The activities of urokinase (uPA) and tissue type plasminogen activator (tPA) are regulated by interaction with their rapidly acting inhibitor, plasmin on acting inhibitor, plasminogen activator inhibitor-1 (PAI-1). The studies outlined in this proposal will probe PAI-1 structure and function using a combination of molecular and genetic approaches. In preliminary genetic studies, a two base pair insertion has been identified in exon 4 in a patient with complete PAI-1 deficiency and a congenital bleeding disorder. Preliminary protein studies have characterized over 200 recombinant PAI-1 variants by a number of mutagenesis approaches, providing preliminary localizations for relevant functional domains. A number of new variants have been identified with unique biologic properties including increased stability, and novel, highly restricted target specificities toward either uPA or tPA. The studies outlined in the first part of the proposal, will further investigate the molecular genetics of human PAI-1 deficiency. In the second part of the proposal, analysis of PAI-1 structural mutations in vitro will characterize the specific regions of PAI-1 critical for (i) target protease specificity, (ii) binding to vitronectin, heparin and a potential """"""""second site"""""""" on tPA, (iii) transformation between the active and latent conformations, and (iv) sensitivity to inactivation by oxidants. al-antitrypsin/PAI-1 chimeric SERPINs will be analyzed and expression libraries containing random amino acid substitutions throughout the PAI-1 coding sequence will be constructed and screened to identify rare, unique mutants demonstrating gain or loss of these specific functions. In the third part of the proposal, these unique mutants will be introduced into transgenic mice in order to evaluate their potential effects in vivo. Selected mutants will be studied in a PAI-1 null background by genetic crosses with a recently developed PAI-1 deficient mouse. In addition, null mice for PAI-2 and vitronectin will be engineered by homologous recombination in embryonic stem cells. Finally, the interplay among PAI-1, PAI-2, and vitronectin in vivo will be studied by genetic crosses among these various transgenic mouse lines. The studies outlined in this proposal should provide important insights into PAI-1 structure and function, its significance in vivo, and the biology of SERPINs in general. In addition, this work will add to the understanding of the molecular pathogenesis for human bleeding and thrombotic disorders, with important diagnostic and therapeutic implications.
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