Kinetic Characterization Of The Fast Acting Plasmin Inhibitor From Human Platelets

Abstract

An inhibitor of the plasma proteinase plasmin (EC 3.4.21.7) was partially purified from washed and lysed human blood platelets. The presence of the known plasma proteinase inhibitors in the preparations investigated was excluded by crossed immunoelectrophoresis and electroimmuno assay. The kinetics of the reaction between the inhibitor preparation and plasmin has been investigated using the synthetic chromogenic substrate S-2251 (D-Val-Leu-Lys-pNA).Residual plasmin activity was measured varying the amounts of inhibitor at various fixed concentrations of substrate (S-2251). The inhibitor is of the noncompetitive type as indicated by a Dixon-plot. The reaction between inhibitor and plasmin is fast with an estimated dissociation constant of approximately. 0.1. nM. The reaction is reversible as evidenced by the following findings: 1. inhibition in the presence of high concentrations of substrate is a fast reaction, but total inhibition of plasmin is not obtained, 2. inactivation of plasmin as a function of inhibitor concentration does not follow a simple titration curve, 3. addition of tranexamic acid increases the apparent Ki, 4. the complex beetween the inhibitor and plasmin is demonstrable in crossed immunoelectrophoresis but not in SDS-polyacrylamide gel electrophoresis. The results suggest that the platelet plasmin inhibitor is a reversible inhibitor with a high affinity for plasmin.