IDENTIFICATION OF TISSUE-TYPE PLASMINOGEN ACTIVATOR-SPECIFIC PLASMINOGEN-ACTIVATOR INHIBITOR-1 MUTANTS - EVIDENCE THAT 2ND SITES OF INTERACTION CONTRIBUTE TO TARGET SPECIFICITY

Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of the plasminogen activators (PAs), tis sue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA). A library of PAL-1 mutants containing substitutions at the P-1 and P-1' 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 P-1 including Asn, Gin, His, Ser, Thr, Leu, Met, and all the aromatic amino acids. This group of mutants also demonstrated a spectrum of substitutions at P-1'. Kinetic analyses of selected variants identified P(1)Tyr and P(1)His as the most efficient tPA-specific inhibitors, with second order rate constants (k(i)) of 4.0 x 10(5) M(-1) s(-1) and 3.6 x 10(5) M(-1) s(-1), respectively. Additional PA specific PAI-1 variants containing substitutions at P-3 through P-1' were constructed. P(3)Tyr-P(2)Ser-P(1)Lys-P-1'Trp and P(3)Tyr-P(2)Ser-P(1)Tyr-P-1'Met had k(i) values of 1.7 x 10(6) M(-1) s(-1) and 2.5 x 10(6) M(-1) s(-1) against tPA, respectively, but both were inactive against uPA. In contrast, P(2)Arg-P(1)Lys-P-1'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 P(1)Arg 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.