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

ESSENTIALS: FV‐Short, a normal splice isoform of Factor V, binds tissue factor pathway inhibitor (TFPIα) with high affinity. FV‐Short functions as a synergistic TFPIα cofactor with protein S in inhibition of Factor Xa. FV‐Short is much more efficient as TFPIα cofactor than full length FV. TFPIα‐cofactor activity of FV‐Short is lost upon activation of coagulation by thrombin‐mediated cleavage. BACKGROUND: 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 aim was to elucidate whether FV‐Short affects TFPIα as inhibitor of coagulation FXa and to test whether the TFPIα‐cofactor activity of protein S is influenced by FV‐Short. METHODS: 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. RESULTS: 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.