The C-Terminus of Tfpiα Inhibits Factor V Activation By Protecting the Arg1545 Cleavage Site
Authors: van Doorn Peter , Rosing Jan , Wielders Simone , Hackeng Tilman M. , Castoldi Elisabetta 
Published: October 2019

Abstract
Coagulation factor V (FV) is the inactive precursor of FVa, which acts as an essential cofactor of factor Xa (FXa) in the prothrombinase complex. FV is maintained in the inactive state by the interaction between a basic and acidic region in the B-domain. The C-terminus of tissue factor pathway inhibitor-α (TFPIα) is highly homologous to the FV basic region and also binds to the acidic region of FV. In fact, a large fraction of plasma TFPIα circulates in complex with FV. Thanks to this interaction, FV acts as a cofactor of TFPIα in the inhibition of FXa and TFPIα inhibits prothrombinase complexes containing forms of FVa that retain the acidic region. However, when FV is activated through cleavage at Arg709, Arg1018 and Arg1545 by FXa or thrombin, it loses its anticoagulant properties and becomes a strong procoagulant. Recently, a FV splicing variant (FV-short) that lacks the basic region and binds TFPIα with high affinity has been described. FV-short is present in all individuals and represents ~5% of all plasma FV.
To gain more insight in the functional implications of the FV-TFPIα interaction, we studied the effects of a peptide identical to the TFPIα C-terminus (TFPIα C-term) on thrombin generation in plasma and on FV activation in model systems. All major findings were confirmed with full-length TFPIα.
TFPIα C-term (0-5 µM) prolonged the lag time and decreased the peak height of tissue factor- and FXa-triggered thrombin generation in a dose-dependent manner. These effects were more pronounced at low procoagulant stimuli and in the presence of plasma TFPIα. TFPIα C-term also inhibited thrombin generation in FV-depleted plasma reconstituted with FV, but not in FV-depleted plasma reconstituted with FVa, suggesting an effect on FV activation and/or prothrombinase. In model systems, TFPIα C-term inhibited the activation of purified FV by FXa and thrombin in a dose-dependent manner. This could be due to inhibition of FV proteolysis and/or to inhibition of prothrombinase in the assay used to quantify FVa activity. Therefore, FV activation was also followed by SDS-PAGE and Western blotting. This showed that TFPIα C-term (1 µM) interferes with FV proteolysis by both FXa and thrombin by selectively impairing cleavage of FV at Arg1545, which is located close to the FV acidic region (residues 1493-1537). The effect of TFPIα C-term on FV activation by thrombin was 3-fold stronger for FV-short than for full-length FV, in line with their respective affinities for the TFPIα C-terminus. Full-length TFPIα (10 nM) also inhibited FV cleavage at Arg1545 and delayed FV activation by thrombin. Its effect was also more pronounced on FV-short than on FV.
In summary, binding of the TFPIα C-terminus to the acidic region of FV inhibits FV activation by FXa or thrombin by blocking access to the Arg1545 cleavage site. Since cleavage at this site marks the transition of FV from an anticoagulant form (TFPIα-cofactor) to a procoagulant form (FXa-cofactor), this may represent an important new anticoagulant function of TFPIα. The main target of this anticoagulant mechanism is presently unclear, but it is unlikely to be intact FV, whose plasma concentration is 100-fold higher than the TFPIα concentration. More likely candidates are low-abundance FV species that lack the basic region but retain the acidic region, and therefore bind TFPIα with high affinity, such as FV-short, early FV activation intermediates and/or platelet FV.
Supported by grant nr. 2014-1 from the Dutch Thrombosis Foundation.

Disclosures
No relevant conflicts of interest to declare.