title: Mechanism of thrombin inhibition by antithrombin and heparin cofactor II in the presence of heparin.
abstract: The kinetics of thrombin inhibition by antithrombin (AT) and heparin cofactor II (HC II) were analysed as a function of the heparin concentration, from 10(-9) to 10(-4) M. The initial concentrations of inhibitor (l) and thrombin (E) were set at equimolar levels (CI = CE = 10(-8) M). The experimental data indicate that the reaction of thrombin inhibition was second-order both in the absence and in the presence of heparin, and that the apparent rate constant increased at heparin concentrations ranging from 10(-9) to 10(-6) M and decreased at higher concentrations. The data fit with the kinetic model established by Jordan et al. [J. Biol. Chem. 1979, 254, 2902-2913] for the catalysis of the thrombin-AT reaction by a low-molecular-weight heparin fraction. In this model, heparin (H) binds quickly to the inhibitor (I) and forms a heparin-inhibitor complex (HI), which is more reactive than the free inhibitor towards thrombin, leading to the formation of an inactive inhibitor-thrombin complex (I*E) and the release of free heparin, in a second step which is rate limiting. KH,I, the dissociation constant of HI, and k, the second-order rate constant of free thrombin inhibition by HI, were found to be 3.7 x 10(-7) M and 1.3 x 10(9) M-1 min-1, respectively, for AT, compared to a KH,I of 2.0 x 10(-6) M and k of 6.4 x 10(9) M-1 min-1 for HC II. These data indicate that heparin-HC II complex reactivity is greater than that of the heparin-AT complex towards thrombin, whereas heparin affinity is stronger for AT. At heparin concentrations higher than 10(-6) M, the decrease in the reaction rate was in keeping with the formation of a heparin-thrombin complex (HE), whose inactivation by the heparin-inhibitor complex (HI) is slower than that of the free protease.
authors.affiliation: Laboratoire d'hématologie, CHU Necker-Enfants Malades, Paris, France.
authors.forename: R M
authors.initials: RM
authors.lastname: Maaroufi
authors.forename: M
authors.initials: M
authors.lastname: Jozefowicz
authors.forename: J
authors.initials: J
authors.lastname: Tapon-Bretaudière
authors.forename: A M
authors.initials: AM
authors.lastname: Fischer
mesh_terms: D000991:Antithrombins / Q000494:pharmacology*; D012103:Batroxobin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology*; D015844:Heparin Cofactor II / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000991:Antithrombins; D015844:Heparin Cofactor II; D005340:Fibrinogen; D006493:Heparin; D012103:Batroxobin; D013917:Thrombin