CONSTRUCTION AND CHARACTERIZATION OF PLASMINOGEN-ACTIVATOR INHIBITOR-1 MUTANTS IN WHICH PART OF THE ACTIVE-SITE LOOP IS DELETED

Plasminogen activator inhibitor-1 (PAI-1) can occur in a labile active inhibitory conformation, a noninhibitory but cleavable substrate conf ormation and a non-reactive latent conformation. The active conformati on is not stable and converts spontaneously into the latent conformati on. To prevent this conversion, we have constructed, purified and char acterized two mutants in which part of the reactive site loop is delet ed: PAI-1-d(P6-P4) lacking residues P6 to P4 (Val-Ile-Val), and PAI-1- d(P9-P4) lacking residues P9 to P4 (Ser-Thr-Ala-Val-Ile-Val). Wild-typ e PAI-1 (wtPAI-1) revealed a specific activity of 54 +/- 8% (mean +/- SD, n = 4) of the theoretical maximum value towards tissue-type plasmi nogen activator (t-PA). Both deletion mutants revealed no inhibitory a ctivity towards t-PA or towards urokinase-type plasminogen activator ( u-PA). Conformational analysis of PAI-1-d(P9-P4) and PAI-1-d(P9-P4) re vealed the formation of a cleaved derivative originating from the subs trate form (55 +/- 3% and 84 +/- 8%, respectively) and nonreactive mat erial (45 +/- 3% and 16 +/- 8%, respectively). Incubation at 37 degree s C resulted in the conversion of the substrate conformation of both P AI-1 mutants into a non-reactive conformation. Reactivation experiment s with guanidinium chloride revealed that for both mutants this non-re active conformation represented a latent form from which the substrate properties could be restored up to 70-90%. In contrast to the latent form of wtPAI-1 and PAI-1-d(P6-P4), the non-reactive conformation of P AI-1-d(P9-P4) is not susceptible to plasmin cleavage outside the react ive site loop. Heat denaturation experiments revealed that the substra te conformation, as well as the latent conformation and the cleaved su bstrate derivative of PAI-1-d(P6-P4) and PAI-1-d(P9-P4), are less resi stant to denaturation compared to wtPAI-1. In conclusion, shortening t he reactive site loop of PAI-1 results in non-inhibitory mutants with an increased substrate behaviour. In contrast to the stable substrate conformation of wtPAI-1, the substrate conformation of these deletion mutants reveals an unexpected transition to the latent form.