PLASMINOGEN ACTIVATOR INHIBITOR/VITRONECTIN INTERACTIONS

纤溶酶原激活物抑制剂-1（派-1）是体内纤溶酶原激活的主要生理抑制剂。它还与细胞外基质蛋白质玻连蛋白（VN）结合，并在这样做时，调节尿激酶受体（uPAR）和整合素介导的细胞粘附到这种粘附糖蛋白。因此，PAI-1是细胞外蛋白水解和细胞粘附/迁移之间的一个新的联系，这两个过程在血管生物学中发挥关键作用。本提案检查派/VN相互作用的生物化学（目的1）、细胞生物学（目的2）、细胞生物学（目的2）和生理学/病理生理学（目的3）。指导性假设是，详细检查派-1/VN在体外和体内的相互作用将提供新的见解，这两种分子的结构和功能，并将有助于澄清各自的作用，在血栓形成和溶解。在目的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派-1、uPAR和整联蛋白与VN的相互作用的系统和定量分析。这些相互作用的基本结构域，动力学常数和生化后果将被确定。在目的2中，将建立uPAR作为细胞粘附受体的一般性。派-1调节uPAR和整合素介导的细胞粘附/迁移的机制将在体外使用在各种VN片段和变体上培养的细胞进行研究，并在体内使用最近描述的PAI-1依赖性小鼠肿瘤血管生成模型进行研究。VN和派-1缺陷小鼠的可用性，以及来自它们的细胞和血清，将有助于这些研究。最后，在目的3中，将研究小鼠血小板和组织N的来源，并将确定派-1对正常和病理条件下血小板和组织中VN的体内结构和功能的影响。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赠款的中心主题，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.