CHARACTERIZATION OF TISSUE-TYPE PLASMINOGEN ACTIVATOR

异常的血栓形成和溶解与几种 心血管疾病，包括动脉粥样硬化和血栓栓塞 和出血情况。 纤维蛋白溶解的一个重要机制是 是通过纤溶酶原的可用性和活性来调节的 活化剂（PA）。 生理相关的血管PA被认为是 是组织型PA（t-PA），因为1）其结合纤维蛋白的能力， 2）其活性依赖于纤维蛋白，3）其通过血管生成， 内皮细胞，以及4）其作为血栓溶解剂的有效和特异性用途 剂 本申请的长期目标是了解 控制生理情况下的纤维蛋白溶解。 具体目标是 这一建议是为了表征结构-功能关系， t-PA的特异性免疫探针的帮助下。 这些探测器将是一个 本研究建立了t-PA单克隆抗体库 来自用完整的天然t-PA免疫的小鼠。 初始表征 将决定它们是否，1）抑制t-PA活性， 2）识别天然的和/或变性的t-PA（构象特异性表位）， 3)识别t-PA的A链或B链，4）识别与纤维蛋白结合的t-PA， 5)识别活性位点“阻断的”t-PA，6）识别与 抗激活剂，和7）针对不同的表位（表位 映射）。 这一程序应允许制定一些人与生物圈计划 沿着整个t-PA分子，其中一些结合但不阻断 t-PA的功能，以及其他结合和阻断其功能的物质。 这些 然后将分析MAB干扰四种细胞的能力。 t-PA的功能活性，包括其1）活性位点，及其 与2）纤维蛋白，3）纤溶酶原和4）抗激活剂的相互作用。 一旦 MAB被鉴定为这四个功能结构域，相应的 将鉴定t-PA分子中的结构域。 这些 实验将需要蛋白水解和/或化学降解 t-PA和用免疫组织化学方法鉴定每种所得肽 MAB。 描述了t-PA和t-PA之间发生的复杂相互作用， 血液中的其他纤维蛋白溶解成分可能导致新的 用于诊断和治疗各种血栓性疾病的方法。

Abnormal thrombus formation and dissolution are associated with several cardiovascular diseases including atherosclerosis and both thromboembolic and hemorrhagic conditions. One important mechanism by which fibrinolysis is regulated is through the availability and activity of plasminogen activators (PAs). The physiologically relevant vascular PA is believed to be tissue-type PA (t-PA) because of 1) its ability to bind to fibrin, 2)\its dependency on fibrin for activity, 3) its production by vascular endothelial cells, and 4) its efficient and specific use as a thrombolytic agent. The long range objective of this application is to understand the control of fibrinolysis in physiological situations. The specific aim of this proposal is to characterize the structure-function relationship of t-PA with the aid of specific immunological probes. These probes will be a library of monoclonal antibodies (MABs) to t-PA developed in this study from mice immunized with intact, native t-PA. The initial characterization of these MABs will determine whether they, 1) inhibit t-PA activity, 2)\recognize native and/or denatured t-PA (conformation specific epitopes), 3) recognize the A- or B-chain of t-PA, 4) recognize t-PA bound to fibrin, 5) recognize active site 'blocked' t-PA, 6) recognize t-PA complexed to the antiactivator, and 7) are directed toward distinct epitopes (epitope mapping). This procedure should permit the development of a number of MABs along the entire t-PA molecule, some of which bind but do not block the function of t-PA, and others which bind and blocks its function. These MABs will then be analyzed for their ability to interfere with four functional activities of t-PA including its 1) active site, and its interaction with 2) fibrin, 3) plasminogen, and 4) the antiactivator. Once MABs are identified to these four functional domains, the corresponding structural domains in the t-PA molecule will be identified. These experiments will require the proteolytic and/or chemical degradation of t-PA and the identification of each of the resulting peptides with the MABs. Delineating the complex interactions that occur between t-PA and other fibrinolytic components in blood may lead to the development of new approaches for the diagnosis and treatment of various thrombotic diseases.