unprovoked DVT.

Topics: Acute Disease; Adult; Blood Coagulation; Cell-Derived Microparticles; Female; Humans; Male; Middle Aged; Prospective Studies; Thromboplastin; Venous Thrombosis

Elevated plasma levels of factor VIII (FVIII) are associated with increased risk of deep venous thrombosis. The aim of this study is to elucidate how elevated FVIII levels affect venous thrombus formation and propagation in vivo. We examined rabbit plasma FVIII activity, plasma thrombin generation, whole blood coagulation, platelet aggregation and venous wall thrombogenicity before and one hour after an intravenous infusion of recombinant human FVIII (rFVIII). Venous thrombus induced by the endothelial denudation of rabbit jugular veins was histologically assessed. Thrombus propagation was evaluated as indocyanine green fluorescence intensity. Argatroban, a thrombin inhibitor, and neutralised antibodies for tissue factor (TF), factor XI (FXI), and von Willebrand factor (VWF) were infused before or after thrombus induction to investigate their effects on venous thrombus formation or propagation. Recombinant FVIII (100 IU/kg) increased rabbit plasma FVIII activity two-fold and significantly enhanced whole blood coagulation and total plasma thrombin generation, but did not affect initial thrombin generation time, platelet aggregation and venous wall thrombogenicity. The rFVIII infusion also increased the size of venous thrombus 1 hour after thrombus induction. Argatroban and the antibodies for TF, FXI or VWF inhibited such enhanced thrombus formation and all except TF suppressed thrombus propagation. In conclusion, elevated plasma FVIII levels enhance venous thrombus formation and propagation. Excess thrombin generation by FXI and VWF-mediated FVIII recruitment appear to contribute to the growth of FVIII-driven venous thrombus.

Topics: Animals; Antibodies, Blocking; Arginine; Blood Platelets; Factor VIII; Factor XI; Hemostasis; Humans; Jugular Veins; Male; Pipecolic Acids; Platelet Aggregation; Rabbits; Sulfonamides; Thrombin; Thromboplastin; Venous Thrombosis; von Willebrand Factor

Ovarian cancer is known to display a particular association with venous thromboembolism (VTE) with reports up to 42% of patients developing thromboembolic complications. Tissue Factor (TF) and its inhibitor Tissue Factor Pathway Inhibitor (TFPI) have been implicated in VTE risk in cancer. The aim of this study was to measure tumour derived TF and TFPI and to investigate their potential role in VTE in ovarian cancer patients.. TF and TFPI mRNA expression was measured using TaqMan real time PCR in 99 ovarian tumour samples. Nineteen cases complicated by VTE were matched to 19 cases without VTE. TF and TFPI protein levels were measured using ELISA and immunohistochemistry was used to localize TF expression. The role of TF expression on overall survival was also determined.. TF mRNA and protein expression was increased in tumours from patients with clear cell carcinoma (p<0.001). TF protein expression was also increased in endometroid carcinoma (P<0.01) compared with benign tumours. TFPI mRNA expression was increased in clear cell carcinoma (P<0.01). TF mRNA and antigen level was increased in malignant tumours of patients who developed VTE compared with matched malignant õtumours of patients who remained thrombosis free (P<0.01). There was no difference in TFPI expression between the two groups.. TF expression in ovarian cancer is significantly higher in patients who develop VTE. TF expression was increased in clear cell ovarian cancer and endometroid cancer and this may explain the higher risk of VTE in these subgroups. TF derived from these tumours may be the trigger for VTE in ovarian cancer.

Topics: Aged; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lipoproteins; Middle Aged; Ovarian Neoplasms; Ovary; RNA, Messenger; Thromboplastin; Up-Regulation; Venous Thrombosis

Circulating tissue factor positive microparticles (MPTF) were reported in a wide range of diseases with thrombotic tendency. Though D-dimer assay had a high negative predictive value for deep venous thrombosis (DVT) recurrence, there are currently no reliable positive predictors for recurrent DVT. We therefore quantified MPTF in patients with acute recurrent DVT to determine whether MPTF levels could be used to predict recurrent DVT.. Microparticles (MPs) were isolated from plasma of initial DVT patients (n=25), recurrent DVT patients (n=25) and sex- and age-matched healthy individuals (n=25), stained with annexin V, cell-specific monoclonal antibodies (MoAbs) and a MoAb directed against tissue factor (TF), and analyzed by flow cytometry. We also determined the plasma procoagulant activity with a Human TF Chromogenic Activity Assay Kit.. We found total MPTF to be elevated in recurrent DVT patients versus normal individuals (P=0.001). The number of monocyte-derived MPTF in both initial and recurrent DVT was higher than in normal individuals (P<0.01, respectively). The platelet and endothelial cell derived MPTF in recurrent DVT were significantly increased relative to other MPTF (P<0.05), although there was no difference between initial DVT patients and normal individuals. We demonstrated elevated procoagulant activity of platelet-free plasma in DVT patients relative to normal individuals, and a positive correlation with MPTF.. The elevated MPTF could be a potentially predictor for DVT recurrence. Further studies are needed to validate its sensitivity and specificity.

Topics: Acute Disease; Adult; Blood Cells; Cell-Derived Microparticles; Coagulants; Female; Humans; Male; Middle Aged; Prognosis; Thromboplastin; Venous Thrombosis

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

Venous thrombosis (VT), a major cause of venous obstruction, is a rather rare but dangerous complication of pacemaker (PM) implantation.. To assess the prognostic value of selected proinflammatory and prothrombotic markers in predicting symptomatic venous obstruction after PM implantation.. The study involved 81 patients (31 females; mean age 71 ± 8 years) divided into 2 groups depending on the occurrence of venous obstruction. Group I included 71 patients (29 females; mean age 71 ± 2 years) without this complication and group II included 10 patients (2 females, mean age 71.6 ± 2) with venous obstruction. All patients were followed up for 19 months. Transthoracic echocardiography and venous ultrasonography were performed before PM implantation and at the time of incident venous obstruction. Interleukin-6 (IL-6), high-sensitivity C-reactive protein (hsCRP), D-dimer, fibrinogen, tissue factor (TF), factor VII and plasminogen activator inhibitor-1 (PAI-1) levels were measured at baseline and within 7 days after PM implantation.. Patients in group II had a significantly lower left ventricular ejection fraction (LVEF), higher left ventricular end--diastolic diameter (LVEDD) and impaired left ventricular filling (Vp) compared to group I. Patients in group II developed VT on average at 13.06 (range 7-18) months following PM implantation. At baseline, IL-6, hsCRP, D-dimer, fibrinogen, TF, factor VII, and PAI-1 levels were significantly higher in group II compared to group I. In all patients, levels of prothrombotic factors were higher following PM implantation compared to baseline values, with the exception of fibrinogen level in group I. Cut-off values indicating increased thrombosis risk were determined for the examined parameters (LVEF, LVEDD, Vp, IL-6, hsCRP, D-dimer, fibrinogen, factor VII, TF, PAI-1) based on the ROC curves. Major predictors of thrombotic risk included LVEF, LVEDD, and D-dimer, fibrinogen and TF levels. Highest predictive values were noted for LVEDD > 58 mm (OR = 52.8) and D-dimer level > 498 mg/L (OR = 3003).. 1. Patients who developed VT after PM implantation had elevated baseline levels of IL-6, hsCRP, fibrinogen, D-dimer, TF, factor VII, and PAI-1. 2. Levels of pro-inflammatory markers increased after the implantation procedure in all patients. 3. Parameters with the highest diagnostic value for predicting incident VT included decreased LVEF, increased LVEDD and elevated D-dimer, fibrinogen and TF levels.

Topics: Aged; Biomarkers; C-Reactive Protein; Female; Fibrin Fibrinogen Degradation Products; Fibrinogen; Humans; Incidence; Interleukin-6; Male; Middle Aged; Pacemaker, Artificial; Plasminogen Activator Inhibitor 1; Risk Factors; ROC Curve; Thromboplastin; Time Factors; Venous Thrombosis

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

Deep vein thrombosis (DVT) is a major cause of cardiovascular death. The sequence of events that promote DVT remains obscure, largely as a result of the lack of an appropriate rodent model. We describe a novel mouse model of DVT which reproduces a frequent trigger and resembles the time course, histological features, and clinical presentation of DVT in humans. We demonstrate by intravital two-photon and epifluorescence microscopy that blood monocytes and neutrophils crawling along and adhering to the venous endothelium provide the initiating stimulus for DVT development. Using conditional mutants and bone marrow chimeras, we show that intravascular activation of the extrinsic pathway of coagulation via tissue factor (TF) derived from myeloid leukocytes causes the extensive intraluminal fibrin formation characteristic of DVT. We demonstrate that thrombus-resident neutrophils are indispensable for subsequent DVT propagation by binding factor XII (FXII) and by supporting its activation through the release of neutrophil extracellular traps (NETs). Correspondingly, neutropenia, genetic ablation of FXII, or disintegration of NETs each confers protection against DVT amplification. Platelets associate with innate immune cells via glycoprotein Ibα and contribute to DVT progression by promoting leukocyte recruitment and stimulating neutrophil-dependent coagulation. Hence, we identified a cross talk between monocytes, neutrophils, and platelets responsible for the initiation and amplification of DVT and for inducing its unique clinical features.

Topics: Animals; Blood Platelets; Cell Communication; Factor XII; Mice; Mice, Inbred C57BL; Monocytes; Neutrophils; P-Selectin; Thromboplastin; Venous Thrombosis

Cancer patients often have an activated clotting system and are at increased risk for venous thrombosis. In the present study, we analyzed tissue factor (TF) expression in 4 different human pancreatic tumor cell lines for the purpose of producing derivative tumors in vivo. We found that 2 of the lines expressed TF and released TF-positive microparticles (MPs) into the culture medium. The majority of TF protein in the culture medium was associated with MPs. Only TF-positive cell lines activated coagulation in nude mice, and this activation was abolished by an anti-human TF Ab. Of the 2 TF-positive lines, only one produced detectable levels of human MP TF activity in the plasma when grown orthotopically in nude mice. Surprisingly, < 5% of human TF protein in plasma from tumor-bearing mice was associated with MPs. Mice with TF-positive tumors and elevated levels of circulating TF-positive MPs had increased thrombosis in a saphenous vein model. In contrast, we observed no difference in thrombus weight between tumor-bearing and control mice in an inferior vena cava stenosis model. The results of the present study using a xenograft mouse model suggest that tumor TF activates coagulation, whereas TF on circulating MPs may trigger venous thrombosis.

Topics: Animals; Blood Coagulation; Cell Line, Tumor; Cell-Derived Microparticles; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Hemostasis; Humans; Mice; Mice, Nude; Pancreatic Neoplasms; RNA, Messenger; Thromboplastin; Venous Thrombosis

Venous thrombosis is a leading cause of morbidity and mortality in industrialized countries, especially in the elderly. Many risk factors have been identified for venous thrombosis that alter blood flow, activate the endothelium, and increase blood coagulation. However, the precise mechanisms that trigger clotting in large veins have not been fully elucidated. The most common site for initiation of the thrombus appears to be the valve pocket sinus, due to its tendency to become hypoxic. Activation of endothelial cells by hypoxia or possibly inflammatory stimuli would lead to surface expression of adhesion receptors that facilitate the binding of circulating leukocytes and microvesicles. Subsequent activation of the leukocytes induces expression of the potent procoagulant protein tissue factor that triggers thrombosis. Understanding the mechanisms of venous thrombosis may lead to the development of new treatments.

Topics: Animals; Blood Coagulation; Endothelium; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Leukocytes; Regional Blood Flow; Risk Factors; Thromboplastin; Veins; Venous Thromboembolism; Venous Thrombosis; Venous Valves

Rivaroxaban is an oral anticoagulant that directly targets both free factor Xa and factor Xa in complex with its protein cofactor, factor Va, in the prothrombinase complex. It is approved in the United States for the prophylaxis of deep vein thrombosis and stroke in patients with atrial fibrillation; however, it also carries a black box warning regarding the risk of thrombosis after discontinuation of treatment. The purpose of this study was to determine the degree to which rivaroxaban, over a range of physiologically relevant free plasma concentrations, inhibits preassembled prothrombinase at a typical venous shear rate (100 s(-1)) and to determine the dynamics of rivaroxaban washout.. Prothrombinase was assembled on phospholipid-coated glass capillaries. Its activity was characterized with respect to the activation of prothrombin (mean plasma concentration, 1.4 μmol/L) in the absence and presence of rivaroxaban (2, 5, and 10 nmol/L). The degree of inactivation of preassembled prothrombinase is sensitive to the solution-phase rivaroxaban concentration; however, prothrombinase unmasking upon removal of rivaroxaban is concentration independent.. The model system presented suggests that when rivaroxaban plasma concentrations decrease after cessation of therapy, there will be an unmasking of thrombus-associated prothrombinase that may be related to the reported rebound phenomena.

Topics: Administration, Oral; Anticoagulants; Blood Flow Velocity; Dose-Response Relationship, Drug; Factor V; Factor Va; Factor Xa; Humans; Models, Biological; Morpholines; Regional Blood Flow; Risk Factors; Rivaroxaban; Thiophenes; Thromboplastin; Venous Thrombosis

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

Circulating tissue factor (TF) is associated with inflammation and may contribute to thrombotic events. Aim of this study was to analyze circulating TF activity and proinflammatory cytokines in patients with deep venous thrombosis.. Forty-eight patients with deep vein thrombosis and 45 control subjects were included. Venous blood samples were obtained at diagnosis for analysis of TF activity, TF antigen, prothrombin fragment F1 + 2, microparticles (expressing phosphatidylserine and supporting FXa generation), Interleukin (IL)-1beta, IL-6, IL-8, IL-10, IL-12 and tumor-necrosis-factor-alpha (TNF).. TF antigen, activity and microparticles were similar in both groups: In contrast, a significant increase in plasma IL-6, IL-8 and F1 + 2 levels was found in thrombosis. This increase in IL-6 and IL-8 as well as F1 + 2 was not correlated with the extent of thrombosis, predisposing factors or onset of symptoms.. Circulating TF and microparticles are not elevated in deep venous thrombosis. The increase in IL-6, IL-8 and F1 + 2 during thrombosis was not proportional to the extent or predisposing risk factors.

Topics: Adult; Aged; Biomarkers; Case-Control Studies; Cell-Derived Microparticles; Cytokines; Female; Germany; Humans; Inflammation Mediators; Linear Models; Male; Middle Aged; Risk Assessment; Risk Factors; Thromboplastin; Venous Thrombosis

Combining thalidomide (Thal) with chemotherapeutic agents or steroid preparations led to improved response rates in the treatment of multiple myeloma. However, deep vein thrombosis (DVT) is one of the most serious side-effects noted with this regimen, and how a Thal-based regimen causes DVT is unclear. We investigated the procoagulant effects of Thal when combined with chemotherapeutic agents in vitro, focusing on tissue factor (TF) and phosphatidylserine. We examined the effects of the chemotherapeutic doxorubicin hydrochloride (Dox) and the steroid dexamethasone (Dex), with or without Thal. Our study used the human vascular endothelial, monocytic, and myeloma cell lines, EAhy926, THP-1, and RPMI8226, respectively. In EAhy926 and THP-1, Dex treatment increased expression of TF, which may induce procoagulant activity (PCA). Upregulation of TF mRNA correlated with activation of the Egr-1 pathway. In Thal and Dex treatments, the increase of PCA induction from phosphatidylserine exposure was modest. In contrast, Dox and Thal-Dox increased phosphatidylserine exposure in both cell types. In THP-1 cells, cell surface phosphatidylserine exposure correlated with increased PCA by Dox. Thal alone showed a modest increase in phosphatidylserine exposure in endothelial cells and monocytes. When Thal is given in combination with chemotherapies or Dex, endothelial cell and monocyte PCA may be induced through phosphatidylserine exposure, or TF expression. Induction may be protracted by Thal, which has an antiangiogenic activity. Therefore, prophylactic anticoagulant strategies should be considered in Thal-based combination regimens.

Topics: Annexin A5; Anticoagulants; Antineoplastic Agents; Blood Coagulation; Blood Coagulation Factors; Cell Line; Dexamethasone; Doxorubicin; Drug Combinations; Drug Interactions; Endothelial Cells; Endothelium, Vascular; Flow Cytometry; Humans; Monocytes; Multiple Myeloma; Phosphatidylserines; Thalidomide; Thromboplastin; Venous Thrombosis

To explore the changes and clinical significances of plasma D-dimer, factor X and tissue factor in acute exacerbation of chronic obstructive pulmonary disease (AECOPD) and analyze the in-depth changes of these indicators in AECOPD with co-current deep venous thrombosis (DVT).. A total of 56 AECOPD patients were divided into the DVT and non-DVT subgroups (n = 28 each). And 60 normal control subjects were recruited according to age and gender. For each subject, 2.7 ml whole blood was drawn and then the plasma levels of D-dimer, factor X and tissue factor were detected. The results were statistically analyzed with the software SPSS 13.0. And the analysis of variance was performed between the groups.. There was no significant difference between the distribution of the AECOPD group and the control group by gender and age. Therefore two groups were comparable. And in the AECOPD group, there was no significant difference between the distribution of DVT and non-DVT subgroups by gender and age. Therefore these two subgroups were comparable as well. The value of D-dimer in the AECOPD patients was significantly higher than that in the normal control [(0.76 ± 0.30) vs (0.29 ± 0.11) mg/L, P < 0.01]; and in the AECOPD group, the value of D-dimer in the DVT subgroup was significantly higher than that in the non-DVT subgroup [(0.85 ± 0.29) vs (0.67 ± 0.28) mg/L, P < 0.05]. In the AECOPD group, the value of tissue factor was (238 ± 68) mg/L and the value of factor X (1181 ± 337) mg/L. While in the normal control group, the values were (124 ± 30) and (998 ± 260) mg/L respectively. As for tissue factor and factor X, there were significant differences between two groups (all P < 0.01). Yet in AECOPD patients, neither indicator had significant differences between the DVT and non-DVT subgroups (all P > 0.05).. The blood of AECOPD patients is in a hypercoagulatory state. And an obvious rise in their plasma level of D-dimer suggests that it may be complicated with DVT.

Topics: Acute Disease; Aged; Aged, 80 and over; Blood Coagulation; Case-Control Studies; Factor X; Female; Fibrin Fibrinogen Degradation Products; Humans; Male; Middle Aged; Prospective Studies; Pulmonary Disease, Chronic Obstructive; Thromboplastin; Venous Thrombosis

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

The mechanism of increased thrombin production has been investigated in children with high-risk neuroblastoma (NB), to detect any possible association between catheter- related venous thrombosis (VT) and prothrombotic factors.. Consecutive children with high-risk NB were studied by color-doppler ultrasonography of the upper vein system and thrombophilia factors assessment. Plasma levels of Tissue Factor (TF), Vascular Endothelial Growth Factor (VEGF), Prothrombin Activation Fragment 1+2, and Thrombin-Antithrombin Complex were evaluated. Moreover, inherited thrombophilia factors (homocystein, antithrombin, protein C, protein S, factor V Leiden, activated protein C resistence, mutation H1299R and G1691A of factor V, mutation G20210A of prothrombin, mutation T677 and A1298C of methylenetetrahydrofolate reductase, and allele 4G of plasminogen activator inhibitor-1) were tested to exclude congenital disorders.. Six patients with mean age: 48.8 months---were studied. Five patients were affected by stage 4 NB and another one by stage 3 NB with Myc-N amplification. All children had a central venous line (mean duration: 8.5 mos). Four patients (67%) had asymptomatic catheter-related VT visualized by color-doppler ultrasonography. No patient had major inherited thrombophilia factors. The levels of plasma TF and plasma VEGF were found elevated in all patients. Mean value of TF (nv 20.3+/-6.6) was 82 pg/mL with a range of 39 to 131 pg/mL. Mean value of VEGF (nv 24.3 pg/mL) was 78.5 pg/mL with a range of 31 to 142 pg/mL.. The increased risk of catheter-related VT detected in our small series of high-risk NB patients, was associated with elevated levels of circulating TF and VEGF. Further studies are needed to evaluate if elevated levels of TF/VEGF are involved both in the hypercoagulable state and in advanced childhood cancer.

Topics: Antithrombin III; Catheterization; Child, Preschool; Female; Humans; Male; Neuroblastoma; Peptide Fragments; Peptide Hydrolases; Prothrombin; Thromboplastin; Vascular Endothelial Growth Factor A; Venous Thrombosis

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

Increasing reports of bleeding and peri- or post-operative blood dyscrasias in Brazil were possibly associated with the use of heparin from bovine instead of porcine intestine. These two pharmaceutical grade heparins were analysed for potential differences. NMR analyses confirmed that porcine heparin is composed of mainly trisulfated disaccharides -->4-alpha-IdoA2S-1-->4-alpha-GlcNS6S-1-->. Heparin from bovine intestine is also composed of highly 2-sulfated alpha-iduronic acid residues, but the sulfation of the alpha-glucosamine units vary significantly: approximately 50% are 6- and N -disulfated, as in porcine heparin, while approximately 36% are 6-desulfated and approximately 14% N -acetylated. These heparins differ significantly in their effects on coagulation, thrombosis and bleeding. Bovine heparin acts mostly through factor Xa. Compared to porcine heparin on a weight basis, bovine heparin exhibited approximately half of the anticoagulant and antithrombotic effects, but similar effect on bleeding. These two heparins also differ in their protamine neutralisation curves. The doses of heparin from bovine intestine required for effective antithrombotic protection and the production of adverse bleeding effects are closer than those for porcine heparin. This observation may explain the increasing bleeding observed among Brazilian patients. Our results suggest that these two types of heparin are not equivalent drugs.

Topics: Animals; Blood Coagulation; Cattle; Disaccharides; Factor Xa; Hemorrhage; Heparin; Intestinal Mucosa; Nuclear Magnetic Resonance, Biomolecular; Protamines; Rats; Rats, Wistar; Swine; Thromboplastin; Venous Thrombosis

We report the effects of a chemically oversulfated chondroitin sulfate and a naturally fucosylated chondroitin sulfate on the coagulation system. The former has been recently identified as a contaminant of heparin preparations and the latter has been proposed as an alternative anticoagulant. The mechanism of action of these polymers on coagulation is complex and target different components of the coagulation system. They have serpin-independent anticoagulant activity, which preponderates in plasma. They also have serpin-dependent anticoagulant activity but differ significantly in the target coagulation protease and preferential serpin. Their anticoagulant effects differ even more markedly when tested as inhibitors of coagulation proteases using plasma as a source of serpins. It is possible that the difference is due to the high availability of fucosylated chondroitin sulfate whereas oversulfated chondroitin sulfate has strong unspecific binding to plasma protein and low availability for the binding to serpins. When tested using a venous thrombosis experimental model, oversulfated chondroitin sulfate is less potent as an antithrombotic agent than fucosylated chondroitin sulfate. These highly sulfated chondroitin sulfates activate factor XII in in vitro assays, based on kallikrein release. However, only fucosylated chondroitin sulfate induces hypotension when intravenously injected into rats. In conclusion, the complexity of the regulatory mechanisms involved in the action of highly sulfated polysaccharides in coagulation requires their analysis by a combination of in vitro and in vivo assays. Our results are relevant due to the urgent need for new anticoagulant drugs or alternative sources of heparin.

Topics: Animals; Anticoagulants; Blood Coagulation; Blood Pressure; Cattle; Chondroitin Sulfates; Chromatography, Affinity; Dogs; Factor XII; Heparin; Horses; Humans; Models, Animal; Partial Thromboplastin Time; Rats; Thromboplastin; Venous Thrombosis

The generation of thrombin is a critical process in the formation of venous thrombi. In isolated plasma under static conditions, phosphatidylserine (PS)-exposing platelets support coagulation factor activation and thrombin generation; however, their role in supporting coagulation factor binding under shear conditions remains unclear. We sought to determine where activated factor X (FXa), (pro)thrombin, and fibrin(ogen) are localized in thrombi formed under venous shear.. Fluorescence microscopy was used to study the accumulation of platelets, FXa, (pro)thrombin, and fibrin(ogen) in thrombi formed in vitro and in vivo. Co-perfusion of human blood with tissue factor resulted in formation of visible fibrin at low, but not at high shear rate. At low shear, platelets demonstrated increased Ca(2+) signaling and PS exposure, and supported binding of FXa and prothrombin. However, once cleaved, (pro)thrombin was observed on fibrin fibers, covering the whole thrombus. In vivo, wild-type mice were injected with fluorescently labeled coagulation factors and venous thrombus formation was monitored in mesenteric veins treated with FeCl(3). Thrombi formed in vivo consisted of platelet aggregates, focal spots of platelets binding FXa, and large areas binding (pro)thrombin and fibrin(ogen).. FXa bound in a punctate manner to thrombi under shear, while thrombin and fibrin(ogen) distributed ubiquitously over platelet-fibrin thrombi. During thrombus formation under venous shear, thrombin may relocate from focal sites of formation (on FXa-binding platelets) to dispersed sites of action (on fibrin fibers).

Topics: Animals; Blood Platelets; Calcium Signaling; Factor Xa; Fibrin; Fibrinogen; Humans; Mice; Perfusion; Phosphatidylserines; Platelet Aggregation; Protein Binding; Thrombin; Thromboplastin; Venous Thrombosis

Topics: Administration, Oral; Blood Coagulation; Blood Coagulation Tests; Clinical Chemistry Tests; Fibrinolytic Agents; Hemorrhage; Humans; International Normalized Ratio; Safety; Stroke; Thrombin; Thromboplastin; Venous Thrombosis

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

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

We have adapted the corn-trypsin inhibitor whole-blood model to include EA.hy926 as an endothelium surrogate to evaluate the vascular modulation of blood coagulation initiated by relipidated recombinant tissue factor (rTf) and a cellular Tf surrogate, lipopolysaccharide (LPS)-stimulated THP1 cells (LPS-THP-1). Compared with bare tubes, EA.hy926 with rTf decreased the rate of thrombin formation, ITS accumulation, and the production of fibrinopeptide A. These phenomena occurred with increased rates of factor Va (fVa) inactivation by cleavages at R(506) and R(306). Thus, EA.hy926 provides thrombin-dependent protein C activation and APC fVa inactivation. Comparisons of rTf with LPS-THP-1 showed that the latter gave reduced rates for TAT formation but equivalent fibrinopeptide A, and fV activation/inactivation. In the presence of EA.hy926, the reverse was obtained; with the surrogate endothelium and LPS-THP-1 the rates of TAT generation, fibrinopeptide release, and fV activation were almost doubled, whereas cleavage at R(306) was equivalent. These observations suggest cooperativity between the 2 cell surrogates. These data suggest that the use of these 2 cell lines provides a reproducible quasi-endothelial quasi-inflammatory cytokine-stimulated monocyte system that provides a method to evaluate the variations in blood phenotype against the background of stable inflammatory cell activator and a stable vascular endothelial surrogate.

Topics: Adult; Blood Coagulation; Cells, Cultured; Cytokines; Endothelium, Vascular; Factor Va; Humans; Lipopolysaccharides; Male; Models, Biological; Monocytes; Protein C; Thromboplastin; Umbilical Veins; Venous Thrombosis

NF-kappaB transcription factors regulate the expression of tissue factor (TF), a principal initiator of the coagulation cascade. Dominant among them is the p50/p65 heterodimer. Here we report that Andrographolide (Andro; a p50 inhibitor) and genetic deletion of p50 attenuated TF activity in stimulated endothelial cells and monocytes/macrophages. Results of the electrophoretic mobility "supershift" assay and chromatin immunoprecipitation demonstrated the direct interaction of the p50/p65 heterodimer with the NF-kappaB site of the human TF promoter. Andro-treated and p50 null mice both exhibited blunted TF expression and reduced venous thrombosis, which were recapitulated by an anti-murine TF antibody in vivo. Our findings thus indicate that regulation of TF by NF-kappaB transcription factor p50 is essential for the pathogenesis of deep vein thrombosis and suggest that specific inhibitors of p50, such as Andro, may be therapeutically valuable for preventing and perhaps treating venous thrombosis.

Topics: Animals; Cells, Cultured; Diterpenes; Endothelial Cells; Gene Expression Regulation; Humans; Macrophages; Mice; Mice, Mutant Strains; Monocytes; NF-kappa B p50 Subunit; Platelet Aggregation Inhibitors; Thromboplastin; Venous Thrombosis

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

Topics: ABO Blood-Group System; Factor VII; Humans; Pancreatic Neoplasms; Risk Assessment; Risk Factors; Thromboplastin; United States; Venous Thrombosis; von Willebrand Factor

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

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

Cancer confers a prothrombotic state and statins are associated with a lowered risk for prostate cancer in vivo by unknown mechanisms. Prostate cancer cells release tissue factor (TF)-bearing, cholesterol-rich prostasomes which are pro-coagulant in vitro and a possible source for the blood-borne TF found in prostate cancer patients. We investigated the effect of cholesterol depletion on the production of prostasomes and on the TF activity in the conditioned medium of simvastatin-treated PC3 cells. Human PC3 prostate cancer cells were treated with high and low concentrations of simvastatin for different time periods. Caspase-3 was detected with the Array Scan microscope, whereas TF mRNA and protein were analyzed by TaqMan and flow cytometry. TF activity was assessed by measuring the cleavage of a chromogenic thrombin substrate. Prostasomes were isolated by repeated centrifugations and detected and quantified by flow cytometry. A micromolar dose of simvastatin caused reduction of TF expression and induction of apoptosis in the PC3 cells. The levels of TF on the prostasomes were also decreased but the TF activity in the conditioned medium of the simvastatin-treated PC3 cells was increased due to apoptosis-dependent release of prostasomes. Treatment with a nanomolar dose of simvastatin did not induce apoptosis or alter the expression of TF but instead decreased the production and release of the prostasomes. The TF activity was reduced in parity with the decline in prostasome release. In conclusion, in prostate cancer, a nanomolar dose of simvastatin may have an anti-thrombotic effect due to decreased levels of circulating TF-bearing prostasomes.

Topics: Anticholesteremic Agents; Apoptosis; Cell Line, Tumor; Culture Media, Conditioned; Dose-Response Relationship, Drug; Down-Regulation; Flow Cytometry; Humans; Male; Particle Size; Prostatic Neoplasms; Simvastatin; Thromboplastin; Venous Thrombosis

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

Topics: Activated Protein C Resistance; Blood Coagulation; Case-Control Studies; Factor V; Factor VIIa; Factor Xa Inhibitors; Humans; Kinetics; Lipoproteins; Multienzyme Complexes; Protein S; Thromboplastin; Venous Thrombosis

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

Blood coagulation and platelet activation are mutually dependent processes, but contribute differently to venous and arterial thrombosis. We investigated the interplay of these processes in vivo in a mouse model of arteriolar and venular thrombus formation.. Thrombus formation was studied by intravital (fluorescence) microscopy after topical application of FeCl3 on mouse mesenteric microvessels.. Both in arterioles and venules, the thrombus-forming process relied on tissue factor-factor VII(a) interaction, collagen exposure, and glycoprotein VI-mediated platelet activation. Arterial thrombus formation was impaired by mild thrombin inhibition or platelet inhibition, while venous thrombosis was only suppressed by strong thrombin inhibition or by mild thrombin inhibition together with platelet inhibition. Phosphatidylserine-exposing platelets were present in thrombi of both vessel types, as detected with fluorescently labeled annexin A5. Injection of annexin A5 to shield exposed phosphatidylserine abolished thrombus formation in arterioles and venules, while mutant M1234-annexin A5 was ineffective. Arterial and venous thrombus formations were only slightly affected in mice carrying the factor V Leiden mutation, suggesting insensitivity to factor Va inactivation.. In this microvascular model, the formation of both arterial and venous thrombi relies on collagen-induced platelet activation and tissue factor-induced thrombin generation. Activated, phosphatidylserine-exposing platelets play a key role in thrombus growth in arterioles and venules.

Topics: Animals; Arterioles; Blood Coagulation; Blood Platelets; Collagen; Enzyme Activation; Factor V; Factor VIIa; Mice; Mice, Knockout; Platelet Activation; Thrombin; Thromboplastin; Venous Thrombosis; Venules

Tissue factor (TF) is the main physiological initiator of blood coagulation; it is membrane-bound on monocytes (mTF) and free in plasma (pTF). Abnormal expression of TF by monocytes has been implicated in various diseases. We therefore quantified monocytes expressing TF and pTF levels in patients with lower-limb deep venous thrombosis (DVT).. DVT was confirmed by Duplex Scan. Blood mTF levels under resting condition (baseline), after incubation without (unstimulated) and with (stimulated) lipopolysaccharide (LPS), and total mTF levels were determined by flow cytometry using two analytical methods (Histogram and Quadrant-Statistics). Plasma TF levels were measured using an enzyme-linked immunoabsorbent assay (ELISA). Results were compared with age-matched controls.. Histogram analysis in patients with DVT showed significantly elevated mTF levels for baseline, unstimulated and total mTF over controls. For Quadrant-Statistics, DVT patients also showed significantly raised baseline, unstimulated, stimulated and total mTF. Similarly, pTF levels were significantly raised in subjects with DVT compared to controls. Baseline mTF levels correlated with pTF levels by Histogram and Quadrant-Statistics analysis. Using the relative operating characteristic (ROC) curve, baseline mTF and pTF assays displayed sensitivity and specificity in detecting DVT. Quadrant-Statistics baseline mTF and pTF gave the best discrimination.. The TF assays used in this study showed acceptable sensitivity and specificity and are cost-effective and practical. Therefore, they should be considered in patients with, or at risk of, DVT.

Topics: Adult; Aged; Case-Control Studies; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Humans; Lipopolysaccharides; Lower Extremity; Male; Middle Aged; Monocytes; Plasma; Sensitivity and Specificity; Thromboplastin; Venous Thrombosis

Cancer, in particular mucinous adenocarcinoma, is associated with venous thromboembolism (VTE). Tissue factor (TF), initiator of coagulation, plays a central role in the paradigm that clotting and tumor growth form a vicious circle, in which hypercoagulability facilitates the aggressive biology of cancer and vice versa. Expression of TF in tumors is associated with poor differentiation and poor prognosis.. We investigated the association between clinically manifest VTE and procoagulant properties of circulating microparticles (MP) isolated from blood of unselected pancreatic and breast adenocarcinoma patients' consecutive subjects, who presented with ultrasound or CT-scan confirmed VTE, and healthy subjects.. Patients with disseminated breast and pancreatic cancer had significantly increased levels of MP-associated TF activity compared with healthy controls, subjects with idiopathic acute VTE and non-metastatic cancer patients. Patients with both high MP-associated TF-activity and MP-associated epithelial mucin (MUC1) had a lower survival rate at 3-9 months follow-up than those with low TF-activity and no MUC1 expression: the likelihood of survival was 0.42 (95% CI: 0.19- 0.94) for an individual with these two predictor variables present, after adjustment for other factors (age cohort, type of cancer, VTE) in the Cox proportional hazards model.. Our results suggest an important role for MP-associated TF and MUC1 in the pathogenesis of thrombosis in disseminated mucinous adenocarcinoma patients. Future studies should reveal the mechanism underlying the observed associations.

Topics: Adenocarcinoma, Mucinous; Adult; Aged; Antigens, Neoplasm; Blood Coagulation; Breast Neoplasms; Case-Control Studies; Cell Differentiation; Cytoplasmic Vesicles; Female; Follow-Up Studies; Humans; Kaplan-Meier Estimate; Likelihood Functions; Male; Middle Aged; Mucin-1; Mucins; Neoplasm Invasiveness; Neoplasm Staging; Pancreatic Neoplasms; Predictive Value of Tests; Prognosis; Proportional Hazards Models; Risk Assessment; Thromboembolism; Thromboplastin; Time Factors; Venous 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

Hemostatic activation is common in pancreatic cancer and may be linked to angiogenesis and venous thromboembolism. We investigated expression of tissue factor (TF), the prime initiator of coagulation, in noninvasive and invasive pancreatic neoplasia. We correlated TF expression with vascular endothelial growth factor (VEGF) expression, microvessel density, and venous thromboembolism in resected pancreatic cancer.. Tissue cores from a tri-institutional retrospective series of patients were used to build tissue microarrays. TF expression was graded semiquantitatively using immunohistochemistry in normal pancreas (n=10), intraductal papillary mucinous neoplasms (n=70), pancreatic intraepithelial neoplasia (n=40), and resected or metastatic pancreatic adenocarcinomas (n=130).. TF expression was observed in a majority of noninvasive and invasive pancreatic neoplasia, including 77% of pancreatic intraepithelial neoplasias, 91% of intraductal papillary mucinous neoplasms, and 89% of pancreatic cancers, but not in normal pancreas. Sixty-six of 122 resected pancreatic cancers (54%) were found to have high TF expression (defined as grade >or=2, the median score). Carcinomas with high TF expression were more likely to also express VEGF (80% versus 27% with low TF expression, P<0.0001) and had a higher median MVD (8 versus 5 per tissue core with low TF expression, P=0.01). Pancreatic cancer patients with high TF expression had a venous thromboembolism rate of 26.3% compared with 4.5% in patients with low TF expression (P=0.04).. TF expression occurs early in pancreatic neoplastic transformation and is associated with VEGF expression, increased microvessel density, and possibly clinical venous thromboembolism in pancreatic cancer. Prospective studies evaluating the role of TF in pancreatic cancer outcomes are warranted.

Topics: Aged; Capillaries; Female; Humans; Male; Middle Aged; Neovascularization, Pathologic; Pancreatic Neoplasms; Survival Analysis; Thromboplastin; Vascular Endothelial Growth Factor A; Venous Thrombosis

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

The case history is presented of a patient with Trousseau's syndrome in which tissue factor originating from lung cancer appeared responsible for recurrent DVT/PE. This is thought to be the first such case to be reported.

Topics: Adenocarcinoma; Adult; Humans; Lung Neoplasms; Male; Pulmonary Embolism; Recurrence; Syndrome; Thromboplastin; Venous Thrombosis

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

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

Behaviour of the vein thrombus is determined by the activity ratio of coagulation factors to factors of fibrinolytic system. The aim of the study is to evaluate activity of some coagulation and fibrinolytic factors in the vein thrombus.. The activity of platelets aggregating factors, tissue factor, thrombin, antithrombins, antiheparin factors, plasminogen activators, plasmin (plasminogen) and antiplasmins of the vein thrombus homogenate was determined using coagulative, fibrinolytic and caseinolytic tests. Retracted blood clot was a compared material.. Tissue factor activity in the vein thrombus was above twofold higher and antiheparin activity was nearly twice higher in comparison to the blood clot. The vein thrombus contains also active thrombin. Plasminogen activators activity in the vein thrombus was twofold higher and activity of plasmin (plasminogen) was threefold higher than in the blood clot. High activity of the tissue factor, substances neutralizing heparin and presence of thrombin intensify the thrombus enlargement. However, the thrombotic tendency may be balanced by a high activity of plasminogen activators and high activity of plasmin (plasminogen).. 1) Vein thrombus is characterized by high activity of tissue factor, presence of active thrombin and high antiheparin activity. 2) High coagulative potential of vein thrombus is balanced to a certain grade by high fibrinolytic potential: high activity of plasminogen activators and high activity of plasmin (plasminogen), as well as absence of antiplasmins activity.

Topics: Adult; Antifibrinolytic Agents; Antithrombins; Blood Coagulation; Blood Coagulation Tests; Female; Fibrinolysin; Fibrinolysis; Humans; Male; Middle Aged; Plasminogen; Plasminogen Activators; Thromboplastin; Venous Thrombosis

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

The range of plasma concentrations of hemostatic analytes in the population is wide. In this study these components of blood coagulation phenotype are integrated in an attempt to predict clinical risk.. We modeled tissue factor (TF)-induced thrombin generation in the control population (N = 473) from the Leiden Thrombophilia Study utilizing a numerical simulation model. Hypothetical thrombin generation curves were established by modeling pro- and anticoagulant factor levels for each individual. These curves were evaluated using parameters which describe the initiation, propagation and termination phases of thrombin generation, i.e. time to 10 nm thrombin (approximate clot time), total thrombin and the maximum rates and levels of thrombin generated.. The time to 10 nm thrombin varied over a 3-fold range (2.9-9.5 min), maximum levels varied over a approximately 4-fold range (200-800 nm), maximum rates varied approximately 4.8-fold (90-435 nm min(-1)) and total thrombin varied approximately 4.5-fold (39-177 microm s(-1)) within this control population. Thrombin generation curves, defined by the clotting factor concentrations, were distinguished by sex, age, alcohol consumption, body mass index (BMI) and oral contraceptive (OC) use (OC > sex > BMI > age). Our results show that the capacity for thrombin generation in response to a TF challenge may represent a method to identify an individual's propensity for developing thrombosis.

Topics: Adult; Age Factors; Algorithms; Anticoagulants; Blood Coagulation Factors; Blood Coagulation Tests; Case-Control Studies; Coagulants; Female; Hemostasis; Hemostatics; Humans; Male; Middle Aged; Models, Theoretical; Proteome; Proteomics; Risk; Risk Factors; Statistics as Topic; Thrombin; Thromboplastin; Time Factors; Venous Thrombosis

Tissue factor (TF) mRNA levels in leukocyte and TF antigen in plasma were examined in patients with deep vein thrombosis (DVT). Although TF mRNA levels in leukocytes were higher in patients with DVT than in healthy volunteers, they were lower in patients with DVT than in those with solid cancer and those with disseminated intravascular coagulation (DIC). On the other hand, the plasma levels of TF antigens were markedly high in patients with DVT/pulmonary embolism (PE). Analysis of the role of underlying disease of DVT showed no significant difference in TF mRNA levels and TF antigens among patients with solid cancer, post-surgical, other diseases and those free of underlying diseases. In patients with VTE, plasma levels of D-dimer, soluble fibrin, GE-XDP and plasminogen activator inhibitor-1 did not correlate with TF mRNA or TF antigen. In analysis of 18 patients with PE with and without DVT, TF mRNA levels in leukocytes correlated with the plasma levels of D-dimer. These findings suggest that TF in leukocytes is more likely to be involved in the development of thrombosis in PE than DVT.

Topics: Adult; Aged; Case-Control Studies; Disseminated Intravascular Coagulation; Female; Fibrin Fibrinogen Degradation Products; Humans; Leukocytes; Male; Middle Aged; Neoplasms; Pulmonary Embolism; RNA, Messenger; Thromboplastin; Up-Regulation; Venous Thrombosis

Protein S is a vitamin K-dependent plasma protein that functions as an APC-cofactor, but also exhibits anticoagulant activity in the absence of APC. The Heerlen polymorphism of protein S is characterized by a Ser460Pro substitution and lacks glycosylation at Asn458. It is associated with decreased protein S levels due to selective deficiency of free protein S Heerlen. To understand the lack of thrombotic complications associated with the protein S Heerlen mutation, we compared recombinant protein S Heerlen, wild type (wt) protein S and plasma-derived protein S. wt-Protein S and protein S Heerlen each bound 1:1 to C4BP with dissociation constants of 0.27 and 0.33 nM, respectively. Both wt-protein S and protein S Heerlen, either free or in complex with C4BP, were equally active as prothrombinase inhibitors in the absence of APC. All three protein S preparations stimulated APC-catalyzed inactivation of normal FVa, FVa Leiden and FVIIIa to the same extent. If extrapolated to plasma, it is not likely that the decreased free protein S levels in carriers of the protein S Heerlen mutation are compensated by an increased anticoagulant activity of protein S Heerlen-C4BP complexes. It is possible that an unrecognized plasma factor selectively enhances the anticoagulant activity of protein S Heerlen. If not, the reduction of free protein S levels in heterozygous protein S Heerlen-carriers combined with (low) normal total protein S levels apparently minimally affects the total anticoagulant activity of protein S (APC-cofactor and APC-independent activity) and hence is not associated with increased risk of venous thrombosis.

Topics: Anticoagulants; Blood Coagulation Factors; Cell Line; Complement C4b; Humans; Mutation, Missense; Protein C; Protein S; Protein S Deficiency; Recombinant Proteins; Risk; Thromboplastin; Transfection; Venous Thrombosis

Fondaparinux and enoxaparin are both effective and safe in preventing post-operative venous thromboembolism. However, neither of them significantly influence the conventional clotting tests. We compared the influence of clinically relevant concentrations of fondaparinux and enoxaparin on normal whole blood (WB) thromboelastographic profiles after triggering TF-pathway with minimal amount of thromboplastin. Diluted thromboplastin was added to WB samples supplemented with buffer (control), fondaparinux (0.25; 0.5; 1 microg/ml), or enoxaparin (0.1; 0.5; 1 anti-Xa IU/ml). Four parameters were analyzed, R: clotting time, K: time required to reach an amplitude of 20 mm, alpha angle: measurement reflecting clot development kinetics and MA: maximal amplitude. At concentrations used in prophylaxis, both enoxaparin (0.1 anti-Xa IU/ml) and fondaparinux (0.25 microg/ml which correspond to 0.27 anti-Xa IU/ml) significantly prolonged the R and K times, but did not significantly modify the alpha angle as compared to the control. At concentrations observed after administration of curative doses for the treatment of DVT (> or = 0.5 anti-Xa IU/ml for enoxaparin and > or = 0.5 microg/ml for fondaparinux) both drugs induced a significant increase of R and K times, and a significant decrease of the alpha angle (p < 0.05). In contrast to fondaparinux, enoxaparin at concentrations equal to or higher than 0.5 anti-Xa IU/ml significantly reduced MA. The present study provides evidence that the whole blood TF-triggered TEG assay is sensitive to the presence of clinically relevant concentrations of enoxaparin or fondaparinux. Moreover, the angle may be used in order to distinguish the effect of prophylactic and therapeutic concentrations, since it was significantly reduced by the later ones. Further studies are needed to evaluate the clinical usefulness of whole blood TF-triggered TEG assay for the monitoring of treatment with enoxaparin or fondaparinux.

Topics: Anticoagulants; Blood Coagulation; Blood Coagulation Tests; Dose-Response Relationship, Drug; Enoxaparin; Fondaparinux; Humans; In Vitro Techniques; Kinetics; Polysaccharides; Thrombelastography; Thrombin; Thromboembolism; Thromboplastin; Time Factors; Venous Thrombosis

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

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

In this study we test the hypothesis that blood/plasma-based prothrombinase assays, rather than inhibition of purified factor Xa (fXa), are predictive of in vivo antithrombotic activity.. Six fXa inhibitors with equivalent nanomolar Ki were studied in thrombin generation assays using human plasma/blood and endogenous macromolecular substrate. In all assays, benzamidine inhibitors were more potent (100 to 800 nmol/L) than the aminoisoquinolines (5 to 58 micromol/L) or neutral inhibitors (3 to 10 micromol/L). A similar rank order of compound inhibition was also seen in purified prothrombinase assays as well as in a rabbit model of deep vein thrombosis.. Assays using prothrombinase with protein substrates are better predictors of in vivo efficacy than fXa Ki using amidolytic substrates.

Topics: Animals; Benzamidines; Binding Sites; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Enzyme Inhibitors; Factor Xa Inhibitors; Fibrinolytic Agents; Humans; Isoquinolines; Male; Molecular Structure; Prothrombin; Sensitivity and Specificity; Substrate Specificity; Thrombin; Thromboplastin; Venous Thrombosis

Topics: Adult; Cerebral Veins; Female; Humans; Middle Aged; Monocytes; Thromboplastin; Venous Thrombosis

Antibody mediated inhibition of tissue factor (TF) function reduces thrombus size in ex vivo perfusion of human blood over a TF-free surface at venous shear rates suggesting that TF might be involved in the mechanism of deep vein thrombosis. Moreover, TF-bearing monocytes and polymorphonuclear (PMN) leukocytes were identified in human ex vivo formed thrombi and in circulating blood. To understand the role of TF in thrombus growth, we applied a rabbit venous thrombosis model in which a collagen-coated thread was installed within the jugular vein or within a silicon vein shunt. The effect of an inhibitory monoclonal antirabbit TF antibody (AP-1) or Napsagatran, a specific inhibitor of thrombin, was quantified by continuously monitoring 125I-fibrinogen incorporation into the growing thrombi. The antithrombotic effect obtained with the anti-TF antibody was comparable to the effect observed with the thrombin inhibitor napsagatran suggesting that in this animal model the thrombus propagation is highly TF dependent. Immunostaining revealed that TF was mostly associated with leukocytes within the thrombi formed in the jugular vein or in the silicon vein shunt. Ex vivo perfusion experiments over collagen-coated coverslips demonstrated the presence of TF-bearing PMN leukocytes in circulating blood. The results suggest that in rabbits venous thrombus growth is mediated by clot-bound TF and that blocking the TF activity can inhibit thrombus propagation.

Topics: Animals; Antibodies, Monoclonal; Blood Vessel Prosthesis; Fibrinogen; Immunohistochemistry; Jugular Veins; Leukocytes; Naphthalenes; Piperidines; Rabbits; Thromboplastin; Venous Thrombosis

The effect of new heparin mimetics (synthetic oligosaccharides) was studied in vitro with regard to thrombin generation (TG) in rat platelet rich plasma (PRP) and whole blood (WB) and in vivo on stasis-induced venous thrombosis in the rat. TG in PRP and in WB was highly dependent on platelet count and strongly influenced by the haematocrit. The peak of TG appeared to be significantly higher in WB than in PRP whereas the endogenous thrombin potential (ETP) was not significantly different under either condition. The effect of hirudin, the synthetic pentasaccharide SR90107/Org31540 (SP) and heparin were measured on TG in PRP and WB. We then compared the effect of two new synthetic heparin mimetics (SR121903A and SanOrg123781) with potent and comparable antithrombin (AT) mediated activity against factor Xa and thrombin. These two compounds were made of a pentasaccharide with a high affinity to AT, prolonged at the non-reducing end by an oligosaccharide chain recognised by thrombin. In SR121903A, the charge density and charge distribution was analogous to that of heparin whereas in SanOrg123781 the charges were only located on the last 5 saccharides of the non-reducing end of the molecule. In PRP and in WB, SR121903A acted on the lag time and on the AUC whereas SanOrg123781 inhibited thrombin formation with no effect on the lag time. SanOrg123781 was more potent in inhibiting TG than SR121903A. This difference was due to the structures of the compounds that differed in their ability to be neutralised by platelet factor 4. The antithrombotic effect of the two compounds was examined in a venous thrombosis model in rats. We observed that SanOrg123781 was more active than SR121903A and heparin. Taken together, these results indicate that the activity of oligosaccharides is greatly influenced by the global charge density of the molecule and show that SanOrg123781 is a potent and promising antithrombotic drug candidate.

Topics: Animals; Anticoagulants; Antithrombin III; Binding Sites; Blood; Blood Platelets; Carbohydrate Conformation; Carbohydrate Sequence; Drug Evaluation, Preclinical; Factor Xa Inhibitors; Fibrinolytic Agents; Hematocrit; Heparin; Hirudins; Male; Models, Animal; Molecular Sequence Data; Molecular Structure; Oligosaccharides; Plasma; Platelet Factor 4; Polysaccharides; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Static Electricity; Structure-Activity Relationship; Thrombin; Thromboplastin; Venous Thrombosis

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

Topics: Humans; Thromboplastin; Thrombosis; Venous Thrombosis

Sulfated D-galactans occur on the red algae Botryocladia occidentalis as three fractions that differ in their sulfate content. Fractions F2 and F3 are potent anticoagulants. Like heparin, they enhance thrombin and factor Xa inhibition by antithrombin and/or heparin cofactor II. The inhibition potency increases simultaneously with the sulfate content of the fractions. The antithrombotic activity of these sulfated D-galactans was investigated on an experimental thrombosis model in which thrombus formation was induced by a combination of stasis and hypercoagulability. In contrast with heparin. the sulfated D-galactans showed a dual dose-response curve preventing thrombosis at doses up to approximately 0.5 mg/ kg body weight but losing the effect at higher doses. This unexpected behavior is probably due to a combined action of the sulfated D-galactan as anticoagulant and also as a strong inducer of platelet aggregation. In platelet-depleted animals the antithrombotic activity at higher dose of sulfated D-galactan is restored and almost total inhibition of thrombus formation is achieved. The sulfated D-galactan has no hemorrhagic effect even at high doses, possibly as a consequence of its effect on platelet aggregation. At comparable dose heparin has an intense bleeding effect. These results indicate that new polysaccharides, with well-defined structures, can help to distinguish events, such as antithrombotic and anticoagulant activities, bleeding and platelet-aggregating effects, which are obscure when induced simultaneously by a single compound.

Topics: Animals; Anticoagulants; Brazil; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Female; Fibrinolytic Agents; Galactans; Hemorrhage; Heparin; Male; Partial Thromboplastin Time; Phytotherapy; Plant Extracts; Platelet Aggregation; Rats; Rats, Wistar; Rhodophyta; Sulfates; Thromboplastin; Vena Cava, Inferior; Venous Thrombosis

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

Tissue factor (TF) is a transmembrane protein considered to be responsible for the initiation of coagulation. TF gene expression may be induced in monocytes and endothelial cells and is present in atherosclerotic plaque to initiate thrombus formation. To investigate whether individual differences in TF gene expression could predispose subjects to thrombosis, we sequenced the 5' domain of the gene up to nucleotide 2732 and found 6 different polymorphisms: 4 of them were completely concordant and defined 2 haplotypes with similar frequencies, designated as 1208 D and 1208 I. Genotyping of patients with myocardial infarction in a case-control study involving 2354 subjects showed no association between the polymorphisms and nonfatal coronary thrombosis. In another study involving 255 patients with venous thromboembolism and 1204 controls, allele D was less common in the cases (P=0.022). The odds ratio associated with the presence of at least 1 D allele was 0.72 (P=0. 031). Comparison of subgroups of control subjects who were homozygous for the D or I allele demonstrated a lower plasma TF concentration in DD homozygotes. These results indicate that the TF gene promoter exists in 2 major forms differing at 4 sites. The 1208 D haplotype is not associated with coronary thrombosis but is associated with reduced plasma TF levels and a lower risk of venous thrombosis.

Topics: 5' Untranslated Regions; Adult; Aged; Base Sequence; Case-Control Studies; DNA Primers; Female; Genetic Predisposition to Disease; Genotype; Haplotypes; Humans; Male; Middle Aged; Molecular Sequence Data; Myocardial Infarction; Polymorphism, Genetic; Prevalence; Promoter Regions, Genetic; Risk Factors; Thromboembolism; Thromboplastin; Venous Thrombosis

Lupus anticoagulant (antiphospholipid antibodies) is associated with venous and arterial thrombosis in patients with and without autoimmune disorders. Vitamin K antagonists are the treatment of choice in patients with thrombosis, of which the dose is titrated by INR monitoring. Several recent reports suggest that the presence of the lupus anticoagulant disturbs the INR test and may lead to unreliable results with a large variation in INR values, dependent on the reagents used.. We studied 11 lupus anticoagulant positive patients and 11 lupus anticoagulant negative patients, all using vitamin K antagonists. The INR value was determined using seven different tests and the variation in INR values was compared between the two groups. The amidolytic Factor X levels were used as an phospholipid independent measure for intensity of warfarin therapy. Factor VII and X activity were measured to assess the stability of warfarin therapy.. The variation of the results with different INR tests within one patient was minimal and comparable in the two groups for INR's in the therapeutic range. The coefficient of variation for the cases and control group was 10.43 and 9.35, respectively. Variation in both groups increased at supratherapeutic levels of anticoagulation and when the anticoagulation was unstable (measured with Factor X/Factor VII ratio). The relationship between INR values and Factor X analysis revealed no influence of the lupus anticoagulant.. In this study, lupus anticoagulant antibodies do not disturb INR laboratory tests. Differences in INR measurements are seen in patients with a high intensity of anticoagulation and in patients who either just started or in whom no stable anticoagulation has been achieved. Abbreviated Abstract. This study investigates the influence of lupus anticoagulant on INR determination tests in patients treated with warfarin. Eleven cases and eleven lupus anticoagulant negative control patients, also on warfarin therapy, were included. Seven INR results per patient were obtained using different laboratory tests. A factor X assay was performed to obtain an independent measure for the intensity of warfarin therapy. The variation of INR results between the cases and controls revealed no difference in these groups. In addition, the relationship between INR values and Factor X analysis indicated no influence of the lupus anticoagulant. What was observed was an increased difference in INR values in patients with a high intensity of anticoagulation and in patients who either just started or in whom no stable anticoagulation has been achieved

Topics: Factor VII; Factor X; Humans; International Normalized Ratio; Lupus Coagulation Inhibitor; Reagent Kits, Diagnostic; Statistics, Nonparametric; Thromboembolism; Thromboplastin; Venous Thrombosis; Vitamin K; Warfarin

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

Leg ulcres can be seen as manifestations of antiphospholipid syndromes but their pathogenic relationship with vascular thrombotic events secondary to antiphospholipid antibodies remains to be defined with precision. A significant association between anticardiolipin antibodies and venous leg ulcers has been described. We conducted this study to determine whether such an association is found in venous ulcers and if it could also be present in cases involving the arterial and/or arteriolar circulation.. From December 1995 to March 1997, 48 patients with leg ulcers involving venous (27 cases), arteriovenous (9 cases) or arteriolar (12 cases) circulations were admitted. The etiologic diagnosis was based on clinical presentation and duplex Doppler findings examining the superficial and deep venous and arterial circuits. Antiphospholipid antibodies were searched for in all cases: VDRL, ELISA for IgG and IgM antiphospholipid antibodies, antiprothrombinase circulating anticoagulant.. Circulating anticoagulants were found in 22 of the 48 patients (46%): 12/27 (44%) involved venous leg ulcers (anticardiolipin antibodies, 5 cases; circulating anticoagulants, 4 cases; both, 2 cases); 1/9 involved arteriovenous ulcers (anticardiolipin antibodies, 5 cases); 9/12 involved arteriolar ulcers (anticardiolipin antibodies, 3 cases; circulating anticoagulants, 6 cases; both, 3 cases). Seven of the 9 patients also had severe arteritis. A past history of venous thrombosis was found in 3 cases with venous ulcers and antiphospholipid antibodies. One patient among the 5 with arteriolar ulcers had a past history of arterial thrombosis.. Our cohort is too small for a formal conclusion but underlines two points: 1. antiphospholipid antibodies can be associated with venous ulcers independently of thrombosis history. The hypothesis that leukocyte stasis and endothelial cell activation causes an immune reaction implicating antiphospholipid antibodies has been put forward. The usefulness of an antiaggregate treatment or an anticoagulant treatment should be discussed, 2. The possible association between arteriolar ulcers and antiphospholipid antibodies requires further large scale studies.

Topics: Aged; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunoglobulin G; Immunoglobulin M; Leg Ulcer; Male; Middle Aged; Prospective Studies; Risk Factors; Thromboplastin; Ultrasonography, Doppler, Duplex; Venous Thrombosis

Topics: Dicumarol; Heparin; Humans; Thrombophlebitis; Thromboplastin; Thrombosis; Venous Thrombosis

Topics: Anticoagulants; Blood Coagulation; Humans; Lower Extremity; Minnesota; Thromboplastin; Venous Thrombosis

Topics: Female; Gynecology; Humans; Obstetrics; Pregnancy; Thromboplastin; Veins; Venous Thrombosis

Topics: Thrombophlebitis; Thromboplastin; Veins; Venous Thrombosis

Topics: Humans; Lower Extremity; Thromboplastin; Veins; Venous Thrombosis

Topics: Humans; Thromboplastin; Veins; Venous Thrombosis