CHARACTERIZATION OF TISSUE-TYPE PLASMINOGEN ACTIVATOR

异常血栓的形成和溶解与几个 心血管疾病，包括动脉粥样硬化和血栓栓塞症 以及出血的情况。纤溶作用的一个重要机制 是通过纤溶酶原的可用性和活性来调节的 激活剂(PA)。生理上相关的血管PA被认为是 是组织型纤溶酶原激活剂(t-PA)，因为1)其与纤维蛋白结合的能力， 2)它的活性依赖于纤维蛋白，3)它是由血管产生的 血管内皮细胞，以及4)其作为溶栓剂的高效和特异性用途 探员。这个应用程序的长期目标是理解 生理性情况下纤溶的控制。的具体目的 这项建议是为了刻画结构与功能的关系 T-PA在特异性免疫探针的辅助下。这些探测器将是一个 本研究建立的抗t-PA单抗(MAB)文库 来自用完整的天然t-PA免疫的小鼠。最初的刻画 这些MAB将确定它们是否，1)抑制t-PA活性， 2)识别天然和/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)识别被阻断的活性部位t-PA，6)识别与t-PA络合的t-PA 抗激活剂和7)针对不同的表位(表位 映射)。这一程序应允许发展若干人与生物圈计划 沿着整个t-PA分子，其中一些结合但不阻止 T-PA的功能，以及其他绑定和阻断其功能的功能。这些 然后将分析单抗对四种抗体的干扰能力 组织型纤溶酶原激活剂的功能活性及其活性部位 与纤维蛋白的相互作用，3)纤溶酶原，以及4)抗激活剂。一次 单抗被识别为这四个功能域，对应的 T-PA分子中的结构域将被确定。这些 实验将需要蛋白质分解和/或化学降解 T-PA和每个所得的多肽的鉴定 单抗。描绘了t-PA和t-PA之间发生的复杂相互作用 血液中的其他纤溶成分可能导致新的 各种血栓性疾病的诊断和治疗方法。

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.