The Role of O-GlcNAc in Platelet Activation

描述(申请人提供)：血小板在止血中的作用是对血管系统的损伤做出反应。功能障碍会导致出血性疾病或致病性血栓形成，导致中风、心脏病发作和其他闭塞事件，这些都是当今社会的主要健康问题。要了解止血，关键是要了解血小板的激活情况。这项建议的重点是最近确定的血小板蛋白的翻译后修饰，似乎是激活所必需的。O-GlcN酰化不是糖基化的标准形式。它是将单一的N-乙酰氨基葡萄糖加到SER或Thr残基上。O-GlcNAc残基似乎与许多蛋白质上的磷酸化是相互作用的，因此与其他类型细胞中的关键细胞内信号事件有关。在血小板中，O-GlcNAc存在于多达100种蛋白质上，其动态周转是血小板功能所必需的，特别是颗粒释放。当O-GlcNAc的循环交换被阻止其清除的特定抑制剂阻断时，血小板对凝血酶、胶原或A23187不起反应。拟议中的实验将扩展这些观察，以确定哪些血小板蛋白被修饰，从而阐明O-GlcN酰化如何影响血小板生理。这些研究将提供初步数据，直接解决我们的长期假设，即特定血小板蛋白O-GlcN酰化的动态变化对血小板功能至关重要。我们提出了一个特定的目标：鉴定O-GlcNAc修饰的血小板蛋白，并确定它们的修饰位点(S)。这一目标将使用O-GlcNAc特异性亲和层析技术和基于质谱学的蛋白质组学分析来鉴定静息和刺激血小板中的O-GlcNacylated蛋白质。一旦完成这两年的实验，R21的应用将提供所需的数据库，以更全面地了解O-GlcN酰化在血小板功能中的作用。这些信息将进一步阐明对止血调节至关重要的事件，并可能导致更好地了解糖尿病等代谢综合征如何影响血小板功能。

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.