Effects of the synthetic fibrinolytic agents ortho-thymotic acid and S-1623 on the reaction between human plasmin and antiplasmin

The effects of two synthetic fibrinolytic agents (SFA), ortho-thymotic acid (methyl-6 isopropyl-3 hydroxy-2 benzoic acid) and S-1623 (α(isobutyl-4 cyclohexene-1 yl) propionic acid) on the reaction between plasmin and antiplasmin, the fast-acting plasmin inhibitor in human plasma, were investigated in purified systems. The fibrinolytic agents were incubated with antiplasmin and/or plasmin and residual plasmin activity measured with the tripeptide substrate D-Val-Leu-Lys-pNA. At concentrations of the SFA between 2 and 7.5 mM, a concentration and time dependent inactivation of antiplasmin was observed but no inhibition of plasmin or of the plasmin-antiplasmin reaction. The inactivation rate followed pseudo-first order kinetics but the rate constant increased rapidly with the concentration of SFA. Hill plots indicated that antiplasmin was inactivated after binding of about 4 mol of the SFA's per mol. Incubation of 14C-S-1623 and antiplasmin showed that approximately 25 molecules of the SFA remained associated with antiplasmin. The activity of the inhibitor was not restored upon removal of excess reagent and the protein was aggregated as indicated by SDS-polyacrylamide gel electrophoresis. At concentrations of the SFA's above 10mM inactivation of plasmin was observed. These findings may explain why these substances are fibrinolytically active in a very narrow concentration range only.