Activated protein C N-linked glycans modulate cytoprotective signaling function on endothelial cells

J Biol Chem. 2011 Jan 14;286(2):1323-30. doi: 10.1074/jbc.M110.159475. Epub 2010 Nov 2.


Activated protein C (APC) has potent anticoagulant and anti-inflammatory properties that limit clot formation, inhibit apoptosis, and protect vascular endothelial cell barrier integrity. In this study, the role of N-linked glycans in modulating APC endothelial cytoprotective signaling via endothelial cell protein C receptor/protease-activated receptor 1 (PAR1) was investigated. Enzymatic digestion of APC N-linked glycans (PNG-APC) decreased the APC concentration required to achieve half-maximal inhibition of thrombin-induced endothelial cell barrier permeability by 6-fold. Furthermore, PNG-APC exhibited increased protection against staurosporine-induced endothelial cell apoptosis when compared with untreated APC. To investigate the specific N-linked glycans responsible, recombinant APC variants were generated in which each N-linked glycan attachment site was eliminated. Of these, APC-N329Q was up to 5-fold more efficient in protecting endothelial barrier function when compared with wild type APC. Based on these findings, an APC variant (APC-L38D/N329Q) was generated with minimal anticoagulant activity, but 5-fold enhanced endothelial barrier protective function and 30-fold improved anti-apoptotic function when compared with wild type APC. These data highlight the previously unidentified role of APC N-linked glycosylation in modulating endothelial cell protein C receptor-dependent cytoprotective signaling via PAR1. Furthermore, our data suggest that plasma β-protein C, characterized by aberrant N-linked glycosylation at Asn-329, may be particularly important for maintenance of APC cytoprotective functions in vivo.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Anticoagulants / metabolism
  • Apoptosis / drug effects
  • Apoptosis / physiology
  • Cells, Cultured
  • Cytoprotection / drug effects
  • Cytoprotection / immunology
  • Endothelial Cells / cytology
  • Endothelial Cells / immunology
  • Endothelial Cells / metabolism*
  • Enzyme Inhibitors / pharmacology
  • Glycosylation / drug effects
  • Humans
  • Inflammation / metabolism
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / metabolism
  • Polysaccharides / metabolism*
  • Protein C / genetics
  • Protein C / immunology
  • Protein C / metabolism*
  • Receptor, PAR-1 / metabolism
  • Signal Transduction / drug effects
  • Signal Transduction / immunology*
  • Staurosporine / pharmacology
  • Thrombin / metabolism
  • Thrombin / pharmacology
  • Thrombosis / immunology
  • Thrombosis / metabolism*


  • Anticoagulants
  • Enzyme Inhibitors
  • Polysaccharides
  • Protein C
  • Receptor, PAR-1
  • Thrombin
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
  • Staurosporine