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J. Biol. Chem., Vol. 282, Issue 30, 21848-21855, July 27, 2007

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Characterization of a Factor Xa Binding Site on Factor Va near the Arg-506 Activated Protein C Cleavage Site^*

Andrew J. Gale¹, Subramanian Yegneswaran, Xiao Xu, Jean-Luc Pellequer, and John H. Griffin

From the Department of Molecular and Experimental Medicine, The Scripps Research Institute, La, Jolla, California 92037 and the Commissariat à l'Energie Atomique Valrhô, Institute of Biotechnology and Environmental Biology, DSV/iBEB/SBTN/LIRM, 30207 Bagnols-sur Cèze, Cedex, France

Prothrombin is proteolytically activated by the prothrombinase complex comprising the serine protease Factor (F) Xa complexed with its cofactor, FVa. Based on inhibition of the prothrombinase complex by synthetic peptides, FVa residues 493–506 were proposed as a FXa binding site. FVa is homologous to FVIIIa, the cofactor for the FIXa protease, in the FX-activating complex, and FVIIIa residues 555–561 (homologous to FVa residues 499–506) are recognized as a FIXa binding sequence. To test the hypothesis that FVa residues 499–505 contribute to FXa binding, we created the FVa loop swap mutant (designated 499–505_VIII FV) with residues 499–505 replaced by residues 555–561 of FVIIIa, which differ at five of seven positions. Based on kinetic measurements and spectroscopic titrations, this FVa loop swap mutant had significantly reduced affinity for FXa. The fully formed prothrombinase complex containing this FVa mutant had fairly normal kinetic parameters (k_cat and K_m) for cleavage of prothrombin at Arg-320. However, small changes in both Arg-320 and Arg-271 cleavage rates result together in a moderate change in the pathway of prothrombin activation. Although residues 499–505 directly precede the Arg-506 cleavage site for activated protein C (APC), the 499–505_VIII FVa mutant was inactivated entirely normally by APC. These results suggest that this A2 domain sequence of the FVa and FVIIIa cofactors evolved to have different specificity for binding FXa and FIXa while retaining compatibility as substrate for APC. In an updated three-dimensional model for the FVa structure, residues 499–505, along with Arg-506, Arg-306, and other previously suggested FXa binding sequences, delineate a continuous surface on the A2 domain that is strongly implicated as an extended FXa binding surface in the prothrombinase complex.

Received for publication, March 13, 2007 , and in revised form, May 24, 2007.

^* This study was supported by National Institutes of Health Grants HL82588 (to A. J. G.), HL07695 (to S. Y.), and HL21544 (to J. H. G.) and by a grant from the Sam and Rose Stein Endowment Fund and the Armstrong McDonald Foundation (to A. J. G.). 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.

¹ To whom correspondence should be addressed: Dept. of Molecular and Experimental Medicine, The Scripps Research Institute, MEM-286, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-2177; Fax: 858-784-7981; E-mail: agale{at}scripps.edu.

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