Thrombotic Occlusion Time Research Articles

Impact of very low dose rivaroxaban in addition to dual antiplatelet therapy on endogenous fibrinolysis in acute coronary syndrome: The VaLiDate-R study

BackgroundImpaired endogenous fibrinolysis is adverse cardiovascular risk factor in acute coronary syndrome (ACS) patients. Addition of very low dose rivaroxaban (VLDR) to dual antiplatelet therapy (DAPT) reduces cardiovascular events but increases bleeding. ObjectiveWe aimed to assess whether addition of VLDR to DAPT can enhance endogenous fibrinolysis. MethodsIn a prospective, open-label trial, we assessed endogenous fibrinolysis in whole blood, in 549 patients with ACS using the Global Thrombosis Test (GTT) and Thromboelastography (TEG). Patients (n = 180) who demonstrated impaired endogenous fibrinolysis (lysis time [LT] >2000s with the GTT) were randomised 1:1:1 to (i) clopidogrel 75 mg daily; (ii) clopidogrel 75 mg daily plus rivaroxaban 2.5 mg twice daily; or (iii) ticagrelor 90 mg twice daily, for 30 days, in addition to aspirin. Fibrinolytic status was assessed at 0, 2, 4 and 8 weeks. The primary outcome was the change in LT from admission to week 4. We also measured thrombotic occlusion time (OT) at high shear, and rivaroxaban level. ResultsThere was no difference between the groups with respect to LT or clot lysis with TEG, and no change in these parameters compared to baseline during study drug allocation. In the rivaroxaban plus clopidogrel group, OT was prolonged compared to the other groups, although rivaroxaban levels were low, suggesting non-compliance. ConclusionAddition of rivaroxaban 2.5 mg twice daily to DAPT does not affect endogenous fibrinolysis of thrombus formed at either high or low shear. Further studies are needed to determine whether higher doses of rivaroxaban can favourably modulate fibrinolysis. Condensed abstractImpaired endogenous fibrinolysis is a strong risk factor in ACS. We aimed to assess whether adding very low dose rivaroxaban (VLDR) to DAPT can enhance fibrinolysis. Fibrin and clot lysis were assessed in whole blood. ACS patients with impaired fibrinolysis were randomised 1:1:1 to clopidogrel 75 mg daily; clopidogrel 75 mg plus VLDR; or ticagrelor 90 mg twice daily, in addition to aspirin. At 30-days, there was no difference in lysis time between the groups, nor change from baseline. VLDR does not improve fibrinolysis at high or low shear. Further studies are needed to determine whether alternative antithrombotic regimens can enhance endogenous fibrinolysis.

The Salutary Effects of Catalpol on Diesel Exhaust Particles-Induced Thrombogenic Changes and Cardiac Oxidative Stress, Inflammation and Apoptosis.

Inhaled particulate air pollution exerts pulmonary inflammation and cardiovascular toxicity through secondary systemic effects due to oxidative stress and inflammation. Catalpol, an iridiod glucoside, extracted from the roots of Rehmannia glutinosa Libosch, has been reported to possess anti-inflammatory and antioxidant properties. Yet, the potential ameliorative effects of catalpol on particulate air pollution—induced cardiovascular toxicity, has not been studied so far. Hence, we evaluated the possible mitigating mechanism of catalpol (5 mg/kg) which was administered to mice by intraperitoneal injection one hour before the intratracheal (i.t.) administration of a relevant type of pollutant particle, viz. diesel exhaust particles (DEPs, 30 µg/mouse). Twenty-four hours after the lung deposition of DEPs, several cardiovascular endpoints were evaluated. DEPs caused a significant shortening of the thrombotic occlusion time in pial microvessels in vivo, induced platelet aggregation in vitro, and reduced the prothrombin time and the activated partial thromboplastin time. All these actions were effectively mitigated by catalpol pretreatment. Likewise, catalpol inhibited the increase of the plasma concentration of C-reactive proteins, fibrinogen, plasminogen activator inhibitor-1 and P- and E-selectins, induced by DEPs. Moreover, in heart tissue, catalpol inhibited the increase of markers of oxidative (lipid peroxidation and superoxide dismutase) and nitrosative (nitric oxide) stress, and inflammation (tumor necrosis factor α, interleukin (IL)-6 and IL-1β) triggered by lung exposure to DEPs. Exposure to DEPs also caused heart DNA damage and increased the levels of cytochrome C and cleaved caspase, and these effects were significantly diminished by the catalpol pretreatment. Moreover, catalpol significantly reduced the DEPs-induced increase of the nuclear factor κB (NFκB) in the heart. In conclusion, catalpol significantly ameliorated DEPs–induced procoagulant events and heart oxidative and nitrosative stress, inflammation, DNA damage and apoptosis, at least partly, through the inhibition of NFκB activation.

Cardiac Inflammation, Oxidative Stress, Nrf2 Expression, and Coagulation Events in Mice with Experimental Chronic Kidney Disease.

Chronic kidney disease (CKD) is known to be associated with cardiovascular dysfunction. Dietary adenine intake in mice is also known to induce CKD. However, in this experimental model, the mechanisms underlying the cardiotoxicity and coagulation disturbances are not fully understood. Here, we evaluated cardiac inflammation, oxidative stress, DNA damage, and coagulation events in mice with adenine (0.2% w/w in feed for 4 weeks)-induced CKD. Control mice were fed with normal chow for the same duration. Adenine increased water intake, urine output, relative kidney weight, the plasma concentrations of urea and creatinine, and the urinary concentrations of kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin. It also decreased the body weight and creatinine clearance, and caused kidney DNA damage. Renal histological analysis showed tubular dilation and damage and neutrophilic influx. Adenine induced a significant increase in systolic blood pressure and the concentrations of troponin I, tumor necrosis factor-α, and interleukin-1β in heart homogenates. It also augmented the levels of markers of lipid peroxidation measured by malondialdehyde production and 8-isoprostane, as well as the antioxidants superoxide dismutase and catalase. Immunohistochemical analysis of the hearts showed that adenine increased the expression of nuclear factor erythroid-derived 2-like 2 by cardiomyocytes. It also caused cardiac DNA damage. Moreover, compared with the control group, adenine induced a significant increase in the number of circulating platelet and shortened the thrombotic occlusion time in pial arterioles and venules in vivo, and induced a significant reduction in the prothrombin time and activated partial thromboplastin time. In conclusion, the administration of adenine in mice induced CKD-associated cardiac inflammation, oxidative stress, Nrf2 expression, and DNA damage. It also induced prothrombotic events in vivo. Therefore, this model can be satisfactorily used to study the cardiac pathophysiological events in subjects with CKD and the effect of drug treatment thereon.

Pulmonary exposure to silver nanoparticles impairs cardiovascular homeostasis: Effects of coating, dose and time

Pulmonary exposure to silver nanoparticles (AgNPs) revealed the potential of nanoparticles to cause pulmonary toxicity, cross the alveolar-capillary barrier, and distribute to remote organs. However, the mechanism underlying the effects of AgNPs on the cardiovascular system remains unclear. Hence, we investigated the cardiovascular mechanisms of pulmonary exposure to AgNPs (10 nm) with varying coatings [polyvinylpyrrolidone (PVP) and citrate (CT)], concentrations (0.05, 0.5 and 5 mg/kg body weight), and time points (1 and 7 days) in BALB/C mice. Silver ions (Ag+) were used as ionic control. Exposure to AgNPs induced lung inflammation. In heart, tumor necrosis factor α, interleukin 6, total antioxidants, reduced glutathione and 8-isoprostane significantly increased for both AgNPs. Moreover, AgNPs caused oxidative DNA damage and apoptosis in the heart. The plasma concentration of fibrinogen, plasminogen activation inhibitor-1 and brain natriuretic peptide were significantly increased for both coating AgNPs. Likewise, the prothrombin time and activated partial thromboplastin time were significantly decreased. Additionally, the PVP- and CT- AgNPs induced a significant dose-dependent increase in thrombotic occlusion time in cerebral microvessels at both time points. In vitro study on mice whole blood exhibited significant platelet aggregation for both particle types. Compared with AgNPs, Ag+ increased thrombogenicity and markers of oxidative stress, but did not induce either DNA damage or apoptosis in the heart. In conclusion, pulmonary exposure to AgNPs caused cardiac oxidative stress, DNA damage and apoptosis, alteration of coagulation markers and thrombosis. Our findings provide a novel mechanistic insight into the cardiovascular pathophysiological effects of lung exposure to AgNPs.

The acute pulmonary and thrombotic effects of cerium oxide nanoparticles after intratracheal instillation in mice.

Cerium oxide nanoparticles (CeO2 NPs), used as a diesel fuel catalyst, can be emitted into the ambient air, resulting in exposure to humans by inhalation. Recent studies have reported the development of lung toxicity after pulmonary exposure to CeO2 NPs. However, little is known about the possible thrombotic effects of these NPs. The present study investigated the acute (24 hours) effect of intratracheal (IT) instillation of either CeO2 NPs (0.1 or 0.5 mg/kg) or saline (control) on pulmonary and systemic inflammation and oxidative stress and thrombosis in mice. CeO2 NPs induced a significant increase of neutrophils into the bronchoalveolar lavage (BAL) fluid with an elevation of tumor necrosis factor α (TNFα) and a decrease in the activity of the antioxidant catalase. Lung sections of mice exposed to CeO2 NPs showed a dose-dependent infiltration of inflammatory cells consisting of macrophages and neutrophils. Similarly, the plasma levels of C-reactive protein and TNFα were significantly increased, whereas the activities of catalase and total antioxidant were significantly decreased. Interestingly, CeO2 NPs significantly and dose dependently induced a shortening of the thrombotic occlusion time in pial arterioles and venules. Moreover, the plasma concentrations of fibrinogen and plasminogen activator inhibitor-1 were significantly elevated by CeO2 NPs. The direct addition of CeO2 NPs (1, 5, or 25 μg/mL) to mouse whole blood, collected from the inferior vena cava, in vitro neither caused significant platelet aggregation nor affected prothrombin time or partial thromboplastin time, suggesting that the thrombotic events observed in vivo may have resulted from systemic inflammation and/or oxidative stress induced by CeO2 NPs. This study concludes that acute pulmonary exposure to CeO2 NPs induces pulmonary and systemic inflammation and oxidative stress and promotes thrombosis in vivo.

Chronic exposure to water-pipe smoke induces cardiovascular dysfunction in mice.

Water-pipe tobacco smoking is becoming prevalent in all over the world including Western countries. There are limited data on the cardiovascular effects of water-pipe smoke (WPS), in particular following chronic exposure. Here, we assessed the chronic cardiovascular effects of nose-only WPS exposure in C57BL/6 mice. The duration of the session was 30 minutes/day, 5 days/week for 6 consecutive months. Control mice were exposed to air. WPS significantly increased systolic blood pressure. The relative heart weight and plasma concentrations of troponin-I and B-type natriuretic peptide were increased in mice exposed to WPS. Arterial blood gas analysis showed that WPS caused a significant decrease in [Formula: see text] and an increase in [Formula: see text] WPS significantly shortened the thrombotic occlusion time in pial arterioles and venules and increased the number of circulating platelet. Cardiac lipid peroxidation, measured as thiobarbituric acid-reactive substances, was significantly increased, while superoxide dismutase activity, total nitric oxide activity, and glutathione concentration were reduced by WPS exposure. Likewise, immunohistochemical analysis of the heart revealed an increase in the expression of inducible nitric oxide synthase and cytochrome c by cardiomyocytes of WPS-exposed mice. Moreover, hearts of WPS-exposed mice showed the presence of focal interstitial fibrosis. WPS exposure significantly increased heart DNA damage assessed by Comet assay. We conclude that chronic nose-only exposure to WPS impairs cardiovascular homeostasis. Our findings provide evidence that long-term exposure to WPS is harmful to the cardiovascular system and supports interventions to control the spread of WPS, particularly amid youths.NEW & NOTEWORTHY No data are available on the chronic cardiovascular effects of water-pipe smoke (WPS). Our findings provide experimental evidence that chronic exposure to WPS increased blood pressure, relative heart weight, troponin I, and B-type natriuretic peptide in plasma and induced hypoxemia, hypercapnia, and thrombosis. Moreover, WPS caused cardiac oxidative stress, DNA damage, and fibrosis.

A Predictive Model of High Shear Thrombus Growth.

The ability to predict the timescale of thrombotic occlusion in stenotic vessels may improve patient risk assessment for thrombotic events. In blood contacting devices, thrombosis predictions can lead to improved designs to minimize thrombotic risks. We have developed and validated a model of high shear thrombosis based on empirical correlations between thrombus growth and shear rate. A mathematical model was developed to predict the growth of thrombus based on the hemodynamic shear rate. The model predicts thrombus deposition based on initial geometric and fluid mechanic conditions, which are updated throughout the simulation to reflect the changing lumen dimensions. The model was validated by comparing predictions against actual thrombus growth in six separate in vitro experiments: stenotic glass capillary tubes (diameter=345µm) at three shear rates, the PFA-100(®) system, two microfluidic channel dimensions (heights=300 and 82µm), and a stenotic aortic graft (diameter=5.5mm). Comparison of the predicted occlusion times to experimental results shows excellent agreement. The model is also applied to a clinical angiography image to illustrate the time course of thrombosis in a stenotic carotid artery after plaque cap rupture. Our model can accurately predict thrombotic occlusion time over a wide range of hemodynamic conditions.

Betaine (N,N,N-trimethylglycine) averts photochemically-induced thrombosis in pial microvessels invivo and platelet aggregation invitro.

Betaine (N,N,N-trimethylglycine) is an important food component with established health benefits through its homocysteine-lowering effects, and is used to lower total homocysteine concentration in plasma of patients with homocystinuria. It is well established that hyperhomocysteinemia is an established risk factor for cardiovascular disease and stroke. However, the possible protective effect of betaine on coagulation events invivo and invitro has thus far not been studied. Betaine was given to mice at oral doses of either 10 mg/kg (n = 6) or 40 mg/kg (n = 6) for seven consecutive days, and control mice (n = 6) received water only. The thrombotic occlusion time in photochemically induced thrombosis in pial arterioles was significantly delayed in mice pretreated with betaine at doses of 10 mg/kg (P < 0.001) and 40 mg/kg (P < 0.01). Similar effects were observed in pial venules with 10 mg/kg (P < 0.05) and 40 mg/kg (P < 0.05) betaine. Invitro, in whole blood samples collected from untreated mice (n = 3-5), betaine (0.01-1 mg/mL) significantly reversed platelet aggregation induced by adenosine diphosphate (5 µM). The number of circulating platelets and plasma concentration of fibrinogen invivo were not significantly affected by betaine pretreament compared with the control group. Lipid peroxidation (LPO) in mice pretreated with betaine was significantly reduced compared with the control group. Moreover, betaine (0.01-1 mg/mL) caused a dose-dependent and significant prolongation of PT (n = 5) and aPTT (n = 4-6). In conclusion, our data show that betaine protected against coagulation events invivo and invitro and decreased LPO in plasma.

Short-Term Nose-Only Water-Pipe (Shisha) Smoking Exposure Accelerates Coagulation and Causes Cardiac Inflammation and Oxidative Stress in Mice

Background/Aim: Water-pipe smoking (WPS) has acquired worldwide popularity, and is disseminating particularly rapidly in Europe and North America. However, little is known about the short-term cardiovascular effects of WPS. Methods: Presently, we assessed the short-term cardiovascular effects of nose-only exposure to mainstream WPS in BALB/c mice for 30 min/day for 5 consecutive days. Control mice were exposed to air. At the end of the exposure period, several cardiovascular endpoints were measured. Results: WPS did not affect the number of leukocytes and the plasma concentrations of C-reactive protein, tumor necrosis factor α (TNFα) and interleukin-6 (IL-6). Likewise, plasma levels of lipid peroxidation (LPO), reduced glutathione (GSH) and catalase were not affected by WPS. By contrast, WPS aggravated in vivo thrombosis by shortening the thrombotic occlusion time in pial arterioles and venules. The number of circulating platelets was reduced by WPS suggesting the occurrence of platelet aggregation in vivo. Elevated concentrations of fibrinogen and plasminogen activator inhibitor-1 were seen after the exposure to WPS. Blood samples taken from mice exposed to WPS and exposed to adenosine diphosphate showed more platelet aggregation. The heart concentrations of IL-6 and TNFα were augmented by WPS. Likewise, heart levels of LPO, reactive oxygen species and the antioxidants catalase and GSH were increased by WPS. However, the systolic blood pressure and heart rate were not affected by WPS. Conclusion: It can be concluded that short-term exposure to WPS exerts procoagulatory effects and induce cardiac inflammation and oxidative stress. At the time point investigated, there was no evidence for blood inflammation or oxidative stress.

The Antidepressant 5-HT2A Receptor Antagonists Pizotifen and Cyproheptadine Inhibit Serotonin-Enhanced Platelet Function

There is considerable interest in defining new agents or targets for antithrombotic purposes. The 5-HT2A receptor is a G-protein coupled receptor (GPCR) expressed on many cell types, and a known therapeutic target for many disease states. This serotonin receptor is also known to regulate platelet function. Thus, in our FDA-approved drug repurposing efforts, we investigated the antiplatelet activity of cyproheptadine and pizotifen, two antidepressant 5-HT2A Receptor antagonists. Our results revealed that cyproheptadine and pizotifen reversed serotonin-enhanced ADP-induced platelet aggregation in vitro and ex vivo. And the inhibitory effects of these two agents were found to be similar to that of EMD 281014, a 5-HT2A Receptor antagonist under development. In separate experiments, our studies revealed that these 5-HT2A receptor antagonists have the capacity to reduce serotonin-enhanced ADP-induced elevation in intracellular calcium levels and tyrosine phosphorylation. Using flow cytometry, we also observed that cyproheptadine, pizotifen, and EMD 281014 inhibited serotonin-enhanced ADP-induced phosphatidylserine (PS) exposure, P-selectin expression, and glycoprotein IIb-IIIa activation. Furthermore, using a carotid artery thrombosis model, these agents prolonged the time for thrombotic occlusion in mice in vivo. Finally, the tail-bleeding time was investigated to assess the effect of cyproheptadine and pizotifen on hemostasis. Our findings indicated prolonged bleeding time in both cyproheptadine- and pizotifen-treated mice. Notably, the increases in occlusion and bleeding times associated with these two agents were comparable to that of EMD 281014, and to clopidogrel, a commonly used antiplatelet drug, again, in a fashion comparable to clopidogrel and EMD 281014. Collectively, our data indicate that the antidepressant 5-HT2A antagonists, cyproheptadine and pizotifen do exert antiplatelet and thromboprotective effects, but similar to clopidogrel and EMD 281014, their use may interfere with normal hemostasis.

Abstract 19091: Platelet mTOR is an Important Mediator of Arterial Thrombosis

Background: Arterial thrombosis is responsible for peripheral vascular disease, ischemic stroke, and myocardial infarction. Mammalian target of rapamycin (mTOR) inhibition promotes arterial thrombosis. To determine the contributions of platelet specific mTOR deficiency to arterial thrombosis, we generated platelet specific mTOR deficient mice. To determine the rescue effect of platelet mTOR deficiency, we also generated platelet specific TSC1 deficient (PF4Cre-TSC1 flox/flox ) mice. Because the effect of mTOR activation on thrombus formation in vivo has not yet been studied, we hypothesize that mTOR signaling pathway in platelets may play an important role in arterial thrombosis. Methods and Results: To determine the role of platelet mTOR in arterial thrombosis, we generated a mouse strain with platelet-specific mTOR deficiency (PF4Cre-mTOR flox/flox ) using conditional mTOR flox/flox mice and platelet-specific PF4Cre mice. PF4Cre-mTOR flox/flox mice did not show any obvious phenotypic abnormalities. Arterial thrombus formation was investigated using in vivo ferric chloride induced thrombosis model. Ferric Chloride (5% FeCl 3 ) induced thrombotic occlusion. Rapamycin treatment and mTOR conditional knockout induced arterial thrombosis significantly. Compared to PF4Cre mice, PF4Cre-TSC1 flox/flox mice exhibited elongated thrombotic occlusion time by 51.7%. Platelet specific mTOR deficiency induced arterial thrombosis quickly compare to the WT via activation of p-S6, a downstream target of mTORC1 and inhibition of p-Rictor of mTORC2. Conclusion: These finding indicate that platelet specific mTOR deficiency resulted in significantly induced arterial thrombosis but delayed at the platelet specific TSC1 deficient mice. These results suggest that activation of mTORC1 may have therapeutic benefits in arterial thrombosis.

Cardiovascular effects of nose-only water-pipe smoking exposure in mice

Water-pipe smoking (WPS) is a major type of smoking in Middle Eastern countries and is increasing in popularity in Western countries and is perceived as relatively safe. However, data on the adverse cardiovascular effects of WPS are scarce. Here, we assessed the cardiovascular effects of nose-only exposure to mainstream WPS generated by commercially available honey-flavored "moasel" tobacco in BALB/c mice. The duration of the session was 30 min/day for 1 mo. Control mice were exposed to air. WPS caused a significant increase of systolic blood pressure (SBP) in vivo (+13 mmHg) and plasma concentrations of IL-6 (+30%) but not that of TNF-α. Heart concentrations of IL-6 (+184%) and TNF-α (+54%) were significantly increased by WPS. Concentrations of ROS (+95%) and lipid peroxidation (+27%) were significantly increased, whereas those of GSH were decreased (-21%). WPS significantly shortened the thrombotic occlusion time in pial arterioles (-46%) and venules (40%). Plasma von Willebrand factor concentrations were significantly increased (+14%) by WPS. Erythrocyte numbers (+15%) and hematocrit (+17%) were significantly increased. Blood samples taken from mice exposed to WPS and exposed to ADP showed significant platelet aggregation compared with air-exposed mice. WPS caused a significant shortening of activated partial thromboplastin time (-45%) and prothrombin time (-13%). We conclude that 1-mo nose-only exposure to WPS increased SBP and caused cardiac inflammation, oxidative stress, and prothrombotic events. Our findings provide plausible elucidation that WPS is injurious to the cardiovascular system.

Impact of experimental type 1 diabetes mellitus on systemic and coagulation vulnerability in mice acutely exposed to diesel exhaust particles

BackgroundEpidemiological evidence indicates that diabetic patients have increased susceptibility to adverse cardiovascular outcomes related to acute increases in exposures to particulate air pollution. However, mechanisms underlying these effects remain unclear.MethodsTo evaluate the possible mechanisms underlying these actions, we assessed the systemic effects of diesel exhaust particles (DEP) in control mice, and mice with streptozotocin–induced type 1 diabetes. Four weeks following induction of diabetes, the animals were intratracheally instilled (i.t.) with DEP (0.4 mg/kg) or saline, and several cardiovascular endpoints were measured 24 h thereafter.ResultsDEP caused leukocytosis and a significant increase in plasma C-reactive protein and 8-isoprostane concentrations in diabetic mice compared to diabetic mice exposed to saline or non-diabetic mice exposed to DEP. The arterial PO2 as well as the number of platelets and the thrombotic occlusion time in pial arterioles assessed in vivo were significantly decreased following the i.t. instillation of DEP in diabetic mice compared to diabetic mice exposed to saline or non-diabetic mice exposed to DEP. Both alanine aminotransferase and aspartate transaminase activities, as well as the plasma concentrations of plasminogen activator inhibitor and von Willebrand factor were significantly increased in DEP-exposed diabetic mice compared to diabetic mice exposed to saline or DEP-exposed non-diabetic mice. The in vitro addition of DEP (0.25-1 μg/ml) to untreated mouse blood significantly and dose-dependently induced in vitro platelet aggregation, and these effects were exacerbated in blood of diabetic mice.ConclusionThis study has shown that systemic and coagulation events are aggravated by type 1 diabetes in mice, acutely exposed to DEP and has described the possible mechanisms for these actions that may also be relevant to the exacerbation of cardiovascular morbidity accompanying particulate air pollution in diabetic patients.

Abstract 19695: Platelet TSC1 is an Important Mediator of Arterial Thrombosis

Background : Arterial thrombosis is responsible for peripheral vascular disease, myocardial infarction, and stroke. Mammalian target of rapamycin (mTOR) inhibition promotes arterial thrombosis. To determine the contributions of platelet specific Tuberous sclerosis 1 (TSC1) deficiency to arterial thrombosis, we generated PF4Cre-TSC1 flox/+ mice. Because the effect of mTOR activation on thrombus formation in vivo has not yet been studied, we hypothesize that mTORC1 signaling pathway in platelets may play an important role in arterial thrombosis. Methods and Results : To determine the role of platelet TSC1 in arterial thrombosis, we generated a mouse strain with platelet-specific TSC1 deficiency (PF4Cre-TSC1 flox/+ ) using conditional TSC1 flox/flox mice and platelet-specific PF4-Cre mice. PF4Cre-TSC1 flox/+ mice did not show any obvious phenotypic abnormalities. Arterial thrombus formation was investigated using in vivo ferric chloride induced thrombosis model. Ferric Chloride (5% FeCl 3 ) induced thrombotic occlusion. Compared to PF4-Cre mice, PF4Cre-TSC1 flox/+ mice exhibited elongated thrombotic occlusion time by 21.7%. Platelet specific TSC1 deficiency promotes arterial thrombosis via activation of mTOR and S6, a downstream target of mTORC1. Conclusion : These finding indicate that platelet specific TSC1 deficiency promotes arterial thrombosis in vivo . These results suggest that activation of mTORC1 may have therapeutic benefits in arterial thrombosis.

Hemostatic effects of recombinant DisBa-01, a disintegrin from Bothrops alternatus

A monomeric RGD-disintegrin was recently identified from a cDNA library from the venom gland of Bothrops alternatus. The corresponding 12 kDa-recombinant protein, DisBa-01, specifically interacted with alpha(v)beta3 integrin and displayed potent anti-metastatic and anti-angiogenic properties. Here, the interaction of DisBa-01 with platelet alphaIIb beta3 integrin and its effects on hemostasis and thrombosis were investigated. DisBa-01 bound to Chinese Hamster Ovary (CHO) cells expressing beta3 or alphaIIb beta3 and promoted their adhesion and the adhesion of resting platelets onto glass coverslips. The disintegrin inhibited the binding of FITC-fibrinogen and FITC-PAC-1 to ADP-stimulated platelets and inhibited ADP-, TRAP- and collagen-induced aggregation of murine, rabbit or human platelets. In a flow chamber assay, DisBa-01 inhibited and reverted platelet adhesion to immobilized fibrinogen. DisBa-01 inhibited the phosphorylation of FAK following platelet activation. The intravenous injection of DisBa-01 in C57Bl6/j mice, prolonged tail bleeding time as well as thrombotic occlusion time in mesenteric venules and arterioles following vessel injury with FeCl3. In conclusion, DisBa-01 antagonizes the platelet alphaIIb beta3 integrin and potently inhibits thrombosis.

Antithrombotic activity of dermatan sulfate in heparin cofactor II-deficient mice

AbstractHeparin cofactor II (HCII) is a plasma protein that inhibits thrombin rapidly in the presence of dermatan sulfate or heparin. We previously reported that the time to thrombotic occlusion of the carotid artery after photochemical injury was shorter in HCII-deficient mice than in wild-type control animals. In this paper, we describe the antithrombotic activity of dermatan sulfate in wild-type and HCII-deficient mice. Intravenous administration of porcine skin dermatan sulfate induced a dose-dependent prolongation of the carotid artery occlusion time in HCII+/+ mice that was not observed in HCII-/- animals. Pharmacokinetic studies suggested that porcine skin dermatan sulfate expresses antithrombotic activity after being transferred from the plasma to sites in the vessel wall. Using invertebrate dermatan sulfate preparations, we showed that N-acetylgalactosamine-4-O-sulfate residues are required for the HCII-dependent antithrombotic effect. Furthermore, the invertebrate dermatan sulfates, which have higher charge densities than mammalian dermatan sulfate, slightly prolonged the thrombotic occlusion time of HCII-/- mice. These results indicate that HCII mediates the antithrombotic effect of porcine skin dermatan sulfate after injury to the carotid arterial endothelium in mice, whereas more highly charged dermatan sulfates possess weak antithrombotic activity independent of HCII. (Blood. 2004;104:3965-3970)

Inhibitory effects of total flavones of Hippophae Rhamnoides L on thrombosis in mouse femoral artery and in vitro platelet aggregation

Total flavones of Hippophae Rhamnoides L (TFH) are extracted from Sea buckthorn, a Chinese herbal medicine. Sea buckthorn has antioxidant, anti-ulcerogenic and hepato-protective actions, and its berry oil is reported to suppress platelet aggregation. Though it is frequently used for patients with thrombosis, the likely mechanism(s) and effects of TFH on thrombogenesis remain unclear. Thus, we have investigated the effect in-vivo of TFH on thrombogenesis and in vitro on platelet aggregation, comparing them to those of aspirin. We measured thrombotic occlusion time in a mouse femoral artery thrombosis model by the photochemical reaction between intravenously injected rose bengal and green light irradiation. In vitro platelet aggregation in whole blood was measured by single platelet counting. Thrombotic occlusion time was 8.5 ± 0.6 min in the control group. TFH at a dose of 300 μg/kg, intravenously administered 15 min before the rose bengal injection, significantly prolonged it to 11.6 ± 1.0 min ( P < 0.05), a similar effect on in-vivo thrombogenesis to that of aspirin. TFH at a concentration of 3.0 μg/ml significantly ( P < 0.01) inhibited in vitro platelet aggregation induced by collagen (2 μg/ml) in a concentration dependent manner, in contrast TFH did not affect aggregation induced by arachidonic acid (80 μM) and ADP (0.3 μM). The results of the present study, in which TFH prevented in-vivo thrombogenesis, probably due to inhibition of platelet aggregation, suggest a possible clinical approach for the prevention of thrombosis.

Genistein, an isoflavone included in soy, inhibits thrombotic vessel occlusion in the mouse femoral artery and in vitro platelet aggregation

Diet can be the most important factor that influences risks for cardiovascular diseases. Genistein included in soy is one candidate that may benefit the cardiovascular system. Here, we investigated the inhibitory effects of genistein on thrombotic vessel occlusion in the mouse femoral artery using a photochemical reaction, and in vitro platelet aggregation in whole blood measured by single platelet counting. Genistein (10 mg/kg), intravenously administered 10 min before the rose bengal injection, significantly prolonged the thrombotic occlusion time from 6.1±0.4 to 8.4±0.8 min ( P<0.05). Genistein at doses higher than 30 μM significantly ( P<0.01) inhibited in vitro platelet aggregation induced by collagen (1 and 3 μg/ml). When 10 mg/kg genistein was intravenously administered, ex vivo platelet aggregation induced by collagen (1 and 3 μg/ml) was significantly suppressed ( P<0.01). In conclusion, genistein prevented in vivo thrombogenesis and suppressed in vitro platelet aggregation. These results suggest that dietary supplementation of soy may prevent the progression of thrombosis and atherosclerosis.

Antithrombotic activity of a symmetrical triglyceride with eicosapentaenoic acid and γ-linolenic acid in guinea pig mesenteric microvasculature

The antithrombotic effect of a synthetic symmetrical triglyceride having eicosapentaenoic acid (EPA) at positions 1 and 3, and γ-linolenic acid (GLA) at position 2 was investigated. Administration of the triglyceride significantly increased thrombus formation time and thrombotic occlusion time induced by light irradiation and a fluorescent dye in guinea pigs after 14 days administration compared to that of soybean oil. The antithrombotic effect of the triglyceride was similar to that of EPA ethyl ester. Administration of the triglyceride increased GLA, dihomo-γ-linolenic acid (DGLA) and EPA contents in plasma and the liver, and the ratio of DGLA to arachidonic acid. These results might be responsible for this antithrombotic effect.