Factor V-short and protein S as synergistic tissue factor pathway inhibitor (TFPIα) cofactors

FV-Short is a normal splice variant of Factor V (FV) having a short B domain, which exposes a high affinity-binding site for tissue factor pathway inhibitor α (TFPIα). FV-Short and TFPIα circulate in complex in plasma. Objectives: The

FV-Short and protein S are highly efficient, synergistic cofactors to TFPIα in the regulation of FXa activity, whereas full length FV has lower activity. Our results suggest the formation of an efficient FXa-inhibitory complex between FV-Short, TFPIα and protein S on the surface of negatively charged phospholipids.

Recombinant FV, wild-type FV-Short and a FV-Short thrombin-cleavage resistant variant were expressed and purified. The influence of FV and FV-Short variants and/or protein S on the FXa inhibitory activity of TFPIα was monitored both in a purified system and in a plasma-based thrombin generation assay.

FV-Short had intrinsically weak TFPIα-cofactor activity but with protein S present, FV-Short yielded efficient inactivation of FXa. Protein S alone did not promote full TFPIα-activity. Intact FV was inefficient at low protein S concentrations and had 10-fold lower activity compared to FV-Short at physiological protein S levels. Activation of FV-Short by thrombin resulted in the loss of the TFPIα-cofactor activity. The synergistic TFPIα-cofactor activity of FV-Short and protein S was also demonstrated in plasma using a thrombin generation assay. Conclusions: FV-Short and protein S are highly efficient, synergistic cofactors to TFPIα in the regulation of FXa activity, whereas full length FV has lower activity. Our results suggest the formation of an efficient FXa-inhibitory complex between FV-Short, TFPIα and protein S on the surface of negatively charged phospholipids.