The TFPIα C-terminal tail is essential for TFPIα-FV-short-protein S complex formation and synergistic enhancement of TFPIα
File(s)
Published version
Author(s)
Gierula, Magdalena
Noakes, Victoria M
Salles-Crawley, Isabelle
Crawley, James
Ahnstroem, Josefin
Type
Journal Article
Abstract
Background
For maximal TFPIα functionality, 2 synergistic cofactors, protein S and FV-short, are required. Both interact with TFPIα, protein S through Kunitz 3 residues Arg199/Glu226 and FV-short with the C-terminus. How these interactions impact the synergistic enhancement remains unclear.
Objectives
To determine the importance of the TFPIα-protein S and TFPIα-FV-short interactions for TFPIα enhancement.
Methods
TFPIα variants unable to bind protein S (K3m [R199Q/E226Q]) or FV-short (ΔCT [aa 1–249]) were generated. TFPIα-FV-short binding was studied by plate-binding and co-immunoprecipitation assays; functional TFPIα enhancement by FXa inhibition and prothrombin activation.
Results
While WT TFPIα and TFPIα K3m bound FV-short with high affinity (Kd∼2nM), TFPIα ΔCT did not. K3m, in contrast to WT, did not incorporate protein S in a TFPIα-FV-short-protein S complex while TFPIα ΔCT bound neither FV-short nor protein S. Protein S enhanced WT TFPIα-mediated FXa inhibition, but not K3m, in the absence of FV-short. However, once FV-short was present, protein S efficiently enhanced TFPIα K3m (EC50: 4.7nM vs 2.0nM for WT). FXa inhibition by ΔCT was not enhanced by protein S alone or combined with FV-short. In FXa-catalyzed prothrombin activation assays, FV-short enhanced TFPIα K3m function in the presence of protein S (5.5 vs 10.4-fold enhancement of WT) whereas ΔCT showed reduced or lack of enhancement by FV-short and protein S, respectively.
Conclusion
Full TFPIα function requires the presence of both cofactors. While synergistic enhancement can be achieved in the absence of TFPIα-protein S interaction, only TFPIα with an intact C-terminus can be synergistically enhanced by protein S and FV-short.
For maximal TFPIα functionality, 2 synergistic cofactors, protein S and FV-short, are required. Both interact with TFPIα, protein S through Kunitz 3 residues Arg199/Glu226 and FV-short with the C-terminus. How these interactions impact the synergistic enhancement remains unclear.
Objectives
To determine the importance of the TFPIα-protein S and TFPIα-FV-short interactions for TFPIα enhancement.
Methods
TFPIα variants unable to bind protein S (K3m [R199Q/E226Q]) or FV-short (ΔCT [aa 1–249]) were generated. TFPIα-FV-short binding was studied by plate-binding and co-immunoprecipitation assays; functional TFPIα enhancement by FXa inhibition and prothrombin activation.
Results
While WT TFPIα and TFPIα K3m bound FV-short with high affinity (Kd∼2nM), TFPIα ΔCT did not. K3m, in contrast to WT, did not incorporate protein S in a TFPIα-FV-short-protein S complex while TFPIα ΔCT bound neither FV-short nor protein S. Protein S enhanced WT TFPIα-mediated FXa inhibition, but not K3m, in the absence of FV-short. However, once FV-short was present, protein S efficiently enhanced TFPIα K3m (EC50: 4.7nM vs 2.0nM for WT). FXa inhibition by ΔCT was not enhanced by protein S alone or combined with FV-short. In FXa-catalyzed prothrombin activation assays, FV-short enhanced TFPIα K3m function in the presence of protein S (5.5 vs 10.4-fold enhancement of WT) whereas ΔCT showed reduced or lack of enhancement by FV-short and protein S, respectively.
Conclusion
Full TFPIα function requires the presence of both cofactors. While synergistic enhancement can be achieved in the absence of TFPIα-protein S interaction, only TFPIα with an intact C-terminus can be synergistically enhanced by protein S and FV-short.
Date Issued
2023-12
Date Acceptance
2023-09-08
Citation
Journal of Thrombosis and Haemostasis, 2023, 21 (12), pp.3568-3580
ISSN
1538-7836
Publisher
Elsevier
Start Page
3568
End Page
3580
Journal / Book Title
Journal of Thrombosis and Haemostasis
Volume
21
Issue
12
Copyright Statement
© 2023 The Author(s). Published by Elsevier Inc. on behalf of International Society on Thrombosis and Haemostasis. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/4.0/).
license (http://creativecommons.org/licenses/by/4.0/).
License URL
Identifier
https://www.jthjournal.org/article/S1538-7836(23)00700-6/fulltext
Publication Status
Published
Date Publish Online
2023-09-19