Molecular Mechanisms of Fibrinolysis

描述（由申请方提供）：拟定研究的长期目标 是确定链激酶（SK）激活 人纤维蛋白溶解系统，这是使用SK作为一种 用于治疗心血管疾病的血栓溶解药物。研究 解决在理解的独特机制的重大差距 SK对纤溶酶原（Pg）的构象激活和偶联的蛋白水解 将Pg转化为纤溶蛋白酶（纤溶酶）的活化途径 （下午）。在待评估的假设中，Pg的构象活化是 由SK的快速结合和sK氨基末端插入到 Pg上的结合口袋，与活化的 酶原的催化结构域的构象，作为其 优先对SK具有更高的亲和力。 通过表达新的结合位点，首先在SK-Pg上， 随后在SK-Pm复合物上，其作为外位点发挥作用， Pg作为底物的特异性结合和裂解。构象和 SK对Pg的蛋白水解激活受以下内在差异的调节： 赖氨酸的参与增强了5 K对[ys]Pg和[ys] Pm的亲和力 结合位点，并在体内由纤维蛋白原和纤维蛋白促进的 生产综合体的集合。这些假设将在 定量平衡结合研究采用独特的荧光 Pg、Pm和SK的衍生物，结合稳态和 快速反应动力学和蛋白质结构方法。具体目标是： (1)为了确定分子事件的顺序， SK结合诱导的Pg构象活化。(2)划定 SK启动的Pm形成的蛋白水解活化途径的机制。 (3)确定SK α、β和γ结构域的功能作用 和SK的单个赖氨酸残基在构象和 （4）为阐明纤维蛋白原和纤维蛋白原的作用机制， 纤维蛋白对SK纤溶的调节作用及其在 SK治疗的纤维蛋白特异性。建议的基本研究 该机制的热力学、动力学和结构方面预计将 改变目前对SK作用机制的概念， 血栓溶解药从这些研究中获得的新信息可能会使更多 纤维蛋白特异性血栓溶解剂有待开发。

DESCRIPTION (provided by applicant): The long-term goal of the proposed studies is to determine the molecular mechanism by which streptokinase (SK) activates the human fibrinolytic system, which is the basis for the use of SK as a thrombolytic drug for the treatment of cardiovascular disease. The studies address significant gaps in the understanding of the unique mechanism of conformational activation of plasminogen (Pg) by SK and the coupled proteolytic activation pathway that converts Pg into the fibrinolytic proteinase, plasmin (Pm). In the hypotheses to be evaluated, conformational activation of Pg is triggered by rapid binding of SK and insertion of the sK aminoterminus into a binding pocket on Pg, in cooperation with stabilization of the activated conformation of the catalytic domain of the zymogen as a result of its preferentially higher affinity for SK. This initiates the proteolytic activation pathway by expression of a new binding site, first on SK-Pg and subsequently on SK-Pm complexes, that functions as an exosite to enable specific binding and cleavage of Pg as a substrate. Conformational and proteolytic activation of Pg by SK are modulated by intrinsic differences in the affinities of 5K for ys]Pg, and ysJPm, enhanced by involvement of lysine binding sites, and regulated in vivo by fibrinogen- and fibrin-promoted assembly of productive complexes. The hypotheses will be evaluated in quantitative equilibrium binding studies employing unique fluorescent derivatives of Pg, Pm, and SK, in combination with steady-state and rapid-reaction kinetics, and protein structural approaches. Specific aims are: (1) To define the sequence of molecular events in the mechanism of conformational activation of Pg induced by SK binding. (2) To delineate the mechanism of the SK-initiated proteolytic activation pathway of Pm formation. (3) To determine the functional roles of the SK alpha, beta, and gamma-domains and individual lysine residues of SK in the mechanisms of conformational and proteolytic activation of Pg. (4) To elucidate the mechanism of fibrinogen and fibrin regulation of SK-initiated fibrinolysis and its role in the fibrin-specificity of SK therapy. The proposed studies of fundamental thermodynamic, kinetic, and structural aspects of the mechanism are expected to change the current conceptualization of the mechanism of action of SK as a thrombolytic drug. New information derived from these studies may enable more fibrin-specific thrombolytic agents to be developed.