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: 6713653
• 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: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6713653
• 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.

纤溶酶原激活物抑制物-1(PAI-1)是体内纤溶酶原激活的主要生理抑制物。它还与细胞外基质蛋白Vitronectin(VN)结合，并在此过程中调节尿激酶受体(UPAR)和整合素介导的细胞与这种粘附性糖蛋白的黏附。因此，PAI-I是细胞外蛋白降解和细胞黏附/迁移之间的一种新的联系，这两个过程在血管生物学中发挥着关键作用。该建议研究了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，详细研究PAI-1/VN在体外和体内的相互作用将为这两个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，我们将从血小板中提纯结构改变或“修饰”的VN，并与VN的单抗和重组变异体一起用于系统和定量地分析PAI-1、uPAR和整合素与VN的相互作用。这些相互作用的基本结构域、动力学常数和生化结果将被确定。在目标2中，将建立uPAR作为细胞黏附受体的一般性。PAI-1调节uPAR和整合素介导的细胞黏附/迁移的机制将在体外用培养在各种VN片段和变体上的细胞来研究，并在体内用最近描述的PAI-I依赖的肿瘤血管生成的小鼠模型来研究。VN和PAI-1缺陷小鼠及其衍生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，将探讨小鼠血小板和组织N的来源，以及正常和病理条件下PAI-1对体内血小板和组织中VN结构和功能的影响。这些研究将依赖于骨髓移植实验，以产生含有来自正常骨髓的血小板的VN缺陷小鼠，反之亦然。这些研究直接涉及本计划项目赠款的中心主题，并涉及与所有项目和核心的广泛合作。