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

Research output: Contribution to journalArticle

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 (<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.

Details

Authors
  • R J S Preston
  • A Villegas-Mendez
  • Yong-Hui Sun
  • J Hermida
  • P Simioni
  • H Philippou
  • Björn Dahlbäck
  • D A Lane
Organisations
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Biochemistry and Molecular Biology

Keywords

  • activated protein C, endothelial cell protein C receptor, protein C
Original languageEnglish
Pages (from-to)97-108
JournalThe FEBS Journal
Volume272
Issue number1
Publication statusPublished - 2005
Publication categoryResearch
Peer-reviewedYes