SNARE PROTEINS IN PLATELET ALPHA-GRANULE SECRETION

富含血小板的动脉血栓在心肌梗死、中风和外周血管疾病中介导组织梗塞，因此是美国最常见的致病和死亡原因。在血栓形成过程中，血小板分泌其颗粒内容物。最丰富的血小板颗粒，阿尔法颗粒，含有黏附分子、凝血因子和血管活性因子，被认为有助于血栓形成和血小板募集。这些观察表明，血小板α颗粒分泌可能是干扰壁性血小板血栓形成传播的有用靶点。然而，对引导α颗粒分泌的分子机制知之甚少。相反，对有核细胞囊泡分泌的分子研究导致发现了一个蛋白质超家族，称为SNARE蛋白，它介导有核细胞中囊泡和质膜之间的膜融合。我们已经建立了一个新的α颗粒分泌的通透性血小板模型，以证明SNARE蛋白介导α颗粒分泌。利用这个模型，我们将确定SNARE蛋白在α颗粒分泌中的作用。在本提案的特定目标1中描述的实验将使用亚细胞分离和免疫沉淀来确定SNARE蛋白在血小板中的亚细胞定位，以纯化α颗粒和表面连接的膜。然后通过流式细胞仪和免疫印迹分析这些膜的SNARE蛋白含量。亚细胞定位也将通过免疫荧光光学显微镜和免疫金电子显微镜来确定。具体目标2中描述的研究是基于初步发现，三磷酸腺苷-伽马-S是一种NEM敏感的融合蛋白的抑制剂，可以刺激通透性血小板分泌α颗粒。NEM敏感的融合蛋白在血小板分泌中的作用将在α颗粒分泌的渗透性模型中使用NSF的其他特异性抑制剂来确定。这个通透性的血小板模型也将用于特定目的3中描述的实验，以确定Rab GTP酶和磷脂酰肌醇(4，5)-二磷酸是否介导α-颗粒的分泌。在特定目标4中描述的实验将使用蛋白质分级来纯化在通透性的血小板中重建α颗粒分泌的蛋白质。这些研究具有根本性的重要性，因为它们将确定阿尔法颗粒分泌的分子基础，这一现象以前仅从形态上描述。

Platelet-rich, arterial thrombi mediate tissue infarction in myocardial infarction, stroke, and peripheral vascular disease and, thus, represent the most common cause of morbity and mortality in the United States. During thrombus formation, platelets secrete their granule contents. The most abundant platelet granule, the alpha-granule, contains adhesion molecules, coagulation factors, and vasoactive factors thought to contribute to thrombus formation and platelet recruitment. Such observations suggest that platelet alpha-granule secretion may be a useful target in interfering with propagation of mural platelet thrombosis. However, little is known about the molecular mechanisms that direct alpha-granule secretion. In contrast, molecular studies of vesicle secretion from nucleated cells has lead to the discovery of a superfamily of proteins, termed SNARE proteins, that mediate membrane fusion between vesicle and plasma membrane in nucleated cells. We have developed a novel permeabilized platelet model of alpha-granule secretion to demonstrate that SNARE proteins mediate alpha-granule secretion. Using this model, we will define the role of SNARE proteins in alpha-granule secretion. Experiments described in the Specific Aim 1 of this proposal will determine the subcellular localization of SNARE proteins within the platelet using subcellular fractionation followed by immunoprecipitation to purify alpha-granules and surface-connected membranes. These membranes will then be analyzed for SNARE protein content by flow cytometry and immunoblotting. The subcellular localization will also be determined by immunofluorescence light microscopy and immunogold electron microscopy. Studies described in the Specific Aim 2 are based on the preliminary findings that ATP-gamma-S, an inhibitor of NEM-sensitive fusion protein, stimulates alpha-granule secretion in permeabilized platelets. The role of NEM-sensitive fusion protein in platelet secretion will be determined using other specific inhibitors of NSF in the permeabilized model of alpha-granule secretion. This permeabilized platelet model will also be used in experiments described in Specific Aim 3 to determine whether Rab GTPases and phosphatidylinositol (4,5)-bisphosphate mediate alpha-granule secretion. Experiments described in Specific Aim 4 will use protein fractionation to purify proteins that reconstitute alpha- granule secretion in permeabilized platelets. These studies are of fundamental importance as they will define the molecular basis of alpha-granule secretion, a phenomenon that has previously been described only morphologically.