The Platelet Secretory Machinery

描述(由申请人提供)：激活的血小板分泌被认为是止血和伤口愈合的血栓后后遗症的关键步骤。控制血小板胞吐为控制血管损伤部位形成的微环境提供了独特的治疗机会。然而，我们对促进血小板颗粒释放的机制的了解仍然不足以确定有价值的药物靶点。我们提出的研究试图通过描绘血小板分泌所需的分子事件来纠正这一点。根据以往的报道，很明显，血小板分泌需要特定的可溶性NSF附着蛋白受体(v-和t-SNARE)。随着这一点的确立，我们的目标现在扩大到定义分泌物在止血中的作用，并确定胞吐机制被调节的机制。我们提出了两个具体的目标：1)评估v-SNARE VAMP-8在血小板功能中的作用。我们的工作证明VAMP-8/endobrevin是血小板胞吐所必需的。目的1将通过特定的实验来确定VAMP-8-/-血小板的体内和体外表型，从而完成对这种v-SNARE作用的表征。其他实验将解决VAMP-8的亚血小板分布问题，并描述其相互作用的伙伴。2)鉴定和鉴定与血小板中的句法蛋白t-陷阱相互作用和控制的圈套调节因子。过去的研究指定SNAP-23、Synaxin 2和4的角色。这一目的集中在定义已知t-SNARES调节因子的角色和确定新的角色。合成素调节因子Munc18、Munc13、DOC2和Tomosyn将使用生化技术和渗透性血小板分泌试验进行研究。这些实验将扩大我们对这些蛋白质如何介导SNAR配对和随后的膜融合的理解。总而言之，从这项提议中获得的信息将确定控制血小板过度活跃的新的治疗靶点，并将提供诊断试剂来确定为什么血小板活性低下。与公共健康相关：中风和心脏病发作是美国的主要杀手。在这两种情况下，损伤都是由于血流丧失造成的，通常是因为血液凝块在错误的位置形成。了解血栓形成的方方面面是治疗这个问题的关键。这项应用侧重于血栓形成的一个方面，即血小板。具体地说，这项应用关注的是血小板如何分泌凝块形成所需的成分。这项工作至关重要，因为它将指导未来减少假性凝块形成风险所需的治疗剂的开发。

DESCRIPTION (provided by applicant): Secretion from activated platelets is thought to be a key step in hemostasis as well as in the post-thrombus sequellae of wound healing. Controlling platelet exocytosis offers a unique therapeutic opportunity to manipulate the microenvironment formed at the site of vascular damage. However, our understanding of the machinery that facilitates platelet granule release is still not sufficient to identify valuable drug targets. Our proposed studies seek to rectify this by delineating the molecular events required for platelet secretion. From past reports, it is clear that specific Soluble NSF Attachment Protein Receptors (v- and t-SNAREs) are required for platelet secretion. With this established, our goals are now expanded to define the roles of secretion in hemostasis and to identify the mechanisms by which the exocytosis machinery is regulated. Two Specific Aims are proposed: 1) To assess the role of the v-SNARE, VAMP-8, in platelet function. Our work demonstrated that VAMP-8/endobrevin is required for platelet exocytosis. Aim 1 will complete the characterization of this v-SNARE's role using specific experiments to define the in vivo and in vitro phenotypes of VAMP-8-/- platelets. Other experiments will address the sub-platelet distribution of VAMP-8 and characterize its interacting partners. 2) To identify and characterize the SNARE regulators that interact with and control the syntaxin t-SNAREs in platelets. Past studies assigned roles for SNAP-23, syntaxin 2, and 4. This aim focuses on defining roles for known t-SNAREs regulators and identifying novel ones. The syntaxin-regulators Munc18, Munc13, DOC2, and tomosyn will be studied using biochemical techniques and a permeabilized platelet secretion assay. The experiments will expand our understanding of how these proteins mediate SNARE pairing and subsequent membrane fusion. In summary, the information gained from this proposal will identify new therapeutic targets to control hyperactive platelets and will provide diagnostic reagents to determine why hypoactive platelets are defective. PUBLIC HEALTH RELEVANCE: Strokes and heart attacks are major killers in the United States. In both cases, the damage is caused by a loss of blood flow, usually because a blood clot has formed in the wrong place. Understanding all aspects of clot formation is critical to treating this problem. This application focuses on one aspect of clot formation, platelets. Specifically this application focuses on how platelets secrete components needed for clot formation. This work is of critical important because it will direct the development of future therapeutic agents needed to decrease the risk of spurious clot formation.