Abstract
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.
Highlights
Thrombin-thrombomodulin complex on endothelial cells and serves to limit clot development [1]
As described previously [17], removal of Activated protein C (APC) N-linked glycans caused a slight increase in plasma APC anticoagulant activity. 5 nM APC reduced the ETP to 47% of the ETP in the absence of APC, whereas an identical concentration of PNGAPC attenuated thrombin generation to 27% of the original ETP (Fig. 1B)
Increased anticoagulant activity was not associated with increased amidolytic activity, as determined by PNGase F-treated APC (PNG-APC) hydrolysis of a short APC-specific chromogenic substrate
Summary
Amino acid substitution of the N-linked glycan attachment site at Asn-97 was found to impair in vitro protein C secretion from mammalian cells. The same study showed that individual mutagenesis of each N-linked glycosylation attachment site on the APC serine protease domain increases APC generation by the thrombin-thrombomodulin complex and improves anticoagulant activity in a modified activated partial thromboplertin time (APTT) assay [17]. N-Linked glycosylation at amino acid position Asn-329 was identified as a key regulator of APC-EPCRPAR1 signaling on endothelial cells. These data indicate that deglycosylated, non-anticoagulant recombinant APC variants may represent a novel tool for treatment of sepsis and other inflammatory diseases
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