title: Inactivation of thrombin by antithrombin is accompanied by inactivation of regulatory exosite I.
abstract: Exosite I of the blood clotting proteinase, thrombin, mediates interactions of the enzyme with certain inhibitors, physiological substrates and regulatory proteins. Specific binding of a fluorescein-labeled derivative of the COOH-terminal dodecapeptide of hirudin ([5F] Hir54-65) to exosite I was used to probe changes in the function of the regulatory site accompanying inactivation of thrombin by its physiological serpin inhibitor, antithrombin. Fluorescence-monitored equilibrium binding studies showed that [5F]Hir54-65 and Hir54-65 bound to human alpha-thrombin with dissociation constants of 26 +/- 2 nM and 38 +/- 5 nM, respectively, while the affinity of the peptides for the stable thrombin-antithrombin complex was undetectable (>/=200-fold weaker). Kinetic studies showed that the loss of binding sites for [5F]Hir54-65 occurred with the same time-course as the loss of thrombin catalytic activity. Binding of [5F] Hir54-65 and Hir54-65 to thrombin was correlated quantitatively with partial inhibition of the rate of the thrombin-antithrombin reaction, maximally decreasing the bimolecular rate constants 1.7- and 2.1-fold, respectively. These results support a mechanism in which thrombin and the thrombin-Hir54-65 complex can associate with antithrombin and undergo formation of the covalent thrombin-antithrombin complex at modestly different rates, with inactivation of exosite I leading to dissociation of the peptide occurring subsequent to the rate-limiting inactivation of thrombin. This mechanism may function physiologically in localizing the activity of thrombin by allowing inactivation of thrombin that is bound in exosite I-mediated complexes with regulatory proteins, such as thrombomodulin and fibrin, without prior dissociation of these complexes. Concomitant with inactivation of thrombin, the thrombin-antithrombin complex may be irreversibly released due to exosite I inactivation.
authors.affiliation: Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA. paul.bock@mcmail.vanderbilt.edu
authors.forename: P E
authors.initials: PE
authors.lastname: Bock
authors.forename: S T
authors.initials: ST
authors.lastname: Olson
authors.forename: I
authors.initials: I
authors.lastname: Björk
mesh_terms: D000990:Antithrombin III / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D006629:Hirudins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000378:metabolism*
chemical_list: D006629:Hirudins; D046911:Macromolecular Substances; D000990:Antithrombin III; D013917:Thrombin