Platelet Receptor Mediated Factor X Activation

描述(申请人提供)：在凝血过程中，血小板可促进两种连续的钙依赖反应的催化作用：FixA和FVIIIa复合体激活凝血因子X(FX)，以及FXA和FVA复合体将凝血酶原转化为凝血酶。由于血小板具有与FixA和FVIII特异的、高亲和力的、可饱和的结合部位，并且受体的占有率与血小板表面Fx的激活速率密切相关，因此，血小板对Fx激活的贡献是由受体介导的。我们最近的研究表明，在没有FVIII和FX的情况下，激活的人血小板暴露于每个血小板500-600个FixA结合位点，其Kd(App)为-2.5 nM，而在FVIII和Fx存在的情况下，具有相同数量的亲和力增强的位点(Kd(App)-0.5 nM)。我们还证实了Nesheim和他的同事的观察结果，他们证明了重组人FVIII(RFVIII)在凝血酶激活的人血小板上存在单一类别的结合位点(450个/血小板，Kd=2.9 nM)。此外，我们还证明了在激活的人血小板上存在与凝血酶原共享的低亲和力、高容量的FX结合位点，以及在FixA和FVIII存在的情况下与FX特异的低亲和力、高亲和力的结合位点。这些观察结果支持这样的假设，即血小板表面的F-X激活复合体由一个三受体复合体组成，该复合体的组装导致FX激活速度加快2400万倍。本申请中提出的研究的目的是更详细地检验这一假说的有效性，并确定血小板表面和组装这一重要凝血复合体所需的酶(FixA)上的结构成分。具体地说，我们建议通过开展与FixA、FVIII(A)和FX的配位结合研究以及FX激活的同步动力学研究，完成对血小板表面FX激活复合体的完整表征。我们建议确定FixA中与其血小板受体结合和组装FX激活复合体所需的结构域，特别是GLA结构域和EGF结构域的作用。我们将确定结合FX激活复合体成分所需的血小板激动剂、受体、信号转导和效应机制。

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.