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J. Biol. Chem., Vol. 283, Issue 11, 6648-6655, March 14, 2008

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Effects of Prothrombin on the Individual Activated Protein C-mediated Cleavages of Coagulation Factor Va^*

From the Department of Laboratory Medicine, Clinical Chemistry, Lund University, The Wallenberg Laboratory, University Hospital, Malmö, SE-205 02 Malmö, Sweden

The factor Va (FVa) inactivation by activated protein C (APC), mediated by cleavages at Arg³⁰⁶ and Arg⁵⁰⁶ in FVa, is inhibited by both factor Xa (FXa) and prothrombin. Although FXa is known to specifically inhibit the Arg⁵⁰⁶ cleavage, the effect of prothrombin has not been confined to one cleavage site. We used recombinant FV variants, FV:R506Q/R679Q and FV:R306Q/R679Q, to investigate the effect of prothrombin on the individual cleavage sites. The APC-mediated FVa inhibition was monitored by a prothrombinase-based FVa assay, and apparent first order rate constants were calculated for each of the cleavage sites both in the presence and absence of prothrombin. Prothrombin impaired cleavages at both Arg³⁰⁶ and Arg⁵⁰⁶ and the inhibition correlated with a delayed appearance of proteolytic products on Western blots. Almost complete inhibition was obtained at around 3 µM prothrombin, whereas half-maximal inhibition was obtained at 0.7 µM prothrombin. After cleavage of prothrombin by thrombin, the inhibitory activity was lost. The inhibitory effect of prothrombin on APC-mediated inhibition of FVa was seen both in the presence and absence of protein S, but in particular for the Arg³⁰⁶ sites, it was more pronounced in the presence of protein S. Thus, prothrombin inhibition of APC inactivation of FVa appears to be due to both impaired APC function and decreased APC cofactor function of protein S. In conclusion, FVa, being part of the prothrombinase complex, is protected from APC by both FXa and prothrombin. Release of products of prothrombin activation from the prothrombinase complex would alleviate the protection, allowing APC-mediated inactivation of FVa.

Received for publication, September 26, 2007 , and in revised form, January 15, 2008.

^* This work was supported by grants from the Swedish Foundation for Strategic Research; Swedish Research Council Grant 07143; a senior investigator's award from the Foundation for Strategic Research; the Söderberg's, Österlund's, and Påhlsson's trusts; and research funds from the University Hospital in Malmö. 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 Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Laboratory Medicine, Clinical Chemistry, Lund University, The Wallenberg Laboratory, University Hospital, Malmö, SE-205 02 Malmö, Sweden. Tel.: 46-40-33-15-01; Fax: 46-40-33-70-44; E-mail: bjorn.dahlback{at}med.lu.se.

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