SNARE PROTEINS IN PLATELET ALPHA-GRANULE SECRETION

血小板α颗粒分泌中的诱捕蛋白

• 批准号: 6390473
• 负责人: Robert C Flaumenhaft
• 金额: $ 26.1万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6390473
• 关键词: adenosine triphosphate; calcium; cell membrane; chimeric proteins; cytoplasm; electron microscopy; enzyme inhibitors; flow cytometry; guanine nucleotide binding protein; guanosinetriphosphatases; human tissue; immunofluorescence technique; immunoprecipitation; intracellular transport; light microscopy; membrane proteins; phosphatidylinositols; platelets; protein purification; protein structure function; secretion; tissue /cell culture; vesicle /vacuole; western blottings

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.

富含血小板的动脉血栓介导心肌梗死、中风和外周血管疾病中的组织梗死，因此代表了美国发病率和死亡率的最常见原因。 在血栓形成期间，血小板分泌其颗粒内容物。 最丰富的血小板颗粒，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-颗粒分泌可能是一个有用的目标，在干扰附壁血小板血栓形成的传播。 然而，很少有人知道的分子机制，直接α-颗粒分泌。 相比之下，从有核细胞的囊泡分泌的分子研究已经导致发现蛋白质的超家族，称为SNARE蛋白，其介导有核细胞中囊泡和质膜之间的膜融合。 我们开发了一种新的透化血小板α颗粒分泌模型，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。 本提案的具体目标1中描述的实验将使用亚细胞分级分离，然后通过免疫沉淀纯化α颗粒和表面连接膜，确定血小板内SNARE蛋白的亚细胞定位。 然后通过流式细胞术和免疫印迹法分析这些膜的SNARE蛋白含量。 还将通过免疫荧光光学显微镜和免疫金电子显微镜确定亚细胞定位。 具体目标2中描述的研究基于初步发现，即ATP-γ-S（一种NEM敏感性融合蛋白的抑制剂）刺激透化血小板中的α颗粒分泌。NEM敏感性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。 该透化血小板模型也将用于具体目标3中所述的实验，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构α颗粒分泌的蛋白质。这些研究具有根本的重要性，因为它们将定义α颗粒分泌的分子基础，这是一种以前仅在形态学上描述的现象。