PLASMINOGEN ACTIVATOR INHIBITOR/VITRONECTIN INTERACTIONS

纤溶酶原激活物抑制剂-1 （PAI-1）是体内纤溶酶原激活的主要生理抑制剂。它还能与细胞外基质蛋白玻璃体连接蛋白（VN）紧密特异性结合，从而调节尿激酶受体（uPAR）和整合素介导的细胞对这种黏附糖蛋白的粘附。因此，pai - 1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起着至关重要的作用。本提案探讨了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）。指导假设是，在体外和体内详细检查PAI-1/VN相互作用将为这两种分子的结构和功能提供新的见解，并将有助于阐明它们在血栓形成和溶解中的各自作用。在Aim 1中，将从血小板中纯化结构改变或“修饰”形式的VN，并与VN的单克隆抗体和重组变体一起用于系统和定量分析PAI-1、uPAR和整合素与VN的相互作用。这些相互作用的基本领域、动力学常数和生化后果将被确定。在Aim 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/迁移的机制将在体外通过多种VN片段和变体培养的细胞进行研究，并在体内使用最近描述的依赖PAI-1的肿瘤血管生成小鼠模型进行研究。VN-和PAI-1缺陷小鼠及其细胞和血清的可用性将有助于这些研究。最后，在Aim 3中，我们将研究小鼠血小板和组织N的来源，并确定在正常和病理条件下，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。这些研究直接关系到本计划项目资助的中心主题，并涉及与所有项目和核心的广泛合作。

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.