氯通道对血小板活化标志物PAC-1和P-选择素的影响

摘要

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ClC-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ClC-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ClC-3蛋白；ADP时间依赖性地处理血小板，ClC-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ClC-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: hTe expression of ClC-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), ClC-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 kD could be observed by Western blot in platelets isolated from healthy adult peripheral blood; using ADP to stimulate platelets by time-dependently, ClC-3 protein expression increased signiifcantly 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:ClC-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.