斑蝥素经PI3K/Akt/PKC通路对血小板功能的调控作用

Regulation of platelet function by cantharidin via PI3K/Akt/PKC pathway

目的 探讨斑蝥素(Cantharidin,CTD)对血小板功能的影响和抗血小板聚集的作用机制。方法 采集健康志愿者静脉血,提取洗涤血小板,聚集仪检测CTD(2.5、5、10μmol·L^(-1))对胶原、凝血酶、ADP和佛波酯(Phorbol-12-myristate-13-acetate,PMA,PKC激动剂)诱导的血小板聚集,和对ATP释放的影响。荧光显微镜拍照观察CTD对纤维蛋白原表面的血小板扩展功能和对血小板斑块回缩的影响。检测CTD联用740Y-P(PI3K特异性激动剂)对胶原诱导血小板的聚集率,Western blot检测PI3K蛋白磷酸化水平。小鼠腹腔注射CTD(0.35、0.7 mg·kg^(-1))和阿司匹林(30 mg·kg^(-1)),观察胶原诱导各组离体血小板聚集率,检测PI3K、Akt和PKC蛋白磷酸化水平,检测凝血指标凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)和小鼠断尾流血时间。结果 CTD抑制胶原、凝血酶和PMA诱导的血小板聚集和抑制ATP释放,对ADP诱导的血小板聚集无影响,CTD抑制血小板扩展和斑块回缩,740Y-P处理能够减弱CTD对胶原诱导的血小板聚集的抑制作用。CTD抑制离体血小板聚集功能和PI3K、Akt和PKC磷酸化水平,延长APTT和小鼠尾巴出血时间。结论 CTD抑制血小板聚集、释放、扩展、斑块回缩和影响止血功能,其机制与调控PI3K、Akt和PKC蛋白磷酸化水平有关。

To investigate the effect of cantharidin(CTD)on platelet function and the mechanism of anti-platelet aggregation.Methods Washed platelets were collected from the venous blood of healthy volunteers.The effect of CTD on platelet aggregation and release was determined by aggregometer.The CTD concentration was 2.5,5,10μmol·L^(-1).The agonists were collagen,thrombin,adenosine diphosphate(ADP),and Phorbol-12-myristate-13-acetate(PMA,PKC activator).The average spreading area of individual platelets on the immobilized fibrinogen was observed by fluorescence microscopy.The digital camera took pictures of fibrin clots.The effect of 740Y-P(PI3K specific agonist)combined with CTD on collagen-induced platelet aggregation was detected.The phosphorylation of PI3K was detected by Western blot.Mice were administered CTD(0.35,0.7 mg·kg^(-1))by intraperitoneal injection.The platelet aggregation rate induced by collagen in each group was observed,and the phosphorylation of PI3K,Akt,and PKC was assessed.The coagulation indexes prothrombin time(PT),activated partial thromboplastin time(APTT),and tail bleeding time were detected.Results CTD suppressed platelet aggregation induced by collagen,thrombin,and PMA and inhibited ATP release,but did not affect platelet aggregation induced by ADP.CTD reduced platelet spreading and clot retraction.740Y-P treatment significantly attenuated the inhibitory effect of CTD on collagen-induced platelet aggregation.CTD suppressed collagen-induced platelet aggregation and the phosphorylation of PI3K,Akt,and PKC ex vivo.CTD prolonged APTT and tail bleeding time.Conclusions It is found that CTD curbs platelet aggregation,release,spreading,and clot retraction and affects hemostatic function.The mechanism is related to the regulation of phosphorylation levels of PI3K,Akt,and PKC.