Targeting Platelet Endocytosis and Exocytosis to Control Thrombosis

在世界范围内，每4例非传染性疾病死亡中就有1例是假性血栓形成。 心血管疾病是美国退伍军人老龄化的头号杀手，也是老年人死亡的主要原因 女退伍军人。了解如何调节血栓形成将大大有助于退伍军人管理局的使命 改善退伍军人健康状况。正常情况下，血小板对血管损伤作出反应，并分泌颗粒货物 这对于招募更多的血小板和形成血栓是必不可少的。这个版本促进了 这是正常的后遗症，但也可能导致中风和心脏病发作等闭塞性疾病。在……里面 血小板、VAMP、SNAP-23和Synaxin-11形成跨膜复合体，介导 胞吐。这种复合体的形成需要许多陷阱调节因子，例如Munc18b， STXBP5/Tomosyn-1和颗粒素/SLP4；然而，这些蛋白控制的机制 血小板释放反应的复杂性(其速度、程度和含量)是不确定的。血小板是 还能进行其他细胞过程(即RNA剪接、翻译、糖基化、自噬)； 然而，它们对血小板功能的影响仍不清楚。我们的数据表明，内吞作用会影响 通过调节血小板扩散和血小板-血小板接触来促进血栓的生长。我们的目标是 控制血小板膜转运，包括内吞和胞吐，以便 调节闭塞性血栓形成，止血效果不大。为了实现这一目标，我们 必须探索胞吐作用的分子机制。我们的重点将放在语法蛋白-11上 监管者。我们还将使用内吞缺陷小鼠品系来确定内吞作用在 血栓形成和止血。提出了两个目标：1)定义蛋白质-蛋白质网络 影响Synaxin-11介导膜融合和颗粒货物释放的相互作用；以及 2)确定血小板内吞作用在止血中的作用。为了实现这些目标，我们将 使用生化分析来确定诱捕调节剂与体外和 使用转基因小鼠进行体内功能分析，以确定特定Synaxin-11的作用 血栓形成和止血中的内吞作用调节剂。我们的结果将扩大我们对 控制血小板的分子需求和蛋白质相互作用的顺序 胞吐。我们还将扩展对血小板细胞过程的机制的理解 执行(即，内吞作用)以及为什么它们很重要。我们的结果将对该领域具有重要意义，因为 他们将提供所需的机械洞察力，以确定潜在的治疗干预目标 并评估指导患者的基因/风险关联的数量不断增加的相关性 治疗策略。这将加强可供护理的抗血栓治疗选择 上了年纪的老兵。

World-wide, spurious thrombosis accounts for 1 of 4 non-communicable disease deaths. Cardiovascular disease is a leading killer of aging US veterans and is the major cause of death in older female veterans. Understanding how to modulate thrombosis will significantly aide the VA's mission to improve veterans' health. Normally platelets respond to vascular damage and secrete granule cargo that are essential for recruiting more platelets and for generating a thrombus. This releasate promotes normal sequelae but can also contribute to occlusive pathologies such as strokes and heart attacks. In platelets, VAMPs, SNAP-23, and Syntaxin-11 form a membrane-spanning complex that mediates exocytosis. Formation of this complex requires a host of SNARE-regulators, e.g., Munc18b, STXBP5/tomosyn-1, and granuphilin/SLP4; however, the mechanisms by which these proteins control the complexity of the platelet release reaction (its rate, extent, and content) is uncertain. Platelets are also capable of other cellular processes (i.e., RNA splicing, translation, glycosylation, autophagy); however, their effects on platelet function are still unknown. Our data suggest that endocytosis affects thrombus growth by modulating platelet spreading and platelet-platelet contacts. Our goal is to manipulate the membrane trafficking in platelets, both endocytosis and exocytosis, in order to modulate occlusive thrombosis with only modest effects on hemostasis. To reach this goal, we must probe the molecular mechanisms of exocytosis. Our specific focus will be on Syntaxin-11 regulators. We will also use an endocytosis defective mouse strain to define the roles of endocytosis in thrombosis and hemostasis. Two aims are proposed: 1) Define the network of protein-protein interactions that affect Syntaxin-11-mediated membrane fusion and granule cargo release; and 2) Determine the roles of platelet endocytosis in hemostasis. To complete these aims, we will employ biochemical assays to define the interactions between the SNARE regulators and in vitro and in vivo functional assays, using transgenic mice, to define the roles of the specific Syntaxin-11 regulators and of endocytosis in thrombosis and hemostasis. Our results will expand the understanding of the molecular requirements and the sequence of protein-protein interactions controlling platelet exocytosis. We will also expand the mechanistic understanding of what cellular processes platelets can perform (i.e., endocytosis) and why they are important. Our results will be significant to the field since they will provide the needed mechanistic insights to identify potential targets for therapeutic intervention and to evaluate the relevance of the increasing volume of gene/risk associations that are guiding patient treatment strategies. This will enhance the anti-thrombotic treatment options available for the care of aging veterans.