ROLE OF FIBRINOGEN IN FIBRINOLYSIS

申请人的长期目标是明确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。该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关键因素。本提案的具体目的是：1)确定γ链延伸在调节纤维蛋白凝块稳定性中的作用。假设是伽马和伽马多聚体形成的速度和程度以及α 2-纤溶酶抑制剂与含有伽马的凝块的结合将会更大。此外，将测试与γ′延伸相对应的合成肽vrpehpaeteydslypedl抑制凝块溶解速率和抑制γ′纤维蛋白原/因子XIII复合物形成的能力。凝血酶激活后，还将测量因子XIII/ γ纤维蛋白原复合物的稳定性，以检验凝血酶激活解离该复合物的假设。II)鉴定γ′链延伸中介导因子XIII结合的关键氨基酸。这将通过测量因子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关联常数来实现。III)确定因子XIII b亚基中与γ′链延伸结合的肽序列。这将通过将蛋白水解因子XIII亚基结合到固定化γ′肽的树脂上来识别结合肽片段来实现。IV)利用含有Lys to Ala突变的重组纤维蛋白原，验证α链Lys残基α 148和α 157和γ链Lys残基γ ma321、γ ma338、γ ma356和γ ma373介导t-PA依赖性纤溶酶原激活、t-PA结合和纤溶酶原结合的假设。本实验的结果应该提供关于纤维蛋白溶解过程的新知识，可能导致调节纤维蛋白凝块稳定性和体内凝块溶解的方法。

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.