Molecular Interactions of Fibrinolysis

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DESCRIPTION (provided by applicant): The overall goal of this project is to elucidate, on a molecular level, the mechanisms of inhibitory control of fibrinolysis by plasminogen activator inhibitor-1 (PAI-1). The following three specific aims will be pursued: (1) Determine the structure/function, mechanisms of inhibition and target specificity for PAI-1 inhibition of serine proteases. This will involve identification of critical residues and specific protein regions which are key to PAI-1 function. (2) Neutralization of PAI-1 by monoclonal antibodies. We will investigate four types of antibodies which inactivate the PAI-1 inhibitory function. The first type accelerates the rate of spontaneous conversion of PAI-1 to a latent form: the second type competes with proteinases for binding to PAI-1; the third type retards the rate of reactive center loop (RCL) insertion: and the fourth type probably prevents the disordering of the proteinase active center by PAI-l after RCL insertion. (3) Identify structural determinants, mechanisms and functional aspects that regulate the molecular interactions of PAI-1 with vitronectin (Vn) and fibrin. Vitronectin is the key molecule involved in interaction of PAI-1 with cell surfaces and fibrin clots. We intend to study the cooperative interactions and conformational changes of PAI-1 and monomeric and polymeric Vn. Our experimental approach will involve site-directed mutagenesis, fluorescence techniques, and rapid reaction kinetics to observe the rates of formation and decay of transient intermediates. This project can provide a foundation at the molecular level for rational development of therapeutic agents to modulate PAI-1 activity.