Dissociation of activated protein C functions by elimination of protein S cofactor enhancement

J Biol Chem. 2008 Nov 7;283(45):30531-9. doi: 10.1074/jbc.M802338200. Epub 2008 Sep 8.


Activated protein C (APC) plays a critical anticoagulant role in vivo by inactivating procoagulant factor Va and factor VIIIa and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor can initiate protease-activated receptor-1 (PAR-1)-mediated cytoprotective signaling. Protein S constitutes a critical cofactor for the anticoagulant function of APC but is not known to be involved in regulating APC-mediated protective PAR-1 signaling. In this study we utilized a site-directed mutagenesis strategy to characterize a putative protein S binding region within the APC Gla domain. Three single amino acid substitutions within the APC Gla domain (D35T, D36A, and A39V) were found to mildly impair protein S-dependent anticoagulant activity (<2-fold) but retained entirely normal cytoprotective activity. However, a single amino acid substitution (L38D) ablated the ability of protein S to function as a cofactor for this APC variant. Consequently, in assays of protein S-dependent factor Va proteolysis using purified proteins or in the plasma milieu, APC-L38D variant exhibited minimal residual anticoagulant activity compared with wild type APC. Despite the location of Leu-38 in the Gla domain, APC-L38D interacted normally with endothelial cell protein C receptor and retained its ability to trigger PAR-1 mediated cytoprotective signaling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to separate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Antigens, CD / genetics
  • Antigens, CD / metabolism
  • Binding Sites / genetics
  • Cell Line
  • Coenzymes / genetics
  • Coenzymes / metabolism*
  • Endothelial Cells / cytology
  • Endothelial Cells / metabolism*
  • Endothelial Protein C Receptor
  • Factor VIIIa / genetics
  • Factor VIIIa / metabolism
  • Factor Va / genetics
  • Factor Va / metabolism
  • Humans
  • Mutagenesis, Site-Directed
  • Peptide Mapping / methods
  • Protein C / genetics
  • Protein C / metabolism*
  • Protein S / genetics
  • Protein S / metabolism*
  • Receptor, PAR-1 / genetics
  • Receptor, PAR-1 / metabolism*
  • Receptors, Cell Surface / genetics
  • Receptors, Cell Surface / metabolism
  • Signal Transduction / physiology*


  • Antigens, CD
  • Coenzymes
  • Endothelial Protein C Receptor
  • PROCR protein, human
  • Protein C
  • Protein S
  • Receptor, PAR-1
  • Receptors, Cell Surface
  • Factor Va
  • Factor VIIIa