thromboplastin and Carotid-Artery-Thrombosis

Arterial thrombosis is the main underlying mechanism of acute atherothrombosis. Combined antiplatelet and anticoagulant regimens prevent thrombosis but increase bleeding rates. Mast cell-derived heparin proteoglycans have local antithrombotic properties, and their semisynthetic dual AntiPlatelet and AntiCoagulant (APAC) mimetic may provide a new efficacious and safe tool for arterial thrombosis. We investigated the in vivo impact of intravenous APAC (0.3-0.5 mg/kg; doses chosen according to pharmacokinetic studies) in two mouse models of arterial thrombosis and the in vitro actions in mouse platelets and plasma.. Platelet function and coagulation were studied with light transmission aggregometry and clotting times. Carotid arterial thrombosis was induced either by photochemical injury or surgically exposing vascular collagen after infusion of APAC, UFH or vehicle. Time to occlusion, targeting of APAC to the vascular injury site and platelet deposition on these sites were assessed by intra-vital imaging. Tissue factor activity (TF) of the carotid artery and in plasma was captured.. APAC inhibited platelet responsiveness to agonist stimulation (collagen and ADP) and prolonged APTT and thrombin time. After photochemical carotid injury, APAC-treatment prolonged times to occlusion in comparison with UFH or vehicle, and decreased TF both in carotid lysates and plasma. Upon binding from circulation to vascular collagen-exposing injury sites, APAC reduced the in situ platelet deposition.. Intravenous APAC targets arterial injury sites to exert local dual antiplatelet and anticoagulant actions and attenuates thrombosis upon carotid injuries in mice. Systemic APAC provides local efficacy, highlighting APAC as a novel antithrombotic to reduce cardiovascular complications.

Topics: Animals; Anticoagulants; Carotid Artery Thrombosis; Collagen; Fibrinolytic Agents; Mice; Platelet Aggregation; Platelet Aggregation Inhibitors; Thromboplastin; Thrombosis; Vascular System Injuries

CKD, characterized by retained uremic solutes, is a strong and independent risk factor for thrombosis after vascular procedures . Urem ic solutes such as indoxyl sulfate (IS) and kynurenine (Kyn) mediate prothrombotic effect through tissue factor (TF). IS and Kyn biogenesis depends on multiple enzymes, with therapeutic implications unexplored. We examined the role of indoleamine 2,3-dioxygenase-1 (IDO-1), a rate-limiting enzyme of kynurenine biogenesis, in CKD-associated thrombosis after vascular injury.. IDO-1 expression in mice and human vessels was examined. IDO-1. Both global IDO-1. Leveraging genetic and pharmacologic manipulation in experimental models and data from human studies implicate IS as an inducer of IDO-1 and a perpetuator of the thrombotic milieu and supports IDO-1 as an antithrombotic target in CKD.

Topics: Animals; Aorta; Carotid Artery Injuries; Carotid Artery Thrombosis; Culture Media; Enzyme Induction; Feedback, Physiological; Female; HEK293 Cells; Humans; Indican; Indoleamine-Pyrrole 2,3,-Dioxygenase; Kynurenine; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Targeted Therapy; Myocytes, Smooth Muscle; Postoperative Complications; Renal Insufficiency, Chronic; Thromboplastin; Thrombosis; Tryptophan; Uremia; Vascular Surgical Procedures

The precise mechanism for reduced thrombosis in prekallikrein null mice (Klkb1(-/-)) is unknown. Klkb1(-/-) mice have delayed carotid artery occlusion times on the rose bengal and ferric chloride thrombosis models. Klkb1(-/-) plasmas have long-activated partial thromboplastin times and defective contact activation-induced thrombin generation that partially corrects upon prolonged incubation. However, in contact activation-induced pulmonary thromboembolism by collagen/epinephrine or long-chain polyphosphate, Klkb1(-/-) mice, unlike F12(-/-) mice, do not have survival advantage. Klkb1(-/-) mice have reduced plasma BK levels and renal B2R mRNA. They also have increased expression of the renal receptor Mas and plasma prostacyclin. Increased prostacyclin is associated with elevated aortic vasculoprotective transcription factors Sirt1 and KLF4. Treatment of Klkb1(-/-) mice with the Mas antagonist A-779, COX-2 inhibitor nimesulide, or Sirt1 inhibitor splitomicin lowers plasma prostacyclin and normalizes arterial thrombosis times. Treatment of normal mice with the Mas agonist AVE0991 reduces thrombosis. Klkb1(-/-) mice have reduced aortic tissue factor (TF) mRNA, antigen, and activity. In sum, Klkb1(-/-) mice have a novel mechanism for thrombosis protection in addition to reduced contact activation. This pathway arises when bradykinin delivery to vasculature is compromised and mediated by increased receptor Mas, prostacyclin, Sirt1, and KLF4, leading to reduced vascular TF.

Topics: Angiotensin II; Animals; Carotid Artery Thrombosis; Epoprostenol; Imidazoles; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Mice; Mice, Knockout; Naphthalenes; Nerve Tissue Proteins; Partial Thromboplastin Time; Peptide Fragments; Prekallikrein; Proto-Oncogene Mas; Proto-Oncogene Proteins; Pyrones; Receptor, Bradykinin B2; Receptors, G-Protein-Coupled; RNA, Messenger; Sirtuin 1; Sulfonamides; Synaptotagmins; Thromboplastin

Topics: Animals; Carotid Artery Thrombosis; Epoprostenol; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Prekallikrein; Proto-Oncogene Mas; Proto-Oncogene Proteins; Receptors, G-Protein-Coupled; Thromboplastin

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

Plant-derived α-linolenic acid (ALA) may constitute an attractive cardioprotective alternative to fish-derived n-3 fatty acids. However, the effect of dietary ALA on arterial thrombus formation remains unknown.. Male C57Bl/6 mice were fed a high-ALA or low-ALA diet for 2 weeks. Arterial thrombus formation was delayed in mice fed a high-ALA diet compared with those on a low-ALA diet (n=7; P<0.005). Dietary ALA impaired platelet aggregation to collagen and thrombin (n=5; P<0.005) and decreased p38 mitogen-activated protein kinase activation in platelets. Dietary ALA impaired arterial tissue factor (TF) expression, TF activity, and nuclear factor-κB activity (n=7; P<0.05); plasma clotting times and plasma thrombin generation did not differ (n=5; P=not significant). In cultured human vascular smooth muscle and endothelial cells, ALA inhibited TF expression and activity (n=4; P<0.01). Inhibition of TF expression occurred at the transcriptional level via the mitogen-activated protein kinase p38 in smooth muscle cells and p38, extracellular signal-regulated kinases 1 and 2, and c-Jun N-terminal kinases 1 and 2 in endothelial cells.. ALA impairs arterial thrombus formation, TF expression, and platelet activation and thereby represents an attractive nutritional intervention with direct dual antithrombotic effects.

Topics: alpha-Linolenic Acid; Animals; Cardiotonic Agents; Carotid Arteries; Carotid Artery Thrombosis; Cells, Cultured; Dietary Supplements; Endothelial Cells; Gene Expression; Humans; Male; MAP Kinase Kinase Kinase 5; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Myocytes, Smooth Muscle; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Platelet Activation; Platelet Aggregation; Thromboplastin; Tumor Necrosis Factor-alpha

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

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

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

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

The phytosterol guggulsterone is a potent anti-inflammatory mediator with less side effects than classic steroids. This study assesses the impact of guggulsterone on tissue factor (TF) expression and thrombus formation.. Guggulsterone inhibited TNF-alpha-induced endothelial TF protein expression and surface activity in a concentration-dependent manner; in contrast, dexamethasone did not affect TNF-alpha-induced TF expression. Guggulsterone enhanced endothelial tissue factor pathway inhibitor and impaired plasminogen activator inhibitor-1 as well as vascular cell adhesion molecule-1 protein. Real-time polymerase chain reaction revealed that guggulsterone inhibited TNF-alpha-induced TF mRNA expression; moreover, it impaired activation of the MAP kinases JNK and p38, while that of ERK remained unaffected. In vivo, guggulsterone inhibited TF activity and photochemical injury induced thrombotic occlusion of mouse carotid artery. Guggulsterone also inhibited TF expression, proliferation, and migration of vascular smooth muscle cells in a concentration-dependent manner.. Guggulsterone inhibits TF expression in vascular cells as well as thrombus formation in vivo; moreover, it impairs vascular smooth muscle cell activation. Hence, this phytosterol offers novel therapeutic options, in particular in inflammatory diseases associated with an increased risk of thrombosis.

Topics: Animals; Anti-Inflammatory Agents; Blotting, Western; Carotid Artery Thrombosis; Cells, Cultured; Endothelial Cells; Enzyme Activation; Gene Expression; Humans; MAP Kinase Kinase 4; Mice; Muscle, Smooth, Vascular; p38 Mitogen-Activated Protein Kinases; Phytosterols; Pregnenediones; Reverse Transcriptase Polymerase Chain Reaction; Thromboplastin; Tumor Necrosis Factor-alpha

Thrombomodulatory factors have been implicated in plaque instability. The aim was to examine the relationship between thrombomodulatory gene expression, timing of clinical events and plaque histology.. Plaques were obtained from 40 consecutive patients undergoing carotid endarterectomy and divided into three groups (group 1, early symptomatic, within 1 month; group 2, late symptomatic, 1-6 months and group 3, asymptomatic). Total RNA was isolated to determine the expression of tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), plasminogen activator inhibitor-1 (PAI-1), tissue factor (TF), tissue factor pathway inhibitor (TFPI), thrombomodulin (TM), CD68 and vascular endothelial-cadherin (VE-Cadherin).. Expression of t-PA, PAI-1, TF, TFPI, TM, CD68 and VE-cadherin were significantly increased in the early symptomatic group (p=0.019, 0.028, 0.018, 0.025, 0.038, 0.016 and 0.027 respectively), but the level of gene expression in the late symptomatic group was indistinguishable from the asymptomatic group. The incidence of plaque rupture and intraplaque haemorrhage was significantly increased in the early symptomatic groups (58% versus 18%/18% group 2/3, and 55% versus 6%/9% respectively, p<0.05 for both).. Expression of thrombomodulatory genes is increased in unstable plaques, though levels after 1 month are comparable to asymptomatic plaques. This transient rise may influence plaque instability, and rapid resolution mirrors the clinical reduction in risk of further thrombo-embolic events.

Topics: Aged; Aged, 80 and over; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Blood Coagulation Factors; Cadherins; Carotid Arteries; Carotid Artery Thrombosis; DNA; Endarterectomy, Carotid; Endothelium, Vascular; Factor Xa Inhibitors; Female; Follow-Up Studies; Gene Expression Regulation; Genetic Predisposition to Disease; Humans; Lipoproteins; Macrophages; Male; Middle Aged; Phenotype; Plasminogen Activator Inhibitor 1; Reverse Transcriptase Polymerase Chain Reaction; Thrombomodulin; Thromboplastin; Tissue Plasminogen Activator

In patients with coronary artery disease and reduced ejection fraction, amiodarone reduces mortality by decreasing sudden death. Because the latter may be triggered by coronary artery thrombosis as much as ventricular arrhythmias, amiodarone might interfere with tissue factor (TF) expression and thrombus formation.. Clinically relevant plasma concentrations of amiodarone reduced TF activity and impaired carotid artery thrombus formation in a mouse photochemical injury model in vivo. PTT, aPTT, and tail bleeding time were not affected; platelet number was slightly decreased. In human endothelial and vascular smooth muscle cells, amiodarone inhibited tumor necrosis factor (TNF)-alpha and thrombin-induced TF expression as well as surface activity. Amiodarone lacking iodine and the main metabolite of amiodarone, N-monodesethylamiodarone, inhibited TF expression. Amiodarone did not affect mitogen-activated protein kinase activation, TF mRNA expression, and TF protein degradation. Metabolic labeling confirmed that amiodarone inhibited TF protein translation.. Amiodarone impairs thrombus formation in vivo; in line with this, it inhibits TF protein expression and surface activity in human vascular cells. These pleiotropic actions occur within the range of amiodarone concentrations measured in patients, and thus may account at least in part for its beneficial effects in patients with coronary artery disease.

Topics: Amiodarone; Animals; Anti-Arrhythmia Agents; Carotid Artery Injuries; Carotid Artery Thrombosis; Cells, Cultured; Endothelial Cells; Humans; Mice; Mice, Inbred C57BL; Myocytes, Smooth Muscle; Protein Biosynthesis; RNA, Messenger; Thromboplastin

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

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

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

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

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

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

Reduced activity of endothelial nitric oxide (NO) may be involved in thrombus formation on atherosclerotic plaques, a major cause of acute coronary syndrome. However, mechanisms of such increase in arterial thrombogenecity have not been fully understood. We previously reported that long-term inhibition of NO synthesis by administration of N(G)-nitro-L-arginine methyl ester (L-NAME) causes hypertension and activates vascular tissue angiotensin-converting enzyme (ACE) activity. We used this model to investigate the mechanism by which long-term impairment of NO activity increases arterial thrombogenecity. We observed cyclic flow variations (CFVs), a reliable marker of platelet thrombi, after the production of stenosis of the carotid artery in rats treated with L-NAME for 4 wk. The thrombin antagonist argatroban suppressed the CFVs. The CFVs were detected in rats receiving L-NAME plus hydralazine but not in rats receiving L-NAME plus an ACE inhibitor (imidapril). Treatment with the ACE inhibitor imidapril, but not with hydralazine, prevented L-NAME-induced increases in carotid arterial ACE activity and attenuated tissue factor expression. These results suggest that long-term inhibition of endothelial NO synthesis may increase arterial thrombogenecity at least in part through angiotensin II-induced induction of tissue factor and the resultant thrombin generation. These data provide a new insight as to how endothelial NO exhibits antithrombogenic properties of the endothelium.

Topics: Administration, Oral; Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Animals; Antithrombins; Arginine; Blood Flow Velocity; Blood Pressure; Carotid Arteries; Carotid Artery Thrombosis; Enzyme Inhibitors; Gene Expression Regulation; Heart Rate; Imidazoles; Imidazolidines; Injections, Intravenous; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Peptidyl-Dipeptidase A; Pipecolic Acids; Platelet Aggregation; Prothrombin Time; Rats; Rats, Inbred WKY; Sulfonamides; Thromboplastin; Transcription, Genetic

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

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

Tissue factor (TF) is a low-molecular-weight glycoprotein that initiates the extrinsic clotting cascade and is considered a major regulator of arterial thrombogenicity. TF pathway inhibitor (TFPI) is a major physiological inhibitor of TF-initiated coagulation. The aim of this study was to define the complex interplay between TF and TFPI and the regulation of vascular thrombogenicity in a model of vascular remodeling. To determine the levels and pattern of vascular expression of TF and TFPI associated with vascular remodeling, a murine model of flow cessation was studied. TF activity of the arteries increased after ligation (P<0.05). Quantitative analysis of homogenates of remodeled carotid arteries revealed increased TF expression but unchanged TFPI expression compared with normal carotid arteries, resulting in enhanced TF activity. To determine the potential therapeutic role of TFPI in this thrombogenic state, mice were treated with intravascular adenoviral delivery of either murine TFPI (Ad-mTFPImyc) or a control adenovirus (Ad-DeltaE1). Overexpression of TFPI decreased vascular TF activity compared with viral control (P<0.01). Overexpression of TFPI inhibited neointimal formation (P=0.038), resulting in enhanced luminal area (P=0.001) 4 weeks after flow cessation. In this murine model of vascular remodeling, an imbalance between TF and TFPI expression is generated, resulting in increased TF activity. Overexpression of TFPI in this model inhibits vascular TF activity and results in attenuation of vascular remodeling associated with flow interruption.

Topics: Animals; Arteriosclerosis; Carotid Artery Thrombosis; Genetic Therapy; Lipoproteins; Mice; Mice, Inbred C57BL; Thromboplastin

Tissue factor (TF)-dependent activation of the coagulation is important in the pathophysiology of intravascular thrombus formation. We tested the effects of a monoclonal antibody against TF (AP-1) on lysis time induced by tissue-type plasminogen activator (TPA) and on reocclusion rate in a rabbit model of carotid artery thrombosis.. Intravascular thrombosis was obtained by placing an external constrictor around carotid arteries with endothelial injury. Carotid blood flow velocity ws measured continuously with a Doppler flow probe. Thirty minutes after thrombus formation, the rabbits received either AP-1 (0.15 mg/kg IV, n=8) or placebo (n=8). All rabbits also received TPA (80 microg/kg bolus plus 8 microg x kg(-1) x min(-1) infusion for up to 90 minutes or until reperfusion was achieved) and heparin (200 U/kg IV as a bolus). At reperfusion, TPA was discontinued, and the rabbits were followed for an additional 90 minutes. AP-1 shortened lysis time from 44+/-8 minutes (mean+/-SEM) in control rabbits to 26+/-7 minutes in AP-1 rabbits (P<.01). Reocclusion occurred in all control rabbits in 10+/-3 minutes, whereas it occurred in only two of eight AP-1 treated rabbits in 72 and 55 minutes (P<.01). No changes in prothrombin time and ex vivo platelet aggregation in response to various agonists were observed after AP-1 administration, indicating the absence of systemic effects by this antibody.. TF exposure and activation of the extrinsic coagulation pathway play an important role in prolonging lysis time and mediating reocclusion after thrombolysis in this model. AP-1, a monoclonal antibody against TF, might be suitable as adjunctive therapy to TPA.

Topics: Animals; Antibodies, Monoclonal; Carotid Artery Thrombosis; Female; Fibrinogen; Fibrinopeptide A; Male; Plasminogen Activators; Platelet Aggregation; Rabbits; Thromboplastin; Tissue Plasminogen Activator