INTERACTION OF PLATELETS WITH COAGULATION FACTORS IX & X

血小板与凝血因子 IX 的相互作用

• 批准号: 3338184
• 负责人: PETER Newton WALSH
• 金额: $ 15.8万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-05-01 至 1993-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3338184
• 关键词: activation product; bioassay; blood coagulation tests; calcium; chemical binding; coagulation factor IX; coagulation factor VIII; coagulation factor X; cytoskeleton; enzyme complex; enzyme mechanism; enzyme substrate; hemostasis; human tissue; kallikreins; membrane activity; monoclonal antibody; peptide structure; phospholipids; platelet activation; platelets; protein purification; protein structure function; proteolysis; radioassay; receptor; receptor binding; synthetic peptide; zymogens

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.

血小板具有特异性、高亲和力、可饱和的受体， 因子XI，因子XIa，因子Xa，凝血酶，高分子量 激肽原和因子Va并促进蛋白水解活化 因子XII、因子XI、因子X和凝血酶原。 到 进一步研究活化的血小板表面作为 凝血蛋白的分子间相互作用， 研究了因子IXa和因子IX与血小板的结合 血小板对X因子激活的贡献。 因子IX， 通过免疫亲和纯化从人血浆中分离 利用鼠单克隆抗体，转化为因子 通过与纯化的因子XIa孵育。 我们有 证明了特异性，高亲和力，可逆结合 凝血因子IXa和因子IX对凝血酶活化的血小板的作用， 钙离子的存在。 饱和结合数据分析 在平衡条件下得到的结果表明， 550 ± 70（平均值± SD）个部位/血小板， 因子IXa，解离常数为2.5 ± 0.5 nM，而因子IX有306 ± 57个位点 Kd为2.68 ± 0.25 nM。 初步结果 提示血小板结合因子IXa促进半最大 0.5 nM浓度下的因子-X活化速率。 的 待检验的中心假设是因子IXa与高水平的 亲和力，特异性血小板受体与X因子加速 激活作为功能性结果。 具体来说，我们 目标是：1）阐明生物化学基础， 因子IX和因子IXa结合的生理意义 血小板，包括因子IX和因子之间的关系， IXa受体; 2）确定因子VIII和血管紧张素Ⅱ受体的作用（如果有的话）。 在因子IX和因子IXa结合至 血小板和血小板对因子-X活化的贡献; 3)确定结合所需的血小板活化状态 因子IX和因子IXa对血小板的影响， 对因子X激活的贡献; 4）阐明结构 结合所需的因子IX和因子IXa的特性 血小板受体和血小板对X因子的贡献 （5）确定因素的功能后果- 通过检查因子X的动力学来检测IXa与血小板的结合 活化，利用显色测定，蛋白水解裂解 研究、凝血测定和激活的释放 3 H标记的X因子肽。 这些研究将提供 关于血小板在促进 酶-辅因子-底物复合物的组装和 凝血酶原的蛋白水解活化。