Molecular Mechanisms of Fibrinolysis

描述(申请人提供)：拟议研究的长期目标是确定链激酶(SK)激活人类纤溶的分子机制。了解这一机制具有重要意义，因为它是使用SK作为溶栓药物治疗心肌梗死的基础。我们假设了SK启动的纤溶酶原(PG)构象和蛋白分解激活到纤溶酶(Pm)的耦合途径有一个统一的机制。PG的构象活化发生在一种快速和可逆的机制中，伴随着SK的IIe1插入PG的N端结合裂隙，导致催化部位的激活，形成SK.Pg*。Pm形成的第一个周期是由胞外介导的PG底物与SK.Pg*结合和分子间裂解形成Pm。初始循环作为一种自限触发机制来产生一个SK-Pm的等价物，它将SK隔离在紧密结合的SK.Pm络合物中，在第二个循环中催化PG完全转化为PmPG活化的偶联途径受SK对[Glu]PG、[Lys]PG和[Lys]Pm的亲和力的内在差异、[Glu]PG在紧密构象和扩展构象之间的平衡以及促进SK.Pg*和SK.Pm催化络合物形成和随后的PG底物识别的赖氨酸结合位点相互作用所控制。SK对PG的激活是由纤维蛋白原(FBG)和纤维蛋白(FBN)促进的产物复合体的组装调节的，这些机制对SK的治疗活性至关重要。针对SK激活PG及其调控机制的主要空白的假说将在平衡结合研究中进行评估，这些研究将使用活性位点标记的PG和PM类似物、稳态和快速反应动力学、突变和蛋白质结构研究。具体目的是：(1)评估偶联PG激活途径的假想机制，明确PG构象和胞外酶介导的PG底物识别的作用。(2)用快速反应动力学描述PG构象活化机制中的分子事件序列。(3)确定SK结构域在该机制中的作用，并鉴定SK的Lys残基和假Lys结构，以增强PG构象激活和底物识别。(4)阐明FBG和FBN对SK诱导的纤溶作用的调节机制。有关SK机制及其调控的新信息可能使设计更有效的溶栓药物成为可能。

DESCRIPTION (provided by applicant): The long-term goal of the proposed studies is to determine the molecular mechanism by which streptokinase (SK) activates human fibrinolysis. Understanding this mechanism is significant because it is the basis for the use of SK as a thrombolytic drug for treatment of myocardial infarction. We hypothesize a unified mechanism for the SK-initiated coupled pathway of conformational and proteolytic activation of plasminogen (Pg) to plasmin (Pm). Conformational activation of Pg occurs in a rapid and reversible mechanism accompanied by insertion of IIe1 of SK into the N-terminal binding cleft of Pg, which induces activation of the catalytic site and forms SK.Pg*. The first cycle of Pm formation is initiated by exosite-mediated Pg substrate binding to SK.Pg* and intermolecular cleavage to Pm. The initial cycle acts as a self-limiting triggering mechanism to produce one SK-equivalent of Pm which sequesters SK in the tightly bound SK.Pm complex that in the second cycle catalyzes full conversion of Pg to Pm. The coupled pathway of Pg activation is controlled by intrinsic differences between the affinities of SK for [Glu]Pg, [Lys]Pg, and [Lys]Pm, the equilibrium of [Glu]Pg between compact and extended conformations, and lysine-binding site interactions that enhance SK.Pg* and SK.Pm catalytic complex formation and subsequent Pg substrate recognition. Pg activation by SK is regulated by fibrinogen (Fbg)- and fibrin (Fbn)-promoted assembly of productive complexes through mechanisms that are crucial to the therapeutic activity of SK. Hypotheses addressing major gaps in the understanding of the mechanism of Pg activation by SK and its regulation will be evaluated in equilibrium binding studies employing active site-labeled fluorescent Pg and Pm analogs, steady-state and rapid-reaction kinetics, mutagenesis, and protein structural studies. Specific aims are: (1) To evaluate the hypothesized mechanism of the coupled Pg activation pathway, and to define the roles of Pg conformation and exosite-mediated Pg substrate recognition. (2) To delineate the sequence of molecular events in the mechanism of Pg conformational activation using rapid-reaction kinetics. (3) To determine the roles of SK domains in the mechanism, and to identify Lys residues and pseudo-Lys structures of SK that enhance Pg conformational activation and substrate recognition. (4) To elucidate the mechanism of Fbg and Fbn regulation of SK-initiated fibrinolysis. New information about the SK mechanism and its regulation may enable more effective thrombolytic drugs to be designed.