佛波酯诱导血小板黏附受体GPⅠbα酶切的机制研究  

Research on molecular mechanisms of PMA-induced platelet GPⅠbα shedding

作　　者：王志成[1] 罗梅宏[1] 谢如锋[2] 张晓峰[1] 

机构地区：[1]上海中医药大学附属市中医医院实验中心,上海200071 [2]上海市血液中心血液工程室,上海200051

出　　处：《检验医学》2013年第5期420-424,共5页Laboratory Medicine

基　　金：上海市自然科学基金(No.12ZR1429900);上海市教育委员会科技创新项目(No.12YZ068);上海市中医医院院级课题(No.124101)

摘　　要：目的佛波酯(PMA)能诱导血小板黏附受体GPⅠbα酶切,但分子机制未完全阐明。本研究主要探讨PMA诱导GPⅠbα酶切的分子机制。方法取健康志愿者静脉血,分离得到洗涤血小板。将洗涤血小板分别与蛋白激酶C(PKC)抑制剂、活性氧(ROS)抑制剂、NAD(P)H氧化酶抑制剂、线粒体ROS拮抗剂或二甲基亚矾(DMSO)对照等预孵育,再与PMA孵育。流式细胞仪检测ROS浓度;Western blot检测GPⅠbα酶切产物;用非同位素标记法检测PKC活性。结果 PKC抑制剂BIM和ROS抑制剂二硫苏糖醇(DTT)单独使用时,都能完全抑制PMA诱导的GPⅠbα酶切。PMA浓度依赖地诱导血小板ROS产生,PKC抑制剂和NAD(P)H氧化酶抑制剂都能抑制PMA诱导的ROS产生,而线粒体ROS拮抗剂没有抑制作用。BIM抑制PMA诱导的PKC活化,DTT则没有抑制作用。结论 PMA可能通过PMA-PKC-NAD(P)H氧化酶-ROS-ADAM17-GPⅠbα信号通路调节GPⅠbα酶切。Objective Phorbol 12-myristate-13-acetate （PMA） could induce platelet GP Ⅰbαshedding, however, the molecular mechanisms are not fully elucidated. This study investigates the molecular mechanisms of PMA-induced platelet GP Ⅰbα shedding. Methods Washed platelets were obtained from anti-coagulated healthy volunteers＇ whole blood by centrifugation and washing. Washed platelets were pre-incubated with protein kinase C （ PKC ） inhibitor, reactive oxygen species （ROS） antagonist, NAD（P） H oxidase inhibitor, mitochondrial ROS antagonist, or dimethyl sulfoxide （DMSO）, and were incubated with PMA. ROS levels were measured by flow cytometry. The production of platelet GP Ⅰbα shedding was detected by Western blot. The nonradioactive detection was used to measure PKC activity. Results PMA-induced platelet GP Ⅰbα shedding was completely inhibited by PKC inhibitor BIM or ROS antagonist dithiothreitol（ DTT）, respectively. PMA did dependently elevate ROS levels. PMA-induced ROS production was inhibited by PKC inhibitor and NAD（P） H oxidase inhibitor, but not mitochondrial ROS antagonist. BIM inhibited PMA-induced PKC activity, however, DTT did not. Conclusions PMA-induced platelet GP Ⅰbα shedding could be regulated by PMA-PKC-NAD（P） H oxidase-ROS-ADAM17-GP Ⅰbαsignaling pathway.

关 键 词：血小板 GPⅠbα酶切 佛波酯 蛋白激酶C 活性氧 

分 类 号：R446.11[医药卫生—诊断学]