Factor V as Anticoagulant Cofactor for Activated Protein C in Factor Va Inactivation

Factor V (FV) is a cofactor that promotes inactivation of activated factor VIII (FVIIIa) by the activated protein C and protein S complex (APC/protein S). Cleavage in FV at Arg506 is required for proteolytic inactivation of FVa, but also for the anticoagulant function of FV as cofactor for APC in the inactivation of FVIIIa. This is demonstrated by the well known FV_Leiden mutant with Arg506 mutated to glutamine (Q506), causing APC resistance due to both impaired sensitivity of Q506FVa to APC and reduced cofactor activity of Q506FV for APC inactivation of FVIIIa. However, FVIIIa loses activity rapidly due to dissociation of the A2 domain, and this may be the primary mechanism of FVIIIa inactivation. Thus, we question whether the APC-mediated inactivation of FVIIIa is relevant to the FV_Leiden thrombophilic phenotype. Rather, we hypothesized that FV can function as an anticoagulant cofactor for the APC/protein S complex in the inactivation of activated FV (FVa). To test this hypothesis, we designed a coagulation assay initiated by tissue factor that was sensitive to FV but was insensitive to FVIII. FV was titrated into FV deficient plasma and clotting times were measured in absence and presence of APC to determine an APC sensitivity ratio (APCsr). An increase in the APCsr was observed as the level of FV was increased, suggesting an anticoagulant function of FV. Similar titrations were done with Q506FV, showing no increase in clotting time when APC was present and an APCsr of 1.0 in the presence of Q506 FV. Control experiments confirmed that this clotting assay was insensitive to the presence or absence of FVIII; thus, these assays were reflecting FVa inactivation. The potential anticoagulant effect of FV as cofactor for APC in FVa inactivation was further investigated by monitoring proteolysis of purified FVa by APC over time using SDS PAGE. Recombinant purified FVa was labeled with a fluorescent dye, and then subjected to proteolysis by APC/protein S in the absence or presence of FV in a time course. The resulting FVa fragments seen on SDS gels reflected the known cleavages at Arg306 and Arg506, and the FVa cleavage products were quantified by digital fluorescent scanning of the gel. FV stimulated a small but statistically insignificant increase in the rate of FVa cleavage by APC/protein S. Thus, in our experimental conditions, we found a significant anticoagulant effect of FV in clotting assays that were sensitive to FV but not sensitive to FVIII whereas in purified reaction mixtures there was not a significant enhancement by FV of APC proteolysis of FVa. These data contrasting FV’s apparent APC-cofactor activities between plasma and purified reaction mixtures lead us to speculate that other factors or mechanisms present in plasma also contribute to the anticoagulant function of APC in a FV dependent manner.