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Selective modulation of protein C affinity for EPCR and phospholipids by Gla domain mutation

Preston, R J S; Villegas-Mendez, A; Sun, Yong-Hui; Hermida, J; Simioni, P; Philippou, H; Dahlbäck, Björn LU and Lane, D A (2005) In The FEBS Journal 272(1). p.97-108
Abstract
Uniquely amongst vitamin K-dependent coagulation proteins, protein C interacts via its Gla domain both with a receptor, the endothelial cell protein C receptor (EPCR), and with phospholipids. We have studied naturally occurring and recombinant protein C Gla domain variants for soluble (s)EPCR binding, cell surface activation to activated protein C (APC) by the thrombin-thrombomodulin complex, and phospholipid dependent factor Va (FVa) inactivation by APC, to establish if these functions are concordant. Wild-type protein C binding to sEPCR was characterized with surface plasmon resonance to have an association rate constant of 5.23x10(5) M-1.s(-1), a dissociation rate constant of 7.61x10(-2) s(-1) and equilibrium binding constant (K-D) of... (More)
Uniquely amongst vitamin K-dependent coagulation proteins, protein C interacts via its Gla domain both with a receptor, the endothelial cell protein C receptor (EPCR), and with phospholipids. We have studied naturally occurring and recombinant protein C Gla domain variants for soluble (s)EPCR binding, cell surface activation to activated protein C (APC) by the thrombin-thrombomodulin complex, and phospholipid dependent factor Va (FVa) inactivation by APC, to establish if these functions are concordant. Wild-type protein C binding to sEPCR was characterized with surface plasmon resonance to have an association rate constant of 5.23x10(5) M-1.s(-1), a dissociation rate constant of 7.61x10(-2) s(-1) and equilibrium binding constant (K-D) of 147 nM. It was activated by thrombin over endothelial cells with a K-m of 213 nM and once activated to APC, rapidly inactivated FVa. Each of these interactions was dramatically reduced for variants causing gross Gla domain misfolding (R-1L, R-1C, E16D and E26K). Recombinant variants Q32A, V34A and D35A had essentially normal functions. However, R9H and H10Q/S11G/S12N/D23S/Q32E/N33D/H44Y (QGNSEDY) variants had slightly reduced (<twofold) binding to sEPCR, arising from an increased rate of dissociation, and increased K-m (358 nM for QGNSEDY) for endothelial cell surface activation by thrombin. Interestingly, these variants had greatly reduced (R9H) or greatly enhanced (QGNSEDY) ability to inactivate FVa. Therefore, protein C binding to sEPCR and phospholipids is broadly dependent on correct Gla domain folding, but can be selectively influenced by judicious mutation. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
activated protein C, endothelial cell protein C receptor, protein C
in
The FEBS Journal
volume
272
issue
1
pages
97 - 108
publisher
Federation of European Neuroscience Societies and Blackwell Publishing Ltd
external identifiers
  • pmid:15634335
  • wos:000227358000009
  • scopus:11444268040
ISSN
1742-464X
DOI
10.1111/j.1432-1033.2004.04401.x
language
English
LU publication?
yes
id
ad8bce9e-5588-4c2b-9e97-ed6125f75018 (old id 250540)
date added to LUP
2007-10-05 12:27:46
date last changed
2017-08-20 04:22:40
@article{ad8bce9e-5588-4c2b-9e97-ed6125f75018,
  abstract     = {Uniquely amongst vitamin K-dependent coagulation proteins, protein C interacts via its Gla domain both with a receptor, the endothelial cell protein C receptor (EPCR), and with phospholipids. We have studied naturally occurring and recombinant protein C Gla domain variants for soluble (s)EPCR binding, cell surface activation to activated protein C (APC) by the thrombin-thrombomodulin complex, and phospholipid dependent factor Va (FVa) inactivation by APC, to establish if these functions are concordant. Wild-type protein C binding to sEPCR was characterized with surface plasmon resonance to have an association rate constant of 5.23x10(5) M-1.s(-1), a dissociation rate constant of 7.61x10(-2) s(-1) and equilibrium binding constant (K-D) of 147 nM. It was activated by thrombin over endothelial cells with a K-m of 213 nM and once activated to APC, rapidly inactivated FVa. Each of these interactions was dramatically reduced for variants causing gross Gla domain misfolding (R-1L, R-1C, E16D and E26K). Recombinant variants Q32A, V34A and D35A had essentially normal functions. However, R9H and H10Q/S11G/S12N/D23S/Q32E/N33D/H44Y (QGNSEDY) variants had slightly reduced (&lt;twofold) binding to sEPCR, arising from an increased rate of dissociation, and increased K-m (358 nM for QGNSEDY) for endothelial cell surface activation by thrombin. Interestingly, these variants had greatly reduced (R9H) or greatly enhanced (QGNSEDY) ability to inactivate FVa. Therefore, protein C binding to sEPCR and phospholipids is broadly dependent on correct Gla domain folding, but can be selectively influenced by judicious mutation.},
  author       = {Preston, R J S and Villegas-Mendez, A and Sun, Yong-Hui and Hermida, J and Simioni, P and Philippou, H and Dahlbäck, Björn and Lane, D A},
  issn         = {1742-464X},
  keyword      = {activated protein C,endothelial cell protein C receptor,protein C},
  language     = {eng},
  number       = {1},
  pages        = {97--108},
  publisher    = {Federation of European Neuroscience Societies and Blackwell Publishing Ltd},
  series       = {The FEBS Journal},
  title        = {Selective modulation of protein C affinity for EPCR and phospholipids by Gla domain mutation},
  url          = {http://dx.doi.org/10.1111/j.1432-1033.2004.04401.x},
  volume       = {272},
  year         = {2005},
}