Antiplatelet Effects Of Clopidogrel DerivativesⅠand The Preliminary Study Of Its Mechanisms

Vascular embolism is one of the highest rates of morbidity and mortality in the world.According to the study,there are nearly 2 billion patients with cardiovascular and cerebrovascular diseases and potential patients globally,and more than half of the global deaths are due to cardiovascular and cerebrovascular diseases every year.Platelets are a key component of the body’s normal coagulation mechanism and play an important role in hemostasis and thrombosis.Therefore,the development of antiplatelet drugs is of great significance.As the second-generation thienopyridine P2Y₁₂2 receptor antagonists,clopidogrel is the most widely used antiplatelet aggregation drug in clinical practice and can inhibit platelet activation.Although its safety and efficacy are among the best among P2Y₁₂2 receptor antagonists,its bioavailability is low,and some patients suffer from the clopidogrel resistance phenomenon and the presence of bleeding side effects,which limits their clinical application.Therefore,it is of great significance to search for antiplatelet drugs that have stronger effects,fewer side effects,and higher bioavailability.We modified the 7-position of clopidogrel to synthesize a series of new clopidogrel derivatives.The pharmacokinetic studies in rats and Beagle dogs have been completed in the previous period and the results show that clopidogrel derivativesⅠhas good pharmacokinetic advantages.Based on the pharmacokinetics,this project conducted in-depth studies on the antiplatelet and antithrombotic effects of clopidogrel derivativesⅠand its mechanism.Methods:Kunming mice were randomly divided into 6 groups with 10 rats in each group:normal group（distilled water）,vehicle control group（0.5%CMC）,clopidogrel group（15mg/kg）,clopidogrel derivativesⅠlow dose group（7.5 mg/kg）,clopidogrel derivativesⅠmedium dose group（15 mg/kg）,clopidogrel derivativesⅠhigh dose group（30 mg/kg）.The effect of clopidogrel derivativesⅠon clotting time of mice was determined by capillary glass tube method;tail cutting method is used to determination of clopidogrel derivativesⅠeffect on bleeding time in mice;determination of clopidogrel derivativesⅠon thrombosis in vivo by injecting Carrageenan to induce tail thrombus formation.Wistar rats were randomly divided into 6 groups with 8 rats in each group:normal group（distilled water）,vehicle control group（0.5%CMC）,clopidogrel group（10 mg/kg）,clopidogrel derivativesⅠlow dose group（5 mg/kg）,clopidogrel derivativesⅠmedium dose（10 mg/kg）,clopidogrel derivativesⅠhigh dose（20 mg/kg）.Born nephelometry was used to determine the effect of clopidogrel derivativesⅠon platelet aggregation in rats;the effect of clopidogrel derivativesⅠon platelet adhesion in rats was determined by rotating glass ball method;and the effect of clopidogrel derivative I on thrombosis was determined by in vitro thrombosis assay.The effect of substance I on thrombosis;the effect of clopidogrel derivativesⅠon serum cAMP,P-selectin,PAC-1,TXB₂,and6-Keto-PGF_1αlevels was determined by enzyme-linked immunosorbent assay.Results:Compared with the vehicle control group:（1）clopidogrel derivativesⅠlow,middle and high dose groups significantly prolonged clotting time in mice（P<0.01,P<0.001）,clotting time in each dose group The prolongation rates were 49.20%,62.12%,and 87.70%,respectively;（2）clopidogrel derivativesⅠmiddle and high dose groups significantly prolonged the bleeding time（P<0.001）;compared with the clopidogrel group,low dose group significantly reduced bleeding time（P<0.001）;（3）clopidogrel derivativesⅠmiddle and high dose groups significantly reduced the maximum platelet aggregation rate（P<0.05,P<0.001）,platelet aggregation inhibition rate was 21.78%and 43.95%.Compared with clopidogrel group,high dose group significantly reduced the maximum platelet aggregation rate（P<0.05）;（4）clopidogrel derivativesⅠlow,middle and high dose groups significantly reduced the platelet adhesion rate（P<0.01,P<0.001）;（5）clopidogrel derivativesⅠlow,middle and high dose groups significantly shortened the thrombus length in in vitro and in vivo thrombosis assays（P<0.01）.Clopidogrel derivativeⅠshortens thrombus length in in vitro thrombosis experiments,reducing thrombus wet weight and thrombus dry weight.The inhibition rates of thrombus dry weight in each administration group were 35.47%,41.74%,and 47.45%,respectively;（6）clopidogrel derivativesⅠmiddle and high dose groups significantly increased serum cAMP levels（P<0.05）;significantly reduced serum P-selectin（P<0.05,P<0.01）and PAC-1（P<0.01,P<0.001）;（7）clopidogrel derivativesⅠhad no significant effect on serum TXB₂content and 6-Keto-PGF_1αcontent（P>0.05）.Conclusion:Clopidogrel derivativesⅠcan prolong clotting time,bleeding time,inhibits platelet activation,aggregation,adhesion and antithrombotic effects.Its mechanism of action is related to the promotion of platelet granule release of cAMP,inhibition of P-selectin,PAC-1 release,and has nothing to do with thromboxane A₂ signaling pathway.