Effects of protein S and factor Xa on peptide bond cleavages during inactivation of factor Va and factor VaR506Q by activated protein C*

Jan Rosing*, Lico Hoekema, Gerry A.F. Nicolaes, M. Christella L. G. D. Thomassen, H. Coenraad Hemker, Katalin Varadi, Hans Peter Schwarz, Guido Tans

*Corresponding author for this work

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    Abstract

    Inactivation of membrane-bound factor Va by activated protein C (APC) proceeds via a biphasic reaction that consists of a rapid and a slow phase, which are associated with cleavages at Arg(506),and Arg(306) Of the heavy chain of factor Va, respectively, We have investigated the effects of protein S and factor Xa on APC-catalyzed factor Va inactivation, Protein S accelerates factor Va inactivation by selectively promoting the slow cleavage at Arg(306) (20-fold), Factor Xa protects factor Va from inactivation by APC by selectively blocking cleavage at Arg(506). Inactivation of factor Va(R506Q), which was isolated from the plasma of a homozygous APC resistant patient and which lacks the Arg(506) cleavage site, was also stimulated by protein S but was not affected by factor Xa, This confirms that the target sites of protein S and factor Xa involve Arg(306) and Arg(506), respectively, Factor Xa completely blocked APC-catalyzed cleavage at Arg(506) in, normal factor Va (1 nM) With a half-maximal effect (K-1/2Xa) at 1.9 nM factor Xa. Expression of cofactor activity of factor Va in prothrombin activation required much lower factor Xa concentrations (K-1/2Xa = 0.08 nM). When the ability of factor Xa to protect factor Va from inactivation by APC was determined at low factor Va concentrations during prothrombin activation much lower amounts of factor Xa were required (K-1/2Xa = 0.03 nM). This indicates 1) that factor Va is optimally protected from inactivation by APC by incorporation into the prothrombinase complex during ongoing prothrombin activation, and 2) that the formation of a catalytically active prothrombinase complex and protection of factor Va from inactivation by APC likely involves the same interaction of factor Xa with factor Va, In accordance with the proposed mechanisms of action of protein S and factor Xa, we observed that the large differences between the rates of APC-catalyzed inactivation of normal factor Va and factor Va(R506Q) Were almost annihilated in the presence of factor Xa and protein S, This observation may explain why, in the absence of other risk factors, APC resistance only results in a weak prothrombotic condition,
    Original languageEnglish
    Pages (from-to)27852-27858
    Number of pages7
    JournalJournal of Biological Chemistry
    Volume270
    Issue number46
    DOIs
    Publication statusPublished - 17 Sept 1995

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