PLATELET RECEPTOR MEDIATED FACTOR X ACTIVATION

血小板促进两种顺序钙依赖的催化作用 凝血反应：凝血因子X(FX)的激活 FixA与FVIIa的络合物及凝血酶原转化为 凝血酶是由FXA和FVA形成的复合体。血小板的作用 对F-X的激活是由受体介导的，因为血小板具有特异性， FixA、FVIII和受体的高亲和力、可饱和的寻找位点 入住率与F-X激活率密切相关 血小板表面。我们最近的研究表明，激活的 人血小板暴露于每个血小板500-600个FixA结合位点 在没有FVIII和Fx的情况下，Kd(App)约为2.5 NM 具有增强亲和力的相同数量的网站(KD(App)) 在FVIII和FX存在的情况下约0.5 NM)。我们有 也证实了Nesheim和他的同事们的观察 已经证明了存在单一类别的结合位点 (450个/血小板，Kd=2.9 nM)用于重组人FVIII(RFVIII) 在凝血酶激活的人血小板上。此外，我们有 展示了低亲和力、高容量结合位点的存在 活化的人血小板对凝血酶原共享的FX的作用 和低容量、高亲和力的站点，该站点专用于 FixA和Fviii的存在。这些观察结果支持 假设血小板表面的F-X激活复合体 由一个三受体复合体组成，它的组装导致 F-X的激活速度加快了2400万倍。这个 本申请书所建议的研究目的是研究 更详细地验证这一假设的正确性，并确定 血小板表面和酶上的结构成分 (固定物)组装这一重要凝血系统所需的 很复杂。具体地说，我们建议完成一个完整的 血小板表面F-X激活复合体的特性研究 通过与FIXA、FVIII(A)和 FX和F-X激活的同步动力学研究。我们建议 确定FixA中与其结合所需的结构域 血小板受体和F-X激活复合物的组装， 特别关注GLA结构域和EGF的作用 域名。我们将确定所需的血小板激活状态 用于结合F-X激活络合物的组分和载体 OUT研究旨在确定亚细胞定位和 血小板膜受体的生化特性 结合F-X激活复合体的组分。

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.