抑素蛋白1（PHB1）抑制剂的设计优化及其对血小板活化、血栓形成的影响

Platelet activation plays a central role in thrombosis and hemostasis. Unappreciated local and systemic platelet activation are implicated in diverse disorder processes, such as atherosclerosis. Prohibitins (PHBs), comprising the two homologous members PHB1 and PHB2, are ubiquitously expressed and highly conserved. Proteomic studies have revealed the presence of PHBs in human platelets, but the roles of PHBs during platelet aggregation are unknown. In our present study, PHB1 and PHB2 were detected on the surface of human platelets, especially in the lipid raft. PHBs were associated with protease-activated receptor 1 (PAR1), as determined by co-immunoprecipitation and confocal microscopy. Human platelet aggregation, granular secretion and calcium mobilization stimulated by low concentrations of thrombin or PAR1-activating peptide (PAR1-AP) were reduced or abolished as a result of the blockade of PHBs by anti-PHB antibodies or their Fab fragments; The calcium mobilization in MEG-01 megakaryocytes stimulated by PAR1-AP was significantly suppressed by PHB depletion using RNA interference against PHB1 and PHB2. Until recently, PHBs were unknown regulators of PAR1 signaling and may be effective targets for anti-platelet therapy. .PHB1 is a multifunctional protein and is related with many disease, however, there is no inhibitor or antagonist of PHB1 was founded. Based on the structure of PHB1, we found a peptide (P5, RHKWK) could interact with PHB1 by molecular docking. Besides, P5 could inhibit PAR1-AP and low concentration of thrombin induced platelet aggregation and calcium mobilization at the concentration of 200 micromole, which IC90 is 400 micromole±10 micromole, and has no effect on PAR4-AP, collagen and ADP induced platelet aggregation. In this study, we thought PHB1 could certainly be the target of antiplatelet therapy.. The aims of our project are to: (1) optimize, modify and screen highly active PHB1 inhibitor base on the peptide sequance of P5 by using structure bioinformatics. （2) anaylize the activity of the highly active inhibitor in human, guinea pig platelet activation mediated by PAR1 and the function in guniea pig thrombus formation induced by FeCl3. This research in this project will investigate and optimize the new inhibitor of PHB1 and aid new sight into therapeutic approach for anti-platelet therapy for a variety of diseases states. .

血小板是人体内具有多种功能的盘状无核细胞，在止血、血栓形成、动脉粥样硬化及各种心血管疾病等生理病理过程中发挥着重要作用。前期研究首次发现与小鼠不同，PHBs分布在人血小板膜上，是血小板PAR1信号通路的新的未知的调节因子，对人血小板激活具有重要的调节功能，因此PHBs可能会成为新的抗血小板治疗的有效靶点。在以血小板为研究对象，基于PHB1的结构，首次筛选出了一种与PHB1结合的小肽分子P5,并且P5能够专一的抑制PAR1-AP（PAR1 activating peptide）或低剂量凝血酶引起的血小板聚集，说明PHB1可以作为靶点调节血小板活性。在这个基础上，本项目将开展如下方面的主要研究内容：1)利用结构生物信息学，筛选、优化PHB1的抑制剂并通过抗血小板聚集实验进行筛选；2)对筛选出的高效PHB1抑制剂进行体外、体内活性检测试验，从而为抗血小板治疗及抗血小板药物的开发提供新的科学依据。

血小板是人体内具有多种功能的盘状无核细胞，在止血、血栓形成、伤口修复、动脉粥样硬化及各种心血管疾病等生理病理过程中发挥着重要的作用。蛋白酶激活受体1( protease activated receptor, PAR1)属于G蛋白偶联受体家族,在血小板上它主要作为凝血酶等蛋白酶的受体，其激活后会迅速启动血小板的聚集及凝血过程。. 前期研究发现人血小板膜上的抑素蛋白1（prohibitin 1，PHB1）参与调节了PAR1介导的血小板的聚集（Journal of Thrombosis and.Haemostasis，2012，10:411-418），提示PHB1 可以作为特异靶点来筛选抑制血小板聚集的有效抑制剂。因此，本项目拟解决的关键科学问题在于：以血小板膜 PHB1 为靶点，筛选一些新型的小分子血小板活化抑制剂，并研究其对血小板活化、血栓形成的影响。本项目开展了以下研究内容：1、结合生物信息学设计和体外血小板聚集实验，筛选出高效的PHB1小分子多肽类抑制剂；2、对获得的高效 PHB1 多肽抑制剂进行体外抑制血小板聚集活性检测；3、对以上获得的PHB1抑制肽进行体内抗血栓效果研究。. 围绕上述科学问题，本项目开展3年来已顺利完成了主要研究内容，并取得了一定的科研成果。首先借助于生物信息学的手段，我们成功筛选到一个特异性与PHB1结合的抑制小肽P5 (RHKWK)，对P5的体外生物活性研究表明：P5具有强大的抑制血小板聚集活性，深入细致的研究发现P5可以显著抑制PAR1激动肽引起的血小板的颗粒释放及钙释放等；有趣的是，在P5发挥功能的浓度下，未观察到其对红细胞、血小板及血管内皮细胞的毒性；体外生物分子相互作用检测发现P5与PHB1具有直接的相互作用，而将P5第五位Lys突变后其活性及与PHB1的相互作用都大幅减弱。最后我们在豚鼠的三氯化铁诱发的动脉血栓模型上进行了体内实验，发现P5可以有效地抑制三氯化铁引起的动脉血栓的形成。. 综上，我们成功的筛选到一个高活性的抑制血小板聚集的PHB1的抑制肽P5，为抗血小板药物的研发提供新的分子模板，为血栓类心血管疾病的预防及治疗提供新的线索和科学依据。

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