Platelet Receptor Mediated Factor X Activation

描述（由申请人提供）：血小板可促进凝血中两个连续钙依赖性反应的催化：通过FIXa和FVIIIa的复合物激活因子X（FX），以及通过FXa和FVa的复合物将凝血酶原转化为凝血酶。血小板对FX活化的贡献是受体介导的，因为血小板对FIXa和FVIII具有特异性、高亲和力、可饱和的结合位点，并且受体占有率与血小板表面上的FX活化速率密切相关。我们最近的研究已经证明，活化的人血小板暴露每个血小板500-600个FIXa结合位点，在不存在FVIII和FX的情况下Kd（app）为~ 2.5 nM，并且在存在FVIII和FX的情况下具有相同数量的具有增强的亲和力的位点（Kd（app）~ 0.5 nM）。我们还证实了Nesheim及其同事的观察结果，他们证明了凝血酶活化的人血小板上存在重组人FVIII（rFVIII）的单一类结合位点（450个/血小板，Kd = 2.9 nM）。此外，我们已经证明了在活化的人血小板上存在与凝血酶原共享的FX的低亲和力、高容量结合位点，以及在FIXa和FVIII存在下对FX具有特异性的低容量、高亲和力位点。这些观察结果支持血小板表面上的FX活化复合物由三受体复合物组成的假设，其组装导致FX活化速率加速2400万倍。本申请中提出的研究的目的是更详细地检查该假设的有效性，并确定血小板表面和该重要凝血复合物组装所需的酶（FIXa）上的结构组分。具体而言，我们建议通过与FIXa、FVIII（a）和FX进行配位结合研究以及同时进行FX活化的动力学研究，完成血小板表面FX活化复合物的完整表征。我们建议确定FIXa的结构域所需的结合到其血小板受体和FX激活复合物的组装，特别是专注于Gla结构域和EGF结构域的作用。我们将确定血小板激动剂，受体，信号转导和效应器的FX激活复合物的成分结合所需的机制。

DESCRIPTION (provided by applicant): 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 FX activation is receptor mediated since platelets possess specific, high-affinity, saturable binding sites for FIXa and FVIII and receptor occupancy is closely correlated with rates of FX 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 -2.5 nM in the absence of FVIII and FX and the same number of sites with enhanced affinity (Kd (app) -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 that 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 FX 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 FX-activating complex on the platelet surface by carrying out coordinate binding studies with FIXa, FVIII(a), and FX and simultaneous kinetic studies of FX activation. We propose to determine the structural domains in FIXa required for binding to its platelet receptor and for assembly of the FX-activating complex, specifically focusing upon the role of the Gla domain and the EGF domains. We will determine the platelet agonists, receptors, signal transduction and effector mechanisms required for binding the components of the FX-activating complex.