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J Biol Chem, Vol. 274, Issue 26, 18635-18643, June 25, 1999

Role of Regulatory Exosite I in Binding of Thrombin to Human Factor V, Factor Va, Factor Va Subunits, and Activation Fragments

Kumudini R. DharmawardanaDagger , Steven T. Olson§, and Paul E. BockDagger

From the Dagger  Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and the § Center for Molecular Biology of Oral Diseases, University of Illinois, Chicago, Illinois 60612

The blood coagulation proteinase, thrombin, converts factor V into factor Va through a multistep activation pathway that is regulated by interactions with thrombin exosites. Thrombin exosite interactions with human factor V and its activation products were quantitatively characterized in equilibrium binding studies based on fluorescence changes of thrombin covalently labeled with 2-anilinonaphthalene-6-sulfonic acid (ANS) linked to the catalytic site histidine residue by Nalpha -[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl ([ANS]FPR-thrombin). Exosite I was shown to play a predominant role in the binding of factor V and factor Va from the effect of the exosite I-specific ligand, hirudin54-65, on the interactions. Factor V and factor Va bound to exosite I of [ANS]FPR-thrombin with similar dissociation constants of 3.4 ± 1.3 and 1.1 ± 0.4 µM and fluorescence enhancements of 182 ± 41 and 127 ± 17%, respectively. Native thrombin and labeled thrombin bound with similar affinity to factor Va. Among factor V activation products, the factor Va heavy chain was shown to contain the site of exosite I binding, whereas exosite I-independent, lower affinity interactions were observed for activation fragments E and C1, and no detectable binding was observed for the factor Va light chain. The results support the conclusion that the factor V activation pathway is initiated by exosite I-mediated binding of thrombin to a site in the heavy chain region of factor V that facilitates the initial cleavage at Arg709 to generate the heavy chain of factor Va. The results further suggest that binding of thrombin through exosite I to factor V activation intermediates may regulate their conversion to factor Va and that similar binding of thrombin to the factor Va produced may reflect a mode of interaction involved in the regulation of prothrombin activation.

Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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