Activated protein C N-linked glycans modulate cytoprotective signaling function on endothelial cells.
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.
Haemostasis Research Group, Institute of Molecular Medicine, Trinity College Dublin, Dublin 2, Ireland.
Fionnuala
F
Ní Ainle
James S
JS
O'Donnell
Jennifer A
JA
Johnson
Laura
L
Brown
Eimear M
EM
Gleeson
Owen P
OP
Smith
Roger J S
RJ
Preston
D019943:Amino Acid Substitution; D000925:Anticoagulants / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D019610:Cytoprotection / Q000187:drug effects / Q000276:immunology; D042783:Endothelial Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D007249:Inflammation / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D011486:Protein C / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D044463:Receptor, PAR-1 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology*; D019311:Staurosporine / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology; D013927:Thrombosis / Q000276:immunology / Q000378:metabolism*
D000925:Anticoagulants; D004791:Enzyme Inhibitors; D011134:Polysaccharides; D011486:Protein C; D044463:Receptor, PAR-1; D013917:Thrombin; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D019311:Staurosporine