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J. Biol. Chem., Vol. 283, Issue 17, 11645-11651, April 25, 2008

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Identification of Residues Contributing to A2 Domain-dependent Structural Stability in Factor VIII and Factor VIIIa^*

From the Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642

Factor VIII circulates as a heterodimer composed of heavy (A1A2B domains) and light (A3C1C2 domains) chains, whereas the contiguous A1A2 domains are separate subunits in the active cofactor, factor VIIIa. Whereas the A1 subunit maintains a stable interaction with the A3C1C2 subunit, the A2 subunit is weakly associated in factor VIIIa and its dissociation accounts for the labile activity of the cofactor. In examining the ceruloplasmin-based factor VIII A domain model, potential hydrogen bonding based upon spatial separations of <2.8Å were found between side chains of 14 A2 domain residues and 7 and 9 residues in the A1 and A3 domains, respectively. These residues were individually replaced with Ala, except Tyr residues were replaced with Phe, and proteins stably expressed to examine the contribution of each residue to protein stability. Factor VIII stability at 55 °C and factor VIIIa activity were monitored using factor Xa generation assays. Fourteen of 30 factor VIII mutants showed >2-fold increases in either or both decay rates compared with wild type; whereas, 7 mutants showed >2-fold increased rates in factor VIIIa decay compared with factor VIII decay. These results suggested that multiple residues at the A1-A2 and A2-A3 domain interfaces contribute to stabilizing the protein. Furthermore, these data discriminate residues that stabilize interactions in the procofactor from those in the cofactor, where hydrogen bonding in the latter appears to contribute more significantly to stability. This observation is consistent with an altered conformation involving new inter-subunit interactions involving A2 domain following procofactor activation.

Received for publication, December 17, 2007 , and in revised form, February 6, 2008.

^* This work was supported by National Institutes of Health Grants HL38199 and HL76213. An account of this work was presented at the 49th Annual Meeting of the American Society of Hematology, December 8, 2007, in Atlanta, GA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure.

¹ To whom correspondence should be addressed: 601 Elmwood Ave., Rochester, NY 14642. Tel.: 585-275-6576; Fax: 585-473-4314; E-mail: philip_fay{at}urmc.rochester.edu.

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