FUNCTION OF PLATELET AND COAGULATION FACTORS

血小板和凝血因子的功能

• 批准号: 2215000
• 负责人: PHILIP W MAJERUS
• 金额: $ 45.57万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2215000
• 关键词: blood proteins; chemical structure function; enzyme mechanism; gene expression; genetic library; human subject; hydrolase; inositol phosphates; laboratory mouse; laboratory rabbit; molecular cloning; monoclonal antibody; phosphorylase phosphatase; platelets; protein C; protein sequence; radioimmunoassay; radiotracer; secretion; site directed mutagenesis; thrombomodulin; thrombosis; tissue /cell culture

The objective of this grant is to elucidate basic mechanisms of hemostasis and to devise strategies to alter the natural history of thrombotic disorders. There are two projects. The first deals with the role of platelets, leukocytes, and endothelial cells in the initiation and control of coagulation reactions. Prothrombin activation on platelets will be studied using homogeneous factor Va and its component peptides. The role of factor Va in protein C activation will be elucidated using the isolated factor Va peptides. The relation between thrombomodulin and factor Va, two proteins that both participate in protein C activation, will be studied using cultured endothelial cells and antibodies to thrombomodulin and factor Va. Monoclonal antibodies to protein C, factor Va and its component chains will be prepared. The platelet protease that activates factor V will be purified and its role in the initiation of platelet surface prothrombin activation will be investigated using antibodies specific for factor Va and the western blotting technique. The second project deals with the mechanism of eicosanoid precursor uptake and release by platelets and cultured cells. The fatty acid specificity of arachidonoyl CoA synthetase will be elucidated using a variety of 14C-labeled polyunsaturated fatty acids and compared to the high affinity fatty acid uptake capacity of platelets. The fatty acid specificity of the agonist-induced fatty acid release mechanism of platelets will also be elucidated. Arachidonoyl CoA synthetase will be purified to homogeneity and antibodies prepared. The technique of suicide selection after mutagenesis will be used to select arachidonate uptake and release mutants of HSD mouse fibrosarcoma cells. Mutants will be isolated by cloning and replica-plating on polyester cloth. Mutant cell lines will be useful in the elucidation of mechanisms of fatty acid uptake and release in these cells.

该基金的目的是阐明止血的基本机制 并设计策略来改变血栓形成的自然史， 紊乱 有两个项目。 第一个问题涉及 血小板、白细胞和内皮细胞 凝结反应。 血小板上的凝血酶原活化将是 使用同质因子Va及其组分肽进行研究。 的作用 的因子Va在蛋白C激活将阐明使用分离的 因子Va肽。 血栓调节蛋白与因子Va、2的关系 蛋白质都参与蛋白C激活，将进行研究 使用培养的内皮细胞和抗血栓调节蛋白的抗体， 因子Va。 抗蛋白C、因子Va及其组分的单克隆抗体 链将准备好。 激活因子V的血小板蛋白酶 将被纯化，并在血小板表面的启动作用 凝血酶原激活将使用特异性抗体进行研究， 因子Va和蛋白质印迹技术。 第二个项目涉及 其机制是类二十烷酸前体被血小板摄取和释放 和培养的细胞。 花生四烯酰辅酶A的脂肪酸特异性 合成酶将使用多种14C标记的 多不饱和脂肪酸和高亲和力脂肪酸相比， 血小板的吸收能力。 脂肪酸的特异性 激动剂诱导的血小板脂肪酸释放机制也将被 阐明。 将花生四烯酸酰CoA合成酶纯化至均一 并制备抗体。 自杀选择技术 诱变将用于选择花生四烯酸摄取和释放突变体 HSD小鼠纤维肉瘤细胞。 将通过克隆分离突变体， 在聚酯布上进行复制电镀。 突变细胞系将用于 阐明脂肪酸摄取和释放的机制， 细胞