Factor V is an anticoagulant cofactor for activated protein C during inactivation of factor Va

Abstract

Coagulation factor V (FV) promotes inactivation of activated factor VIII (FVIIIa) by activated protein C (APC) and protein S. Loss of this APC cofactor activity is proposed to be partially responsible for the APC resistance phenotype of FV(Leiden). However, FVIIIa loses activity rapidly due to dissociation of the A2 domain, and this may be the primary mechanism of FVIIIa inactivation. APC/protein S also readily inactivates activated FV (FVa). We therefore hypothesized that FV can function as an anticoagulant cofactor for APC/protein S in the inactivation of FVa. FV was titrated into FV-deficient plasma, and the APC sensitivity ratio (APCsr; a measure of APC activity) was measured in a clotting assay that was not sensitive to FVIII. Our results showed an increase in APCsr as the FV concentration increased, suggesting an anticoagulant function for FV in this assay. FV(Leiden) showed APC resistance with an APCsr of 1.0. Therefore, under our experimental conditions, FV acted as an anticoagulant cofactor for APC in the inactivation of FVa.

Fig. 1

FV titrations in FV-deficient plasma in the dilute prothrombin time clotting assay. a, c Clotting time in seconds of WT-FV (a) and Q506-FV (c) titrations in the absence (▵, □) and presence (▴, ▪) of APC. b APCsr of WT-FV (▴) and Q506-FV (▪). n = 5. Error bars represent the SEM.

Fig. 2

FV titrations in FV-deficient plasma in the presence of 2 nM FVa in the dilute prothrombin time clotting assay. Clotting time in seconds of WT-FV (▴, ▵) and Q506-FV (▪, □) titrations in the absence (▵, □) and presence (▴, ▪) of APC. Inset: APCsr. n = 2. Error bars represent the SEM.

Fig. 3

Control experiments for FVIII sensitivity of the dilute prothrombin time clotting assay. a Pooled normal human plasma was incubated in the absence or presence of 1 μg/ml BO2C11 for 2 h at 37°C and then used in the APTT assay and dilute prothrombin time clotting assay. n = 4. Error bars represent the SEM. b FV-deficient plasma was incubated in the absence (▴, ▵) or presence (•, ○) of 20 μg/ml BO2C11 for 80 min at 37°C and then used in FV titrations, in the absence (▵, ○) or presence (▴, •) of APC. Inset: APCsr. Final concentration of BO2C11 was 1.3 μg/ml. n = 1. c M662C/D1828C-FVIII was titrated in FVIII-deficient plasma and clotting time was measured in the absence (⋄) or presence (♦) of APC. n = 1. d M662C/D1828C-FVIII was titrated in FV-deficient plasma and clotting time was measured in the absence (⋄) or presence (♦) of APC. The FVIII concentration for each data point represents total FVIII present in the final reaction, which included added FVIII plus 0.067 U/ml FVIII present in 15-fold diluted FV-deficient plasma. n = 1.