title: Activated protein C β-glycoform promotes enhanced noncanonical PAR1 proteolysis and superior resistance to ischemic injury.
abstract: Activated protein C (APC) is an anticoagulant protease that initiates cell signaling via protease-activated receptor 1 (PAR1) to regulate vascular integrity and inflammatory response. In this study, a recombinant APC variant (APC(N329Q)) mimicking the naturally occurring APC-β plasma glycoform was found to exhibit superior PAR1 proteolysis at a cleavage site that selectively mediates cytoprotective signaling. APC(N329Q) also enhanced integrin αMβ2-dependent PAR1 proteolysis to exert significantly improved antiinflammatory activity on macrophages compared with wild-type APC. Recent therapeutic applications of recombinant APC in ischemic stroke models have used APC variants with limited anticoagulant activity to negate potential bleeding side effects. Using a mouse model of ischemic stroke and late t-PA intervention, the neuroprotective activity of a murine APC variant with limited anticoagulant activity (mAPC(PS)) was compared with an identical APC variant except for the absence of glycosylation at the APC-β sequon (mAPC(PS/N329Q)). Remarkably, mAPC(PS/N329Q) limited cerebral ischemic injury and reduced brain lesion volume significantly more effectively than mAPC(PS). Collectively, this study reveals the importance of APC glycosylation in controlling the efficacy of PAR1 proteolysis by APC and demonstrates the potential of novel APC variants with superior cytoprotective signaling function as enhanced therapeutic agents for the treatment of ischemic stroke.
authors.affiliation: Department of Clinical Medicine, School of Medicine, Trinity College Dublin, Dublin, United Kingdom; National Children's Research Centre, Our Lady's Children's Hospital Crumlin, Crumlin, Dublin, United Kingdom;
authors.forename: Eimear M
authors.initials: EM
authors.lastname: Gleeson
authors.affiliation: Atherothrombosis Laboratory, Center for Applied Medical Research, University of Navarra, Pamplona, Spain;
authors.forename: María G
authors.initials: MG
authors.lastname: Dichiara
authors.affiliation: Atherothrombosis Laboratory, Center for Applied Medical Research, University of Navarra, Pamplona, Spain;
authors.forename: Agustina
authors.initials: A
authors.lastname: Salicio
authors.affiliation: Department of Clinical Medicine, School of Medicine, Trinity College Dublin, Dublin, United Kingdom; National Children's Research Centre, Our Lady's Children's Hospital Crumlin, Crumlin, Dublin, United Kingdom;
authors.forename: Louise M
authors.initials: LM
authors.lastname: Quinn
authors.affiliation: Department of Clinical Medicine, School of Medicine, Trinity College Dublin, Dublin, United Kingdom; National Children's Research Centre, Our Lady's Children's Hospital Crumlin, Crumlin, Dublin, United Kingdom;
authors.forename: Clive
authors.initials: C
authors.lastname: Drakeford
authors.affiliation: Department of Clinical Medicine, School of Medicine, Trinity College Dublin, Dublin, United Kingdom; National Children's Research Centre, Our Lady's Children's Hospital Crumlin, Crumlin, Dublin, United Kingdom;
authors.forename: Shane E
authors.initials: SE
authors.lastname: Russell
authors.affiliation: Department of Clinical Medicine, School of Medicine, Trinity College Dublin, Dublin, United Kingdom; National Children's Research Centre, Our Lady's Children's Hospital Crumlin, Crumlin, Dublin, United Kingdom;
authors.forename: Patrick T
authors.initials: PT
authors.lastname: Walsh
authors.affiliation: Atherothrombosis Laboratory, Center for Applied Medical Research, University of Navarra, Pamplona, Spain;
authors.forename: Josune
authors.initials: J
authors.lastname: Orbe
authors.affiliation: Atherothrombosis Laboratory, Center for Applied Medical Research, University of Navarra, Pamplona, Spain;
authors.forename: José
authors.initials: J
authors.lastname: Hermida
authors.affiliation: National Children's Research Centre, Our Lady's Children's Hospital Crumlin, Crumlin, Dublin, United Kingdom; Department of Haematology, Our Lady's Children's Hospital Crumlin, Crumlin, Dublin, United Kingdom; and.
authors.forename: Owen P
authors.initials: OP
authors.lastname: Smith
authors.affiliation: Haemostasis Research Group, School of Medicine, Trinity College Dublin, Dublin, United Kingdom.
authors.forename: James S
authors.initials: JS
authors.lastname: O'Donnell
authors.affiliation: Atherothrombosis Laboratory, Center for Applied Medical Research, University of Navarra, Pamplona, Spain;
authors.forename: Ramón
authors.initials: R
authors.lastname: Montes
authors.affiliation: Department of Clinical Medicine, School of Medicine, Trinity College Dublin, Dublin, United Kingdom; National Children's Research Centre, Our Lady's Children's Hospital Crumlin, Crumlin, Dublin, United Kingdom;
authors.forename: Roger J S
authors.identifier: http://orcid.org/0000-0003-0108-4077
authors.initials: RJ
authors.lastname: Preston
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D002545:Brain Ischemia / Q000378:metabolism* / Q000628:therapy; D002403:Cathepsins / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D000074283:Endothelial Protein C Receptor; D014644:Genetic Variation; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D009844:Oligosaccharides; D011486:Protein C / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use; D059748:Proteolysis; D044463:Receptor, PAR-1 / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use; D015398:Signal Transduction
chemical_list: D015703:Antigens, CD; C410987:Cts7 protein, mouse; D000074283:Endothelial Protein C Receptor; D009844:Oligosaccharides; C478370:PROCR protein, human; D011486:Protein C; D020033:Protein Isoforms; D044463:Receptor, PAR-1; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C488960:lycotetraose; D002403:Cathepsins