EFFECTS OF PROTEIN-S AND FACTOR XA ON PEPTIDE-BOND CLEAVAGES DURING INACTIVATION OF FACTOR VA AND FACTOR VA(R506Q) BY ACTIVATED PROTEIN-C

Inactivation of membrane-bound factor Va by activated protein C (APC) proceeds via a biphasic reaction that consists of a rapid and a slow p hase, which are associated with cleavages at Arg(506),and Arg(306) Of the heavy chain of factor Va, respectively, We have investigated the e ffects of protein S and factor Xa on APC-catalyzed factor Va inactivat ion, Protein S accelerates factor Va inactivation by selectively promo ting the slow cleavage at Arg(306) (20-fold), Factor Xa protects facto r Va from inactivation by APC by selectively blocking cleavage at Arg( 506). Inactivation of factor Va(R506Q), which was isolated from the pl asma 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 fa ctor Xa involve Arg(306) and Arg(506), respectively, Factor Xa complet ely 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. Express ion of cofactor activity of factor Va in prothrombin activation requir ed much lower factor Xa concentrations (K-1/2Xa = 0.08 nM). When the a bility of factor Xa to protect factor Va from inactivation by APC was determined at low factor Va concentrations during prothrombin activati on much lower amounts of factor Xa were required (K-1/2Xa = 0.03 nM). This indicates 1) that factor Va is optimally protected from inactivat ion by APC by incorporation into the prothrombinase complex during ong oing prothrombin activation, and 2) that the formation of a catalytica lly active prothrombinase complex and protection of factor Va from ina ctivation by APC likely involves the same interaction of factor Xa wit h factor Va, In accordance with the proposed mechanisms of action of p rotein S and factor Xa, we observed that the large differences between the rates of APC-catalyzed inactivation of normal factor Va and facto r Va(R506Q) Were almost annihilated in the presence of factor Xa and p rotein S, This observation may explain why, in the absence of other ri sk factors, APC resistance only results in a weak prothrombotic condit ion,