The Role of O-GlcNAc in Platelet Activation

描述（由申请方提供）：血小板在止血中的作用是对血管系统中的病变做出反应。功能障碍导致出血素质或致病性血栓形成，导致中风、心脏病发作和其他闭塞性事件，这些是当今社会的主要健康问题。为了了解止血，了解血小板活化至关重要。这项建议的重点是最近定义的血小板蛋白的翻译后修饰，似乎不可或缺的激活。O-GlcNAc化不是糖基化的标准形式。它是在SER或THR残基上添加单个N-乙酰葡糖胺。O-GlcNAc残基似乎与许多蛋白质上的磷酸化相互作用，因此与其他细胞类型中的关键细胞内信号传导事件有关。在血小板中，O-GlcNAc存在于多达100种蛋白质上，并且其动态周转是血小板功能，特别是颗粒释放所需的。当O-GlcNAc的循环交换被阻止其清除的特异性抑制剂阻断时，血小板无法对凝血酶、胶原蛋白或A23187作出反应。拟议的实验将扩大这些观察，以确定哪些血小板蛋白被修饰，从而阐明O-GlcNAc酰化如何影响血小板生理学。这些研究将提供初始数据，以直接解决我们的长期假设，即特定血小板蛋白的O-GlcNAc酰化的动态变化对血小板功能至关重要。提出了一个具体目的：鉴定用O-GlcNAc修饰的血小板蛋白并确定其修饰位点。本目标将使用O-GlcNAc特异性亲和层析技术和基于质谱的蛋白质组学分析来鉴定静息和刺激血小板中的O-GlcNAc酰化蛋白。一旦完成/这两年的实验，R21应用程序将提供更全面地了解O-GlcNAc酰化在血小板功能中的作用所需的数据库。这些信息将进一步阐明止血调节所必需的事件，并可能导致更好地理解血小板功能如何受到代谢综合征（如糖尿病）的影响。

DESCRIPTION (provided by applicant): A platelet's role in hemostasis is to respond to lesions in the vasculature. Dysfunction leads to bleeding diatheses or to the pathogenic thrombosis that causes strokes, heart attacks and other occlusive events which are major health issues in today's society. To understand hemostasis, it is critical to understand platelet activation. This proposal focuses on a recently defined post-translational modification of platelet proteins that seems integral to activation. O-GlcNAcylation is not a standard form of glycosylation. It is the addition of a single N-acetylglucosamine to SER or THR residues. O-GlcNAc residues appear to be reciprocal with phosphorylation on a number of proteins and thus have been tied to key intracellular signaling events in other cell types. In platelets, O-GlcNAc is present on as many as 100 proteins and its dynamic turnover is required for platelet function, specifically granule release. When cyclical exchange of O-GlcNAc is blocked by a specific inhibitor that prevents its removal, platelets fail to respond to thrombin, collagen, or A23187. The proposed experiments will expand these observations to determine which platelet proteins are modified and thus shed light on how O-GlcNAcylation affects platelet physiology. These studies will provide the initial data to directly address our long term hypothesis that dynamic changes in O-GlcNAcylation of specific platelet proteins are critical for platelet function. One Specific Aim is proposed: To identify platelet proteins which are modified with O-GlcNAc and to determine their site(s) of modification. This Aim will use O-GlcNAc-specific affinity chromatography techniques and mass spectroscopy-based proteomic analysis to identify O-GlcNAcylated proteins in both resting and stimulated platelets. Once completed/the experiments of this two year, R21 application will provide the database needed to more fully understand the role of O- GlcNAcylation in platelet function. This information will further elucidate the events essential to the regulation of hemostasis and perhaps lead to a better understanding of how platelet function can be affected by metabolic syndromes such as diabetes.