MOLECULAR MECHANISMS OF FIBRINOLYSIS

描述：（研究者摘要） 提出了链激酶激活人纤溶酶原， 长期目标是确定SK激活的分子机制 人纤维蛋白溶解系统，这是其用作 用于治疗心血管疾病的溶栓药物。 在 目前的假设，SK激活Pg涉及两种相关模式， 互动 Pg激活由SK的独特功能启动， 通过特异性非蛋白水解诱导Pg蛋白酶活性 结合和可逆诱导构象变化， 酶原的催化位点。 Pg的构象激活在此 交互模式被假设为在 将Pg蛋白水解活化为纤溶酶（Pm）。 Pg的蛋白水解裂解是 被认为最初由SK-Pg中构象活化的Pg催化 复合物，并随后通过高浓度的磷酸化形成的SK-Pm复合物传播。 Pm的亲和结合SK对Pg的构象激活作用及其对蛋白质构象的影响 随后的蛋白水解活化过程由蛋白质中的差异调节。 SK与Pg及其蛋白水解活化产物的相互作用， 这些物种上的赖氨酸结合位点参与相互作用。 为了诱导Pg的蛋白水解激活，SK急剧地重定向Pg的蛋白水解位点。 Pm的底物特异性与不 将Pg激活为特定的Pg激活剂。 变化的起源 假设底物特异性涉及构象变化 伴随SK与Pm的结合，其影响活性位点以增强 Pg中裂解位点序列的特异性。此外， Pg激活的特异性被假定涉及第二种模式， Pg相互作用在SK-Pm和SK-Pg复合物上表达的位点， 作为一种特异性的蛋白质底物识别外位点， Pg的结合和切割。特异性的Pg的新型衍生物 在酶原的催化位点用荧光探针标记， 这些工具为拟议的评估 Pg激活的机制，通过使用荧光光谱，蛋白质 化学和酶动力学技术。 具体目标是：（1） SK诱导的Pg构象活化的定量表征 在可逆的平衡条件下。 (2)原产地的确定 SK诱导的底物特异性的变化和机制， SK诱导的Pg蛋白水解激活（3）反应的定义 SK激活Pg构象的机理。（4）SK的结构鉴定 SK-Pg相互作用结构位点及其功能测定 激活机制中的角色。 建议的结果 这些研究可望对阐明基本原理具有重要意义 SK作为溶栓药物作用中的分子事件， 为改善SK治疗提供了基础，并使设计更多 临床有效的血栓溶解剂。

DESCRIPTION: (Investigator's abstract) Investigation of the mechanism of activation of human plasminogen by streptokinase is proposed with the long-term goal of determining the molecular mechanism by which SK activates the human fibrinolytic system, which is the basis for its use as a thrombolytic drug for the treatment of cardiovascular diseases. In the current hypotheses, activation of Pg by SK involves two linked modes of interaction. Pg activation is initiated by the unique function of SK to induce proteinase activity in Pg nonproteolytically, through specific binding and reversible induction of a conformational change that activates the catalytic site of the zymogen. Conformational activation of Pg in this mode of interaction is hypothesized to act as the triggering event in the proteolytic activation of Pg to plasmin (Pm). Proteolytic cleavage of Pg is thought to be catalyzed initially by conformationally activated Pg in SK-Pg complexes and propagated subsequently by SK-Pm complexes formed by high affinity binding of Pm. Conformational activation of Pg by SK and the ensuing proteolytic activation process are modulated by differences in the interactions of SK with Pg and its proteolytic activation products, and the involvement of lysine binding sites on these species in the interactions. To induce proteolytic activation of Pg, SK redirects drastically the substrate specificity of Pm from that of a proteinase which does not activate Pg to a specific Pg activator. The origin of the change in substrate specificity is hypothesized to involve a conformational change accompanying SK binding to Pm which affects the active site to enhance specificity for the cleavage-site sequence in Pg. Moreover, the acquisition of specificity for Pg activation is postulated to involve a second mode of Pg interaction at a site expressed on SK-Pm and SK-Pg complexes that functions as a specific, protein-substrate-recognition exosite to facilitate binding and cleavage of Pg. Novel derivatives of Pg that are specifically labeled with fluorescence probes at the catalytic site of the zymogen have been developed which provide new tools for the proposed evaluation of the mechanism of Pg activation, by the use of fluorescence spectroscopy, protein chemistry, and enzyme kinetic techniques. Specific aims are: (1) Quantitative characterization of SK-induced conformational activation of Pg under reversible, equilibrium conditions. (2) Determination of the origin of the SK-induced change in substrate specificity of Pm and the mechanism of SK-induced proteolytic activation of Pg. (3) Definition of the reaction mechanism of conformational activation of Pg by SK. (4) Identification of structural sites of SK-Pg interactions and determination of their functional roles in the activation mechanism. The results of the proposed investigation are expected to have significance in elucidating fundamental molecular events in the action of SK as a thrombolytic drug, which may provide the basis for improving SK therapy and enable the design of more clinically effective thrombolytic agents.