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Volume 272, Number 32, Issue of August 8, 1997 pp. 19837-19845
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Inactivation of Thrombin by Antithrombin Is Accompanied by Inactivation of Regulatory Exosite I

(Received for publication, April 30, 1997)

Paul E. Bock , Steven T. Olson ^¶ and Ingemar Björk

From the  Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, the ^¶ Center for Molecular Biology of Oral Diseases, University of Illinois-Chicago, Chicago, Illinois 60612, and the  Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, S-75123 Uppsala, Sweden

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] Hir^54-65) 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]Hir^54-65 and Hir^54-65 bound to human -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]Hir^54-65 occurred with the same time-course as the loss of thrombin 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 results support a mechanism in which thrombin and the thrombin-Hir^54-65 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.

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