PLATELET RECEPTOR MEDIATED FACTOR X ACTIVATION

血小板受体介导的 X 因子激活

• 批准号: 6485293
• 负责人: PETER Newton WALSH
• 金额: $ 29万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6485293
• 关键词: coagulation factor X; human subject; phlebotomy; platelet activation; protein structure function; prothrombin; receptor binding

Platelets promote the catalysis of two sequential calcium-dependent reactions in blood coagulation: the activation of factor X (FX) by a complex of FIXa and FVIIIa and the conversion of prothrombin to thrombin by a complex of FXa and FVa. The contribution of platelets to F-X activation is receptor-mediated since platelets posses specific, high-affinity, saturable finding sites for FIXa and FVIII and receptor occupancy is closely correlated with rates of F-X activation on the platelet surface. Our recent studies have demonstrated that activated human platelets expose 500-600 FIXa binding sites per platelet with a Kd(app) of approximately 2.5 nM in the absence of FVIII and FX and the same number of sites with enhanced affinity (Kd(app) approximately 0.5 nM) in the presence of FVIII and FX. We have also confirmed the observation of Nesheim and his colleagues who have demonstrated the presence of a single class of binding sites (450/platelet, Kd = 2.9 nM) for recombinant human FVIII (rFVIII) on thrombin-activated human platelets. Moreover, we have demonstrated the presence of a low-affinity, high-capacity binding site on activated human platelets for FX which is shared with prothrombin and a lower capacity, higher affinity site that is specific for FX in the presence of FIXa and FVIII. These observations support the hypothesis that the F-X activating complex on the platelet surface consists of a three-receptor complex, the assembly of which results in a 24 million-fold acceleration of the rate of F-X activation. The purpose of the studies proposed in this application is to examine in more detail the validity of this hypothesis and to determine the structural components on the platelet surface and on the enzyme (FIXa) required for the assembly of this important coagulation complex. Specifically, we propose to accomplish a complete characterization of the F-X activating complex on the platelet surface by carrying out coordinate binding studies with FIXa, FVIII(a), and FX and simultaneous kinetic studies of F-X activation. We propose to determine the structural domains in FIXa required for binding to its platelet receptor and for assembly of the F-X activating complex, specifically focusing upon the role of the Gla domain and the EGF domains. We will determine the state of platelet activation required for binding the components of the F-X activating complex and carry out studies aimed to determined the subcellular localization and biochemical characterization of the platelet receptors essential for binding the components of the F-X activating complex.

血小板促进两个顺序的钙依赖性的催化 凝血反应：凝血因子X（FX）的激活， FIXa和FVIIIa的复合物以及凝血酶原向 凝血酶通过FXa和FVa的复合物。 血小板的贡献 F-X激活是受体介导的，因为血小板是特异性的， FIXa和FVIII及受体的高亲和力、可饱和发现位点 占有率与F-X激活率密切相关， 血小板表面 我们最近的研究表明， 人血小板每个血小板暴露500-600个FIXa结合位点， 在不存在FVIII和FX的情况下，Kd（app）约为2.5 nM， 相同数量的具有增强的亲和力（Kd（app））的位点 约0.5 nM）。 我们有 也证实了Nesheim和他的同事的观察， 已经证明了存在单一类型的结合位点 (450/血小板，Kd = 2.9 nM），用于重组人FVIII（rFVIII） 凝血酶激活的人类血小板上。 而且我们 证明存在低亲和力、高容量的结合位点 与凝血酶原共享的FX对活化的人血小板的影响 和在所述细胞中特异于FX的较低容量、较高亲和力位点。 存在FIXa和FVIII。 这些观察结果支持 假设血小板表面的F-X活化复合物 由三受体复合物组成，其组装导致 F-X的激活速度加快了2400万倍 的 本申请中提出的研究的目的是检查 更详细地说明这一假设的有效性，并确定 血小板表面和酶上的结构成分 （FIXa）组装这一重要凝血所需的 复杂. 具体而言，我们建议完成一个完整的 血小板表面F-X活化复合物的表征 通过与FIXa、FVIII（a）和 FX和F-X活化的同步动力学研究。 我们建议 确定FIXa中与其结合所需的结构域 血小板受体和用于组装F-X活化复合物， 特别关注Gla结构域和EGF的作用， 域. 我们将确定所需的血小板活化状态 用于结合F-X活化复合物的组分，并携带 我们的研究旨在确定亚细胞定位， 血小板受体的生物化学特性 结合F-X激活复合物的成分。