ENDOTHELIAL CELL PLASMINOGEN ACTIVATOR INHIBITOR TYPE 2

血栓的异常形成和溶解与 几种心血管疾病，包括动脉粥样硬化和 血栓栓塞症和出血。纤溶活性物质 因此，系统在维护 止血平衡。很明显，许多纤溶活性物质 血浆成分要么来自内皮细胞 (ECS)(例如，组织型纤溶酶原激活剂)或与ECs结合 (例如，尿激酶型纤溶酶原激活剂)，并且EC- 因此，必须对介导的纤溶进行精确的调控。这个 意外发现，培养的内皮细胞也会产生两种 免疫学上独特的纤溶酶原激活物抑制物，包括 类型1(PAI-1)和类型2(PAL-2)都引入了以前的 意想不到的复杂程度影响了我们对此的理解 系统，并增加了改变的纤溶系统的可能性 不同处理后的细胞活性，或血液的活性 某些人类疾病，反映了这两种疾病中的任何一种 抑制剂。虽然PAI-1最近得到了广泛的研究， 有关PAI-2生产的信息很少 被ECS。这个应用程序的长期目标是开发 准确理解PAI-2在人类疾病中的作用。 这项提案的具体目的是定义这一角色 抑制血管内皮细胞介导的纤溶。PAI-2将是 以足够的数量提纯，以允许开发 PAI-2的特异性抗体和免疫学检测 活性和抗原。这些技术将在 结合免疫荧光研究，以确定其 细胞定位(例如，存在于胞浆、膜组分或 细胞外基质)在融合的脐静脉内皮细胞以及在 其他类型的EC。几个结构特征(即，分子 PAI-2的重量、等电点、碳水化合物的存在 由融合的人类ECs产生的将建立和 与从组织细胞淋巴瘤细胞中纯化的PAI-2的比较 线路，U-937。这些研究将提供一个框架来确定 免疫学方法检测纤溶酶原激活物-2合成分泌的基础速率 辅以免疫沉淀实验的检测 使用生物合成标记的PAI-2。几个因素的影响 因素(例如，细胞生长、细胞传代、血清、内毒素、 单基尼等。)关于PAI-2的生产将是 调查过了。这些研究对于描述人类免疫系统的作用至关重要 PAI-2在需要EC介导的各种过程中 蛋白质分解，如凝块溶解、组织修复、血管生成、 等，并准确定义PAI-2对改变的贡献 纤溶状态。

Abnormal thrombus formation and dissolution are associated with several cardiovascular diseases including atherosclerosis and both thromboembolic and hemorrhogic conditions. The fibrinolytic system thus assumes pivotal importance in maintaining the hemostatic balance. It is clear that many of the fibrinolytic components of plasma either originate from endothelial cells (ECs) (e.g., tissue-type plasminogen activator) or bind to ECs (e.g., urokinase-like plasminogen activator), and that EC- mediated fibrinolysis must therefore be precisely regulated. The unexpected finding that cultured ECs also produce two immunological distinct plasminogen activator inhibitors, including both type 1 (PAI-1) and type 2 (PAL-2), introduces a previously unsuspected level of complexity to our understanding of this system, and raises the possibility that the altered fibrinolytic activity of cells following various treatments, or of blood in certain human diseases, reflect changes in either one of these inhibitors. Although PAI-1 has been studied extensively recently, little information is available concerning the production of PAI-2 by ECs. The long term objective of this application is to develop an accurate understanding of the role of PAI-2 in human disease. The specific aim of this proposal is to define the role of this inhibitor in regulating EC-mediated fibrinolysis. PAI-2 will be purified in sufficient quantities to permit the development of specific antibodies and immunologic assays for both PAI-2 activity and antigen. These techniques will by employed, in conjunction with immunofluorescence studies, to define its cellular location (e.g., presence in cytosol, membrane fraction, or extracellular matrix) in confluent umbilical vein ECs, as well as in other types of ECs. Several structural features (i.e., molecular weight, isoelectric point, presence of carbohydrate) of PAI-2 produced by confluent human ECs will be established and compared to PAI-2 purified from the histocytic lymphoma cell line, U-937. These studies will provide a framework to determine the basal rate of PAI-2 synthesis and secretion using immunologic assays complemented by immunoprecipitation experiments employing biosynthetically labeled PAI-2. The effect of several factors (e.g., cell growth, cell passage, serum, endotoxin, monokines, etc.) on the production of PAI-2 will then be investigated. These studies are essential to delineate the role of PAI-2 in a variety of processes that require EC-mediated proteolysis, such as clot dissolution, tissue repair, angiogenesis, etc., and to accurately define the contribution of PAI-2 to altered fibrinolytic states.