佛波酯诱导血小板黏附受体GPⅠbα酶切的机制研究

doi:10.3969/j.issn.1673-8640.2013.05.019

检验医学 ›› 2013, Vol. 28 ›› Issue (5): 420-424.DOI: 10.3969/j.issn.1673-8640.2013.05.019

佛波酯诱导血小板黏附受体GPⅠbα酶切的机制研究

王志成¹,罗梅宏¹,谢如锋²,张晓峰¹

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

收稿日期:2012-03-23 修回日期:2012-04-17 出版日期:2013-05-15 发布日期:2013-05-14
通讯作者: 张晓峰，电话：021-56639828-4520。
作者简介:王志成，男，1975年生，博士，助理研究员，主要从事血小板活化和凋亡以及血小板储存损伤机制研究。
基金资助:
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）

Research on molecular mechanisms of PMA-induced platelet GPⅠbα shedding

1. Laboratory Center，Shanghai Traditional Chinese Medicine Hospital， Shanghai University of Traditional Chinese Medicine，Shanghai 200071， China; 2. Blood Engineering Laboratory，Shanghai Blood Center，Shanghai 200051， China

Received:2012-03-23 Revised:2012-04-17 Online:2013-05-15 Published:2013-05-14

摘要/Abstract

摘要： 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。

关键词: 血小板, GPⅠbα酶切, 佛波酯, 蛋白激酶C, 活性氧

Abstract: Objective　Phorbol 12-myristate-13-acetate (PMA) could induce platelet GPⅠbα shedding，however，the molecular mechanisms are not fully elucidated. This study investigates the molecular mechanisms of PMA-induced platelet GPⅠbα 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 GPⅠbα shedding was detected by Western blot. The nonradioactive detection was used to measure PKC activity. Results　PMA-induced platelet GPⅠbα 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 GPⅠbα shedding could be regulated by PMA-PKC-NAD(P)H oxidase-ROS-ADAM17-GPⅠbα signaling pathway.

Key words: Platelet, GPⅠb&alpha, shedding, Phorbol 12-myristate-13-acetate, Protein kinase C, Reactive oxygen species

王志成1，罗梅宏1，谢如锋2，张晓峰1. 佛波酯诱导血小板黏附受体GPⅠbα酶切的机制研究[J]. 检验医学, 2013, 28(5): 420-424.

WANG Zhicheng 1，LUO Meihong 1，XIE Rufeng 2，ZHANG Xiaofeng 1. Research on molecular mechanisms of PMA-induced platelet GPⅠbα shedding[J]. , 2013, 28(5): 420-424.

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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

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