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 (Sherma n, 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. 2 67, 7588-7595) had significant inhibitory activity toward tPA. This se t of tPA specific PAI-1 mutants contained a wide range of amino acid s ubstitutions at P-1 including Asn, Gin, His, Ser, Thr, Leu, Met, and a ll the aromatic amino acids. This group of mutants also demonstrated a spectrum of substitutions at P-1'. Kinetic analyses of selected varia nts 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 spec ific 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)T yr-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 r apidly than tPA. The mutant PAI-1 Library was also screened for inhibi tory activity toward thrombin in the presence and absence of the cofac tor heparin. While wild-type PAI-1 and several P(1)Arg variants inhibi ted thrombin in the absence of heparin, a number of variants were thro mbin inhibitors only in the presence of heparin. These results demonst rate the importance of the reactive center residues in determining PAI -1 target specificity and suggest that second sites of interaction bet ween inhibitors and proteases can also contribute to target specificit y. Finally, the PA-specific mutants described here should provide nove l reagents for dissecting the physiological role of PAI-1 both in vitr o and in vivo.