ROLE OF FIBRINOGEN IN FIBRINOLYSIS

纤维蛋白原在纤维蛋白溶解中的作用

• 批准号: 6343307
• 负责人: DAVID Henry FARRELL
• 金额: $ 9.24万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-15 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6343307
• 关键词: blood coagulation; fibrin stabilizing factor; fibrinogen; fibrinolysis; plasminogen activator; protein binding; protein isoforms; protein sequence; protein structure function; recombinant proteins

The broad, long-term objectives of the applicant are to define the roles of fibrinogen and fibrin in angiogenesis, hemostasis, and thrombosis. This proposal addresses the issue of fibrin clot stability, which is a critical factor in the outcome of fibrinolytic therapy. The specific aims of this proposal are to: I) Determine the role of the gamma' chain extension in modulating the stability of fibrin clots. The hypothesis is that the rate and extent of gamma and gamma' multimer formation and the incorporation of alpha2-plasmin inhibitor into gamma'-containing clots will be greater. In addition, the ability of a synthetic peptide corresponding to the gamma' extension, VRPEHPAETEYDSLYPEDDL, to inhibit clot lysis rates and inhibit gamma' fibrinogen/factor XIII complex formation will be tested. The stability of the factor XIII/gamma' fibrinogen complex will also be measured following thrombin activation to test the hypothesis that thrombin activation dissociates the complex. II) Identify the critical amino acids in the gamma' chain extension that mediate factor XIII binding. This will be accomplished by measuring the equilibrium association constant between factor XIII and recombinant gamma' fibrinogen mutants containing alanine-scanning substitutions. III) Identify the peptide sequence(s) in factor XIII b-subunit that bind to the gamma' chain extension. This will be accomplished by binding proteolyzed factor XIII subunits to a resin of immobilized gamma' peptide to identify the binding peptide fragment(s). IV) Test the hypothesis that alpha chain Lys residues alpha148 and alpha157 and gamma chain Lys residues gamma321, gamma338, gamma356, and gamma373 mediate t-PA-dependent plasminogen activation, t-PA binding, and plasminogen binding, using recombinant fibrinogens containing Lys to Ala mutations. The results from the experiments in this proposal should provide new knowledge about the process of fibrinolysis that may lead to ways of modulating fibrin clot stability and clot lysis in vivo.

申请人的广泛和长期目标是确定纤维蛋白原和纤维蛋白在血管生成、止血和血栓形成中的作用。这项建议解决了纤维蛋白凝块稳定性的问题，这是纤溶治疗结果的一个关键因素。这项建议的具体目的是：i)确定伽马‘链延伸在调节纤维蛋白凝块稳定性中的作用。假设伽马和伽马多聚体形成的速度和程度以及α2-纤溶酶抑制物掺入含伽马‘的血栓中的速度和程度将会更大。此外，还将测试与伽马‘延伸相对应的合成肽VRPEHPAETEYDSLYPEDDL抑制血栓溶解速率和抑制伽马’纤维蛋白原/凝血因子XIII复合体形成的能力。凝血酶激活后，还将测量凝血酶XIII/γ‘纤维蛋白原复合体的稳定性，以验证凝血酶激活解离复合体的假设。Ii)确定介导因子XIII结合的伽马‘链延伸中的关键氨基酸。这将通过测量凝血因子XIII和含有丙氨酸扫描取代的重组γ‘纤维蛋白原突变体之间的平衡结合常数来实现。Iii)鉴定因子XIIIb-亚基中与γ‘链延伸相结合的肽序列(S)。这将通过将蛋白分解的因子XIII亚单位结合到固定化的伽马‘肽的树脂上来实现，以鉴定结合的多肽片段(S)。Iv)使用含有Lys to Ala突变的重组纤维蛋白原，验证阿尔法链赖氨酸残基α148和α157以及伽马链赖氨酸残基Gamma321、Gamma338、Gamma356和Gamma373介导t-PA依赖的纤溶酶原激活、t-PA结合和纤溶酶原结合的假设。这项建议中的实验结果应该会提供关于纤溶过程的新知识，这可能会导致在体内调节纤维蛋白凝块稳定性和凝块溶解的方法。