CONVERSION OF PLASMINOGEN-ACTIVATOR INHIBITOR-1 FROM INHIBITOR TO SUBSTRATE BY POINT MUTATIONS IN THE REACTIVE-SITE LOOP

Plasminogen activator inhibitor-1 (PAI-1), the main physiological inhi bitor of tissue-type plasminogen activator (t-PA), may occur in three interconvertible conformations: active, latent, and substrate. To deli neate specific domains in the PAI-1 molecule responsible for its confo rmational flexibility and associated functional diversity, four mutant s of PAI-1 (with the amino acids at positions P-12, P-10, P-8, and P-6 , respectively, substituted with proline) were expressed in Escherichi a coli, purified, and characterized. Wild-type PAI-1 (wtPAI-1) had a s pecific activity of 21 +/- 10% (mean +/- S.D., n = 3) of the theoretic al maximum value. PAI-1-P-12 (Ala --> Pro at P-12), PAI-1-P-10 (Ser -- > Pro at P-10), and PAI-1-P-8 (Thr --> Pro at P-8) had specific activi ties of 0.06 +/- 0.03% (n = 3), 2.6 +/- 1.0% (n = 4), and 2.7 +/- 1.1% (n = 3), respectively (p < 0.03 versus wtPAI-1). PAI-1-P-6 (Val --> P ro at P-6) had a specific activity of 12 +/- 3.3% (n = 3) of the theor etical maximum value (p = not significant versus wtPAI-1). SDS-polyacr ylamide gel electrophoresis of mixtures of wtPAI-1 or PAI-1-P-6 with a 2-fold molar excess of t-PA yielded a mixture of a covalent 110-kDa t -PA PAI-1 complex (15-25%), nonreactive 45 kDa material (44-67%), and a 41-kDa band (18-31%) representing cleaved PAI-1. PAI-1-P-12, PAI-1-P -10, and PAI-1-P-8 behaved as substrates, yielding predominantly the 4 1-kDa cleavage product (85-91%) and a small amount (9-15%) of nonreact ive material. NH2-terminal amino acid sequencing revealed that cleavag e occurred at the P-1-P-1' bond (Arg(346)-Met(347)). Incubation of PAI -1-P-12, PAI-1-P-10, or PAI-1-P-8 with a 2-fold molar excess of urokin ase-type plas minogen activator, plasmin, or thrombin also primarily g enerated a 41-kDa cleavage product (62-89%). Incubation of wtPAI-1 and PAI-1-P-6 at 37 degrees C resulted in a loss of inhibitory activity, whereas the substrate behavior of PAI-1-P-12, PAI-1-P-10, and PAI-1-P- 8 remained unaltered. Treatment of the three substrate-like mutants wi th guanidinium Cl did not induce inhibitory activity. In conclusion, p oint mutations at positions P-12, P-10, and P-8 yield PAI-1 variants w ith stable substrate properties, which may facilitate more detailed st ructure/function studies.