Abstract
Background: Inhibition of factor Xa (FXa) by tissue factor pathway inhibitor α (TFPIα) is potentiated by FV-Short and protein S. The C-terminus of TFPIα is important for formation of the TFPIα/protein S/FV-Short complex. It contains a hydrophobic sequence (LIKT). The purpose was to investigate the importance of the LIKT sequence.
Methods: Two TFPIα C-terminal peptides were synthesized, one wild-type and the other with LIKT replaced by AAKA. The effect of the peptides on TFPIα function was tested. An AAKA-mutant TFPIα was created and compared with wild-type TFPIα. AlphaFold was used to elucidate a mechanism for the role of the LIKT sequence.
Results: The wild-type peptide efficiently inhibited the FXa-inhibitory activity of TFPIα/protein S/FV-Short complex, whereas the AAKA peptide did not. The LIKT sequence in TFPIα was crucially important for the synergistic TFPIα-cofactor activity between protein S and FV-Short. AlphaFold (DeepMind, London, UK) suggested an interaction between LIKT and FV-Short B-loop 1510-1517 mediated through a network of hydrogen bonds and hydrophobic interactions.
Conclusions: The C-terminal TFPIα-peptide induced complex formation between protein S and FV-Short. The LIKT sequence in TFPIα was crucially important for the ability of protein S and FV-Short to function as synergistic TFPIα cofactors.