Molecular Interactions of Fibrinolysis

描述(由申请人提供)：本项目的总体目标是 从分子水平阐明抑制控制的机制 纤溶酶原激活物抑制物-1(PAI-1)的纤溶作用。以下三项 将追求具体目标：(1)确定结构/功能、机制 PAI-1抑制丝氨酸蛋白酶的抑制作用和靶标特异性。 这将涉及关键残基和特定蛋白质的鉴定。 PAI-1功能的关键区域。(2)中和PAI-1 单克隆抗体。我们将研究四种类型的抗体 失活PAI-1抑制功能。第一种类型加速了速度 PAI-1自发转化为潜伏型：第二类竞争 与PAI-1结合的蛋白酶；第三种类型延缓了 反应性中心环(RCL)插入：第四种类型可能阻止 PAI-L在RCL植入后对蛋白酶活性中心的干扰。(3) 确定结构决定因素、机制和功能方面 调节PAI-1与玻璃连结蛋白(VN)和纤维蛋白的分子相互作用。 Vitronectin是PAI-1与细胞相互作用的关键分子 表面和纤维蛋白凝块。我们打算研究合作互动和 PAI-1与单体和聚合物VN的构象变化。 我们的实验方法将涉及定点突变、荧光 技术和快速反应动力学，以观察形成速度和 瞬变中间体的衰变。这一项目可以为 分子水平上合理开发调理治疗剂 PAI-1活性。

DESCRIPTION (provided by applicant): The overall goal of this project is to elucidate, on a molecular level, the mechanisms of inhibitory control of fibrinolysis by plasminogen activator inhibitor-1 (PAI-1). The following three specific aims will be pursued: (1) Determine the structure/function, mechanisms of inhibition and target specificity for PAI-1 inhibition of serine proteases. This will involve identification of critical residues and specific protein regions which are key to PAI-1 function. (2) Neutralization of PAI-1 by monoclonal antibodies. We will investigate four types of antibodies which inactivate the PAI-1 inhibitory function. The first type accelerates the rate of spontaneous conversion of PAI-1 to a latent form: the second type competes with proteinases for binding to PAI-1; the third type retards the rate of reactive center loop (RCL) insertion: and the fourth type probably prevents the disordering of the proteinase active center by PAI-l after RCL insertion. (3) Identify structural determinants, mechanisms and functional aspects that regulate the molecular interactions of PAI-1 with vitronectin (Vn) and fibrin. Vitronectin is the key molecule involved in interaction of PAI-1 with cell surfaces and fibrin clots. We intend to study the cooperative interactions and conformational changes of PAI-1 and monomeric and polymeric Vn. Our experimental approach will involve site-directed mutagenesis, fluorescence techniques, and rapid reaction kinetics to observe the rates of formation and decay of transient intermediates. This project can provide a foundation at the molecular level for rational development of therapeutic agents to modulate PAI-1 activity.