趋化因子CCL2通过影响血小板聚集和活化功能参与动脉血栓的发生发展

Platelet aggregation and activation play important roles in the plaque formation and thrombosis of AMI. Dual antiplatelet therapy with clopidogrel and aspirin can effectively relieve the clinical symptoms of AMI patients, however, the response to clopidogrel in 4-30% AMI patients is low, which is called clopidogrel resistance(CR). Increasing the dose of clopidogrel or using the new antiplatelet drug ticagrelor can effectively reduce the risk of cardiovascular events in CR patients. However, they could not absolutely eliminate thrombus events, indicating that a certain degree of platelet aggregation and activation is still existed in CR patients after optimized antiplatelet therapy. Therefore, to search for the key molecules and signaling pathway regulating platelet function could provide the basis and strategies for AMI and other cardiovascular diseases. Chemokine CCL2 is the most widely studied CC chemokine, CCL2 play important roles in atherosclerosis, inflammation, cardiac injury and repair. Our previous work showed that CCL2 expressions in the plasma and coronary artery thrombus tissues of CR patients were significantly higher than NCR group. CCL2 could induce the increase of platelet aggregation in vitro experiments. The above data suggested that CCL2 may play an important role in the atherosclerotic plaque formation and thrombosis of AMI by affecting platelet aggregation and activation. Therefore, this study aims to investigate the roles of CCL2 on the platelet function and arterial thrombosis, and elucidate the relevant mechanism, which not only clarify the pathogenesis of AMI, but also provides the basis for the diagnosis and treatment of AMI and other cardiovascular diseases.

血小板聚集和活化在AMI斑块及血栓形成中有重要作用。双联抗血小板治疗能有效改善AMI患者的临床症状，然而，4-30%患者对氯吡格雷反应低下，称为氯吡格雷抵抗（CR）。增加药物剂量或更换替格瑞洛后，能显著降低但不能完全消除CR患者发生心血管事件的风险，提示CR患者在优化治疗后仍存在血小板聚集和活化。因此，从血小板功能角度入手，寻找对抗血小板功能的关键调控分子至关重要。趋化因子CCL2在动脉粥样硬化和炎症反应中发挥重要作用。我们前期工作表明，CCL2在CR组STEMI患者血浆和冠状动脉血栓组织的表达显著高于NCR组；体外实验证实CCL2促进血小板聚集升高，提示CCL2可能通过影响血小板聚集和活化功能参与AMI血栓的发生发展。因此，本研究拟从体内外水平探讨CCL2对血小板聚集和活化功能及动脉血栓形成的影响及明确相关机制，为深入阐明AMI的发病机制以及为AMI等心血管疾病的防治提供新的策略和依据。

项目背景：研究表明趋化因子CCL2在炎症反应和动脉粥样硬化等疾病中发挥重要作用，然而，CCL2对血小板功能以及动脉血栓形成的影响尚不清楚。.主要研究内容：通过ELISA、western blot、免疫组化和免疫荧光染色检测ST段抬高型心肌梗死患者（STEMI）血浆、血小板和冠状动脉血栓组织中CCL2和受体CCR2表达。通过光学比浊法、流式细胞术和ELISA方法检测CCL2对血小板聚集、活化和分泌的影响。通过蛋白质芯片筛选CCL2刺激后血小板变化的蛋白激酶，明确CCL2调控血小板功能的信号通路。.重要结果及关键数据：与血小板反应正常的STEMI患者相比，CCL2及CCR2在血小板高反应性的STEMI患者血浆及血小板中明显增高。与正常血凝块相比，CCL2及CCR2在STEMI患者罪犯冠状动脉血栓中高表达。体外实验发现，人重组CCL2因子可以增加正常人血小板聚集、活化和颗粒分泌，并且这种效应可以被CCL2中和抗体和CCR2受体拮抗剂所阻断。此外，CCL2通过增加正常人血小板PKCα(Thr638)，P38MAPK(Thr180/Tyr182)和HSP27(S78/S82)表达，并且可以被PKCα抑制剂(RO 318220)和P38MAPK抑制剂(SB 203580)阻断。与野生型小鼠相比，CCL2基因敲除小鼠的血小板聚集和血小板颗粒分泌能力降低，磷酸化PKCα(Thr638)，P38MAPK(Thr180/Tyr182)和HSP27(S78/S82)降低。在动脉血栓形成的模型中发现，与野生型小鼠相比，CCL2基因敲除的小鼠颈动脉闭塞时间延长。.结论：CCL2通过调控PKCα-P38MAPK-HSP27信号通路参与对血小板功能和动脉血栓形成的调控，对于挖掘新型抗血小板药物来治疗心血管疾病提供理论依据和线索。