氯通道对血小板活化标志物PAC-1和P-选择素的影响

The role of chloride channel on platelet activation markers PAC-1 and P-selectin

目的:探讨氯通道对血小板活化标志物PAC-1和P-选择素的影响。方法:采用Western blot和免疫荧光技术检测Cl C-3在人外周血来源的血小板上的表达;ADP(20μM)不同时间点(15、30、60、120、180 min)处理血小板后,Western blot检测Cl C-3蛋白的表达变化;利用流式细胞术检测空白对照组,ADP组,DIDS(100μM)+ADP组,NFA(100μM)+ADP组,NPPB(100μM)+ADP和低氯对照组,低氯+ADP组的血小板活化分子标志物PAC-1和P-选择素的表达变化。结果:在健康成人外周血分离的血小板上表达Cl C-3蛋白;ADP时间依赖性地处理血小板,Cl C-3蛋白表达呈增加趋势,15 min相较于空白对照组已有统计学意义(P<0.05);ADP组PAC-1(32.13%±2.0%)及P-选择素(52.32%±5.31%)表达均高于空白对照组(1.76%±0.41%,1.89%±0.32%,P<0.01);低氯对照组PAC-1(8.01%±1.36%)和P-选择素(12.19%±0.84%)的表达与空白对照组相比,均上调(P<0.05);与ADP组PAC-1相比,DIDS+ADP(5.62%±1.22%),NFA+ADP(1.96%±0.54%)和NPPB+ADP(3.56%±0.79%)组表达降低,低氯+ADP组(45.26%±4.43%)表达增加(P<0.01);与ADP组P-选择素相比,DIDS+ADP(16.64%±1.52%),NFA+ADP(4.97%±0.64%)和NPPB+ADP(8.56%±2.04%)组表达均降低,低氯+ADP组(73.33%±3.80%)表达增加(P<0.01)。结论:人血小板上Cl C-3氯通道参与血小板的活化,氯通道阻断剂抑制ADP诱导的血小板活化,降低血小板胞外氯浓度可促进ADP诱导的血小板的活化。

Objective： To explore the role of chloride channel on platelet activation markers PAC-1 and P-selectin. Methods： The expression of Cl C-3 on platelets isolated from human peripheral blood was detected by Western blotting and confocal microscopy. Using ADP（20 μM） to stimulate platelets at different points（15, 30, 60, 120, 180 min）, Cl C-3 protein expression was detected by Western-blot; the seven groups： the control groups, ADP（20 μM）, DIDS（100 μM）＋ADP, NFA（100 μM）＋ADP, NPPB（100 μM）＋ADP, low chloride control group and low chloride ＋ADP（20 μM）, the expressions of active platelets markers—PAC-1 and P-selectin were detected by flow cytometry. Results： The corresponding band of 85 k D could be observed by Western blot in platelets isolated from healthy adult peripheral blood; using ADP to stimulate platelets by time-dependently, Cl C-3 protein expression increased significantly at 15 min compared to the control group（P〈0.05）; the expressions of PAC-1（32.13%±2.0%） and P-selectin（52.32%±5.31%） in ADP group were more than those in the control group（1.76%±0.41%, 1.89%±0.32%, P〈0.01）, while compared with the control group, the expressions of PAC-1（8.01%±1.36%） and P-selectin（12.19%±0.84%） in low chloride control group increased; the expression levels of PAC-1 in DIDS＋ADP（5.62%±1.22%）, NFA＋ADP（1.96%±0.54%） and NPPB ＋ADP（3.56%±0.79%） groups decreased comparing with ADP group（P〈0.01） and the expression levels of P-selectin in DIDS＋ADP（16.64%±1.52%）, NFA＋ADP（4.97%±0.64%） and NPPB＋ADP（8.56%±2.04%） groups decreased too（P〈0.01）. Compared with ADP group, PAC-1（45.26%±4.43%） and P-selectin（73.33%±3.80%） in low chloride＋ADP group increased（P〈0.01）. Conclusion： Cl C-3 chloride channel took participation in human platelet activation and chloride channel blockers could inhibit platelet activation induced by ADP, decreasing chloride level outside cells could active platelets induced by ADP.