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Importance of individual residues in hydrophobic patch PLVIVGL (1481-1487) in FV-Short for synergistic TFPIα cofactor activity with protein S, an alanine-scanning study: AlphaFold-mediated prediction of FV-Short/TFPIα/protein S trimolecular complex structure

Dahlbäck, Björn LU ; Tran, Sinh LU and Draczkowski, Piotr (2025) In Journal of thrombosis and haemostasis : JTH 23(3). p.849-862
Abstract
Background
In the splice variant factor (F)V-Short, 702 residues are deleted from the B domain, resulting in exposure of an acid region (AR2; 1493-1537) that binds TFPIα. FV-Short and protein S serve as synergistic TFPIα cofactors in inhibition of FXa. In the preAR2 region, a hydrophobic patch PLVIVGL (1481-1487) is crucial for synergistic TFPIα-cofactor activity and assembly of FV-Short, TFPIα, and protein S.
Objectives
To elucidate the importance of individual residues in the PLVIVGL patch for synergism between FV-Short and protein S as TFPIα cofactors.
Methods
An alanine scanning of the hydrophobic patch was performed in which 7 FV-Short variants were created. The synergistic TFPIα-cofactor activity was analyzed by... (More)
Background
In the splice variant factor (F)V-Short, 702 residues are deleted from the B domain, resulting in exposure of an acid region (AR2; 1493-1537) that binds TFPIα. FV-Short and protein S serve as synergistic TFPIα cofactors in inhibition of FXa. In the preAR2 region, a hydrophobic patch PLVIVGL (1481-1487) is crucial for synergistic TFPIα-cofactor activity and assembly of FV-Short, TFPIα, and protein S.
Objectives
To elucidate the importance of individual residues in the PLVIVGL patch for synergism between FV-Short and protein S as TFPIα cofactors.
Methods
An alanine scanning of the hydrophobic patch was performed in which 7 FV-Short variants were created. The synergistic TFPIα-cofactor activity was analyzed by FXa inhibition and a microtiter-based assay tested binding between the proteins. AlphaFold 3 was used to predict protein–protein interactions between FV-Short, protein S, and TFPIα.
Results
Five of the 7 variants (V1483A, I1484A, V1485A, G1486A, and L1487A) demonstrated decreased synergistic TFPIα cofactor activity; in particular, G1486A and L1487A were severely affected. Neither wild-type FV-Short nor any of the mutants bound protein S in the absence of TFPIα. In the presence of TFPIα, wild-type FV-Short, P1481A, L1482A, and V1485A bound protein S, whereas V1483A, I1484A, G1486A, and L1487A did not. AlphaFold predicted an interaction between the hydrophobic patch in FV-Short and a hydrophobic patch in protein S involving residues 268-276 and 422-426.
Conclusion
Individual residues (V1483, I1484, G1486, and L1487) in the hydrophobic patch are demonstrated to be important for the synergistic TFPIα-cofactor activity and for the assembly of a trimolecular FXa-inhibitory complex. (Less)
Abstract (Swedish)
Background
In the splice variant factor (F)V-Short, 702 residues are deleted from the B domain, resulting in exposure of an acid region (AR2; 1493-1537) that binds TFPIα. FV-Short and protein S serve as synergistic TFPIα cofactors in inhibition of FXa. In the preAR2 region, a hydrophobic patch PLVIVGL (1481-1487) is crucial for synergistic TFPIα-cofactor activity and assembly of FV-Short, TFPIα, and protein S.
Objectives
To elucidate the importance of individual residues in the PLVIVGL patch for synergism between FV-Short and protein S as TFPIα cofactors.
Methods
An alanine scanning of the hydrophobic patch was performed in which 7 FV-Short variants were created. The synergistic TFPIα-cofactor activity was analyzed by... (More)
Background
In the splice variant factor (F)V-Short, 702 residues are deleted from the B domain, resulting in exposure of an acid region (AR2; 1493-1537) that binds TFPIα. FV-Short and protein S serve as synergistic TFPIα cofactors in inhibition of FXa. In the preAR2 region, a hydrophobic patch PLVIVGL (1481-1487) is crucial for synergistic TFPIα-cofactor activity and assembly of FV-Short, TFPIα, and protein S.
Objectives
To elucidate the importance of individual residues in the PLVIVGL patch for synergism between FV-Short and protein S as TFPIα cofactors.
Methods
An alanine scanning of the hydrophobic patch was performed in which 7 FV-Short variants were created. The synergistic TFPIα-cofactor activity was analyzed by FXa inhibition and a microtiter-based assay tested binding between the proteins. AlphaFold 3 was used to predict protein–protein interactions between FV-Short, protein S, and TFPIα.
Results
Five of the 7 variants (V1483A, I1484A, V1485A, G1486A, and L1487A) demonstrated decreased synergistic TFPIα cofactor activity; in particular, G1486A and L1487A were severely affected. Neither wild-type FV-Short nor any of the mutants bound protein S in the absence of TFPIα. In the presence of TFPIα, wild-type FV-Short, P1481A, L1482A, and V1485A bound protein S, whereas V1483A, I1484A, G1486A, and L1487A did not. AlphaFold predicted an interaction between the hydrophobic patch in FV-Short and a hydrophobic patch in protein S involving residues 268-276 and 422-426.
Conclusion
Individual residues (V1483, I1484, G1486, and L1487) in the hydrophobic patch are demonstrated to be important for the synergistic TFPIα-cofactor activity and for the assembly of a trimolecular FXa-inhibitory complex. (Less)
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author
; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
AlphaFold, blood coagulation, coagulation factor V, coagulation factor Xa, protein S, tissue factor pathway inhibitor
in
Journal of thrombosis and haemostasis : JTH
volume
23
issue
3
pages
849 - 862
publisher
Elsevier
external identifiers
  • pmid:39617184
  • scopus:85212332344
ISSN
1538-7933
DOI
10.1016/j.jtha.2024.11.013
language
English
LU publication?
yes
id
0c6f1a76-f72f-4e3d-bcd5-d7b8b242a5b1
date added to LUP
2025-01-26 11:14:32
date last changed
2025-10-14 13:16:05
@article{0c6f1a76-f72f-4e3d-bcd5-d7b8b242a5b1,
  abstract     = {{Background<br/>In the splice variant factor (F)V-Short, 702 residues are deleted from the B domain, resulting in exposure of an acid region (AR2; 1493-1537) that binds TFPIα. FV-Short and protein S serve as synergistic TFPIα cofactors in inhibition of FXa. In the preAR2 region, a hydrophobic patch PLVIVGL (1481-1487) is crucial for synergistic TFPIα-cofactor activity and assembly of FV-Short, TFPIα, and protein S.<br/>Objectives<br/>To elucidate the importance of individual residues in the PLVIVGL patch for synergism between FV-Short and protein S as TFPIα cofactors.<br/>Methods<br/>An alanine scanning of the hydrophobic patch was performed in which 7 FV-Short variants were created. The synergistic TFPIα-cofactor activity was analyzed by FXa inhibition and a microtiter-based assay tested binding between the proteins. AlphaFold 3 was used to predict protein–protein interactions between FV-Short, protein S, and TFPIα.<br/>Results<br/>Five of the 7 variants (V1483A, I1484A, V1485A, G1486A, and L1487A) demonstrated decreased synergistic TFPIα cofactor activity; in particular, G1486A and L1487A were severely affected. Neither wild-type FV-Short nor any of the mutants bound protein S in the absence of TFPIα. In the presence of TFPIα, wild-type FV-Short, P1481A, L1482A, and V1485A bound protein S, whereas V1483A, I1484A, G1486A, and L1487A did not. AlphaFold predicted an interaction between the hydrophobic patch in FV-Short and a hydrophobic patch in protein S involving residues 268-276 and 422-426.<br/>Conclusion<br/>Individual residues (V1483, I1484, G1486, and L1487) in the hydrophobic patch are demonstrated to be important for the synergistic TFPIα-cofactor activity and for the assembly of a trimolecular FXa-inhibitory complex.}},
  author       = {{Dahlbäck, Björn and Tran, Sinh and Draczkowski, Piotr}},
  issn         = {{1538-7933}},
  keywords     = {{AlphaFold; blood coagulation; coagulation factor V; coagulation factor Xa; protein S; tissue factor pathway inhibitor}},
  language     = {{eng}},
  number       = {{3}},
  pages        = {{849--862}},
  publisher    = {{Elsevier}},
  series       = {{Journal of thrombosis and haemostasis : JTH}},
  title        = {{Importance of individual residues in hydrophobic patch PLVIVGL (1481-1487) in FV-Short for synergistic TFPIα cofactor activity with protein S, an alanine-scanning study: AlphaFold-mediated prediction of FV-Short/TFPIα/protein S trimolecular complex structure}},
  url          = {{http://dx.doi.org/10.1016/j.jtha.2024.11.013}},
  doi          = {{10.1016/j.jtha.2024.11.013}},
  volume       = {{23}},
  year         = {{2025}},
}