PLATELET SECRETION--MOLECULAR AND REGULATION

在心血管疾病中，由激活的血小板分泌的分子(生长因子、黏附分子、凝血因子、细胞激活剂等)。在导致心脏病发作和中风的血液凝块形成中起关键作用。不幸的是，尽管最近在血小板生物学的其他方面取得了进展，但人们对血小板分泌(胞吐作用)的分子机制知之甚少。对血小板胞吐机制的基本科学见解可以转化为通过治疗调节血小板分泌的新的、特定的方法，以帮助预防导致中风和心脏病发作的血栓形成。由于其关键的生理重要性，我们的研究旨在发现负责血小板分泌的分子机制，并阐明该机制是如何通过信号机制连接到细胞激活过程的。血小板是一种独特的终末期分泌细胞，其胞吐作用是通过特定的细胞外受体与一些“激活”细胞诱导分泌或“钝化”(即减少反应)细胞抑制分泌的物质相互作用而诱导的。我们假设，血小板的分泌过程使用囊泡膜融合的分子机制，这与其他专门的分泌细胞，如神经元所使用的机制是同源的。血小板胞吐作用的独特方面包括一种选择血小板的分子机制，以及该机制与细胞外膜受体的独特偶联，后者与激活的(如凝血酶)或钝化的(如PGI2)配体相互作用。这一假说基于我们之前的发现，即血小板：1)有完整的相互作用的SNARE分子，这是通过复合体形成介导神经元(和其他细胞)分泌的蛋白质的同系物；2)含有Sec1同系物，它似乎在功能上与凝血酶激活血小板有关(通过蛋白激酶C信号)，并且能够调节这些SNARE分子之间的复合体的形成；以及3)需要一个关键的酶复合体(αSNAP/N-乙基马来酰亚胺敏感融合蛋白(NSF))的活性，它调节SNARE分子的相互作用，诱导α和致密的颗粒分泌。其具体目的是：1.通过a)确定胞吐所需的v-和t-SNARE蛋白，b)确定它们的细胞分布，以及c)检查它们的功能相互作用是否通过磷酸化事件被细胞激活来调节，以阐明负责血小板分泌的分子机制。2.鉴定调节血小板v-and-SNARE相互作用的人类Sec1分子，揭示其作用机制，并确定其功能如何受细胞激活事件的调节。3.确定血小板Doc2α(一种可能的钙敏感蛋白)与核心分泌机制的相互作用，并确定这种相互作用如何通过细胞信号转导血小板激活而在功能上联系起来。预计对血小板胞吐的机制及其与细胞激活的联系的深入了解将扩大我们对大脑、胰腺等其他重要分泌细胞分泌的分子控制的了解。鉴于血小板分泌分子在心脏病发作和中风中的关键作用，我们希望这些研究的见解将被应用于治疗调节分泌，以降低心血管疾病的风险。

In cardiovascular disease, molecules secreted by activated platelets (growth factors, adhesion molecules, coagulation factors, cell activators, etc.) play key roles in the blood clot formation that causes heart attacks and strokes. Unfortunately, the molecular mechanisms of platelet secretion (exocytosis) are poorly understood, despite recent progress in other aspects of platelet biology. Fundamental scientific insights into the mechanisms of platelet exocytosis could translate into novel, specific approaches to therapeutically regulating platelet secretion, in order to help prevent the thrombosis that causes strokes and heart attacks. Because of its key physiologic importance, our research is directed towards discovering the molecular machinery responsible for platelet secretion and towards elucidating how that machinery is coupled through signaling mechanisms to the process of cell activation. Platelets are unique, end-stage secretory cells in which exocytosis is induced through the interactions of specific extracellular receptors with agents that "activate" cells to induce secretion or "passivate" (i.e. make less reactive) cells to inhibit secretion. We hypothesize that the platelet secretory processes using molecular mechanisms for vesicle membrane fusion that are homologous to those used by other specialized secretory cells such as neurons. The unique aspects of platelet exocytosis include a platelet- selective molecular machinery and a distinctive coupling of that machinery to extracellular membrane receptors that interact with activating (e.g. thrombin or passivating (e.g., PGI2) ligands. This hypothesis is based on our previous findings that platelets: 1) have a full complement of interacting SNARE molecules, that are homologues of proteins that mediate secretion in neurons (and other cells) by complex formation; 2) contain a Sec1 homologue that appears to be functionally linked to platelet activation by thrombin (via protein kinase C signaling) and which is capable of regulating complex formation among these SNARE molecules, and 3) require the activity of a key enzyme complex (alphaSNAP/N-ethylmaleimide sensitive fusion protein (NSF)), which modulates SNARE molecule interactions, to induce alpha and dense granule secretion. The Specific Aims are: 1. To elucidate the molecular machinery responsible for platelet secretion by a) identifying the v- and t-SNARE proteins that are required for exocytosis, b) by determining their cellular distribution and c) by examining whether their functional interactions are modulated by cell activation through phosphorylation events. 2. To identify the human Sec1 molecules that modulate these v- and - SNARE interaction in platelets, to uncover their mechanism of action and to ascertain how their function is regulated by cell activation events. 3.To determine the interactions of platelet Doc2alpha, a putative Ca2+- sensor protein, with the core secretory machinery and to define how that interaction is functionally linked through cell signaling to platelet activation. It is anticipated that insights into the mechanisms of platelet exocytosis and its linkage to cell activation, will enlarge our understanding of the molecular control of secretion in other important secretory cells in the brain, pancreas, etc. Given the key roles of platelet secretory molecules in heart attacks and strokes, we hope that insights from these studies will be applied to therapeutically regulate secretion in order to reduce the risk of cardiovascular disease.