ERK5在急性心肌梗死患者中的磷酸化水平及对体外血小板激活的影响

Phosphoryaltion levels of ERK5 in acute myocardial infarction patients and its role in platelet activation in vitro

目的观察急性心肌梗死(acute myocardial infarction,AMI)患者血小板中细胞外信号调节激酶5(extracellular signal-regulated kinase 5,ERK5)的活化水平及其特异性抑制剂XMD8-92对血小板功能的影响,探索ERK5调节血小板活性的分子机制。方法运用Western bolt法检测AMI患者(n=34)和稳定性心绞痛患者(n=33)血小板ERK5、Akt473及Akt308的磷酸化水平;通过检测体外血小板的聚集,测定不同浓度XMD8-92对胶原(collagen)刺激引起的血小板聚集的影响;利用荧光素酶同期检测XMD8-92对血小板致密颗粒分泌的影响;通过检测血小板在纤维蛋白原上的铺展、在血浆中的收缩,评价血小板整合素ɑIIbβ3的活化水平;运用Western bolt法检测聚集反应中XMD8-92对Akt473、Akt308及PTEN370磷酸化的影响。结果 AMI患者组ERK5及Akt473、Akt308磷酸化水平显著升高(P<0.05);ERK5抑制剂XMD8-92可抑制胶原诱导的血小板聚集、分泌、栓块收缩及在纤维蛋白原上的铺展等活化指标;XMD8-92处理后血小板Akt473、Akt308及PTEN370磷酸化水平减低。结论 ERK5的激活参与了AMI过程中血小板的活化;ERK5可通过调节PTEN的活性,从而调节Akt的磷酸化水平,进而调控血小板的功能。其特异性抑制剂有望成为新的抗栓药物。

Objective To observe the phosphorylation levels of extracellular signal-regulated kinase 5 （ERK5） in acute myocardial infarction （AMI） patients and the effects of ERK5 selective inhibitor XMD892 on human platelet activation in vitro, and to explore its mechanism. Methods Western blot was applied to detect the phosphorylation levels of ERK5, Akt473 and Akt308 in AMI patients （n=34） and stable angina patients （n=33,control）. The effects of different concentration of XMD8-92 on human platelet aggregation induced by collagen was tested by aggregometer in vitro. The release of ATP was measured simultaneously by luciferase detection. The effects of XMD8-92 on integrin ɑIIbβ3 were detected by platelet spreading on immobilized fibrinogen and clot retraction. The effects of XMD8-92 on phosphorylation levels of Akt473, Akt308 PTEN370 and ERK5 were detected by Western blot. Results The levels of phosphor-Akt473, Akt308 and phosphor-ERK5 were significantly higher in AMI patients than that in control group （P〈0.05）. ERK5 inhibitor XMD8-92 diminished collagen-induced platelet aggregation, ATP secretion, the average area of platelet spreading on immobilized fibrinogen and the clot retraction extent. The levels of phosphor-Akt （Ser-473/Thr-308） and phosphor-PTEN （Ser370） were significantly down-regulated in the presence of XMD8-92. Conclusions ERK5 plays a role in platelet activation in AMI process. It regulates platelet activation by regulating PTEN and Akt phosphorylation. Its specific inhibitor is hoped to be new antithrombotic drug.