Characterization of the Interaction between Tissue Factor Pathway Inhibitor α (TFPIα) and Factor V Species
Authors: Petrillo Teodolinda , Van 't Veer Cornelis , Camire Rodney M. 
Published: February 2020

Activation of factor V (FV) involves removal of its central B-domain following proteolysis at R709, R1018 and R1545. Two evolutionary conserved regions (basic region; BR; residues 964-1008 and acidic region 2; AR2; residues 1493-1538) of the B-domain play an essential role in keeping FV inactive. FV derivatives lacking the BR but retaining AR2 (FVAR) have cofactor-like properties while the BR fragment added in trans blocks their procoagulant function. Physiological important forms of FVAR include: platelet FV, FXa activated FV and FV-short. The latter is a splice variant lacking most of the B-domain, including the BR, yet retains AR2. In normal plasma, FV-short represents &lt;2% of the total FV but is overexpressed in patients affected by the East-Texas bleeding disorder due to a single point mutation or deletion in exon 13. In plasma, FV-short forms a complex with tissue factor pathway inhibitor α (TFPIα) through a high affinity interaction between AR2 and the basic C-terminal region of TFPIα (TFPIα-BR; residues 249-264) which is homologous to FV-BR. It has also been found that FV interacts with TFPIα via its BR, albeit with reduced affinity compared to FVAR. Furthermore, TFPIα and FV levels in plasma appear linked suggesting FV may act as carrier for TFPIα. Collectively these results are puzzling considering the mechanism by which these proteins are thought to interact. How can FV, with its endogenous BR engaged in interactions with AR2, simultaneously interact with TFPIα? To gain more insight into this question, we characterized the binding of TFPIα to different physiologic FV species including full-length (fl) FV, FVa, FV-short and other FVAR species. In direct binding measurements, we found that fluorescently labelled TFPIα-BR (OG488-TFPIα-BR) bound FV-short with high affinity (Kd = 0.66 nM). Unlabeled TFPIα and TFPIα-BR displaced OG488-TFPIα-BR from FVshort equivalently indicating specific binding of the BR region of TFPIα to FV-short. No detectable binding was observed to FVa and the OG488-TFPIα-BR also failed to bind fl-FV. These data indicated that AR2 is required for binding to TFPIα-BR and that the endogenous BR in fl-FV is associated with AR2 and precludes binding to TFPIα-BR. In support of this, thrombin cleavage of FV-short over time during binding measurements showed a gradual and marked decreased in fluorescence which correlated with cleavage at R1545 and release of AR2 as observed by western blotting. Cleavage of fl-FV by thrombin during the binding assay transiently increased fluorescence, indicating that TFPIα-BR binds to cleaved FV which correlated with removal of the endogenous BR (cleavage at R709 and R1018) as shown by western blotting. Subsequent cleavage at R1545 resulted in a decrease in fluorescence and hence binding. Using a FV-derivative that cannot be cleaved at R1018 (R1018Q), no binding of TFPIα-BR could be detected upon thrombin incubation, despite cleavage at R709. Together these data indicate that 1) cleavage of FV at R709 has little, if any influence on disrupting the BR-AR2 interaction; 2) cleavage at R1018 releases endogenous FV BR allowing TFPIα to engage via AR2; and 3) cleavage at R1545 removes AR2 eliminating TFPIα binding. Our data suggests that intramolecular binding of FV BR to AR2 has high affinity. To further assess the difference in apparent affinity of the intramolecular BR for AR2 compared to TFPIα-BR, we compared rates of FV-short activation (± TFPIα-BR) by thrombin to fl-FV and monitored cleavage at R1545. Based on the data, we estimate that intramolecular FV BR binds at least 25-50-fold tighter compared to TFPIα-BR binding to FV-short. Overall, we conclude that TFPIα via its BR binds to FV-short and cleaved forms of FV which retain AR2 but have its BR removed. TFPIa binding to these FV species not only blocks procoagulant function but also delays further cleavage at R1545. FVa and fl-FV do not bind TFPIα and are not regulated by this anticoagulant. Fl-FV must first be cleaved at R709 and R1018 prior to any possible TFPIα binding/regulation. Our data support the findings that TFPIα regulates the procoagulant function of FV-short and dampens thrombin generation by delaying the generation of FVa by tuning the activity of FVAR during the initiation of coagulation. This is especially evident when the coagulation stimulus is weak (e.g. low tissue factor), and much less important with a strong stimulus (e.g. high tissue factor) where other anticoagulant mechanisms dominate.

Disclosures
Camire: Pfizer: Research Funding.