Platelet Receptor Mediated Factor X Activation

血小板受体介导的 X 因子激活

• 批准号: 6507578
• 负责人: PETER Newton WALSH
• 金额: $ 37.5万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6507578
• 关键词: binding sites; biological signal transduction; blood coagulation; cell component structure /function; cell membrane; chimeric proteins; clinical research; coagulation factor X; enzyme activity; enzyme complex; hemostasis; human subject; phlebotomy; platelet disorder; platelets; protein protein interaction; protein structure function; receptor; receptor binding; synthetic peptide

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.

描述（由申请人提供）：血小板促进血液凝固中两个连续钙依赖性反应的催化：FIXa和FVIIIa复合物激活X因子（FX）以及FXa和FVa复合物将凝血酶原转化为凝血酶。血小板对 FX 激活的贡献是受体介导的，因为血小板具有针对 FIXa 和 FVIII 的特异性、高亲和力、可饱和结合位点，并且受体占据与血小板表面上的 FX 激活速率密切相关。我们最近的研究表明，在不存在 FVIII 和 FX 的情况下，活化的人血小板每个血小板暴露 500-600 个 FIXa 结合位点，Kd (app) 为 -2.5 nM，而在 FVIII 和 FX 存在的情况下，相同数量的位点具有增强的亲和力 (Kd (app) -0.5 nM）。我们还证实了 Nesheim 及其同事的观察结果，他们证明在凝血酶激活的人血小板上存在重组人 FVIII (rFVIII) 的单一类别结合位点（450/血小板，Kd = 2.9 nM）。此外，我们还证明了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 存在的情况下对 FX 具有特异性的低容量、高亲和力位点。这些观察结果支持这样的假设：血小板表面的 F-X 激活复合物由三受体复合物组成，其组装导致 FX 激活速率加速 2400 万倍。本申请中提出的研究的目的是更详细地检验该假设的有效性，并确定血小板表面和组装这一重要凝血复合物所需的酶（FIXa）上的结构成分。具体来说，我们建议通过进行 FIXa、FVIII(a) 和 FX 的配位结合研究以及 FX 激活的同时动力学研究来完成血小板表面 FX 激活复合物的完整表征。我们建议确定 FIXa 中结合其血小板受体和组装 FX 激活复合物所需的结构域，特别关注 Gla 结构域和 EGF 结构域的作用。我们将确定结合 FX 激活复合物成分所需的血小板激动剂、受体、信号转导和效应机制。