Effect of anti-thrombotic agents on various experimental models of thrombosis and to elucidate their mechanism of action

Abstract

Thrombosis is the leading cause of morbidity and mortality worldwide. Despite the existing anti-thrombotic treatment more than 15% of patients with acute coronary syndrome suffer from ischemic stroke. The present study was undertaken to delineate the pathogenesis or prognosis of arterial thrombosis in normal and dyslipidemic/insulin resistant animal models and their modulation by standard and test agents to identify more efficacious candidate molecules with lesser side effects. FeCl3 solution (5- 80%; w/v) was topically applied on the carotid artery of rats and mice to measure the time to occlusion (TTO). In these models 10% and 20% of FeCl3 was identified as an optimal concentration for inducing thrombosis in mice and rat respectively. Amongst all the antithrombotic drugs tested in this model, Warfarin and Clopidogrel were found to be most efficacious. The antithrombotic drugs were further evaluated against extracorporeal thrombus formation over the thrombogenic silk thread following arteriovenous shunt in rats. All the tested standard antithrombotic agents were found effective to reduce stent thrombosis under examined experimental conditions. Amongst the antithrombotic candidate molecules screened on the above models, S007-867 was found most effective over the other molecules tested. Since, platelet play an important role in the prognosis of thrombosis, we further evaluated S007-867 on myocardial ischemia reperfusion (MI-RP) injury model of thrombosis along with neuroprotective Curcuma oil (C.oil) and a cardioprotective agent CDRI-93/48. S007-867 and C.oil did not exhibit any infarct sparing effect, while CDRI-93/48 found cardioprotective following MI-RP injury. However, S007-867 and C.oil showed significant reversal of adenosine 5'-diphosphate (ADP) induced platelet aggregation following MI-RP. Moreover, C.oil remarkably reduced the ADP, collagen and thrombin induced platelet aggregation (ex vivo) in rats, which was further corroborated by suppression of elevated tyrosine phosphorylation of various proteins following C.oil treatment and thus prevented platelet activation. Moreover, C.oil prolonged TTO without affecting blood coagulation parameters. While, hyperlipedimia predisposes to thrombotic complications we further assessed the chronology of major pathological events associated with high cholesterol (HC) diet and their modulation by anti-platelet drugs. Hamsters exhibited a significant increase in plasma lipid levels after 15 days of high cholesterol (HC) diet feeding, however reduction in thrombin time and potentiation of thrombotic events were observed only at 90 days of HC feeding. Thus, pathological changes induced by HC diet depend on the duration and extent of hyperlipidemia. However, hamsters treated with anti-platelet agents S007-867, Aspirin or Clopidogrel along with HC feeding exhibited reduction in thrombotic events associated with hyperlipedimia. Increased lipogenesis leads to hyperlipidemia coupled with insulin resistance which further augments the risk for thrombotic events. However Silibinin and Atorvastatin treatment ameliorated insulin resistance and its associated disease conditions like inflammation and endothelial dysfunction. Moreover, Silibinin significantly inhibited the expression of lipogenic genes and ameliorated thrombotic events associated with hyperlipidemia. Since, endothelial dysfunction and inflammation plays important role in the pathogenesis or prognosis of thrombotic events, future studies are warranted to further explore the effect of antiplatelet agents on such mediators.