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J. Biol. Chem., Vol. 279, Issue 51, 53691-53698, December 17, 2004

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Surface-exposed Hemophilic Mutations across the Factor VIII C2 Domain Have Variable Effects on Stability and Binding Activities^*

P. Clint Spiegel, Paul Murphy¶, and Barry L. Stoddard¶||

From the Graduate Program in Biomolecular Structure and Design and the Medical Scientist Training Program, University of Washington, Seattle, Washington 98195 and the ^¶Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109

Factor VIII (fVIII) is a plasma glycoprotein that functions as an essential cofactor in blood coagulation. Its carboxyl-terminal "C2" domain is responsible for binding to both activated platelet surfaces and von Willebrand factor. We characterized the effect of 20 hemophilia-associated missense mutations across this domain (that all occur in patients in vivo) on its stability and its binding activities. At least six of these mutations were severely destabilizing, and another four caused moderate destabilization and corresponding reductions in both binding functions. One mutant (A2201P) displayed a significant reduction in its membrane binding activity but normal von Willebrand factor binding, while two others (P2300S and R2304H) caused the opposite effect. Several mutations (including L2210P, V2223M, M2238V, and R2304C) displayed near wild-type stabilities and binding activities and may instead affect mRNA splicing or alternative properties or functions of the protein. This study demonstrated that von Willebrand factor and membrane binding activities can be uncoupled and uniquely disrupted by different mutations and that either effect can lead to similar reductions in clotting activity. It also illustrated how a heterogeneous genetic disorder causes diverse molecular phenotypes that result in similar disease states.

Received for publication, August 16, 2004 , and in revised form, October 1, 2004.

^* 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.

^|| To whom correspondence should be addressed: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Mailstop A3-025, 1100 Fairview Ave. N., Seattle, WA 98109. Tel.: 206-667-4031; Fax: 206-667-6877; E-mail: bstoddar{at}fhcrc.org.

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