Sterile-20激酶MINK1调控血小板功能并参与止血和血栓形成的机制研究

发布时间：2018-01-11 06:28

本文关键词：Sterile-20激酶MINK1调控血小板功能并参与止血和血栓形成的机制研究　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：血小板在生理止血和血栓形成中扮演了重要角色,血小板的功能不足会导致出血,而血小板过度活化则会引起血栓性疾病。因此对血小板活化分子机理的深入研究,能够阐明出血或血栓性疾病的发病机制,并为该类疾病寻求新的治疗靶点和策略。在血小板活化过程中,丝裂原活化蛋白激酶(MAPK)作为重要的信号通路节点分子,参与调控血小板粘附、释放、聚集等各项功能,以促进血栓的形成。经典的MAPK通路是包含三个激酶的级联信号反应,MAP3K磷酸化激活MAP2K,MAP2K再磷酸化激活MAPK。然而,在血小板信号网络中对MAPK通路具体的细致调控还不清楚。以往研究表明,Sterile-20激酶在信号通路中能够扮演MAP4K的角色来调控MAPK通路的活化。Sterile-20激酶超家族根据结构可分为两大家族:p21活化激酶(PAK)家族和生发中心激酶(GCK)家族。MINK1属于GCK Ⅳ亚家族,在2000年被首次报道,在多种细胞活动过程中发挥重要作用,并能够作为MAP4K参与调控Ras致癌基因诱导的卵巢上皮细胞生长抑制以及T细胞发育过程中的阴性选择。MINK1在止血与血栓领域的作用在此之前未见报道,鉴于MAPK通路在血小板活化以及血栓形成中的作用,我们提出假设:MINK1能够通过调控MAPK来介导血小板活化,并参与止血和血栓形成过程。本研究利用转基因小鼠模型,首次研究了MINK1在血小板功能,止血以及血栓形成中的作用。结果可以概括为以下几点:(1)在小鼠出血实验中,MINK1敲除小鼠尾部出血时间明显延长,说明MINK1的缺失使得小鼠止血功能受损。(2)在三氯化铁诱导的肠系膜动脉血栓模型中,MINK1敲除小鼠的动脉堵塞时间显著延长,两倍于野生型小鼠。我们通过体外全血微灌流实验模拟血栓形成,发现动脉流速条件下,MINK1缺失血小板在胶原表面形成的血栓面积明显减小,证明MINK1对血栓形成的正向促进作用依赖于其在血小板中发挥的作用。(3)在对血小板各项功能进行检测后发现,MINK1的缺失不影响血小板在胶原表面的粘附,然而当用低浓度胶原和凝血酶刺激血小板发生聚集时,MINK1的缺失使得血小板的聚集水平显著降低,并且伴随有致密颗粒的释放障碍,但是血小板α颗粒的释放以及血栓烷A2的合成并不受MINK1缺失的影响。另外,MINK1缺失血小板在纤维蛋白原表面的铺展面积较野生型血小板明显减小,但是其血块回缩功能则表现正常。(4)当继发释放的ADP被apyrase水解后,野生型血小板与MINK1缺失血小板在聚集和铺展功能上的差异基本消失,并且,外源性补充ADP能够使MINK1缺失血小板的聚集和铺展功能恢复到正常水平。因此,MINK1的缺失导致的ADP分泌减少是血小板聚集和铺展功能发生障碍的原因。(5)在分子机制研究方面,我们发现MINK1的缺失不影响整合素αⅡbβ3的由内向外和由外向内信号本身,而在低浓度胶原和凝血酶所诱导的聚集反应中,MINK1的缺失使得ERK,p38和Akt的磷酸化水平显著降低,且MINK1对上述分子的调控不依赖于继发释放的ADP以及整合素αⅡbβ3介导的由外向内信号。进一步研究发现,MINK1也能够调控ERK和p38上游的MAP2K分子——MEK1/2和MKK3/6的活化。在用ERK抑制剂U0126或p38抑制剂SB203580孵育血小板后发现,它们能够消除野生型血小板与MINK1缺失血小板在应对低浓度胶原和凝血酶刺激时聚集和致密颗粒释放上的差异,说明MINK1缺失导致的ERK和p38磷酸化水平的降低,是致密颗粒释放减少以及聚集受损的原因。综上所述,MINK1是参与血小板活化以及止血和血栓形成的新信号分子,其能够通过介导MAPK通路以及Akt的活化,来特异调控致密颗粒的释放。
[Abstract]:Platelet formation plays an important role in hemostasis and thrombosis, platelet function deficiency can lead to bleeding, and excessive platelet activation can cause thrombotic diseases. Therefore further study the molecular mechanism of the activation of platelets, to elucidate the pathogenesis of hemorrhagic or thrombotic disease, and the disease for therapeutic targets and new strategies in the process of platelet activation, mitogen activated protein kinase (MAPK) signaling node as important molecules involved in platelet adhesion, regulation, release, aggregation and other functions, to promote the formation of thrombus. MAPK pathway is the classic cascade reaction consists of three protein kinase MAP3K phosphorylation, MAP2K phosphorylation and activation of MAP2K. The activation of MAPK. however, detailed regulation in platelet signaling network on MAPK pathway specific is not clear. Previous studies showed that Sterile-20 kinase in the signaling pathway can Enough to play the role of MAP4K to regulate MAPK pathway activation of.Sterile-20 kinase superfamily according to the structure can be divided into two families: p21 activated kinase (PAK) family and germinal center kinase (GCK) family.MINK1 belongs to the GCK subfamily IV, was first reported in 2000, in a variety of cytokines play an important role in the process of moving. It can be used as MAP4K is involved in the regulation of Ras gene induced by ovarian epithelial cells and negative growth inhibition of T cell development in the process of selecting.MINK1 reported before hemostasis and thrombosis role in this field, in view of the activation of MAPK pathway and the role in the formation of a thrombus in the blood plate, we hypothesized that MINK1 can regulate MAPK mediated guide and participate in the formation process of platelet activation, hemostasis and thrombosis. This study used transgenic mouse model for the first time on the MINK1 in platelet function and hemostasis and thrombosis in the Use. The results can be summarized as follows: (1) hemorrhage in mice experiment, MINK1 knockout mouse tail bleeding time was prolonged, MINK1 explained that the lack of hemostatic function in mice injured. (2) in the mesenteric artery thrombosis model induced by FeCl3, MINK1 knockout mice blocked arteries significantly longer. Two times in wild-type mice by in vitro blood perfusion. We found experimental thrombosis, arterial flow conditions, the lack of MINK1 platelet formation on the surface of collagen thrombus area significantly reduced, prove the positive effect of MINK1 on thrombus formation depends on the play in the role of platelet. (3) found in the detection of the platelet function, MINK1 deletion does not affect platelet adhesion on the surface of collagen, however, when using low concentration of collagen and thrombin stimulated platelet aggregation, MINK1 that the lack of blood In the aggregation level decreased significantly, and with the release disorder of dense particles, but the release of platelet alpha granule and synthesis of thromboxane A2 was not affected by the deletion of MINK1. In addition, deletion of MINK1 decreased significantly in the spreading area of platelet fibrinogen surface than the wild-type platelets, but the clot retraction function is normal. (4) when the secondary release of ADP was apyrase after hydrolysis, differences in aggregation and spreading on the function of the wild-type platelets and MINK1 deletion platelet disappeared, and exogenous ADP can make MINK1 lack of platelet aggregation and spreading function recovered to the normal level. Therefore, the reason is to reduce platelet aggregation and spreading the functional disorder of MINK1 due to the lack of secretion of ADP. (5) in the study of the molecular mechanism, we found that loss of MINK1 does not affect integrin alpha B beta 3 II from inside to outside and the In the signal itself, and the aggregation induced by collagen and thrombin in low concentration of MINK1, that the lack of ERK, p38 and Akt phosphorylation level were significantly decreased, and the molecular regulation of MINK1 does not depend on the secondary release of ADP and integrin alpha B beta 3 II mediated by inward signal further. The study found that MINK1 can activate the MAP2K molecular regulation of ERK and p38 upstream of MEK1/2 and MKK3/6. Found in ERK inhibitor U0126 or p38 inhibitor SB203580 after incubation of platelets, they can eliminate the wild-type platelets and MINK1 deletion in response to low concentrations of platelet collagen and thrombin stimulated aggregation and dense granule release the difference MINK1, due to the lack of ERK and the decrease of p38 phosphorylation, is dense granule release is reduced and the cause of aggregation damaged. In summary, MINK1 is involved in platelet activation and bleeding and The new signalling molecules of thrombosis can regulate the release of dense particles specifically by mediating the MAPK pathway and the activation of Akt.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R54

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