Molecular Interactions of Fibrinolysis

描述（由申请人提供）：本项目的总体目标是 阐明，在分子水平上，抑制控制的机制， 纤溶酶原激活物抑制剂-1（派-1）的纤溶作用。以下三 具体目标如下：（1）确定结构/职能、机制 派-1抑制丝氨酸蛋白酶的抑制和靶特异性。 这将涉及关键残留物和特定蛋白质的鉴定 这些区域是派-1功能的关键。(2)派-1的中和 克隆抗体我们将研究四种类型的抗体， 派-1抑制功能。第一种是加快速度 派-1自发转化为潜伏形式：第二种类型竞争 与派-1结合的蛋白酶;第三种类型延缓了 反应性中心回路（RCL）插入：第四种类型可能会阻止 RCL插入后PAI-1对蛋白酶活性中心的紊乱。（三） 确定结构决定因素、机制和功能方面， 调节派-1与玻连蛋白（Vn）和纤维蛋白的分子相互作用。 玻连蛋白是派-1与细胞相互作用的关键分子 表面和纤维蛋白凝块。我们打算研究合作的相互作用， 派-1和单体和聚合物Vn的构象变化。 我们的实验方法将包括定点突变，荧光 技术和快速反应动力学，以观察形成速率， 瞬态中间体的衰变。该项目可以提供一个基础 合理开发治疗剂的分子水平 派-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.