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J. Biol. Chem., Vol. 281, Issue 13, 8773-8779, March 31, 2006

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Role of Hirudin-like Factor Va Heavy Chain Sequences in Prothrombinase Function^*

Raffaella Toso and Rodney M. Camire¹

From the Department of Pediatrics, Division of Hematology, The Children's Hospital of Philadelphia and University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Proexosite I on prothrombin has been implicated in providing a recognition site for factor Va within prothrombinase. To examine whether hirudin-like sequences (659–698) on the cofactor contribute to this interaction, we expressed and purified two-chain FVa derivatives that were intracellularly truncated at the C terminus of the heavy chain: FVa⁷⁰⁹ (des710–1545), FVa⁶⁹⁹ (des700–1545), FVa⁶⁹² (des693–1545), FVa⁶⁷⁸ (des679–1545), and FVa⁶⁵⁸ (des659–1545). We found that FVa⁷⁰⁹, FVa⁶⁹⁹, FVa⁶⁹², and FVa⁶⁷⁸ exhibited specific clotting activities that were comparable with plasma-derived and recombinant FVa. Additionally, kinetic studies using prothrombin revealed that the K_m and k_cat values for these derivatives were unaltered. Fluorescent measurements and chromatography studies indicated that FVa⁷⁰⁹, FVa⁶⁹⁹, FVa⁶⁹², and FVa⁶⁷⁸ bound to FXa membranes and thrombin-agarose in a manner that was comparable with the wild-type cofactors. In contrast, FVa⁶⁵⁸ had an 1% clotting activity and reduced affinity for FXa membranes (20-fold) and did not bind to thrombin-agarose. Surprisingly, however, FVa⁶⁵⁸ exhibited essentially normal kinetic parameters for prothrombin when the variant was fully saturated with FXa membranes. Overall our results are consistent with the interpretation that any possible binding interactions between prothrombin and the C-terminal region of the FVa heavy chain do not contribute in a detectable way to the enhanced function of prothrombinase.

Received for publication, October 20, 2005 , and in revised form, December 9, 2005.

^* This work was supported by National Institutes of Health Grants P01 HL-74124-01, Project 2 (to R. M. C.), and National Research Service Award T32 HL-07439-26 (to R. T.). 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: Div. of Hematology, 302E Abramson Research Center, The Children's Hospital of Philadelphia, 3615 Civic Center Blvd., Philadelphia, PA 19104. Tel.: 215-590-9968; Fax: 215-590-3660; E-mail: rcamire{at}mail.med.upenn.edu.

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