The Antiplatelet Effect And Mechanisms Of W1 As A Novel Compound

Coronary heart disease?CHD?is harmful to human health,the most common cause of death in CHD are related to thromboembolism and associated complications.The excessive activation of platelets play an important role in the formation of cardiovascular thrombosis.Therefore,antiplatelet therapy is important to improve the prognosis of patients with CHD.In clinic,clopidogrel is regular used in the treatment for acute coronary syndromes.However,due to CYP450 metablic enzymes genetic polymorphisms and drug interactions,"clopidogrel resistance"is often investigated in clinical usage.Therefore,it is necessary to develop novel antiplatelet drugs with better efficacy and less bleeding events for the treatment of CHD.The P2Y12 receptor antagonist,represented by clopidogrel,is a hot spot of research in recent years.W1 is composed of the first step of the oxidative metabolite of clopidogrel,2-o-clopidogrel,and aspirin in a molecular ratio of 1:1,two of them is combined through the covalent bond.Previous studies have shown that W1 has a positive antiplatelet effect.In the first part of this reseach,we examined the maximum efficacy and potency of W1 using an arteriovenous thrombosis model in rats,then,observed the antithrombotic effect of W1 in the FeCl₃-induced mesenteric microthrombosis mice model,and explored the antiplatelet mechanism of W1;in the second part,we studied the effect of omeprazole combination with W1 on antiplatelet activities and antithrombotic effects,and the possible mechanisms of W1,to verify whether W1 could extert the resistance of clopidogrel.In part one,we established the rat carotid arteriovenous thrombosis model to analysis the antithrombotic effects of W1,clopidogrel and aspirin.The results showed that the maximum effect was achieved when the dose of W1 was 3 mg/kg in rats.The ED50 values of W1,clopidogrel and aspirin were 0.55,1.5,2.18 mg/kg,which means that W1 is the best in antithrombotic.We observed the process of mesenteric microthrombosis in FeCl₃-induced C57BL/6 mice using the laser confocal microscope,the results showed that,compared with the control group,the formation time of the first emboli?>20?m?was significantly delayed?P<0.01?,the number of emboli decreased significantly?P<0.01?,and there was no significant difference between W1 group?5 mg/kg?and clopidogrel group?15 mg/kg?in the above 2 indicators?P>0.05?,indicating that W1 has similar antithrombotic effects to clopidogrel in mice.We also detected the TXB2 content and expression of P2Y12 receptor using the ELISA and western blotting.We found that when compared with ADP induction group,W1 didn't affect the changes of the TXB2 and P2Y12receptor?P>0.05?,which indicates that the main ingredient works in W1should be 2-o-clopidogrel.In part two,we focused on the antiplatelet effect and P2Y12 receptor signaling molecules of W1 when combined with omeprazole to simulate the clinical“clopidogrel resistance”.We first examined the changes in platelet aggregation rate and found that when omeprazole combined with W1 or clopidogrel,the platelet aggregation rate increased significantly,and the platelet aggregation rate of W1 combination group was significantly lower than that of clopidogrel combination group?P<0.01?;then,we explored the effect of omeprazole combination on antithrombotic effects by establishing a FeCl₃-induced mesenteric microartery thrombosis model in mice,the formation time of the first emboli?>20?m?was shorted?P<0.01?,the number of emboli was significantly increased?P<0.01?,and omeprazole had a stronger inhibitory effect on clopidogrel?P<0.01?.We observed the expression of P2Y12 receptor and its downstream signal molecules AKt and Erk in phosphorylation level using western blotting.Compared with W1 or clopidogrel group,the combination of omeprazole had no effect on the expression of P2Y12 receptor,but increased the phosphorylation level of AKt and Erk protein,and the effect on W1 was significantly weaker than of clopidogrel?P<0.01?.The results showed that omeprazole combination could inhibit the antiplatelet activities of clopidogrel or W1,with the inhibitory effect weaker in W1 group than in clopidogrel group.In conclusions,we have proven W1 possessed the properties of antiplatelet and antithrombotic formation using different animal models.W1 not only exterted stronger antiplatelet effect in comparision with clopidogrel,W1 had less influence by the liver enzyme than clopidogrel,indicating W1 can reduce the clopidogrel resistance.