CHARACTERIZATION OF PLASMINOGEN-ACTIVATOR INHIBITOR-1 MUTANTS CONTAINING THE P13 TO P10 REGION OF OVALBUMIN OR ANTITHROMBIN-III - EVIDENCE THAT THE P13 RESIDUE CONTRIBUTES SIGNIFICANTLY TO THE ACTIVE TO SUBSTRATE TRANSITION

The serpin plasminogen activator inhibitor 1 (PAI-I) can occur, in vit ro, in both an inhibitory and a non-inhibitory but cleavable substrate form. In the present study, we have evaluated the effect of replacing the P13 to P10 region of PAI-1 (Val-Ala-Ser-Ser), with the P13 to P10 region of either the non-inhibitory serpin ovalbumin (Glu-Val-Val-Gly ; PAI-l-ovalbumin) or the inhibitory serpin antithrombin III (Glu-Ala- Ala-Ala; PAI-l-antithrombin III). In addition, we have replaced Val at position P13 with Glu (PAI-1-P13 (Val --> Glu)). Wild-type (wt) PAI-1 revealed specific activities of 80 +/- 9% (mean +/- S.D., n=4) of the theoretical maximum value towards t-PA. PAI-l-ovalbumin, PAI-l-antith rombin III and PAI-1-P13 (Val-->Glu) revealed specific activities of 8 6+/-15%, 77+/-11%, and 100+/-30% respectively, towards t-PA and simila r inhibitory properties towards u-PA. Surprisingly, upon inactivation at 37 degrees C, the active conformation of the PAI-1 mutants converte d partly into a substrate conformation (i.e. 52+/-52%, 55+/-8.2% and 4 6+/-4.6% for PAI-I-ovalbumin, PAI-1-antithrombin III and PAI-1-P13 (Va l - Glu), respectively) and partly into a latent conformation. This is in contrast to active wtPAI-1 which, as expected, is converted to the latent conformation (i.e. 86 +/- 1.0%), In conclusion, even though re placement of the P13 to P10 region of PAI-1 by the corresponding regio n of a non-inhibitory serpin or of an inhibitory serpin, does not dire ctly affect its inhibitory properties, the nature of the amino acids i n this region and of P13 in particular, contributes to its conformatio nal transitions. (C) 1998 Elsevier Science B.V. All rights reserved.