Abstract
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.
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.
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.
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.