The Platelet Secretory Machinery

描述（由申请人提供）：活化血小板的分泌被认为是止血和伤口愈合后血栓后遗症的关键步骤。控制血小板胞吐提供了一个独特的治疗机会来操纵在血管损伤部位形成的微环境。然而，我们对促进血小板颗粒释放的机制的理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述血小板分泌所需的分子事件来纠正这一点。从过去的报道来看，血小板分泌需要特异性可溶性NSF附着蛋白受体（v-和t-SNAREs）。有了这一点，我们的目标现在扩大到定义在止血中分泌的作用，并确定胞吐机制的调节机制。提出了两个具体目的：1)评估v-SNARE， VAMP-8在血小板功能中的作用。我们的研究表明，VAMP-8/内啡肽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v-SNARE作用的表征。其他实验将解决VAMP-8的亚血小板分布，并表征其相互作用的伙伴。2)鉴定和表征血小板中与syntaxin t-SNAREs相互作用和控制的SNARE调节因子。过去的研究确定了SNAP-23、syntaxin 2和syntaxin 4的作用。该目标侧重于定义已知t-SNAREs调节因子的作用并识别新的调节因子。syntaxin调节剂Munc18， Munc13， DOC2和tomosyn将使用生化技术和渗透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SNARE配对和随后的膜融合的理解。总之，从该建议中获得的信息将确定新的治疗靶点来控制血小板过度活跃，并将提供诊断试剂来确定血小板过度活跃的原因。公共卫生相关性：中风和心脏病是美国的主要杀手。在这两种情况下，损伤都是由血流减少引起的，通常是因为血凝块在错误的地方形成。了解血栓形成的各个方面是治疗这个问题的关键。这个应用集中在凝块形成的一个方面，血小板。具体来说，这个应用侧重于血小板如何分泌凝块形成所需的成分。这项工作至关重要，因为它将指导未来治疗药物的发展，以降低假性血块形成的风险。

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.