摘要
目的:探究酰基甘油激酶(AGK)在血小板活化中的作用及可能的调控机制。方法:分别提取野生型小鼠(WT)和AgkG126E点突变(AGK激酶活性丧失)小鼠(PM)的血小板。将等体积、等浓度的WT或PM小鼠的血小板放入聚集仪,分别利用3种刺激剂(α-Thrombin、ADP、U46619)刺激,检测每种刺激剂作用下相应的血小板聚集水平;每管聚集曲线达到最大稳定值后,加入等体积的ATP荧光检测剂,检测相应血小板活化过程中释放的ATP水平。分别在静息态WT或PM小鼠血小板(不加α-Thrombin刺激)、活化态WT或PM小鼠血小板(加入α-Thrombin刺激)中加入等量荧光偶联的纤维蛋白原(Fg),利用流式细胞仪检测血小板的Fg结合水平。Western blot法分别检测静息态(不加刺激剂)和活化态(加入α-Thrombin、ADP、U46619刺激)WT或PM小鼠血小板中关键信号通路蛋白的磷酸化水平。结果:在刺激剂作用于血小板后,PM小鼠血小板的聚集程度、ATP释放水平及Fg结合能力均明显低于WT小鼠的血小板(P均<0.01)。与WT小鼠相比,PM小鼠的血小板在活化过程中张力蛋白同源性磷酸酶(PTEN)、磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(Akt-Thr308)和糖原合成激酶3β(Gsk3β-Ser9)的磷酸化水平均明显降低(P均<0.01)。结论:酰基甘油激酶可能通过影响PTEN/PI3K/Akt/Gsk3β信号通路,参与调控血小板的活化。
Objective:To explore the role and possible regulatory mechanism of acylglycerol kinase(AGK) during platelet activation. Methods: Platelets were extractedfrom wild-type mice(WT) and Agk G126E point mutant mice(deprivation of AGK activity),respectively. Levels of aggregation, ATP secretion and fibrinogen(Fg) binding during platelet activation were detected using platelet agonists, aggregator and flow cytometer. The phosphorylation levels of key signaling pathway proteins during platelet activation were assessed by western blot assays. Results: Upon stimulation with the agonists at same concentration, levels of aggregation, ATP secretion and Fg binding of platelets were significantly lower in Agk G126E mice than those in WT mice(all P<0.01). Western blotting showed that, compared with WT mice, phosphorylation levels of phosphatase and tensin homolog(PTEN), phosphatidylinositol-3-kinase(PI3K), protein kinase B(AkT-Thr308), glycogen synthase kinase 3β(Gsk3β-Ser9) were decreased during platelets activation in Agk G126E mice(all P<0.01). Conclusion: This study suggests that AGK may regulate platelet activation through mediating PTEN/PI3K/Akt/GSK3β signaling pathway.
作者
张鹏
毕昌龙
魏萌
姜哲轶
张田田
张俊峰
ZHANG Peng;BI Changlong;WEI Meng;JIANG Zheyi;ZHANG Tiantian;ZHANG Junfeng(Department of Cardiology,Shanghai Ninth People’s Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200011,China)
出处
《国际心血管病杂志》
2023年第1期46-51,共6页
International Journal of Cardiovascular Disease
基金
国家自然科学基金(81670316,81970289)。
