The C-Terminus Basic Region of TFPIα Dynamically Regulates FV(a) Function: Evaluation of FV-Short

Coagulation Factor V (FV) is present in plasma as an inactive procofactor. Following B-domain removal, the active cofactor FVa, enhances the catalytic efficiency of FXa by several orders of magnitude. Previous findings have established that evolutionary conserved regions within the B-domain play a key role in keeping FV in an inactive state by, in part, concealing FXa binding site(s). These regions of the FV B-domain consist of basic and acidic elements and define the minimal sequence necessary to maintain FV as a procofactor. Recent data have shown that removal of either one of these elements results in FVa-like activity and that B-domain fragments spanning the basic region act in-trans to suppress the activity of FV variants bearing only the acidic region (J Biol Chem. 287:26342-51, 2012, J Biol Chem. 288:30151-60, 2013). Physiologically, forms of FV that are missing a basic region but harbor an acidic region are released by activated platelets. Interestingly, another form of FV that only harbors an acidic region has been described. The variant FV-East Texas results in an alternatively spliced form of FV that has most of the B-domain removed but retains the acidic region (FV-short; J Clin. Invest. 123:3777-87; 2013). Together these forms of FV should be constitutively active. However, it is possible that physiologic ligands that mimic the basic region could inhibit their activity. Previous studies have identified tissue factor pathway inhibitor (TFPIα) as one of these potential ligands. Remarkably, the C-terminal segment of TFPIα shares substantial sequence homology with the FV basic region and binds forms of FV that only harbor the acidic region (PNAS, 110:17838-43; 2013). While there are several remaining unanswered questions, the FV(a)-TFPIα interaction has the potential to fundamentally alter our understanding of cofactor regulation at the site of injury.

In order to investigate this fascinating prospect, we expressed and purified recombinant FV-short and a protein fragment containing the basic region of TFPIα. As anticipated, FV-short exhibited FVa-like activity, however this cofactor function in the prothrombinase complex was greatly impaired in the presence of TFPIα basic region. Similar results were obtained in clotting assays, supporting the idea of a trans-acting function of TFPIα on FV derivatives missing the acidic region. To better understand the mechanism of interaction, direct binding measurements by fluorescence were established using a labeled TFPIα basic region fragment. Changes in anisotropy were monitored as a function of the FV-short concentration. Analysis of the data revealed a high affinity interaction between FV-short and the TFPIα basic region (K_d = 3.42 ± 0.39 nM). Based on this high affinity, a proportion of FV-short should be largely bound to TFPIα; an observations consistent with the FV-East Texas family. Thus instead of FV-short being constitutively active, when bound to TFPIα it would effectively revert to a procofactor state. To investigate the implications of this circulating complex on function, we next evaluated whether FV-short could be normally converted to FVa by thrombin (IIa) through proteolysis at Arg⁷⁰⁹ and Arg¹⁵⁴⁵ (Arg¹⁰¹⁸ is missing). Surprisingly, in the presence of TFPIα basic region, cleavage at Arg¹⁵⁴⁵ was significantly delayed suggesting TFPIα modulates both the activity of FV and its interaction with IIa. TFPIα could either directly and/or allosterically interfere with IIa binding sites on FV. Based on previous unpublished work in our lab, we believe that the basic region of TFPIα provides a trans-acting sequence that causes FV-short to revert to a procofactor state both functionally and structurally. The absence of Arg¹⁰¹⁸ abrogates proteolysis necessary to alleviate these structural constraints, hence resulting in delayed cleavage at Arg¹⁵⁴⁵.

This study shows direct evidence of a high affinity interaction between the TFPIα c-terminal basic region and the naturally-occurring truncated form of FV responsible for the East Texas bleeding disorder. We now have unique tools to investigate the role of full length TPFIα not only in this interesting case, but also in relation to partially activated forms of FV released from platelets. This work sets the stage to achieve a better understanding of the early hemostatic events occurring at the site of injury, providing the bases for potential therapeutic regulation.