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


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.


CONCLUSIONS
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.
Ben Mansour
Mohamed
M
Laboratoire de Pharmacologie 04/UR/01-09, Faculté de Médecine, Monastir, Tunisia.
Dhahri
Manel
M
Vénisse
Laurence
L
Jandrot-Perrus
Martine
M
Chaubet
Frédéric
F
Maaroufi
Raoui M
RM
D000818:Animals; D000991:Antithrombins / Q000494:pharmacology*; D001777:Blood Coagulation / Q000187:drug effects; D003871:Dermatan Sulfate / Q000302:isolation & purification / Q000494:pharmacology*; D005343:Fibrinolytic Agents / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*; D015844:Heparin Cofactor II / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D012860:Skates, Fish / Q000378:metabolism*; D012867:Skin / Q000737:chemistry; D013917:Thrombin / Q000037:antagonists & inhibitors*
D000991:Antithrombins; D005343:Fibrinolytic Agents; D003871:Dermatan Sulfate; D015844:Heparin Cofactor II; D013917:Thrombin