ERK5对体外血小板活化及在体血栓的影响

Role of ERK5 in platelet activation in vitro and arterial thrombosis in vivo

目的:探讨细胞外信号调节激酶5(ERK5)对体外血小板聚集及在体血栓的影响及机制。方法:采用Western blot对人血小板中ERK5的表达及其在血小板活化后的磷酸化水平进行检测;采用血小板聚集仪检测ERK5特异性抑制剂XMD8-92对血小板聚集及致密颗粒释放的影响;采用Fe Cl3颈动脉血栓模型检测ERK5对在体血栓的影响;采用Western blot检测XMD8-92对蛋白激酶B(Akt)和人第10号染色体缺失的磷酸酶及张力蛋白同源蛋白(PTEN)磷酸化的影响。结果:人血小板中存在ERK5的稳定表达,其磷酸化水平在血小板活化后显著升高(P<0.05)。XMD8-92可抑制多种血小板激活剂引起的血小板聚集和致密颗粒释放(P<0.05)。Western blot结果表明,XMD8-92可通过下调PTEN Ser370位点磷酸化而增强PTEN的活性,从而抑制Akt的磷酸化,这种抑制效果也通过血小板特异PTEN基因敲除小鼠得到了验证。在体血栓研究表明,XMD8-92经尾静脉给药,可显著延长小鼠第一次颈动脉血栓的形成时间。结论:ERK5可通过影响PTEN的磷酸化调节Akt的活化,进而影响到体外血小板的聚集和在体血栓的形成。

AIM：To investigate the role of extracellular signal-regulated kinase 5（ERK5） in platelet aggregation in vitro and arterial thrombosis in vivo.METHODS：The expression and phosphorylation levels of ERK5 in human platelet were detected by Western blot.The effects of ERK5 selective inhibitor XMD8-92 on platelet aggregation and dense granule secretion were detected by Chrono-Log aggregometer.The effect of ERK5 on in vivo thrombosis was analyzed using an Fe Cl3 artery thrombosis model.The effects of XMD8-92 on protein kinase B（PKB/Akt） and phosphatase and tensin homolog deleted on chromosome ten（PTEN） phosphorylation levels were determined by Western blot.RESULTS：ERK5 was stably expressed in human platelets and its phosphorylation level increased significantly after platelet activation（P 〈0.05）.XMD8-92,a selective inhibitor of ERK5,inhibited platelet aggregation and dense granule secretion in response to several platelet stimulators（P 〈 0.05）.The results of Western blot showed that XMD8-92 inhibited Akt phosphorylation level by down-regulating PTEN Ser370 phosphorylation and enhancing PTEN activity.The pathway was further confirmed using platelet specific PTEN deficiency mice.The first occlusion time was obviously extended in the mice intravenously given XMD8-92 in the Fe Cl3-induced carotid artery injury model.CONCLUSION：ERK5 plays a role in platelet activation and arterial thrombosis by influencing PTEN and Akt phosphorylation.