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

Abstract

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.ObjectivesThe 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.MethodsRecombinant 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.

New functional test for the TFPIα cofactor activity of Protein S working in synergy with FV‐Short

“…Addition of increasing concentrations of protein S gave a dose‐dependent inhibition of FXa with 50% inhibition at approximately 1.25 n m and maximal inhibition at 5 n m protein S. Similarly, the inclusion of diluted plasma in the assay instead of protein S yielded a dose‐dependent inhibition with maximum inhibition observed at 1/50 dilution and around 50% inhibition at 1/200. As we previously have reported , the TFPI α ‐cofactor effect of protein S in the assay depended on the presence of FV‐Short and in the absence of added FV‐Short, neither protein S (up to 10 n m ) (Figure S1) nor plasma (up to 1/100 dilution) yielded any stimulation of FXa inhibition.…”

“…We have recently shown that the interaction between TFPIa and FV-Short affects the function of TFPIa as inhibitor of FXa [3,26]. FV-Short in itself only weakly stimulates the activity of TFPIa but it strongly supports the TFPIa-cofactor activity of protein S. The results suggest that FV-Short and protein S act as synergistic TFPIa cofactors.…”

“…The assay is based on the inhibition of FXa by TFPI α in a purified system using a technique we previously described . In this assay, FV‐Short (2 n m final concentration) was incubated for 10 min at 37 °C with phospholipid (20 : 20 : 60 of PS:PE:PC, 25 μ m ), TFPI α (0.25 n m final concentration), plasma diluted in HNBSACa 2+ buffer (25 Hepes, 0.15 m NaCl, 5 m m CaCl 2 pH 7.7, containing 0.5 mg mL −1 bovine serum albumin and 5 Units per mL of Hirudin) as source of protein S. The reaction was initiated by addition of S2765 (0.7 m m ) and FXa (0.3 n m ) and followed for 15 min by monitoring absorbance at 405 n m in a Tecan Infinite 200 system.…”

“…FV‐Short in itself only weakly stimulates the activity of TFPI α but it strongly supports the TFPI α ‐cofactor activity of protein S. The results suggest that FV‐Short and protein S act as synergistic TFPI α cofactors. In a model system with purified components, the presence of FV‐Short (just a few n m ) and negatively charged phospholipid vesicles, protein S is highly efficient and just a few n m yields maximum TFPI α cofactor activity . In contrast, in the absence of FV‐Short, not even up to 100 n m protein S yields equally efficient TFPI α cofactor activity.…”

Anticoagulant protein S—New insights on interactions and functions

“…9,74,89 Recent studies have shown that TFPIα circulates as a complex with FV and FV-short in circulation and that protein S can function as a synergistic cofactor, together with FV/FV-short, in the inhibition of FXa. 69,86,88 We have shown that protein S alone as well as protein S in complex with FV does this by enhancing the initial encounter complex binding between TFPIα and FXa. 69 It is therefore likely that a similar mechanism is involved in the enhancement by protein S and FV/FV-short.…”