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Amino acid residues in the laminin G domains of protein S involved in tissue factor pathway inhibitor interaction.

Somajo, Sofia LU ; Ahnström, Josefin LU ; Fernandez-Recio, J; Gierula, M; Villoutreix, B O and Dahlbäck, Björn LU (2015) In Thrombosis and Haemostasis 113(5). p.976-987
Abstract
Protein S functions as a cofactor for tissue factor pathway inhibitor (TFPI) and activated protein C (APC). The sex hormone binding globulin (SHBG)-like region of protein S, consisting of two laminin G-like domains (LG1 and LG2), contains the binding site for C4b-binding protein (C4BP) and TFPI. Furthermore, the LG-domains are essential for the TFPI-cofactor function and for expression of full APC-cofactor function. The aim of the current study was to localise functionally important interaction sites in the protein S LG-domains using amino acid substitutions. Four protein S variants were created in which clusters of surface-exposed amino acid residues within the LG-domains were substituted. All variants bound normally to C4BP and were... (More)
Protein S functions as a cofactor for tissue factor pathway inhibitor (TFPI) and activated protein C (APC). The sex hormone binding globulin (SHBG)-like region of protein S, consisting of two laminin G-like domains (LG1 and LG2), contains the binding site for C4b-binding protein (C4BP) and TFPI. Furthermore, the LG-domains are essential for the TFPI-cofactor function and for expression of full APC-cofactor function. The aim of the current study was to localise functionally important interaction sites in the protein S LG-domains using amino acid substitutions. Four protein S variants were created in which clusters of surface-exposed amino acid residues within the LG-domains were substituted. All variants bound normally to C4BP and were fully functional as cofactors for APC in plasma and in pure component assays. Two variants, SHBG2 (E612A, I614A, F265A, V393A, H453A), involving residues from both LG-domains, and SHBG3 (K317A, I330A, V336A, D365A) where residues in LG1 were substituted, showed 50-60 % reduction in enhancement of TFPI in FXa inhibition assays. For SHBG3 the decreased TFPI cofactor function was confirmed in plasma based thrombin generation assays. Both SHBG variants bound to TFPI with decreased affinity in surface plasmon resonance experiments. The TFPI Kunitz 3 domain is known to contain the interaction site for protein S. Using in silico analysis and protein docking exercises, preliminary models of the protein S SHBG/TFPI Kunitz domain 3 complex were created. Based on a combination of experimental and in silico data we propose a binding site for TFPI on protein S, involving both LG-domains. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Thrombosis and Haemostasis
volume
113
issue
5
pages
976 - 987
publisher
F K Schattauer Verlag Gmbh
external identifiers
  • pmid:25716664
  • wos:000354758000009
  • scopus:84931275092
ISSN
0340-6245
DOI
10.1160/TH14-09-0803
language
English
LU publication?
yes
id
383d39aa-0ad4-4103-94ed-586e5b40de20 (old id 5142677)
alternative location
http://www.ncbi.nlm.nih.gov/pubmed/25716664?dopt=Abstract
date added to LUP
2015-03-11 16:53:45
date last changed
2017-06-25 03:59:16
@article{383d39aa-0ad4-4103-94ed-586e5b40de20,
  abstract     = {Protein S functions as a cofactor for tissue factor pathway inhibitor (TFPI) and activated protein C (APC). The sex hormone binding globulin (SHBG)-like region of protein S, consisting of two laminin G-like domains (LG1 and LG2), contains the binding site for C4b-binding protein (C4BP) and TFPI. Furthermore, the LG-domains are essential for the TFPI-cofactor function and for expression of full APC-cofactor function. The aim of the current study was to localise functionally important interaction sites in the protein S LG-domains using amino acid substitutions. Four protein S variants were created in which clusters of surface-exposed amino acid residues within the LG-domains were substituted. All variants bound normally to C4BP and were fully functional as cofactors for APC in plasma and in pure component assays. Two variants, SHBG2 (E612A, I614A, F265A, V393A, H453A), involving residues from both LG-domains, and SHBG3 (K317A, I330A, V336A, D365A) where residues in LG1 were substituted, showed 50-60 % reduction in enhancement of TFPI in FXa inhibition assays. For SHBG3 the decreased TFPI cofactor function was confirmed in plasma based thrombin generation assays. Both SHBG variants bound to TFPI with decreased affinity in surface plasmon resonance experiments. The TFPI Kunitz 3 domain is known to contain the interaction site for protein S. Using in silico analysis and protein docking exercises, preliminary models of the protein S SHBG/TFPI Kunitz domain 3 complex were created. Based on a combination of experimental and in silico data we propose a binding site for TFPI on protein S, involving both LG-domains.},
  author       = {Somajo, Sofia and Ahnström, Josefin and Fernandez-Recio, J and Gierula, M and Villoutreix, B O and Dahlbäck, Björn},
  issn         = {0340-6245},
  language     = {eng},
  number       = {5},
  pages        = {976--987},
  publisher    = {F K Schattauer Verlag Gmbh},
  series       = {Thrombosis and Haemostasis},
  title        = {Amino acid residues in the laminin G domains of protein S involved in tissue factor pathway inhibitor interaction.},
  url          = {http://dx.doi.org/10.1160/TH14-09-0803},
  volume       = {113},
  year         = {2015},
}