Department of Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92037, and Department of Chemistry, University of California, San Diego, LaJolla, California 92093 Received June 12, 1987; Revised Manuscript Received December 23, 1987 abstract: The plasminogen activatorinhibitor 1 (PAI-1) synthesized and released by cultured bovine aortic endothelial cells is present in conditioned medium in a latent form that can be activated by guanidine hydrochloride [Hekman, C. M., & Loskutoff, D. J.(1985) J. Biol. Chem. 260, 11581-11587]. The purified, guanidine-activated PAI-1 was shown to inhibit both plasmin and trypsin in a dose-andtime-dependent manner. Second-order rate constants for these interactions were calculated to be 6.6 X 105 and 7.0 X 106 M-1 s_1 for plasmin and trypsin, respectively. Experiments were conducted to compare the inherently active and the guanidine-activated forms of PAI-1. The two active forms had similar kinetic parameters for interaction with urokinase (ATd, 0.3 pM; fcassoc, 1.5 X 108 M™ 1 s_1) and were both inactivated upon treatment with acid or base andby incubation at 37 C. The latent form was relatively stablewhen incubated under similar conditions. Thedecrease in PAI-1 activity upon incubation at 37 C was partially restored by a second treatment with guanidine hydrochloride. However, the degree of recovery decreased as a function of incubation time at 37 C. These data suggest that active and guanidine-activated PAI-1 represent a single form of PAI-1. Incubation of this form at 37 C yields two distinct populations of inactive PAI-1, one capable of reactivation and anotherthat appears to be irreversibly inactivated. e specific lysis of fibrin is catalyzed by the protease plasmin that exists in plasma as the inactive zymogen, plas-minogen (Collen, 1980). The generation of plasmin occurs through the limited cleavage of plasminogen by the plasmi-nogen activators, urokinase (UK), 1 and tissue plasminogen activator (tPA). In addition to their role in fibrinolysis, the plasminogen activators (PA’s) have been implicated in various other biological processes including ovulation (Beers et al.,