title: Mechanism of thrombin inhibition by heparin cofactor II and antithrombin in the presence of the ray (Raja radula) skin dermatan sulfate.
abstract: 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.
authors.affiliation: Laboratoire de Pharmacologie 04/UR/01-09, Faculté de Médecine, Monastir, Tunisia.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Ben Mansour
authors.forename: Manel
authors.initials: M
authors.lastname: Dhahri
authors.forename: Laurence
authors.initials: L
authors.lastname: Vénisse
authors.forename: Martine
authors.initials: M
authors.lastname: Jandrot-Perrus
authors.forename: Frédéric
authors.initials: F
authors.lastname: Chaubet
authors.forename: Raoui M
authors.initials: RM
authors.lastname: Maaroufi
mesh_terms: 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*
chemical_list: D000991:Antithrombins; D005343:Fibrinolytic Agents; D003871:Dermatan Sulfate; D015844:Heparin Cofactor II; D013917:Thrombin