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J. Biol. Chem., Vol. 280, Issue 29, 27393-27401, July 22, 2005

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Incorporation of Factor Va into Prothrombinase Is Required for Coordinated Cleavage of Prothrombin by Factor Xa^*

Michael A. Bukys, Melissa A. Blum, Paul Y. Kim, Nicole Brufatto, Michael E. Nesheim, and Michael Kalafatis¶||

From the Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115, the Department of Biochemistry, Queen's University, Kingston, Ontario Canada, and the ^¶Department of Molecular Cardiology, The Lerner Research Institute, The Cleveland Clinic Foundation Cleveland, Ohio 44195

Prothrombin is activated to thrombin by two sequential factor Xa-catalyzed cleavages, at Arg²⁷¹ followed by cleavage at Arg³²⁰. Factor Va, along with phospholipid and Ca²⁺, enhances the rate of the process by 300,000-fold, reverses the order of cleavages, and directs the process through the meizothrombin pathway, characterized by initial cleavage at Arg³²⁰. Previous work indicated reduced rates of prothrombin activation with recombinant mutant factor Va defective in factor Xa binding (E323F/Y324F and E330M/V331I, designated factor Va^FF/MI). The present studies were undertaken to determine whether loss of activity can be attributed to selective loss of efficiency at one or both of the two prothrombin-activating cleavage sites. Kinetic constants for the overall activation of prothrombin by prothrombinase assembled with saturating concentrations of recombinant mutant factor Va were calculated, prothrombin activation was assessed by SDS-PAGE, and rate constants for both cleavages were analyzed from the time course of the concentration of meizothrombin. Prothrombinase assembled with factor Va^FF/MI had decreased k_cat for prothrombin activation with K_m remaining unaffected. Prothrombinase assembled with saturating concentrations of factor Va^FF/MI showed significantly lower rate for cleavage of plasma-derived prothrombin at Arg³²⁰ than prothrombinase assembled with saturating concentrations of wild type factor Va. These results were corroborated by analysis of cleavage of recombinant prothrombin mutants rMz-II (R155A/R284A/R271A) and rP2-II (R155A/R284A/R320A), which can be cleaved only at Arg³²⁰ or Arg²⁷¹, respectively. Time courses of these mutants indicated that mutations in the factor Xa binding site of factor Va reduce rates for both bonds. These data indicate that the interaction of factor Xa with the heavy chain of factor Va strongly influences the catalytic activity of the enzyme resulting in increased rates for both prothrombin-activating cleavages.

Received for publication, March 29, 2005 , and in revised form, May 12, 2005.

^* This work was supported by Grant 5R01HL-073343 from the NHLBI, National Institutes if Health (NIH) (to M. K.) and by U. S. Public Health Services, NIH Grant HL-46703-6 (to M. E. N.). 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: Dept. of Chemistry, Cleveland State University, 2121 Euclid Ave., Science Bldg. SR370, Cleveland, OH 44115. Tel.: 216-687-2460; Fax: 216-687-9298; E-mail: m.kalafatis{at}csuohio.edu.

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