瞬时电位受体TRPM8在血小板中的表达和功能

Platelets play an important role in cardiovascular thromboembolic disease, antiplatelet therapy is crucial to treat the artery thrombosis disease. Transient receptor potential melastatin 8 (TRPM8) is a voltage-dependent calcium channel mainly expressed in the neurons. Upon activation by innocuous cooling or its ligand menthol, TRPM8 triggers Ca2+ influx to serve as the thermal sensor. The expression and function of TRPM8 in neuron cells has been fully investigated. However, its expression and function in platelets still remain unclear. Here, for the first time, we reported that the TRPM8 is highly expressed in human platelet. We also found that TRPM8 agonists potentiate platelet activation and Ca2+ influx; TRPM8 antagonists in clinical trials inhibit platelets activation and Ca2+ influx. TRPM8 initiates extracellular Ca2+ influx, which plays an important role in platelet activation signal transduction. Based on our previous work, we hypothesized that TRPM8 is functionally expressed in platelets. We will first investigate the effects and underlying mechanism of TRPM8 agonists/antagonists on platelet adhesion, aggregation, spreading and retraction. Furthermore, we intend to study the role of TRPM8 and its agonists/antagonists in platelet activation and thrombosis formation using TRPM8 knockout mice. Finally, we will investigate whether TRPM8 activation by cooling shorten platelet preservation time. Our project will provide important new knowledge in platelet activation induced by the calcium channel TRPM8. Our project also provides a new potential target of anti-platelet therapy. Considering that there are three TRPM8 antagonists which ongoing clinical trials in neurological disorders, our project will expand the new pharmacological effects of TRPM8 antagonists.

血小板激活在生理性止血和病理性血栓形成中起重要作用。血小板异常激活导致的动脉血栓是冠心病、中风等动脉血栓性疾病的病理基础，目前临床上使用的抗血小板药仍然存在严重副作用，拓展抗血小板激活的新靶点及相关分子机制研究十分重要。TRPM8作为一种主要表达在神经元细胞上的钙离子通道蛋白，是感知寒冷温度的外周神经传感器，但其在血小板的表达、功能均不清楚。我们前期工作中首次发现血小板表达TRPM8，并发现特异性激动剂活化TRPM8显著增强人血小板活化和钙离子内流，已进入临床试验的TRPM8特异性抑制剂抑制血小板活化和钙离子内流。本课题拟在此基础上，对TRPM8通道蛋白参与血小板活化的作用进行深入研究，为TRPM8在血小板上的表达、功能、血小板激活、血栓形成等方面提供新的认知，为完善血小板激活机制提供新的理论基础，并为TRPM8特异性抑制剂作为抗血小板药的临床应用提供重要的理论和实验依据。

本课题按基金任务书要求系统研究了TRPM8-Ca2+信号通路对血小板的活化作用及探讨活化机制。在课题研究中我们采用了多种体外血小板功能实验和体内动物模型探索TRPM8抑制剂GWP-42006抑制血小板活化的药理学作用和分子机制。课题的主要结果为：1、使用RT-PCR和western blot实验明确在mRNA水平和蛋白质水平上血小板表达TRPM8。2、利用体外血小板聚集实验明确TRPM8的三种抑制剂都具有血小板抑制作用，其中GWP-42006的抑制作用最强。GWP-42006对多种激动剂诱导的血小板聚集和ATP释放都有抑制作用，但是对TRPM8敲除小鼠无抑制作用。GWP-42006也可以抑制血小板铺展，血块回缩和JON/A表达。3、利用小鼠体内实验明确TRPM8 抑制剂GWP-42006可以延长野生型小鼠断尾出血时间，减少动脉血栓形成，以及抑制小鼠大脑中动脉阻塞损伤；但是TRPM8 抑制剂GWP-42006对TRPM8敲除小鼠无明显作用。4、利用多种分子生物学实验方法明确TRPM8激活可以升高血小板内Ca2浓度，同时升高其下游的TXA2生成，ERK磷酸化，Rap1活化，PKC活化和PLEK升高发挥增强血小板活化作用。另一方面，我们也进一步明确TRPM8 抑制剂GWP-42006是通过降低血小板内Ca2+浓度，以及减少下游TXA2生成，抑制ERK磷酸化，Rap1活化，PKC活化和减少PLEK表达发挥抑制血小板活化的药理作用。另外，利用免疫荧光共定位实验发现TRPM8与DAG无共定位现象。5、最后我们进一步探索研究发现TRPM8 抑制剂GWP-42006无法升高低温诱导的血小板减少和血小板活性降低。课题调整情况：由于时间限制，本课题的实验结果有待进一步总结和发表。课题已培养博士研究生1名、硕士研究生3 名。公开发表SCI论文4 篇,待发表论文1篇，其它的结果在进一步整理中。