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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 inhibitors in GP Ⅰ bα digestion and its molecular mechanism. Methods 5 ml of venous blood was collected from healthy volunteers [(10 ml / (human)], and the platelets were washed with 3 ml each. The washed platelets were incubated with Calpain inhibitor, ROS antagonist, NAD (P) H oxidase inhibitor, mitochondrial ROS antagonist or solvent control (DMSO), and then incubated with calmodulin inhibitor W7; GP Ⅰ bα expression, intracellular Ca 2 + and ROS concentrations were detected by flow cytometry; GP Ⅰ bα enzyme Digestion of Calpain substrate.Results W7 concentration-dependently induced intracellular Ca2 + concentration increase, Ca2 + average fluorescence intensity of DMSO was 52 ± 9, while W7 at 25, 50 and 100μmol / L was 121 ± 17,225 ± 23 and 308 ± 25, respectively. Compared with DMSO, the relative concentrations of ROS induced by W7 at 25, 50 and 100 μmol / L were 150 ± 11, 209 ± 20 and 297 ± 18; The antagonists of both ROS and Calpain partially inhibited W7-induced GPIbα cleavage when used alone, and the combined use of them suppressed W7-induced GPIbα cleavage.Conclusion Calmodulin inhibitors co-regulate ADAM17 by activating Calpain and generating ROS Mediated GPIbα digestion.