Regulation of Platelet Shape Changes

描述（由申请人提供）：血小板的形状变化对于防止出血的损伤控制功能至关重要，并且依赖于肌动蛋白丝的重塑和组装，以响应结扎受体产生的信号。血小板表面形状变化差异的细节可能对体内伴随止血和血栓反应的血小板分层有重要影响，并可能为未来的抗血栓治疗提供靶点。我们之前定义了以净肌动蛋白组装/拆卸结束的某些血小板信号级联，并关注了钙、gtp酶和磷酸肌苷产生在形状变化调控中的核心作用以及这些药物的下游靶点。我们还努力将肌动蛋白组装反应置于一个结构框架中，首先定义了肌动蛋白的组织和静息血小板的膜骨架，然后剖析了这个结构中的变化，这些变化是启动和维持形状变化反应所必需的。该项目的总体目标是了解血小板如何利用大片层和丝状足扩散，并将这些信息整合到介导血小板形状变化的信号通路和蛋白质中。这项建议有三个具体目标。首先，我们强调了rac， ralA和钙在板足组装中的核心作用。我们采用了一种新的结构方法，将选择性渗透细胞分散在确定的表面上，我们可以引入外源分子。在第二个目标中，我们关注cdc42在血小板丝状结构细化中的作用。第三个目标详细介绍了基于珠状蛋白的肌动蛋白组装系统，依赖于GTPase活性，我们将使用它开始重建片层和丝状肌动蛋白组装，目标是生化简化，然后重建基本成分。

DESCRIPTION (provided by applicant): Shape change is essential for the damage control functions of blood platelets that prevent bleeding and is dependent on the remodeling and assembly of actin filaments in response to signals generated by ligated receptors. Details in differences of platelet surface shape changes may have important ramifications for the layering of platelets accompanying hemostatic and thrombotic reactions in vivo and may provide targets for future antithrombotic therapies. We previously defined certain platelet signaling cascades that end in net actin assembly/disassembly and have focused on the central roles of calcium, GTPases, and phosphoinositide production in the regulation of shape change and on the downstream targets of these agents. We have also labored to put the actin assembly reactions within a structural framework by first having defined the organization of actin and the membrane skeleton of the resting platelet and then dissecting the changes in this architecture that are required to initiate and sustain the shape change reaction. The overall goal of this project is to understand how platelets spread using large lamellae and filopods and integrate this information into the signaling pathways and proteins that mediate platelet shape change. This proposal has three specific aims. In the first, we highlight the central roles of rac, ralA and calcium in lamellapodial assembly. We take a novel structural approach that combines selectively permeabilized cells spread on defined surfaces to which we can introduce exogenous molecules. In the second aim, we focus on the role of cdc42 in platelet filopodial elaboration. The third aim details a bead-based actin assembly system, dependent on GTPase activity, which we will use to begin to reconstruct lamellar and filopodial actin assembly with the goal of biochemical simplification and then reconstitution of the essential components.