Soluble fibrin preparations inhibit the reaction of plasmin with x2-macroglobulin

The kinetics of plasmin inhibition by cx2-antiplasmin (az2AP), a2-macroglobulin (a2M) 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 (KF) 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 a2AP; 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 s1to 1.2 x 106 and 5.3 x I05 M-1 * s-s respectively. The effect of Fn and Fg-CNBr on the rate of plasmin inhibition by a2M 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, a2AP or a2M. When 125I-plasmin was incubated with Fg-CNBr and then allowed to react with a premixed solution of a2AP and a2M, the Fg-CNBr did not significantly change the percentage of plasmin bound to a2AP. These experiments demonstrate that the reaction of plasmin with a2M 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 a2M as well as by a2AP.