REGULATION OF PLATELET SHAPE CHANGES

描述（申请人逐字描述）：血小板形状变化是 对封闭受损血管表面至关重要。血小板差异详情 表面形状的变化可能会有重要的分歧分层 血小板伴随体内止血和血栓形成反应，并且可能 为未来的抗血栓治疗提供了靶点。血小板循环 血液作为小圆盘，响应于栓系和可溶性激动剂， 变成活跃的形式，迅速生长丝状和片状的，从他们的 表面。突起生长依赖于肌动蛋白组装和空间结构。 引发长丝组装和信号传导的成核位点的位置 导致这些位点激活的途径。我们已经证明， 血小板形状改变的信号通路从PAR-1通过 快速转化成GTP形式的小GTP酶cdc 42和rac PAR-1连接后。GTP酶的下游是磷酸肌醇（ppl） 将大量合成PPL的激酶转化为加帽蛋白， 有倒钩的细丝末端。倒钩末端加帽蛋白对于 在凝溶胶蛋白缺乏的小鼠中已经证实血小板形状改变 血小板活性差。然而，尽管血小板扩散不良。小鼠 只有轻微的出血缺陷。最近，PPL和小GTP酶已被 连接到Arp 2/3复合物，其可以形成一些成核位点， 血小板肌动蛋白装配。 该提案将定义导致Arp 2/3激活的途径， 血小板，并确定Arp 2/3通路是否以及如何与丝状伪足连接 和/或由PAR-1受体发出信号的层状肌动蛋白生长。互连 之间的小GTP酶将进行研究，并与丝脱帽或脱 新生成核和形成的肌动蛋白结构的类型。在目标1中，我们 建立小GTP酶rac之间的链接。cdc42. ralA和ARF 6在 血小板，确定上游活化因子（GEFS）和下游靶点 外消旋体和P15-KI-导致倒刺末端暴露，并确定它们如何 调节加帽蛋白和Arp 2/3。在目标2中。我们将探讨 G蛋白偶联受体中Arp 2/3成核系统和细丝蛋白 与IgG-抗体和整联蛋白受体（vWFR，GPVI. Fc RIIa。 GPIIb/IIIa）。我们将确定是否每一个途径产生一个独特的肌动蛋白 细胞骨架结构以及Arp 2/3和细丝蛋白如何参与这些 结构.在最后的目标，我们将识别，纯化和表征因素 激活cdc 42下游的血小板Arp 2/3。

DESCRIPTION (Applicant's Description Verbatim): Platelet shape change is essential to seal damaged vascular surfaces. 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. Platelets circulate in blood as small discs that in response to tethered and soluble agonists convert into active forms which rapidly grow filopods and lamellipods from their surfaces. Process growth is dependent on actin assembly and on the spatial location of nucleating sites that initiate filament assembly and signaling pathways that lead to activation of these sites. We have shown that the signaling pathway for platelet shape change connects from PAR-1 through the small GTPases cdc42 and rac which are rapidly converted into their GTP forms following PAR-1 ligation. Dowstream of GTPases are phosphoinositide (ppl) kinases that synthesize ppls in bulk to inactivate capping proteins and expose barbed filament ends. The importance of barbed end capping proteins for platelet shape change has been demonstrated in gelsolin deficient mouse platelets that activate poorly. However, despite poor platelet spreading. mice show only minor bleeding defects. Recently, ppls and small GTPases have been linked to the Arp2/3 complex which may form some of the nucleation sites for platelet actin assembly. The proposal will define the pathway(s) leading to Arp2/3 activation in platelets and determine if, and how the Arp2/3 pathway links to filopodial and/or lamellar actin growth signaled by the PAR-1 receptor. Interconnections between small GTPases will be studied and related to filament uncapping or de novo nucleation and to the types of actin structure formed. In Aim 1, we will establish the links between the small GTPases rac. cdc42. ralA and ARF6 in platelets, identify upstream activating factors (GEFS) and downstream targets of rac and P15-K I - that lead to barbed end exposure and determine how they regulate capping proteins and Arp2/3. In Aim 2. we will explore the roles of the Arp2/3 nucleation system and filamin in G-protein coupled receptors compared to IgG-farnily and integrin receptors (vWFR, GPVI. Fc RIIa. GPIlb/IIIa). We will determine whether each pathway generates a unique actin cytoskeletal architecture and how Arp2/3 and filamin are involved in these structures. In the last aim, we will identify, purify and characterize factors that activate platelet Arp2/3 downstream of cdc42.