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.