MAPK信号通路新调控分子MINK对血小板活化血栓形成的功能影响和调控机制研究

The role of Ste20 kinase MINK in hemostasis and thrombosis remains unclear. With MINK-deficient (MINK(-/-)) mice, we have shown that MINK plays an important role in platelet biology and thrombus formation. In tail-bleeding assays, MINK(-/-) mice exhibited longer bleeding times than wild-type mice (589.0 +/- 68.1 seconds vs 406.6 +/-70.5 seconds). In an in vivo model of thrombosis induced by photochemical injury to cecum vessels, occlusion times were 2 times longer in MINK(-/-) arterioles than in wild-type arterioles. In an in vitro whole-blood perfusion assays on a collagen matrix under arterial shear conditions, thrombus formation was significantly reduced for MINK(-/-) platelets. Moreover, in vitro platelet aggregation studies demonstrated that, at low doses of agonists (thrombin, collagen, TXA2 analogue U46619), platelet secretion was impaired in MINK(-/-) platelets, leading to altered integrin alphaIIbbeta3 activation and reduced platelet aggregation. Furthermore, platelet spreading on fibrinogen was largely hampered in MINK(-/-) platelets, the difference could also be attributed to impaired ADP secretion upon MINK deficiency. The current project will focus on the signal transduction pathway of MINK molecule, trying to strengthen the undstanding of molecular regulation of platelet activation, especially the granule secretion, and provide basis for developing novel therapeutic targets of anti-thrombotic diseases.

Ste20激酶家族成员MINK是本实验室最近发现的在生理性止血和病理性血栓形成中起重要作用的新信号分子。在MINK敲除（MINK-/-）小鼠中，我们已发现正常止血时间和诱导肠系膜动脉血栓形成的时间均显著长于野生型小鼠。提取血小板进行的体外血小板功能实验中，由低浓度凝血酶引起的血小板聚集在MINK-/-小鼠血小板中受到显著抑制，与ADP释放相关并且伴随有明显的α颗粒释放的障碍。胶原表面的高剪切应力灌流实验也发现了显著的血小板粘附的降低。Apyrase水解ADP后MINK-/-血小板和野生型血小板铺展的差异基本消失，提示了MINK可能是通过调控血小板颗粒释放参与血小板活化和动脉血栓的重要分子。本课题拟利用分子生物学手段对血小板MINK介导的信号通进行阐明，以期加深对血小板活化尤其对颗粒释放调控的分子机理的理解并为寻找血栓性疾病的新干预靶点打下基础。

通过本课题的实施，完成了预定的研究目标。首先，获得了MINK敲除小鼠，以此为研究工具，检测了体内血栓，体外刺激剂作用下的血小板聚集和分泌等功能，发现MINK特异调控小鼠血小板致密颗粒的释放。MINK不影响整合素αIIbβ3的inside-out和outside-in信号本身，也不影响α颗粒的释放。血小板在应对刺激时，致密颗粒内ADP释放在MINK1缺失时显著降低，致使血小板在应对低浓度凝血酶和胶原刺激时聚集功能下降，以及在纤维蛋白原表面的铺展功能受损。除此之外，MINK1调控血小板中致密颗粒释放的信号转导通过ERK，p38以及Akt的活化而进行。因此，该研究深入阐释了MINK在血小板中的功能和发挥作用的分子机制，加深了对血栓性疾病的分子病理机制的理解，为寻找新的有效干预靶点提供了理论依据和支持。