INTERACTION OF PLATELETS WITH COAGULATION FACTORS IX & X

血小板具有特异的、高亲和力的、可饱和的受体 凝血因子XI、Xia、Xa、凝血酶、高分子量 激肽原和Va因子促进蛋白水解酶活性 凝血因子XII、凝血因子XI、凝血因子X和凝血酶原。至 进一步研究活化的血小板表面作为 凝血蛋白的分子相互作用，我们有 还研究了凝血因子IXa和凝血因子IX与血小板的结合 作为血小板对凝血因子-X激活的贡献。九号因子， 免疫亲和纯化技术从人血浆中分离纯化 利用鼠源性单抗，转化为因子 IXA与纯化的XIA因子孵育。我们有 展示了特异性、高亲和力、可逆结合 凝血因子IXa和凝血因子IX对凝血酶激活的血小板的影响 钙离子的存在。饱和结合数据的分析 在平衡条件下得到的结果表明存在 每个血小板550个加或减70个(平均加或减SD)位点 离解常数为2.5正负0.5的因子IXa NM，而因子IX有306个正负57个位点 Kd值为2.68，正负0.25 nm。初步结果 提示血小板结合因子IXa促进半衰期 0.5 nM浓度下凝血因子-X的激活速率。这个 需要检验的中心假设是，因子IXa必然会很高- 亲和力、特异性血小板受体与凝血因子X的加速 作为功能结果的激活。具体地说，我们的 目标是：1)阐明生物化学基础和 凝血因子-IX和凝血因子-IXa结合的生理学意义 血小板，包括凝血因子-IX和凝血因子- IXA受体；2)确定因子VIII和von的作用(如果有的话) 凝血因子IX和凝血因子IXa结合的Willebrand因子 血小板和血小板对凝血因子-X活化的贡献； 3)确定结合所需的血小板激活状态 凝血因子IX和凝血因子IXa对血小板和血小板的作用 对因子-X激活的贡献；4)阐明结构 凝血因子IX和凝血因子IXa的结合特性 血小板受体及其对凝血因子-X的作用 激活；5)确定因子的功能后果- 凝血因子-X与血小板结合的动力学研究 活化，利用显色分析，蛋白水解性切割 研究、凝血试验和活化的释放 来自~3H标记因子X的多肽。这些研究将提供 关于血小板在促进血管生成中的作用的基本信息 酶-辅因子-底物复合体的组装和 凝血酶原的蛋白水解性激活。

Platelets possess specific, high-affinity, saturable receptors for factor XI, factor XIa, factor Xa, thrombin, high molecular weight kininogen, and factor Va and promote the proteolytic activation of factor XII, factor XI, factor X, and prothrombin. To investigate further the activated platelet surface as a locus for the molecular interactions of coagulation proteins, we have studied the binding of factor IXa and factor IX to platelets as well as the contribution of platelets to factor-X activation. Factor IX, isolated from human plasma by immunoaffinity purification utilizing a murine monoclonal antibody, was converted to factor IXa by incubation with purified factor XIa. We have demonstrated the specific, high-affinity, reversible binding of both factor IXa and factor IX to thrombin-activated platelets in the presence of calcium ions. Analysis of saturation binding data obtained under equilibrium conditions indicated the presence of 550 plus or minus 70 (mean plus or minus SD) sites per platelet for factor IXa with a dissociation constant of 2.5 plus or minus 0.5 nM, whereas there were 306 plus or minus 57 sites for factor IX with a Kd of 2.68 plus or minus 0.25 nM. Preliminary results suggest that platelet-bound factor IXa promotes half-maximal rates of factor-X activation at a concentration of 0.5 nM. The central hypothesis to be tested is that factor IXa is bound to high- affinity, specific platelet receptors with acceleration of factor-X activation as a functional consequence. Specifically, our objectives are to: 1) elucidate the biochemical basis and physiological significance of factor-IX and factor-IXa binding to platelets including the relationship between factor-IX and factor- IXa receptors; 2) determine the role (if any) of factor VIII and von Willebrand fator in binding of factor IX and factor IXa to platelets and for the platelet contribution to factor-X activation; 3) determine the state of platelet activation required for binding of factor IX and factor IXa to platelets and for the platelet contribution to factor-X activation; 4) elucidate the structural characteristics of factor IX and factor IXa required for binding to platelet receptors and for the platelet contribution to factor-X activation; 5) determine the functional consequences of factor- IXa binding to platelets by examining the kinetics of factor-X activation, utilizing chromogenic assays, proteolytic cleavage studies, coagulation assays and the release of an activation peptide from 3H-labeled factor X. These studies will provide essential information about the role of platelets in promoting the assembly of enzyme-cofactor-substrate complexes and the proteolytic activation of coagulation zymogens.