USP9X对Akt磷酸化水平及血小板活化的影响

Effect of USP9X on Akt phosphorylation and platelet function

目的 探讨血小板中去泛素化酶USP9X的表达及其对血小板功能的影响。方法 通过聚合酶链式反应和免疫印记方法检测人和小鼠血小板中USP9X的表达情况;分离制备年轻小鼠和老年小鼠的血小板,检测USP9X的表达变化;采用USP9X特异性抑制剂检测USP9X对血小板聚集释放及铺展功能的影响;收集激活不同时间点的血小板蛋白裂解液,并进行免疫印记检测磷酸化Akt的变化。结果 人血小板与小鼠血小板在mRNA水平和蛋白水平均表达USP9X。相对于年轻小鼠,老年小鼠血小板中USP9X的表达明显升高(0.87±0.099 vs 1.05±0.980,P<0.05)。使用USP9X的特异性抑制剂提前30 min孵育血小板,之后检测血小板的聚集、释放及铺展功能,结果显示,相比于对照组,USP9X抑制剂处理组的血小板的聚集和释放水平明显下降(P<0.05);相比于对照组,抑制剂处理组45 min的铺展面积显著降低。最后,免疫印记结果显示,USP9X抑制剂处理组的血小板的Akt磷酸化水平明显下降。结论 人和小鼠的血小板表达USP9X,USP9X可促进血小板的聚集释放及铺展,并可影响Akt的磷酸化水平,USP9X的抑制剂或可成为潜在的临床血栓干预靶点。

Objective To explore the expression of USP9X in platelets and its effect on platelet function.Methods The expression of USP9X in human and mouse was evaluated by PCR and Western blot.Platelets from young and old mice were separated and prepared,and the expression of USP9X was detected.USP9X inhibitos were used to assess the regulation of USP9X in platelet function,including aggregation,ATP release and spreading.Platelet lysates were collected in different time points to evaluate the change of phosphorylation of Akt in USP9X inhibitors treated platelets.Results Both human and mouse platelets expressed USP9X.Compared to the young mice,the old mice showed significantly enhanced expression of USP9X(P<0.05).To assess the effect of USP9X on platelet function,USP9X inhibitor was used to pre-incubate platelets for 30 min and platelet function were examined later.Results showed that USP9X inhibitor significantly decreased platelet activation including aggregation,ATP release and spreading(P<0.05).Compared to the control group,the inhibitor treated group showed a significant decrease in the spreading area after 45 minutes.The Western blot results showed a significant decrease in Akt phosphorylation levels of platelets in the USP9X inhibitor treated group.Conclusion Both human and mouse platelet express USP9X,and inhibition of USP9X decreased platelet function including aggregation,ATP release and spreading.USP9X can also influence the phosphorylation of Akt.The inhibitor of USP9X may become a potential therapeutic target for thrombosis intervention.