Soluble fibrin preparations inhibit the reaction of plasmin with α2-macroglobulin. Comparison with α2-antiplasmin and leupeptin

Abstract

The kinetics of plasmin inhibition by α2-antiplasmin (α2AP), α2-macroglobulin (α2M) and leupeptin were studied in the presence of fibrin monomer (Fn) and CNBr fragments of fibrinogen (Fg-CNBr). Active plasmin was detected in continuous and discontinuous assays using the chromogenic substrate D-Val-L-Leu-L-Lys p-nitroanilide hydrochloride (S-2251). The two 'fibrin-like' preparations functioned as hyperbolic mixed-type inhibitors of S-2251 hydrolysis. The dissociation constants [K(F)] for the binding of plasmin to Fn and Fg-CNBr were 22 nM and 17 nM respectively. Fn and Fg-CNBr inhibited the reaction of plasmin with α2AP; the extent of inhibition depended on the fibrin concentration. In the presence of 800 nM-Fn or 800 nM-Fg-CNBr, the experimental second-order rate constant [k''(app.)] was decreased from 2.4 x 107 M-1·s-1 to 1.2 x 106 and 5.3 x 105 M-1·s-1 respectively. The effect of Fn and Fg-CNBr on the rate of plasmin inhibition by α2M was even greater. The k''(app.) value was decreased from 4.0 x 105 M-1·s-1 to 8.0 x 102 and 1.3 x 103 M-1·s-1 in the presence of 800 nM-Fn and -Fg-CNBr respectively. By contrast, the fibrin preparations caused only a small change in the rate of plasmin inhibition by leupeptin. The maximum change in k''(app.) was 3-fold. All plasmin inhibition curves were linear, suggesting that free and fibrin-bound forms of plasmin remained in equilibrium during the course of reaction with proteinase inhibitors. Fn and Fg-CNBr had no effect on the reaction of miniplasmin with S-2251, α2AP or α2M. When 125I-plasmin was incubated with Fg-CNBr and then allowed to react with a premixed solution of α2AP and α2M, the Fg-CNBr did not significantly change the percentage of plasmin bound to α2AP. These experiments demonstrate that the reaction of plasmin with α2M is inhibited by the non-covalent binding of plasmin to fibrin. We propose that plasmin bound to the surface of a clot is protected from inhibition by α2M as well as by α2AP.