MECHANISM OF ACTIVATION OF PLATELET GPIIB AND GPIIIA

本研究的长期目标是了解心力衰竭的机制。 活化血小板整合素GPIIb-IIIa。GPIIb-IIIa是 血小板黏附和聚集。对其规制的一种理解 可能对治疗许多心血管疾病很重要 包括心肌梗塞和中风。上存在GPIIb-IIIa 处于休眠状态的血小板不能结合可溶性的 纤维蛋白原。在血小板刺激下，IIb-IIIa变得能够 结合纤维蛋白原和介导血小板聚集。虽然 IIb-IIIa激活是血小板功能的关键，其机制 激活问题尚未解决。 这项研究的一个假设是，蛋白水解性切割 IIb-IIIa的胞质结构域控制着细胞的激活状态 整合素。这一假设将通过刻画 两种纯化形式胞质结构域的结构差异 激活状态不同的IIb-IIIa。 这项研究的第二个目标是了解 激活依赖的配体与IIb-IIIa结合。噬菌体展示将是 用于选择优先与休眠和 整合素的活性形式。这项研究的结果可能会 提供解释依赖于激活的结构-活动系列 配基结合。 这项研究的第三个目标是了解 整合素激活。激活是由配体的增加引起的吗 缔合率还是配体解离率的降低？这些 研究将在全血小板上进行，并使用纯化形式的 休眠和活跃的IIb-IIIa。 最终目标是了解IIb上的二价离子结合位点是如何- IIIA影响激活事件。将进行约束性研究 钙与休眠和活性IIb-IIIa的纯化构象之间的关系。 这一分析的结果应该确定哪一类离子结合 结合位点调节整合素的激活。

The long term objective of this study is to understand the mechanism of activation of the platelet integrin GPIIb-IIIa. GPIIb-IIIa is key to platelet adhesion and aggregation. An understanding of its regulation is likely to be important for treating many cardiovascular diseases including myocardial infarction and stroke. GPIIb-IIIa exists on resting platelets in a dormant conformation unable to bind soluble fibrinogen. Upon platelet stimulation, IIb-IIIa becomes capable of binding fibrinogen and mediating platelet aggregation. Although activation of IIb-IIIa is key to platelet function, the mechanism its activation has not been solved. One hypothesis of the study is that proteolytic cleavage of the cytoplasmic domains of IIb-IIIa control the activation state of the integrin. This hypothesis will be tested by characterizing the structural differences in the cytoplasmic domain of two purified forms of IIb-IIIa which differ in activation state. A second goals of the study is to understand the structural basis of activation-dependent ligand binding to IIb-IIIa. Phage-display will be used to select ligands that bind preferentially to the dormant and active forms of the integrin. Results from this study are likely to provide a structure-activity series explaining activation-dependent ligand binding. A third aim of the study is to understand the kinetic aspects of integrin activation. Does activation result from an increase in ligand association rate or a decrease in ligand dissociation rate? These studies will be performed on whole platelets and with purified forms of dormant and active IIb-IIIa. A final goal is to understand how the divalent ion binding sites on IIb- IIIa influence the activation event. Binding studies will be performed between Ca2+ and purified conformers of dormant and active IIb-IIIa. Results from this analysis should determine which class of ion binding sites regulate activation of the integrin.