Abstract Number: OC 36.5
Meeting: ISTH 2022 Congress
Background: TFPIα is dependent on its synergistic cofactors, protein S (PS) and factor (F)V-short, for efficient inhibition of FXa. Both cofactors interact with TFPIα, PS through Kunitz 3 residues including Arg199 and Glu226 and FV-short with the TFPIα C-terminus. While the TFPIα-PS interaction is needed for enhancement by PS alone, it is not clear whether direct interactions between TFPIα and both its cofactors are needed for the synergistic enhancement.
Aims: Determine the importance of the TFPIα-PS and TFPIα-FV-short interactions for synergistic enhancement of FXa inhibition.
Methods: FXa inhibition assays were used to study TFPIα enhancement in the presence and absence of PS and/or FV-short. TFPIα variants unable to bind either PS (R199Q/E226Q) or FV-short (ΔCT, aa 1–249), were used to investigate how abolished interactions with either cofactor affected their ability to enhance TFPIα, alone or in synergy.
Results: Both TFPIα R199Q/E226Q and TFPIα ΔCT inhibited FXa, with ΔCT showing moderately reduced activity ( < 2-fold) compared to WT. In the absence of FV-short, PS enhanced WT TFPIα with an EC50 of 42±12nM with only minimal enhancement of R199Q/E226Q. However, when PS was titrated in the presence of FV-short (2nM), FXa inhibition by TFPIα R199Q/E226Q was efficiently enhanced by PS (EC50: 2.9±0.77 and 1.55±0.29 for WT and R199Q/E226Q, respectively), suggesting that FV-short rescues the synergistic enhancement when the TFPIα-PS interaction is absent. The inhibition of FXa by TFPIα ΔCT was not enhanced by increasing concentration of FV-short (0-4nM). In contrast to TFPIα R199Q/E226Q, the addition of PS had little effect on the TFPIα ΔCT-mediated inhibition of FXa.
Conclusion(s): The lack of enhancement of TFPIα by PS alone, observed in the absence of a TFPIα-PS interaction, can be rescued by the presence of FV-short. In contrast, an interaction between FV-short and TFPIα is essential for the synergetic enhancement by PS and FV-short and full anticoagulant function of TFPIα.
To cite this abstract in AMA style:Gierula M, Noakes V, Salles-Crawley I, Crawley J, Ahnström J. The TFPIα C-terminal tail is essential for the synergistic enhancement of TFPIα mediated inhibition of FXa by protein S and FV-short. [abstract]. https://abstracts.isth.org/abstract/the-tfpi%ce%b1-c-terminal-tail-is-essential-for-the-synergistic-enhancement-of-tfpi%ce%b1-mediated-inhibition-of-fxa-by-protein-s-and-fv-short/. Accessed February 28, 2024.
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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/the-tfpi%ce%b1-c-terminal-tail-is-essential-for-the-synergistic-enhancement-of-tfpi%ce%b1-mediated-inhibition-of-fxa-by-protein-s-and-fv-short/