PLASMINOGEN ACTIVATOR INHIBITOR/VITRONECTIN INTERACTIONS

纤溶酶原激活剂抑制剂/玻连蛋白相互作用

• 批准号: 6564878
• 负责人: DAVID J LOSKUTOFF
• 金额: $ 32.14万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6564878
• 关键词: atherosclerosis; bone marrow transplantation; enzyme activity; fibrinolysis; genetically modified animals; human subject; laboratory mouse; monoclonal antibody; plasminogen activator inhibitors; protein binding; protein protein interaction; protein structure function; receptor expression; recombinant proteins; thrombosis; vitronectin

Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of plasminogen activation in vivo. It also bind avidly and specifically too the extracellular matrix protein vitronectin (VN), and in so doing, regulates urokinase receptor (uPAR)- and integrin-mediated cell adhesion to this adhesive glycoprotein. Thus, PAI-I is a novel link between extracellular proteolysis and cell adhesion/migration, two processes that play critical roles in vascular biology. This proposal examines the biochemistry (Aim 1), cell biology (Aim 2), cell biology (Aim 2) and physiology/pathophysiology (Aim 3) of the PAI/VN interaction. The guiding hypothesis is that detailed examination of the PAI-1/VN interaction in vitro and in vivo will provide novel insights into the structure and function of both molecules, and will help to clarify their respective roles in thrombus formation and dissolution. In Aim 1, the structurally altered or "modified" forms of VN will be purified from platelets, and employed together with monoclonal antibodies and recombinant variants of VN in a systematic and quantitative analysis of the interaction of PAI-1, uPAR and integrins with VN. The essential domains, kinetic constants, and biochemical consequence of these interactions will be determined. In Aim 2, the generality of uPAR as a cell adhesion receptor will be established. The mechanisms by which PAI-1 regulates uPAR- and integrin-mediated cell adhesion/migration will be investigated in vitro using cells cultured on a variety of VN fragments and variants, and in vivo using a recently described, PAI-I dependent, murine model of tumor angiogenesis. The availability of VN- and PAI-1- deficient mice, and of cells and serum derived from them, will aid these studies. Finally, in Aim 3, the origin of murine platelet and tissue N will be investigated, and the effects of PAI-1 on the structure and function of VN in vivo in platelets and tissues under normal and pathological conditions will be determined. These studies will rely on bone marrow transplantation experiments to generate VN-deficient mice containing platelets derived from normal marrow and vice versa. These studies directly relate to the central theme of this Program Project grant and involve extensive collaborations with all projects and cores.

纤溶酶原激活剂抑制剂-1 (PAI-1) 是体内纤溶酶原激活的主要生理抑制剂。它还强烈且特异性地结合细胞外基质蛋白玻连蛋白 (VN)，从而调节尿激酶受体 (uPAR) 和整合素介导的细胞与这种粘附糖蛋白的粘附。因此，PAI-I是细胞外蛋白水解和细胞粘附/迁移之间的新联系，这两个过程在血管生物学中发挥着关键作用。该提案研究了 PAI/VN 相互作用的生物化学（目标 1）、细胞生物学（目标 2）、细胞生物学（目标 2）和生理学/病理生理学（目标 3）。指导性假设是，对体外和体内 PAI-1/VN 相互作用的详细检查将为这两种分子的结构和功能提供新的见解，并有助于阐明它们各自在血栓形成和溶解中的作用。在目标 1 中，结构改变或“修饰”形式的 VN 将从血小板中纯化，并与单克隆抗体和 VN 重组变体一起用于 PAI-1、uPAR 和整合素与 VN 相互作用的系统定量分析。这些相互作用的基本域、动力学常数和生化结果将被确定。在目标 2 中，将建立 uPAR 作为细胞粘附受体的普遍性。 PAI-1 调节 uPAR 和整合素介导的细胞粘附/迁移的机制将使用在各种 VN 片段和变体上培养的细胞进行体外研究，并使用最近描述的 PAI-1 依赖性小鼠模型进行体内研究肿瘤血管生成。 VN 和 PAI-1 缺陷小鼠以及源自它们的细胞和血清的可用性将有助于这些研究。最后，在目标3中，将研究小鼠血小板和组织N的起源，并确定PAI-1对正常和病理条件下体内血小板和组织中VN的结构和功能的影响。这些研究将依靠骨髓移植实验来产生含有来自正常骨髓的血小板的 VN 缺陷小鼠，反之亦然。这些研究与该计划项目拨款的中心主题直接相关，并涉及与所有项目和核心的广泛合作。