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.
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