Mechanism of thrombin inhibition by heparin cofactor II and antithrombin in the presence of the ray ( Raja radula) skin dermatan sulfate

Abstract

Introduction The kinetics of the thrombin inhibition by heparin cofactor II (HCII) and antithrombin (AT) have been studied as a function of the concentration of a dermatan sulfate (DS) from the skin of the ray Raja radula. Materials and methods The initial concentrations of inhibitor (I), HCII or AT, and thrombin (E) were set at equimolecular levels (3.10 - 9 M). Analysis of the experimental data obtained for DS concentrations ranging from 10 - 8 to 10 - 4 M was performed according to a previously described model in which DS binds quickly to the inhibitor and forms a complex more reactive than the free inhibitor towards thrombin. Results The apparent rate constant of the thrombin inhibition, k app, by either HCII or AT, increased in a concentration-dependent manner for DS concentrations up to 10 - 5 M or 10 - 6 M, respectively. At higher DS concentrations, k app remained unchanged for thrombin inhibition by HCII whereas a decrease in k app was observed for the thrombin-AT reaction. The dissociation constant of the polysaccharide-inhibitor complex, K DSI, and the rate constant of the thrombin inhibition by this complex, k, were (7.81 ± 0.75).10 - 7 M and (2.84 ± 0.42).10 9 M - 1 .min - 1 , whereas they were (4.93 ± 0.31).10 - 7 M and (2.47 ± 0.28).10 8 M - 1 .min - 1 , when the inhibitor was either HCII or AT, respectively. Conclusion DS from ray skin catalyzes the thrombin inhibition by HCII or AT primarily by forming a DS-inhibitor complex more reactive than the free inhibitor towards the protease. The affinity of DS for HCII was approximately 2-fold higher whereas the catalyzed reaction rate constant was approximately 20-fold higher when compared to AT.