PLATELET SECRETION--MOLECULAR AND REGULATION

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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.