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Volume 270, Number 16, Issue of April 21, pp. 9301-9306, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.

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Patti M. Sherman , Daniel A. Lawrence , Ingrid M. Verhamme , Dell Paielli , Joseph D. Shore , David Ginsburg

Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of the plasminogen activators (PAs), tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA). A library of PAI-1 mutants containing substitutions at the Pand P` positions was screened for functional activity against tPA and thrombin. Several PAI-1 variants that were inactive against uPA in a previous study (Sherman, P. M., Lawrence, D. A., Yang, A. Y., Vandenberg, E. T., Paielli, D., Olson, S. T., Shore, J. D., and Ginsburg, D. (1992) J. Biol. Chem. 267, 7588-7595) had significant inhibitory activity toward tPA. This set of tPA-specific PAI-1 mutants contained a wide range of amino acid substitutions at Pincluding Asn, Gln, His, Ser, Thr, Leu, Met, and all the aromatic amino acids. This group of mutants also demonstrated a spectrum of substitutions at P`. Kinetic analyses of selected variants identified PTyr and PHis as the most efficient tPA-specific inhibitors, with second-order rate constants ( k) of 4.0 10 Msand 3.6 10 Ms, respectively. Additional PA-specific PAI-1 variants containing substitutions at Pthrough P` were constructed. PTyr-PSer-PLys-P`Trp and PTyr-PSer-PTyr-P`Met had kvalues of 1.7 10 Msand 2.5 10 Msagainst tPA, respectively, but both were inactive against uPA. In contrast, PArg-PLys-P`Ala inhibited uPA 74-fold more rapidly than tPA. The mutant PAI-1 library was also screened for inhibitory activity toward thrombin in the presence and absence of the cofactor heparin. While wild-type PAI-1 and several PArg variants inhibited thrombin in the absence of heparin, a number of variants were thrombin inhibitors only in the presence of heparin. These results demonstrate the importance of the reactive center residues in determining PAI-1 target specificity and suggest that second sites of interaction between inhibitors and proteases can also contribute to target specificity. Finally, the PA-specific mutants described here should provide novel reagents for dissecting the physiological role of PAI-1 both in vitro and in vivo.

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