COLLAGEN RECEPTOR SIGNALING IN PLATELETS

在动脉粥样硬化斑块破裂和正常血管损伤期间，血小板暴露于胶原蛋白。暴露的胶原蛋白不仅作为直接的血小板激动剂，而且还提供了与血小板的粘连表面，从而促进血栓形成。血小板上的两种主要胶原受体是糖蛋白(GP)VI和α-2β-1整合素。GPVI和Alpha-2 Beta-1都是胶原介导的血小板黏附和激活所必需的，可能涉及GPVI介导的Alpha？2 Beta-1的激活，正如我们的实验室和其他实验室所证明的那样。然而，GPVI或其他激动剂受体激活Alpha-2 Beta-1的信号还不是很清楚。与此相关的是，小的GTP酶Rap1和R-RAS分别被认为是整合素激活的正调控因子，而RAS被认为是负调控因子。我们最近证实，血小板中的GPVI信号导致Rap1的激活依赖于P2Y12受体的分泌型ADP激活以及P2Y12非依赖的途径。我们还利用可转导的小鼠原代巨核细胞系统提供了初步证据，表明Rap1可能促进Alpha-2 β-1激活。在单独的研究中，我们发现R-RAS促进了几个α-2β-1介导的事件，并且RAS存在于血小板中，并被激动剂刺激激活。然而，Rap1与R-RAS或RAS的相互关系尚不清楚。在本提案中，我们的目标是通过以下步骤进一步定义GPVI和Alpha-2 Beta-1之间的通讯：首先，阐明Rap1和R-RAS在GPVI诱导的Alpha-2 Beta-1激活中的作用；其次，关于R-RAS和其他分子在这一事件中的潜在作用，定位导致Rap1激活的上游通路；第三，定位激活的Rap1下游导致Alpha-2 Beta-1整合素激活的信号通路；最后，确定RAS在调节Alpha-2 Beta-1整合素激活中的作用。这些研究的结果将提供关于这些重要的胶原受体如何通过血小板和巨核细胞中的这些小G蛋白相互通信的基本信息。

Platelets become exposed to collagen during rupture of the atherosclerotic plaque and during normal vascular injury. The exposed collagen serves not only as a direct platelet agonist but also provides an adhesive surface to platelets, thus contributing to thrombosis. Two of the major collagen receptors on platelets are glycoprotein (GP) VI and the Alpha-2 Beta-1 integrin. GPVI and Alpha-2 Beta-1 are both required for full collagen mediated platelet adhesion and activation, likely involving a GPVI-mediated activation of Alpha?2 Beta-1 as demonstrated by our lab and others. However, signals leading to Alpha-2 Beta-1 activation by GPVI or other agonist receptors are not well understood. Related to this, the small GTPases Rap1 and R-Ras have each been proposed as positive regulators of integrin activation whereas Ras has been proposed as a negative regulator. We recently demonstrated that GPVI signaling in platelets leads to Rap1 activation in a manner dependent upon secreted ADP activation of the P2Y12 receptor as well as a P2Y12-independent pathway. We also provide preliminary evidence using a transducible primary mouse megakaryocyte system, that Rap1 may promote Alpha-2 Beta-1 activation. In separate studies we found that R-Ras promotes several Alpha-2 Beta-1 mediated events, and that Ras is present in platelets and activated by agonist stimulation. However, the interrelationship of Rap1 to R-Ras or Ras is not understood. In the present proposal, we aim to further define the communication between GPVI and Alpha-2 Beta-1 by first, clarifying the roles of Rap1 and R-Ras in GPVI-induced Alpha-2 Beta-1 activation, second, mapping upstream pathways leading to Rap1 activation with regard to the potential role of R-Ras and other molecules in this event, third, mapping signaling pathways downstream of activated Rap1 leading to Alpha-2 Beta-1 integrin activation, and finally, determining the role of Ras in regulating Alpha-2 Beta-1 integrin activation. Results from these studies will provide fundamental information on how these important collagen receptors communicate with one another via these small G-proteins in platelets and megakaryocytes.