INACTIVATION OF THROMBIN BY ANTITHROMBIN IS ACCOMPANIED BY INACTIVATION OF REGULATORY EXOSITE-I

Exosite I of the blood clotting proteinase, thrombin, mediates interac tions of the enzyme with certain inhibitors, physiological substrates and regulatory proteins, Specific binding of a fluorescein-labeled der ivative of the COOH-terminal dodecapeptide of hirudin ([5F]Hir(54-65)) to exosite I was used to probe changes in the function of the regulat ory site accompanying inactivation of thrombin by its physiological se rpin inhibitor, antithrombin, Fluorescence-monitored equilibrium bindi ng studies showed that [5F]Hir(54-65) and Hir(54-65) bound to human cu -thrombin with dissociation constants of 26 +/- 2 nM and 38 +/- 5 nM, respectively, while the affinity of the peptides for the stable thromb in-antithrombin complex was undetectable (greater than or equal to 200 -fold weaker), Kinetic studies showed that the loss of binding sites f or [5F]Hir(54-65) occurred with the same time-course as the loss of th rombin catalytic activity, Binding of [5F]Hir(54-65) and Hir(54-65) 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 resu lts support a mechanism in which thrombin and the thrombin-Hir(54-65) complex can associate with antithrombin and undergo formation of the c ovalent thrombin-antithrombin complex at modestly different rates, wit h inactivation of exosite I leading to dissociation of the peptide occ urring subsequent to the rate-limiting inactivation of thrombin, This mechanism may function physiologically in localizing the activity of t hrombin 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.