Elucidation of the physiological roles of DOCK180 family proteins in platelets and megakaryocytes.

血小板在正常止血和病理性血栓形成(如心肌梗死)中起关键作用。它们的功能受复杂的信号转导通路的调节，而这些信号转导通路又是抗血小板药物的靶点。因此，对血小板内信号转导的研究对临床医学具有重要意义。Rac是一种小分子G蛋白，对多种血小板功能是必不可少的。与GTP绑定的RAC在功能上处于活动状态，而与GDP绑定的RAC处于非活动状态。RAC-鸟嘌呤核苷酸交换因子(RAC-Global)负责激活RAC。令人惊讶的是，人们对RAC-Global在血小板中的作用知之甚少。在这项研究中，我们发现，除了先前发现的Vav1外，血小板还表达两种RAC-GEF，DOCK180和DOCK5。DOCK180是DOCK180家族全环蛋白的原型，也是一个真正的RAC-全环基金。Dock5与DOCK180密切相关，但一直没有得到很好的描述。利用特异性抗体，我们发现人类DOCK5有两种不同的亚型。一种是在血小板和Hela细胞中表达。另一种是在A549细胞中表达，而在血小板中不表达。当DOCK5在COS7细胞中强制表达时，DOCK5确实是一种特异的RAC-Global。免疫荧光研究显示DOCK5在铺展的血小板胞浆中呈点状分布。已知DOCK180可与几种蛋白质结合，如Crk和ELMO1。我们发现DOCK5也是如此。因此，我为进一步研究DOCK180家族RAC-gef在血小板和巨核细胞中的作用奠定了基础，因为血小板蛋白大多是从前体细胞携带过来的。

Platelets play a critical role in normal hemostasis and pathological thrombosis like myocardial infarction. Their functions are regulated by complex signal transduction pathways, which, in turn, are targets of anti-platelet drugs. Thus, the studies on signal transduction in platelets are important for clinical medicine. Rac, a small molecular G-protein, is essential for several platelet functions. GTP-bound rac is functionally active, while GDP-bound one is inactive. Rac-Guanine nucleotide exchange factor (rac-GEF) is responsible for activation of rac. Surprisingly, little is known of rac-GEF in platelets. In this study, we found that platelets express two rac-GEFs, DOCK180 and DOCK5, in addition to previously identified Vav1. DOCK180 is a well characterized prototype of DOCK180 family GEF proteins and a bona fide rac-GEF. DOCK5 is closely related to DOCK180, but has been poorly characterized. Using specific antibodies, we found that there are two different isoforms of human DOCK5. One is expressed in platelets and Hela cells. The other is expressed in A549 cells and not in platelets. When forcedly expressed, in COS7 cells, DOCK5 is indeed a specific rac-GEF. Immunofluorescent studies revealed dot-like distribution of DOCK5 in the cytoplasm of spreading platelets. DOCK180 is known to bind to several proteins like crk and ELMO1. We found that the same is true for DOCK5. Thus, I have set the basis for further studies for DOCK180 family rac-GEF in platelets and megakaryocytes, since platelets proteins are mostly carried over from the precursor cells.