钙调蛋白抑制剂诱导血小板GPⅠbα酶切的分子机制研究  

Molecular mechanism of calmodulin inhibitor-induced platelet GPⅠbα shedding

作　　者：王志成[1,2] 谢如锋[3] 张晓峰[1] 

机构地区：[1]上海中医药大学附属上海市中医医院实验中心,上海200071 [2]复旦大学华山医院 [3]上海市血液中心

出　　处：《中国输血杂志》2012年第12期1253-1257,共5页Chinese Journal of Blood Transfusion

基　　金：上海市教育委员会科技创新项目(12YZ068);上海市自然科学基金(12ZR1429900);上海市博士后科学基金A类资助(12R21411700);上海市中医医院院级课题(124101)

摘　　要：目的探讨钙调蛋白抑制剂在GPⅠbα酶切中的作用和分子机制。方法取健康志愿者静脉血5份[(10 ml/(人)份],分离得到洗涤血小板3 ml/份,将洗涤血小板分别与Calpain抑制剂、活性氧(ROS)拮抗剂、NAD(P)H氧化酶抑制剂、线粒体ROS拮抗剂或溶剂对照(DMSO)等预孵育,再与钙调蛋白抑制剂W7孵育;流式细胞仪检测GPⅠbα表达、胞内Ca2+和ROS浓度;Western blot检测GPⅠbα酶切产物、Calpain底物talin的酶切。结果W7浓度依赖地引起血小板胞内Ca2+浓度升高,Ca2+平均荧光强度DMSO为52±9,而W7在25、50和100μmol/L时分别为121±17、225±23和308±25;W7浓度依赖地诱导血小板ROS产生,与DMSO比较,W7在25、50和100μmol/L时诱导的ROS相对浓度分别为150±11、209±20和297±18;ROS拮抗剂和Calpain抑制剂单独使用时都部分抑制W7诱导的GPⅠbα酶切,联合使用时则完全抑制W7诱导的GPⅠbα酶切。结论钙调蛋白抑制剂通过活化Calpain和产生ROS共同调控ADAM17介导的GPⅠbα酶切。Objective To investigate the role of calmodulin in GP I bet shedding. Methods Three milliliter of washed platelets were obtained from ten milliliter of anti-coagulated healthy volunteers whole blood by centrifugation and wash- ing. Washed platelets were pre-incubated with ealpain inhibitors, ADAM17 inhibitor, ROS antagonist, NAD （P）H oxidase in- hibitor,mitoehondrial ROS antagonist, or DMSO, and then incubated with calmodulin inhibitor. GP I bet expression and ey- tosolie calcium levels were analyzed by flow eytometry. Cleavage of GP I bα and talin was detected by western blot. ROS lev- els were measured with fluorescence spectrometer. Results W7 dose-dependently elevated eytosolie calcium levels, and the levels of calcium fluorescence geo mean in DMSO and various concentrations of W7（25,50 and 100 μM） were 52 ± 9,121±17,225 ± 23 and 308 ± 25 ,respectively. Meanwhile ,W7 dose-dependently induced platelet ROS production, and the relative ROS production compared with DMSO were 150± 11,209±20 and 297 ±18 in platelets treated with W7 at 25,50 and 100 μM, respectively. W7-indueed GP I bet shedding was partially inhibited by ealpain inhibitor MDL or ROS antagonist DTT re- spectively. However,when used in tandem, MDL and DTT completely reduced GP I bet shedding. Conclusion Calpain and ROS together regulated ADAM17 mediated-GP I bet shedding in ealmodulin inhibitor-treated platelets.

关 键 词：血小板 钙调蛋白 GPⅠbα酶切 活性氧 钙离子 

分 类 号：R446.112[医药卫生—诊断学] R331.143[医药卫生—临床医学]