AHV-PI抗血小板聚集的PI3K/Akt信号通路机制研究

Study on the mechanism of AHV-PI on platelet aggregation byPI3K/Akt signal pathway

目的 探讨蝮蛇毒血小板抑制因子（AHV-PI）抗血小板聚集的细胞内信号转导通路及其可能机制。方法 通过蛋白质免疫印迹（Western blot）检测AHV-PI对血小板内Akt蛋白激酶磷酸化水平情况,XS-1000I五分类血球计数仪进行血小板计数;酶联免疫吸附法（ELISA）测定家兔血浆中5’-核苷酸酶（5’-NT）,血小板膜糖蛋白Ib（GPIb）含量;流式细胞术（FCM）观察AHV-PI对荧光标记的单克隆抗体CD61（FITC-CD61）与血小板膜糖蛋白IIb/IIIa（GPIIb/IIIa）结合率的影响。结果 AHV-PI能降低Akt磷酸化水平,减少血小板数量,升高血浆中5’-NT含量,降低血小板GPIb的表达;AHV-PI对单克隆抗体CD61与血小板的结合率没有影响。结论 AHV-PI可以通过抑制蛋白激酶Akt磷酸化来阻断Akt通路的信号转导,限制细胞生长,减少血小板数量;具有5’-核苷酸酶的活性,能够降解ADP,防止血小板性血栓的形成。

Objective To investigate the mechanism of platelet inhibitor from Agkistrodon halys venom （AHV-PI） on platelet aggregation. Methods Protein kinase Akt phosphorylation levels in platelet were measured by Western blot. XS-10001 blood cell counter was used for platelet count. The plasma content of S^NT and platelet membrane GPIb were determined by Enzyme-Linked Immunosorbnent Assay （ELISA）. The effect of AHV-PI on binding rate between the fluorescence labeled monoclonal antibody CD61 （FITC-CD61） and platelet membrae glycoprotein nb/nia （GPIIb/ Ilia） was observed by flow cytometry （FCM）. Results AHV-PI can reduce the level o f Akt-phosphorylation level and the number o f platelet. AHV- PI can increase the content of S^NT in plasma, reduce the expression of platelet GPIb. Flow cytometry displayed AHV-PI can not affect the rate of combination between platelet GPIIb/IIIa and FITC-CD61. Conclusion The mechanism o f inhibition o f platelet aggregation may be inhibit protein kinase Akt phosphorylation to block the signal transduction pathway of Akt. Limit cell grouth and reduce platelet number, also it may be related to its 5’-NT activity, it can degradate ADP to prevent the formation of platelet thrombus.