PLATELET SECRETION--MOLECULAR AND REGULATION

在心血管疾病中，活化血小板分泌的分子（生长因子、粘附分子、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，但血小板分泌（胞吐）的分子机制仍知之甚少。对血小板胞吐机制的基本科学见解可以转化为新的、特定的治疗方法来调节血小板分泌，以帮助预防引起中风和心脏病发作的血栓形成。由于其在生理学上的重要意义，我们的研究方向是发现负责血小板分泌的分子机制，并阐明该机制如何通过信号机制耦合到细胞活化过程。血小板是一种独特的终末期分泌细胞，通过特定的细胞外受体与“激活”细胞以诱导分泌或“钝化”（即使活性降低）细胞以抑制分泌的药物相互作用，诱导胞外分泌。我们假设血小板分泌过程使用囊泡膜融合的分子机制，这与其他特殊分泌细胞（如神经元）使用的分子机制相似。血小板胞外分泌的独特方面包括血小板选择性分子机制和与活化（如凝血酶或钝化（如PGI2）配体）相互作用的胞外膜受体的独特偶联。这一假设基于我们之前的发现，即血小板：1)具有完整的相互作用的SNARE分子，这些分子是通过复杂形成介导神经元（和其他细胞）分泌的蛋白质的同源物；2)含有一个Sec1同源物，该同源物似乎在功能上与凝血酶激活血小板（通过蛋白激酶C信号传导）有关，并且能够调节这些SNARE分子之间的复合物形成；3)需要一个关键酶复合物（alphaSNAP/ n -乙酰酰亚胺敏感融合蛋白（NSF））的活性，该酶复合物调节SNARE分子的相互作用，以诱导α和致密颗粒分泌。具体目标是：1。为了阐明血小板分泌的分子机制，a)鉴定胞吐所需的v-和t-SNARE蛋白，b)确定它们的细胞分布，c)检查它们的功能相互作用是否通过磷酸化事件被细胞活化调节。2. 鉴定在血小板中调节这些v-和- 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活化事件调节。3.确定血小板Doc2alpha（一种假定的Ca2+传感蛋白）与核心分泌机制的相互作用，并定义这种相互作用如何通过细胞信号传导与血小板激活在功能上联系起来。预计对血小板胞吐的机制及其与细胞活化的联系的深入了解，将扩大我们对脑、胰腺等其他重要分泌细胞分泌的分子控制的理解。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应用于治疗性调节分泌，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.