SNARE PROTEINS IN PLATELET ALPHA-GRANULE SECRETION

富含血小板的动脉血栓介导心肌梗塞、中风和外周血管疾病中的组织梗塞，因此是美国发病和死亡的最常见原因。 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，即α颗粒，含有粘附分子、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。 这些观察结果表明，血小板α颗粒分泌可能是干扰壁式血小板血栓形成扩散的有用目标。 然而，人们对指导α颗粒分泌的分子机制知之甚少。 相比之下，对有核细胞囊泡分泌的分子研究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。 我们开发了一种新型的 α 颗粒分泌透化血小板模型，以证明 SNARE 蛋白介导 α 颗粒分泌。使用这个模型，我们将定义 SNARE 蛋白在 α 颗粒分泌中的作用。 本提案具体目标 1 中描述的实验将使用亚细胞分级分离和随后的免疫沉淀来纯化 α 颗粒和表面连接膜，从而确定 SNARE 蛋白在血小板内的亚细胞定位。 然后通过流式细胞术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测定。 具体目标 2 中描述的研究基于以下初步发现：ATP-gamma-S（NEM 敏感融合蛋白的抑制剂）可刺激透化血小板中的 α 颗粒分泌。 NEM敏感融合蛋白在血小板分泌中的作用将在α颗粒分泌的透化模型中使用NSF的其他特异性抑制剂来确定。 该透化血小板模型还将用于特定目标 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 颗粒分泌。 具体目标 4 中描述的实验将使用蛋白质分级分离来纯化在透化血小板中重建 α 颗粒分泌的蛋白质。这些研究具有根本重要性，因为它们将定义α颗粒分泌的分子基础，这种现象以前仅在形态学上被描述过。

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.