thromboplastin and ferric-chloride

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

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

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

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

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

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

Objective- Mutations in Krüppel like factor-11 ( KLF11), a gene also known as maturity-onset diabetes mellitus of the young type 7, contribute to the development of diabetes mellitus. KLF11 has anti-inflammatory effects in endothelial cells and beneficial effects on stroke. However, the function of KLF11 in the cardiovascular system is not fully unraveled. In this study, we investigated the role of KLF11 in vascular smooth muscle cell biology and arterial thrombosis. Approach and Results- Using a ferric chloride-induced thrombosis model, we found that the occlusion time was significantly reduced in conventional Klf11 knockout mice, whereas bone marrow transplantation could not rescue this phenotype, suggesting that vascular KLF11 is critical for inhibition of arterial thrombosis. We further demonstrated that vascular smooth muscle cell-specific Klf11 knockout mice also exhibited significantly reduced occlusion time. The expression of tissue factor (encoded by the F3 gene), a main initiator of the coagulation cascade, was increased in the artery of Klf11 knockout mice, as determined by real-time quantitative polymerase chain reaction and immunofluorescence. Furthermore, vascular smooth muscle cells isolated from Klf11 knockout mouse aortas showed increased tissue factor expression, which was rescued by KLF11 overexpression. In human aortic smooth muscle cells, small interfering RNA-mediated knockdown of KLF11 increased tissue factor expression. Consistent results were observed on adenovirus-mediated overexpression of KLF11. Mechanistically, KLF11 downregulates F3 at the transcriptional level as determined by reporter and chromatin immunoprecipitation assays. Conclusions- Our data demonstrate that KLF11 is a novel transcriptional suppressor of F3 in vascular smooth muscle cells, constituting a potential molecular target for inhibition of arterial thrombosis.

Topics: Animals; Antithrombin III; Apoptosis Regulatory Proteins; Bone Marrow Transplantation; Cells, Cultured; Chlorides; Chromatin Immunoprecipitation; Down-Regulation; Female; Ferric Compounds; Gene Expression Regulation; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Peptide Hydrolases; Platelet Aggregation; Recombinant Proteins; Repressor Proteins; RNA Interference; Thromboplastin; Thrombosis; Transcription, Genetic

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

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

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

Selective inhibitors of cyclooxygenase (COX)-2 increase the risk of myocardial infarction and thrombotic events, but the responsible mechanisms are not fully understood.. We found that ferric chloride-induced arterial thrombus formation was significantly greater in COX-2 knockout compared with wild-type mice. Cross-transfusion experiments excluded the likelihood that COX-2 knockout platelets, despite enhanced aggregation responses to collagen and thrombin, are responsible for increased arterial thrombus formation in COX-2 knockout mice. Importantly, we observed that COX-2 deletion decreased prostacyclin synthase and production and peroxisome proliferator-activated receptor- and sirtuin-1 (SIRT1) expression, with consequent increased upregulation of tissue factor (TF), the primary initiator of blood coagulation. Treatment of wild-type mice with a prostacyclin receptor antagonist or a peroxisome proliferator-activated receptor-δ antagonist, which predisposes to arterial thrombosis, decreased SIRT1 expression and increased TF activity. Conversely, exogenous prostacyclin or peroxisome proliferator-activated receptor-δ agonist completely reversed the thrombotic phenotype in COX-2 knockout mice, restoring normal SIRT1 levels and reducing TF activity. Furthermore, inhibition of SIRT1 increased TF expression and activity and promoted generation of occlusive thrombi in wild-type mice, whereas SIRT1 activation was sufficient to decrease abnormal TF activity and prothrombotic status in COX-2 knockout mice.. Modulation of SIRT1 and hence TF by prostacyclin/peroxisome proliferator-activated receptor-δ pathways not only represents a new mechanism in controlling arterial thrombus formation but also might be a useful target for therapeutic intervention in the atherothrombotic complications associated with COX-2 inhibitors.

Topics: Animals; Blood Platelets; Carotid Artery Thrombosis; Chlorides; Cyclooxygenase 2; Epoprostenol; Ferric Compounds; Incidence; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Animal; PPAR delta; Receptors, Epoprostenol; Risk Factors; Signal Transduction; Sirtuin 1; Thromboplastin

Studies have correlated elevated plasma factor VIII (FVIII) with thrombosis; however, it is unclear whether elevated FVIII is a proinflammatory biomarker, causative agent, or both. We raised FVIII levels in mice and measured the time to vessel occlusion (TTO) after ferric chloride-induced injury. Compared with control (saline-infused) mice, elevated FVIII had no effect after longer (3-minute) carotid artery injury, but it shortened the TTO after shorter (2-minute) injury (P < .008). After injury, circulating thrombin-antithrombin (TAT) complexes were lower after short versus long injury (P < .04), suggesting short treatment produced less coagulation activation. TAT levels in FVIII-infused mice were higher than in controls after short, but not longer, injury. Accordingly, elevated FVIII had no effect on in vitro thrombin generation or platelet aggregation triggered by high tissue factor, but it increased thrombin generation rate and peak (2.4- and 1.5-fold, respectively), and it accelerated platelet aggregation (up to 1.6-fold) when initiated by low tissue factor. Compared with control mice, elevated FVIII stabilized thrombi (fewer emboli) after short injury, but it had no effect after longer injury. TTO and emboli correlated with TATs. These results demonstrate dependence of FVIII activity on extent of vascular injury. We propose elevated plasma FVIII is an etiologic, prothrombotic agent after moderate but not extensive vascular damage.

Topics: Animals; Antithrombin III; Carotid Artery Injuries; Cells, Cultured; Chlorides; Factor VIII; Ferric Compounds; Humans; Male; Mice; Mice, Inbred C57BL; Peptide Hydrolases; Platelet Aggregation; Thrombin; Thromboplastin; Thrombosis; Vascular System Injuries

Tissue factor (TF) initiates coagulation, regulates hemostasis, and plays a critical role in mediating arterial thrombosis. TF is up-regulated in vascular smooth muscle cells (VSMCs) in atherosclerosis and arterial injury. To examine the biologic role of VSMC-derived TF, we crossed TF(flox/flox) mice with SM22alphaCre(+/-) mice. TF mRNA and activity were decreased in the aortic media of TF-deficient mice by 96% and 94.8%, respectively. There were no differences in TF activity measured in plasma or concentrated microparticles. TF-deficient mice were generated with the expected frequency, showed no evidence of bleeding or increased mortality, and had similar activated partial thromboplastin and tail vein bleeding times. Thrombus-mediated flow reduction in response to ferric chloride injury of the carotid arteries was significantly attenuated in VSMC-specific TF-deficient. Stable occlusion was seen in 11 of 12 wild-type mice, but in only 6 of 16 VSMC-specific TF-deficient mice (P = .001). These data suggest that VSMC-derived TF is critical in a macrovascular model of arterial thrombosis. This mouse model should be valuable in determining the contribution of VSMC-derived TF in other TF-mediated phenomena, such as restenosis.

Topics: Animals; Aorta; Carotid Artery Thrombosis; Chlorides; Enzyme-Linked Immunosorbent Assay; Female; Ferric Compounds; Fibrosis; Immunohistochemistry; Male; Mice; Mice, Mutant Strains; Muscle, Smooth, Vascular; Myocardium; Myocytes, Smooth Muscle; Noxae; Reverse Transcriptase Polymerase Chain Reaction; Thromboplastin; Thrombosis

Diclofenac, like selective cyclooxygenase-2 inhibitors, which alter vascular levels of platelet active prostaglandins, has been reported to increase rates of acute myocardial infarction.. The study was performed to investigate, in an animal model of arterial thrombosis in vivo, whether diclofenac differentially influences platelet activation and thrombosis in vessels under non-stimulated conditions or during acute systemic inflammation, such as induced by tumor necrosis factor-alpha (TNF-alpha).. Platelet-vessel wall interaction (PVWI), firm platelet adhesion and arterial thrombosis following vessel injury were analyzed by intravital microscopy in arterioles of hamsters in the dorsal skinfold chamber model. Prostacyclin [prostaglandin I(2) (PGI(2))] and thromboxane A(2) (TxA(2)) metabolites were measured. In vitro, endothelial adhesion molecule expression in cultured human microvascular endothelial cells was analyzed.. Under non-stimulated conditions, diclofenac (1 mg kg(-1)) enhanced PVWI, which was not mediated by increased adhesion molecule expression, but by decreased systemic PGI(2) levels. Following ferric chloride-induced endothelial injury, diclofenac accelerated thrombotic vessel occlusion time, an effect that was reversed by the stable PGI(2) analog iloprost. TNF-alpha, through induction of endothelial adhesion molecule expression, also enhanced PVWI, firm adhesion, and arterial thrombosis, but simultaneous treatment with TNF-alpha and diclofenac did not have an additive effect.. By decreasing levels of PGI(2) without, at the same time, altering prothrombotic TxA(2) levels, diclofenac can exert prothrombotic effects. However, this is not the case when an inflammatory situation is created by TNF-alpha treatment. These data may explain the enhanced risk of acute myocardial infarction observed in patients taking diclofenac.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arterioles; Cells, Cultured; Chlorides; Cricetinae; Cyclooxygenase Inhibitors; Diclofenac; Endothelium, Vascular; Ferric Compounds; Humans; Mesocricetus; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Platelet Activation; Platelet Adhesiveness; Skin Window Technique; Thromboplastin; Thrombosis; Thromboxane B2; Tumor Necrosis Factor-alpha

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 present study was designed to elucidate the role of p38 mitogen-activated protein kinase (p38) in thrombus formation. We used p38alpha heterozygous (p38alpha+/-) mice and used ferric chloride (FeCl3)-induced carotid artery injury as a model of thrombus formation. The time to thrombotic occlusion induced by FeCl3 in p38alpha+/- mice was prolonged compared to that in wild-type (WT) mice. Platelets prepared from p38alpha+/- mice showed impairment of the aggregatory response to a low concentration of U46619, a thromboxane A2 analogue. Furthermore, platelets prepared from p38alpha+/- mice and activated by U46619 were poorly bound to fibrinogen compared with those from WT mice. Both the expression and activity of tissue factor induced by FeCl3 in WT mice were higher than those in p38alpha+/- mice. These results suggest that p38 plays an important role in thrombus formation by regulating platelet function and tissue factor activity.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Alleles; Animals; Blood Coagulation; Blood Platelets; Chlorides; Ferric Compounds; Fibrinogen; Heterozygote; Homozygote; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; p38 Mitogen-Activated Protein Kinases; Placenta; Platelet Adhesiveness; Protein Binding; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thromboplastin; Thrombosis; Thromboxane A2; Time Factors; Vasoconstrictor Agents

The recent observation that knock-out of protease-activated receptor-4 (PAR4) ablates thrombin signaling in mouse platelets and protects against ferric chloride-induced thrombosis of mouse mesenteric arterioles suggests that thrombin's actions on platelets can play an important role in thrombosis. Complete ablation of thrombin signaling would be difficult to achieve in human beings because human platelets have 2 thrombin receptors that are each capable of mediating transmembrane signaling. However, it is possible that complete ablation of thrombin signaling in platelets is not necessary for an antithrombotic effect. In mouse platelets, PAR3 functions as a cofactor that binds thrombin and promotes productive cleavage of PAR4, and thrombin responses are decreased but not absent in Par3(-/-) platelets. We now report that Par3(-/-) mice were protected against ferric chloride-induced thrombosis of mesenteric arterioles and against thromboplastin-induced pulmonary embolism. Surprisingly, Par3(-/-) and Par4(-/-) mice showed similar degrees of protection in these models and similar prolongation of tail bleeding times. Thus, even a partial decrease in mouse platelet responsiveness to thrombin protected against thrombosis and impaired hemostasis in some settings. These results demonstrate the importance of PAR3's unusual cofactor function and underscore the relative importance of thrombin's actions on platelets in vivo. They also suggest that PAR inhibition might be explored for the prevention or treatment of thrombosis in human beings.

Topics: Animals; Arterioles; Bleeding Time; Chlorides; Enzyme Activation; Female; Ferric Compounds; Genotype; Lung; Mesentery; Mice; Mice, Inbred C57BL; Mice, Knockout; Platelet Activation; Pulmonary Embolism; Receptors, Thrombin; Single-Blind Method; Thrombin; Thromboplastin; Thrombosis