fibrin and Carotid-Artery-Thrombosis

The role of neutrophil extracellular traps (NETs) in procoagulant activity (PCA) in stroke patients caused by thromboembolic occlusion of the internal carotid artery (ICA) remains unclear. Our objectives were to evaluate the critical role of NETs in the induction of hypercoagulability in stroke and to identify the functional significance of NETs during atherothrombosis.. The levels of NETs, activated platelets (PLTs), and PLT-derived microparticles (PMPs) were detected in the plasma of 55 stroke patients and 35 healthy controls. NET formation and thrombi were analysed using immunofluorescence. Exposed phosphatidylserine (PS) was evaluated with flow cytometry and confocal microscopy. PCA was analysed using purified coagulation complex, thrombin, and fibrin formation assays.. The plasma levels of NETs, activated PLTs, and PMP markers in the carotid lesion site (CLS) were significantly higher than those in the aortic blood. NETs were decorated with PS in thrombi and the CLS plasma of ICA occlusion patients. Notably, the complementary roles of CLS plasma and thrombin-activated PLTs were required for NET formation and subsequent PS exposure. PS-bearing NETs provided functional platforms for PMPs and coagulation factor deposition and thus increased thrombin and fibrin formation. DNase I and lactadherin markedly inhibited these effects. In addition, NETs were cytotoxic to endothelial cells, converting these cells to a procoagulant phenotype. Sivelestat, anti-MMP9 antibody, and activated protein C (APC) blocked this cytotoxicity by 25%, 39%, or 52%, respectively.. NETs played a pivotal role in the hypercoagulability of stroke patients. Strategies that prevent NET formation may offer a potential therapeutic strategy for thromboembolism interventions.. This study was supported by grants from the National Natural Science Foundation of China (61575058, 81873433 and 81670128) and Graduate Innovation Fund of Harbin Medical University (YJSKYCX2018-58HYD).

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Blood Coagulation; Blood Platelets; Carotid Artery Thrombosis; Carotid Artery, Internal; Cell-Derived Microparticles; Extracellular Traps; Female; Fibrin; Glycine; Human Umbilical Vein Endothelial Cells; Humans; Male; Middle Aged; Neutrophils; Phosphatidylserines; Platelet Activation; Stroke; Sulfonamides; Thrombin

Topics: Animals; Boronic Acids; Carotid Artery Thrombosis; CD40 Ligand; Cell Survival; Chlorides; Drug Carriers; Drug Liberation; Endothelial Cells; Ferric Compounds; Fibrin; Fibrinolytic Agents; Fluorescent Dyes; Humans; Hydrogen Peroxide; Lipopeptides; Mice; Nanoparticles; Optical Imaging; Polymers; RAW 264.7 Cells; Theranostic Nanomedicine; Thrombosis; Tirofiban; Tumor Necrosis Factor-alpha

Computed tomography (CT) is the current standard for time-critical decision-making in stroke patients, informing decisions on thrombolytic therapy with tissue plasminogen activator (tPA), which has a narrow therapeutic index. We aimed to develop a CT-based method to directly visualize cerebrovascular thrombi and guide thrombolytic therapy. Glycol-chitosan-coated gold nanoparticles (GC-AuNPs) were synthesized and conjugated to fibrin-targeting peptides, forming fib-GC-AuNP. This targeted imaging agent and non-targeted control agent were characterized in vitro and in vivo in C57Bl/6 mice (n = 107) with FeCl3-induced carotid thrombosis and/or embolic ischemic stroke. Fibrin-binding capacity was superior with fib-GC-AuNPs compared to GC-AuNPs, with thrombi visualized as high density on microCT (mCT). mCT imaging using fib-GC-AuNP allowed the prompt detection and quantification of cerebral thrombi, and monitoring of tPA-mediated thrombolytic effect, which reflected histological stroke outcome. Furthermore, recurrent thrombosis could be diagnosed by mCT without further nanoparticle administration for up to 3 weeks. fib-GC-AuNP-based direct cerebral thrombus imaging greatly enhance the value and information obtainable by regular CT, has multiple uses in basic / translational vascular research, and will likely allow personalized thrombolytic therapy in clinic by a) optimizing tPA-dosing to match thrombus burden, b) enabling the rational triage of patients to more radical therapies such as endovascular clot-retrieval, and c) potentially serving as a theranostic platform for targeted delivery of concurrent thrombolysis.

Topics: Animals; Brain; Carotid Artery Thrombosis; Fibrin; Gold; Humans; Male; Mice; Mice, Inbred C57BL; Nanoparticles; Stroke; Tomography, X-Ray Computed

Fibrin is a major component of arterial and venous thrombi and represents an ideal candidate for molecular imaging of thrombosis. Here, we describe imaging properties and target uptake of a new fibrin-specific positron emission tomographic probe for thrombus detection and therapy monitoring in 2 rat thrombosis models.. The fibrin-binding probe FBP7 was synthesized by conjugation of a known short cyclic peptide to a cross-bridged chelator (CB-TE2A), followed by labeling with copper-64. Adult male Wistar rats (n=26) underwent either carotid crush injury (mural thrombosis model) or embolic stroke (occlusive thrombosis model) followed by recombinant tissue-type plasminogen activator treatment (10 mg/kg, IV). FBP7 detected thrombus location in both animal models with a high positron emission tomographic target-to-background ratio that increased over time (>5-fold at 30-90 minutes, >15-fold at 240-285 minutes). In the carotid crush injury animals, biodistribution analysis confirmed high probe uptake in the thrombotic artery (≈0.5%ID/g; >5-fold greater than blood and other tissues of the head and thorax). Similar results were obtained from ex vivo autoradiography of the ipsilateral versus contralateral carotid arteries. In embolic stroke animals, positron emission tomographic-computed tomographic imaging localized the clot in the internal carotid/middle cerebral artery segment of all rats. Time-dependent reduction of activity at the level of the thrombus was detected in recombinant tissue-type plasminogen activator-treated rats but not in vehicle-injected animals. Brain autoradiography confirmed clot dissolution in recombinant tissue-type plasminogen activator-treated animals, but enduring high thrombus activity in control rats.. We demonstrated that FBP7 is suitable for molecular imaging of thrombosis and thrombolysis in vivo and represents a promising candidate for bench-to-bedside translation.

Topics: Animals; Carotid Artery Thrombosis; Carrier Proteins; Disease Models, Animal; Fibrin; Intracranial Thrombosis; Male; Molecular Imaging; Positron-Emission Tomography; Rats; Rats, Wistar; Reproducibility of Results; Tissue Distribution; Tomography, X-Ray Computed

Topics: Animals; Carotid Artery Thrombosis; Fibrin; Intracranial Thrombosis; Male; Molecular Imaging; Positron-Emission Tomography

Epidemiological, biological and clinical links between periodontal and cardiovascular diseases are now well established. Several human studies have detected bacterial DNA corresponding to periodontal pathogens in cardiovascular samples. Intraplaque hemorrhage has been associated with a higher risk of atherosclerotic plaque rupture, potentially mediated by neutrophil activation. In this study, we hypothesized that plaque composition may be related to periodontal pathogens.. Carotid culprit plaque samples were collected from 157 patients. Macroscopic characterization was performed at the time of collection: presence of blood, lipid core, calcification and fibrosis. Markers of neutrophil activation released by carotid samples were quantified (myeloperoxidase or MPO, cell-free DNA and DNA-MPO complexes). PCR analysis using specific primers for Porphyromonas gingivalis, Aggregatibacter actinomycetemcommitans, Treponema denticola, Prevotella intermedia and Tannerella forsythia was used to detect DNA from periodontal pathogens in carotid tissues. In addition, bacterial lipopolysaccharide (LPS) and Immunoglobulins G against T. forsythia were quantified in atherosclerotic carotid conditioned medium.. Intraplaque hemorrhage was present in 73/157 carotid samples and was associated with neutrophil activation, reflected by the release of MPO, cell-free DNA and MPO-DNA complexes. LPS levels were also linked to intraplaque hemorrhage but not with the neutrophil activation markers. Seventy-three percent of the carotid samples were positive for periodontal bacterial DNA. Furthermore, hemoglobin levels were associated with the detection of T. forsythia and neutrophil activation/inflammation markers.. This study suggests a potential role of periodontal microorganisms, especially T. forsythia, in neutrophil activation within hemorrhagic atherosclerotic carotid plaques.

Topics: Aged; Aged, 80 and over; Antibodies, Bacterial; Bacteroidaceae; Carotid Artery Diseases; Carotid Artery Thrombosis; Dental Plaque; DNA, Bacterial; Endarterectomy, Carotid; Extracellular Traps; Female; Fibrin; Hemorrhage; Humans; Lipids; Male; Middle Aged; Neutrophil Infiltration; Neutrophils; NF-kappa B; Periodontitis; Peroxidase; Plaque, Atherosclerotic; Respiratory Burst

The α(1,3)-fucosyltransferases, types IV and VII (FUT4 and FUT7, respectively), are required for the synthesis of functional selectin-type leukocyte adhesion molecule ligands. The selectins and their ligands modulate leukocyte trafficking, and P-selectin and its ligand, P-selectin glycoprotein ligand-1, can modulate hemostasis and thrombosis. Regulation of thrombosis by FUT4 and/or FUT7 activity was examined in mouse models of carotid artery thrombosis and collagen/epinephrine-induced thromboembolism. Mice lacking both FUT4 and FUT7 (Fut(-/-) mice) had a shorter time to occlusive thrombus formation in the injured carotid artery and a higher mortality due to collagen/epinephrine-induced pulmonary thromboemboli. Mice lacking P-selectin or P-selectin glycoprotein ligand-1 did not have a prothrombotic phenotype. Whole blood platelet aggregation was enhanced, and plasma fibrinogen content, clot weight, and clot strength were increased in Fut(-/-) mice, and in vitro clot lysis was reduced compared with wild type. Fut4(-/-), but not Fut7(-/-), mice had increased pulmonary thromboembolism-induced mortality and decreased thromboemboli dissolution in vivo. These data show that FUT4 and FUT7 activity regulates thrombosis in a P-selectin- and P-selectin glycoprotein ligand-1-independent manner and suggest that FUT4 activity is important for thrombolysis.

Topics: Animals; Blood Coagulation; Carotid Artery Thrombosis; Disease Susceptibility; Fibrin; Fibrinogen; Fibrinolysis; Fucosyltransferases; Kaplan-Meier Estimate; Mice; Mice, Knockout; Partial Thromboplastin Time; Platelet Aggregation; Prothrombin Time; Pulmonary Embolism; Thrombin; Time Factors

Fibrin targeting is an attractive strategy for nuclear imaging of thrombosis, atherosclerosis and cancer. Recently, FibPep, an (111)In-labeled fibrin-binding peptide, was established as a tracer for fibrin SPECT imaging and was reported to allow sensitive detection of minute thrombi in mice using SPECT. In this study, we developed EPep, a novel (111)In-labeled fibrin-binding peptide containing the fibrin-binding domain of the clinically verified EP-2104R peptide, and sought to compare the potential of EPep and FibPep as tracers for fibrin SPECT imaging. In vitro, both EPep and FibPep showed high stability in serum, but were less stable in liver and kidney homogenate assays. Both peptide probes displayed comparable affinities toward human and mouse derived fibrin (Kd ≈ 1 μM), and similarly to FibPep, EPep showed fast blood clearance, low nontarget uptake and high thrombus uptake (6.8 ± 1.2% ID g(-1)) in a mouse carotid artery thrombosis model. Furthermore, EPep showed a similar affinity toward rat derived fibrin (Kd ≈ 1 μM), displayed high thrombus uptake in a rat carotid artery thrombosis model (0.74 ± 0.39% ID g(-1)), and allowed sensitive detection of thrombosis in rats using SPECT. In contrast, FibPep displayed a significantly lower affinity toward rat derived fibrin (Kd ≈ 14 μM) and low uptake in rat thrombi (0.06 ± 0.02% ID g(-1)) and did not allow clear visualization of carotid artery thrombosis in rats using SPECT. These results were confirmed ex vivo by autoradiography, which showed a 7-fold higher ratio of activity in the thrombus over the contralateral carotid artery for EPep in comparison to FibPep. These findings suggest that the FibPep binding fibrin epitope is not fully homologous between humans and rats, and that preclinical rat models of disease should not be employed to gauge the clinical potential of FibPep. In conclusion, both peptides showed approximately similar metabolic stability and affinity toward human and mouse derived fibrin, and displayed high thrombus uptake in a mouse carotid artery thrombosis model. Therefore, both EPep and FibPep are promising fibrin targeted tracers for translation into clinical settings to serve as novel tools for molecular imaging of fibrin.

Topics: Animals; Carotid Artery Thrombosis; Fibrin; Humans; Indium Radioisotopes; Peptides; Rats; Thrombosis; Tomography, Emission-Computed, Single-Photon

Atherothrombosis is a major cause of cardiovascular events. However, animal models to study this process are scarce.. We describe the first murine model of acute thrombus formation upon plaque rupture to study atherothrombosis by intravital fluorescence microscopy.. Localized rupture of an atherosclerotic plaque in a carotid artery from Apoe(-/-) mice was induced in vivo using ultrasound. Rupture of the plaque and formation of localized thrombi were verified by two-photon laser scanning microscopy (TPLSM) in isolated arteries, and by immunohistochemistry. The thrombotic reaction was quantified by intravital fluorescence microscopy.. Inspection of the ultrasound-treated plaques by histochemistry and TPLSM demonstrated local damage, collagen exposure, luminal thrombus formation as well as intra-plaque intrusion of erythrocytes and fibrin. Ultrasound treatment of healthy carotid arteries resulted in endothelial damage and limited platelet adhesion. Real-time intravital fluorescence microscopy demonstrated rapid platelet deposition on plaques and formation of a single thrombus that remained subocclusive. The thrombotic process was antagonized by thrombin inhibition, or by blocking of collagen or adenosine diphosphate receptor pathways. Multiple thrombi were formed in 70% of mice lacking CD40L.. Targeted rupture of murine plaques results in collagen exposure and non-occlusive thrombus formation. The thrombotic process relies on platelet activation as well as on thrombin generation and coagulation, and is sensitive to established and novel antithrombotic medication. This model provides new possibilities to study atherothrombosis in vivo.

Topics: Animals; Atherosclerosis; Blood Coagulation; Blood Platelets; Carotid Artery Thrombosis; Collagen; Disease Models, Animal; Erythrocytes; Fibrin; Mice; Microscopy, Fluorescence; Thrombosis

We studied the antithrombotic and thrombolytic effects of Trombovazim, a highly-purified proteolytic enzyme preparation obtained by immobilization of bacterial proteinases (Bacillus) on polyethylene oxide with a molecular weight of 1.5 kDa. Blood absorption of the preparation was evaluated after intragastric administration. In vitro experiments showed that Trombovazim produces anticoagulant and thrombolytic effects, which manifested in inhibition of fibrin clot formation and acceleration of its lysis. Drug concentration in the blood was elevated from the 4th to the 7th hour after intragastric administration of Trombovazim in a dose of 2250 U/kg, being maximum by the 5th hour (0.044+/-0.011 U/ml). Course treatment with Trombovazim (1000 U intragastrically, twice daily for 3 days) had a thrombolytic effect on rats with experimental intravascular thrombosis. This effect was manifested in a decrease in thrombus weight and increase in the percent of rats with recanalization of the occluded carotid artery.

Topics: Animals; Anticoagulants; Bacterial Proteins; Blood Coagulation; Carotid Arteries; Carotid Artery Thrombosis; Cerebrovascular Circulation; Ferrous Compounds; Fibrin; Male; Peptide Hydrolases; Rats; Rats, Wistar; Time Factors

The recombinant protein SAK-RGD-K2-Hir is characterized by its fibrin-specific properties of plasminogen activation combined with antithrombin and antiplatelet activities. It was previously shown in our in-vitro studies to be a more potent and faster-acting thrombolytic agent compared with standard r-SAK. In order to document the effects of the thrombolytic potential of SAK-RGD-K2-Hir we examined this protein in an electrically induced carotid artery thrombosis model and stasis-induced venous model in rats. In the arterial thrombosis model, a bolus injection of SAK-RGD-K2-Hir was less effective than rt-PA and r-SAK. However, the most effective in the improvement and maintenance of carotid patency and in arterial thrombus mass reduction was SAK-RGD-K2. In contrast, all r-SAK derivatives reduced venous thrombus weight significantly in comparison to r-SAK and r-Hir. However, the most observable decrease in thrombus weight was obtained after application of recombinant proteins containing the r-Hir. The bleeding time was significantly prolonged in the animals treated with proteins containing r-Hir at a dose of 1.0 mg/kg. There were no observable changes in plasma fibrinogen concentration. In conclusion, our findings show thrombolytic activity in intravenous bolus injection of the novel thrombolytic agent SAK-RGD-K2-Hir in rats. Although this protein compares favourably with r-SAK in rat venous thrombolysis, we were unable to confirm the beneficial effects of SAK-RGD-K2-Hir over r-SAK and rt-PA in the carotid artery thrombolysis model. Furthermore, our results also suggest that SAK-RGD-K2-Hir bears a risk of bleeding, but this may be true for higher doses.

Topics: Animals; Bleeding Time; Blood Coagulation; Carotid Artery Thrombosis; Disease Models, Animal; Dose-Response Relationship, Drug; Electric Stimulation; Fibrin; Fibrinolytic Agents; Hemorrhage; Hirudins; Ligation; Male; Metalloendopeptidases; Partial Thromboplastin Time; Rats; Rats, Wistar; Recombinant Fusion Proteins; Thrombin Time; Time Factors; Tissue Plasminogen Activator; Vascular Patency; Venae Cavae; Venous Thrombosis

Plaque rupture leading to thrombosis and occlusion is a major source of acute coronary syndromes. Methods for accurate detection of thrombosis in veins or arteries may expand our capacity to predict clinical complications and guide therapeutic decisions. We sought to demonstrate the feasibility of in vivo acute thrombus detection using a fibrin-targeted gadolinium based magnetic resonance contrast agent (EP-1242).. Carotid thrombosis was induced in 12 guinea pigs by external injury and blood stasis. MR images were obtained after thrombus formation pre- and post- EP-1242 injection, using a T1-weighted high-resolution fast spin-echo sequence.. An occlusive fibrin-rich thrombus was achieved in all animals. Correlation for thrombus location was excellent between MRI and histology (R=0.94; P<0.001). Contrast-enhanced MRI significantly improved thrombus detection when compared to non contrast-enhanced MRI (100% versus 41.6%; p<0.001). In addition, thrombus signal intensity (SI) was significantly increased after injection (SI(30 min-post)=4.39+/-0.12 versus 1.0; p<0.001). Contrast-to-noise ratio (CNR) was 43.8+/-7.2, 30 min post-injection (P<0.001). No enhancement was seen in the uninjured control arteries.. We demonstrate the feasibility of in vivo MRI for carotid thrombus detection using a novel fibrin-targeted contrast agent. This technique significantly improves detection of small size thrombi in an animal model of occlusive fibrin-rich thrombosis.

Topics: Acute Disease; Animals; Carotid Artery Thrombosis; Contrast Media; Disease Models, Animal; Fibrin; Guinea Pigs; Magnetic Resonance Imaging; Peptides, Cyclic

Fibrin coatings on prosthetic vascular graft, which are conventionally produced by fibrinogen and thrombin, are expected to improve antithrombogenicity and healing characteristics. Thrombin is one of the factors of blood coagulation cascade; however, it has a possibility to play a negative role in the graft antithrombogenicity. The purpose of this study was to evaluate the performance of our new grafts, thrombin-free fibrin-coated small caliber vascular prostheses. Knitted polyester fabric vascular prostheses 2 mm in internal diameter were coated with fibrin coating with thrombin (Graft I) or without thrombin (Graft II). Both grafts were implanted in bilateral common carotid arteries of 35 Japanese white rabbits, with Graft I in one side and Graft II in the contralateral side. Graft patency, histology, thrombin activity, and platelet deposition were compared between both grafts on postoperative days (PODs) 1, 3, 7, 10, 14, 30, and 60. Both grafts were patent without thrombus or stenosis at each end point (maximal period, POD 60). Macro- and microscopic findings revealed that no obvious difference was observed between both grafts. Before graft implantation, thrombin activities in Grafts I and II were 0.711 +/- 0.086 and 0.009 +/- 0.007 optical density at 405 nm, respectively. Thrombin activity of Graft II was significantly less than that of Graft I in every period after graft implantation, and platelet deposition of Graft II was significantly less than that of Graft I until POD 30. Thrombin-free fibrin-coated vascular prostheses have superior performance of antithrombogenicity to conventional fibrin-coated vascular prostheses with thrombin.

Topics: Animals; Blood Vessel Prosthesis; Carotid Artery Thrombosis; Coated Materials, Biocompatible; Drug Implants; Equipment Failure Analysis; Fibrin; Fibrinolytic Agents; Miniaturization; Platelet Activation; Prosthesis Design; Rabbits; Thrombin; Treatment Outcome

In this study, we investigated if elevation of endogenous plasminogen activator inhibitor type 1 (PAI-1) by lipopolysaccharide (LPS) can retard thrombolysis in both a rat model of lung vasculature fibrin deposition and a platelet-rich thrombus model induced by endothelial injury. By 3 h following an intravenous bolus injection of 0.5 mg/kg LPS, the plasma PAI-1 level had increased to approximately 8 ng/ml. 125I-labeled fibrinogen was injected intravenously followed by an injection of batroxobin. Batroxobin converts fibrinogen into insoluble fibrin, which was then deposited in the lungs within 5 min, followed by spontaneous fibrinolysis that completely cleared fibrin deposition in the lungs by 30 min. In rats pre-treated with LPS, spontaneous fibrinolysis was significantly retarded. In the endothelial injury model, topical application of FeCl2 on the carotid artery induced an occlusive platelet-rich thrombus, which was not sensitive to endogenous thrombolysis. Exogenous tissue-type plasminogen activator (tPA) was required to recanalize the occlusive thrombus in a dose-dependent manner. Pre-treatment with LPS did not alter the dose-response curve of exogenous tPA-induced thrombolysis. These data indicate that batroxobin-induced lung vasculature fibrin deposition in rats, unlike the FeCl2 model, is sensitive to the impact of endogenous PAI-1 on fibrinolysis.

Topics: Animals; Batroxobin; Carotid Arteries; Carotid Artery Thrombosis; Ferrous Compounds; Fibrin; Lipopolysaccharides; Lung Diseases; Male; Plasminogen Activator Inhibitor 1; Rats; Rats, Sprague-Dawley; Tissue Plasminogen Activator

Platelet integrin alpha IIb beta 3 (GPIIb/IIIa) plays a central role in the initiation of arterial thrombosis, but its contribution to disseminated microvascular thrombosis is less well defined. Therefore, wild-type mice (beta 3(+/+)), beta 3-integrin-deficient mice (beta 3(-/-)), and wild-type mice treated with a hamster monoclonal antibody (1B5) that blocks murine alpha IIb beta 3 function were tested in models of large-vessel and microvascular thrombosis. In the large-vessel model, ferric chloride was used to injure the carotid artery, and the time to thrombosis was measured. In beta 3(+/+) mice, the median time to occlusion was 6.7 minutes, whereas occlusion did not occur in any of the beta 3(-/-) mice tested (P <.001). Fab and F(ab')(2) fragments of 1B5 increased the median time to occlusion. To initiate systemic intravascular thrombosis, prothrombotic agents were administered intravenously, and platelet thrombus formation was monitored by the decrease in circulating platelet count. Three minutes after the injection of adenosine diphosphate (ADP), collagen + epinephrine, or tissue factor, the platelet counts in beta 3(+/+) mice decreased by 289, 424, and 429 x 10(3)/microL, respectively. beta 3(-/-) mice and wild-type mice pretreated with 1B5 Fab (1 mg/kg, IP) were nearly completely protected from the effects of ADP. In contrast, beta 3(-/-) mice were only partially protected from the effects of collagen + epinephrine and minimally protected from the effects of tissue factor. In all cases, less fibrin became deposited in the lungs of beta 3(-/-) mice than in wild-type mice. These results suggest that though alpha IIb beta 3 plays a dominant role in large-vessel thrombosis, it plays a variable role in systemic intravascular thrombosis. (Blood. 2001;98:1055-1062)

Topics: Animals; Antibodies, Monoclonal; Antigens, CD; Blood Platelets; Carotid Artery Thrombosis; Chlorides; Disease Models, Animal; Female; Ferric Compounds; Fibrin; Immunohistochemistry; Integrin beta3; Male; Mice; Mice, Knockout; Microcirculation; Microscopy, Electron; Platelet Glycoprotein GPIIb-IIIa Complex; Platelet Membrane Glycoproteins; Receptors, Vitronectin; Thrombosis

Mutations in the gene encoding thrombomodulin (TM), a thrombin regulator, are suspected risk factors for venous and arterial thrombotic disease. We have previously described the generation of TM(Pro/Pro) mice carrying a TM gene mutation that disrupts the TM-dependent activation of protein C. Here, it is shown that inbred C57BL/6J TM(Pro/Pro) mice exhibit a hypercoagulable state and an increased susceptibility to thrombosis and sepsis. Platelet thrombus growth after FeCl(3)-induced acute endothelial injury was accelerated in mutant mice. Vascular stasis after permanent ligation of the carotid artery precipitated thrombosis in mutant but not in normal mice. Mutant mice showed increased mortality after exposure to high doses of endotoxin and demonstrated altered cytokine production in response to low-dose endotoxin. The severity of the hypercoagulable state and chronic microvascular thrombosis caused by the TM(Pro) mutation is profoundly influenced by mouse strain-specific genetic differences between C57BL/6 and 129SvPas mice. These data demonstrate that in mice, TM is a physiologically relevant regulator of platelet- and coagulation-driven large-vessel thrombosis and modifies the response to endotoxin-induced inflammation. The phenotypic penetrance of the TM(Pro) mutation is determined by as-yet-uncharacterized genetic modifiers of thrombosis other than TM.

Topics: Animals; Blood Coagulation; Carotid Artery Thrombosis; Chlorides; Cytokines; Ferric Compounds; Fibrin; Genetic Predisposition to Disease; Ligation; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Sepsis; Survival Analysis; Thrombomodulin; Thrombosis

The assumption that fibrin and crosslinked fibrin impart irreversibility to arterial thrombi is explored with procedure developed for measuring changes in platelet function, morphology and fibrinogen metabolism in aging occlusive thrombi, in which the condition of stasis is imposed uniformly. Arterial thrombi containing autologous (111)In labeled platelets were generated in vivo by bilateral mechanical injury of porcine carotid arteries. Vessels containing the platelet-rich thrombi were harvested and incubated intact (37 degrees C) for intervals ranging from 30 min to 12 h. The isolated vessels were then bisected and agitated in culture medium containing tick anticoagulant and hirudin for 60 min. Disaggregated platelets were evaluated for yield (from (111)In radioactivity) viability (dense body ATP secretion) and morphology (electron microscopy). Western analysis of fibrin(ogen) in thrombus extracts was performed using anti-fibrinogen Bbeta- and gamma-chain monoclonal antibodies for thrombi at each time point. A stable recovery of nearly 50% of platelets was observed during 12 h of thrombus aging. As thrombi aged, viability of disaggregated platelets gradually decreased with platelet necrosis the predominant feature beyond 6 h. By western analysis of thrombus extracts, nearly 50% of fibrinogen was cleaved to fibrin and extensively crosslinked within 30 min of injury with no evidence of fibrinolysis. With the exception of a declining proportion of gamma-monomer, these features remain relatively constant during 12 h of thrombus maturation. It is concluded that neither fibrin nor crosslinked fibrin are dominant factors imparting cohesion within platelet thrombi. Furthermore, under conditions of complete arterial occlusion imposed by this experimental design, there is no evidence of endogenous fibrinolysis.

Topics: Adenosine Triphosphate; Animals; Arthropod Proteins; Blood Platelets; Carotid Artery Injuries; Carotid Artery Thrombosis; Cytoplasmic Granules; Fibrin; Fibrin Fibrinogen Degradation Products; Fibrinogen; Fibrinolysis; Fibrinolytic Agents; Hirudins; Intercellular Signaling Peptides and Proteins; Necrosis; Peptides; Recombinant Proteins; Swine; Time Factors

To assess in a swine model the in vivo thrombogenicity of various microcatheters and guiding catheters as a function of catheter material, catheter coating, and duration of implantation.. Microcatheters (Tracker 18 and Fastracker 18, Target Therapeutics, Fremont, Calif; Magic 1.8, Balt, Montmorency, France; and Transit, Cordis Endovascular Systems, Miami Lakes, Fla) were placed through 6F guiding catheters (Fasguide, Target Therapeutics, and Envoy, Cordis Endovascular Systems) into the common carotid arteries of swine for 30 minutes (short term), 90 minutes (medium term), and 35 days (long term). Guiding catheters were implanted for 5 hours. At the end of the implantation periods the catheters were retracted and fixed for scanning electron microscopy.. The surface of the Fastracker microcatheter was devoid of debris after both short- and medium-term implantation. The Tracker microcatheter had minimal accumulation of cellular elements whereas the Transit microcatheter showed moderate accumulation of nondeformed red blood cells. Neither the Tracker nor the Transit microcatheter showed evidence of increasing debris accumulation after medium-term implantation as compared with short-term implantation. The Magic microcatheter was coated with gross thrombus after both short- and medium-term implantation. The Fasguide guiding catheter was nearly devoid of debris, while the Envoy guiding catheter had moderate thrombus formation. Long-term implantation of the Fastracker microcatheter was well tolerated whereas that of the Transit catheter resulted in vessel occlusion.. Hydrophilic microcatheters and guiding catheters are less thrombogenic than their nonhydrophilic counterparts, but not all hydrophilic coatings are equally hypothrombogenic. Degree of thrombogenicity depends on catheter material rather than surface morphology. Medium-term implantation did not yield increasing thrombus formation relative to short-term implantation.

Topics: Animals; Carotid Artery Thrombosis; Carotid Artery, Common; Catheters, Indwelling; Cerebral Angiography; Equipment Design; Fibrin; Microscopy, Electron, Scanning; Platelet Aggregation; Surface Properties; Swine; Thromboembolism

The procoagulant effect of anionic phospholipid may play a major role in the development of arterial thrombosis.. Annexin V, a calcium-dependent anionic-phospholipid-binding protein, was expressed and isolated from Escherichia coli and its antithrombotic effect examined in a rabbit carotid artery thrombosis model. A partially occlusive thrombus was formed in the left carotid artery by application of electric current to produce an approximately 50% occlusion of the lumen. After the current was discontinued, flow ceased completely within 42+/-12 minutes (n=6) because of continuing platelet/fibrin thrombus formation. When annexin V was given at doses of 2.8 to 16.6 microg x kg(-1) x min(-1) for a period of 180 minutes, starting at the time the current was stopped, there was a dose-dependent inhibition of thrombus formation. At a dose of 5.6 microg x kg(-1) x min(-1), blood flow remained patent throughout the infusion and for an additional 60 minutes after the infusion was stopped. In addition, there was a decrease in thrombus weight (16+/-7.4 versus 2.0+/-1.0 g), (125)I-fibrin deposition (approximately 45% reduction, P<.001), and (111)In-labeled platelet accumulation (approximately 43% reduction, P<.001). Prior mixing of annexin V with phosphatidylserine micelles abolished the antithrombotic effect of annexin V, whereas mixing with phosphatidylcholine micelles had no effect. The antithrombotic effect of annexin V was not associated with bleeding tendency, as judged by the amount of blood absorbed in a gauze pad placed in a surgical incision extending to the muscle tissue and by the standard template bleeding time.. These observations support a potentially important role for anionic phospholipid exposure in platelets in arterial thrombosis, and inhibition of this activity could be a novel target for therapy in coronary thrombosis and stroke and after angioplasty.

Topics: Animals; Annexin A5; Antibodies, Monoclonal; Blood Platelets; Carotid Arteries; Carotid Artery Injuries; Carotid Artery Thrombosis; Cloning, Molecular; Drug Carriers; Electric Stimulation; Escherichia coli; Fibrin; Heparin; Indium Radioisotopes; Iodine Radioisotopes; Male; Microscopy, Electron, Scanning; Partial Thromboplastin Time; Phosphatidylserines; Platelet Glycoprotein GPIIb-IIIa Complex; Rabbits; Recombinant Proteins

In order to specifically detect the localization of thrombus in vivo, we have recently developed two monoclonal antibodies (SZ-58, SZ-63) which can specifically bind to cross-linked fibrin. The binding rates of the two monoclonal antibodies (MoAbs) to human plasma clots in vitro were 46.4 +/- 2.3% for 125I-SZ-58, 50.1 +/- 1.7% for 125I-SZ-63 and 3.4 +/- 1.6% for 125I-SZ-53 (control, MoAb against TM). It was shown that both SZ-58 and SZ-63 possess properties of inhibiting the polymerization of fibrin, and SZ-58 could also inhibit the aggregation of platelets induced by ADP. These characteristics make the two MoAbs suitable in the detection of thrombus in vivo. According to the cross reaction tests, thrombi in the jugular veins and carotid arteries in rabbits were made. After injection of the 125I-labeled MoAbs (100,000 cpm/ml of blood), the thrombi and the blood were taken and weighed at various time intervals and radioactivities were measured by an autogamma counter. The ratios of thrombus to blood radioactivity (T/B) of thrombi in jugular veins were 3.0, 5.6 and 3.0 for 125I-SZ-58, 1.5, 3.0 and 5.2 for 125I-SZ-63 and 1.2, 1.0 and 0.7 for control (125I-SZ-53) at the 3rd, 12th and 24th hour after the injection of the radiolabled MoAbs, while the radioactivities of arterial thrombi were almost the same as that in blood after the injection of the two radiotracers. Therefore, it can be concluded that both SZ-58 and SZ-63 can be used in venous thrombus imaging in vivo and the optimal times of imaging are at the 12th hour for SZ-58, 24th hour for SZ-63 after the injection of the radiolabled MoAbs.

Topics: Animals; Antibodies, Monoclonal; Carotid Artery Thrombosis; Dogs; Fibrin; Guinea Pigs; Humans; Jugular Veins; Platelet Aggregation; Rabbits; Radioimmunoassay; Thrombosis

A rat thrombosis model was developed to assess the efficacity of antithrombotic drugs. It had the following characteristics: controlled hemodynamic and rheological conditions corresponding to arterial flow, a collagen coated surface as a relevant thrombogenic stimulus, a method of measurement allowing dynamic monitoring of thrombus formation and the possibility to assess the thrombus structure. A shunt composed of polyethylene and silicone catheters, including in the middle of the shunt a collagen coated glass capillary, was inserted between the two primitive carotids of the rat. The duration of patency of the shunt was recorded using a thermic probe fixed on its central part. In this model, the patency of the shunt was 539 +/- 55 s. Platelet and fibrinogen-fibrin accumulation in successive one centimeter segments along the shunt were measured using 111In labeled platelets and 125I labeled fibrinogen. Platelet accumulation occurred on the collagen coated surface and at the junctions between the different components of the shunt, where flow was disturbed. The effects of four antithrombotic agents were measured: aspirin, clopidogrel, heparin and r-hirudin. Clopidogrel, heparin and hirudin significantly prolonged patency duration of the shunt, whereas aspirin was inactive. Aspirin did not reduce platelet or fibrinogen-fibrin accumulation on the collagen coated surface. Platelet accumulation on the collagen surface was significantly lower in the clopidogrel group (50 mg/kg) than in the group treated with heparin (500 U/kg), demonstrating the direct antiplatelet effect of clopidogrel. Hirudin at doses giving similar values of APTT as heparin (500 U/kg) prolonged the occlusion time to over 2 h while the heparin occlusion time was only 20 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Anticoagulants; Arterio-Arterial Fistula; Aspirin; Carotid Artery Thrombosis; Clopidogrel; Collagen; Disease Models, Animal; Drug Evaluation, Preclinical; Fibrin; Fibrinogen; Fibrinolytic Agents; Glass; Heparin; Hirudin Therapy; Hirudins; Male; Platelet Adhesiveness; Platelet Aggregation Inhibitors; Polyethylenes; Rats; Rats, Wistar; Recombinant Proteins; Regional Blood Flow; Silicones; Thrombin; Ticlopidine

The effect of heparin and the synthetic irreversible antithrombin D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone (FPRCH2Cl) was studied on FeCl3-induced thrombotic occlusion of rat carotid arteries. Thrombocytopenia prevented occlusion in five of 7 rats for the 60 min observation period after FeCl3 injury demonstrating platelet dependence in this model of thrombosis. Intravenous injection of heparin (250 units/kg) followed by continuous infusion (250 units/kg/hr) failed to prevent occlusion in four of 6 rats whereas intravenous FPRCH2Cl infusion prevented occlusion at a dose of 200 nmol/kg/min during infusion in 6/6 rats. These findings indicate that thrombin plays a principle role in the platelet-dependent process of arterial thrombosis in FeCl3-damaged rat carotid arteries. Neutralization of the thrombogenic stimulus in this model by the thrombin inhibitor FPRCH2Cl suggests selective thrombin inhibition may be useful in the treatment of arterial thrombosis.

Topics: Amino Acid Chloromethyl Ketones; Animals; Blood Platelets; Carotid Artery Thrombosis; Disease Models, Animal; Ferrous Compounds; Fibrin; Heparin; Male; Rats; Rats, Inbred Strains; Thrombin; Thrombocytopenia

We evaluated 40 consecutive carotid endarterectomy specimens for the presence of fibrin. Intraplaque hemorrhage was noted in 93% of specimens. At the plaque surface, there were two patterns of fibrin distribution. Type I, suggesting a lumen thrombus, was found in 7 specimens. Type II, suggesting an intraplaque hemorrhage at the lumen surface, was found in 15 specimens. These changes were not significantly associated with the presence of ischemic symptoms or the use of antiplatelet or anticoagulant medications. All specimens with Type I change had arteriographic evidence of at least 70% diameter stenosis. The frequent lack of fibrin at the plaque surface suggests that there may be inherent limitations of standard medical treatment for carotid artery disease.

Topics: Arteriosclerosis; Carotid Arteries; Carotid Artery Diseases; Carotid Artery Thrombosis; Endarterectomy; Female; Fibrin; Hemorrhage; Histocytochemistry; Humans; Immunoenzyme Techniques; Male; Middle Aged; Prospective Studies

Thrombus formation in the rabbit carotid artery has been studied kinetically in vivo using a minimally invasive technique utilising radioisotopes. Clamping of the carotid artery for 5 min resulted in the simultaneous accumulation of platelets and fibrin at the site of injury over the next 45 min. Under the electron microscope the response was seen to range from platelet monolayer adhesion to mural thrombus formation with fibrin deposition. In animals rendered thrombocytopenic, fibrin deposition was impaired during the first 15-20 minutes after injury. Basic coagulation times and fibrinogen concentration were within normal limits. In addition the injured vessels in these animals accumulated more radiolabelled albumin, but not erythrocytes, than injured vessels in control animals. The results may imply a role for platelets in the enhancement of fibrin deposition during the early part of the response to injury and in contributing to the maintenance of normal permeability following vessel injury.

Topics: Animals; Blood Platelets; Carotid Arteries; Carotid Artery Thrombosis; Chromium Radioisotopes; Erythrocytes; Fibrin; Kinetics; Microscopy, Electron; Neuraminidase; Platelet Adhesiveness; Platelet Aggregation; Platelet Count; Rabbits; Serum Albumin

Two types of 4 mm ID prostheses were studied in the carotid arteries of the dog. These were noncrimped polypropylene-supported filamentous velour knitted Dacron (PPSFV) and expanded polytetrafluoroethylene (e-PTFE, Gore-Tex). Thrombus-"Free" Surface TFS) areas and patency rates were determined at the end of the implant periods. One series of implants was subjected to controlled low flow rates for six hours; another was exposed to physiologic flow rates and observed at seven days, 14 days, and 12 weeks. At six hours the filamentous Dacron, preclotted according to a specific regimen utilizating heparin, performed as well as, and possibly better than, e-PTFE. The Gore-Tex developed surface coagulum in an irregular fashion which was related to graft wetting and blood soakage. Seven-day TFS scores and patency rates of the two graft types were comparable at physiologic flow rates. At two weeks, TFS scores and patency rates of the two graft types were comparable at physiologic flow rates. At two weeks, TFS scores and patency rates dropped. This was sufficiently marked in the case of e-PTFE that longer-term implants were not done. However, PPSFV grafts were implanted for 12 weeks, and all grafts examined at that time had closed. It appears that patency of 4 mm ID grafts of this construction will not be reliably attained in the dog carotid artery without the use of platelet-inhibitory drugs until complete healing has occurred.

Topics: Animals; Blood Flow Velocity; Blood Vessel Prosthesis; Carotid Arteries; Carotid Artery Thrombosis; Dogs; Fibrin; Polyethylene Terephthalates; Polytetrafluoroethylene

Topics: Animals; Blood Platelets; Carotid Arteries; Carotid Artery Thrombosis; Erythrocytes; Female; Fibrin; Hemolysis; Leukocytes; Microscopy, Electron; Phagocytosis; Rats; Time Factors

Report on a case of amaurosis fugax during which the wandering of a fibrin platelet embolus through the vessels of the central arteria was photographed.

Topics: Blindness; Carotid Artery Thrombosis; Embolism; Fibrin; Fluorescein Angiography; Humans; Male; Middle Aged; Platelet Aggregation; Retinal Vessels

Topics: Animals; Blood Cell Count; Blood Platelets; Carotid Artery Thrombosis; Chromium Isotopes; Dogs; Erythrocytes; Female; Fibrin; Fibrinogen; Hematocrit; In Vitro Techniques; Iodine Isotopes; Iron Isotopes; Jugular Veins; Male; Microscopy, Phase-Contrast; Radiometry; Scintillation Counting; Thrombophlebitis; Thrombosis

Topics: Animals; Carotid Artery Thrombosis; Cattle; Dogs; Electricity; Femoral Vein; Fibrin; Fibrinolysis; Jugular Veins; Thrombin; Thrombophlebitis

Topics: Animals; Blood Platelets; Carotid Artery Thrombosis; Collagen; Fibrin; Macrophages; Microscopy, Electron; Muscle, Smooth; Phagocytosis; Staining and Labeling; Swine

Topics: Animals; Blood Platelets; Carotid Artery Thrombosis; Fibrin; Methods; Models, Biological; Polyethylenes; Rats

Topics: Blindness; Blood Platelets; Carotid Artery Thrombosis; Carotid Artery, Internal; Cerebral Infarction; Embolism; Endarteritis; Fibrin; Hemiplegia; Humans; Intracranial Embolism; Intracranial Embolism and Thrombosis; Ischemic Attack, Transient; Leukocytes; Retinal Vessels; Thrombosis