title: Dissociation of activated protein C functions by elimination of protein S cofactor enhancement.
abstract: 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.
authors.affiliation: Haemostasis Research Group, Institute of Molecular Medicine, St James's Hospital, Trinity College, Dublin 8, Ireland.
authors.forename: Shona
authors.initials: S
authors.lastname: Harmon
authors.forename: Roger J S
authors.initials: RJ
authors.lastname: Preston
authors.forename: Fionnuala
authors.initials: F
authors.lastname: Ni Ainle
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Johnson
authors.forename: Moya S
authors.initials: MS
authors.lastname: Cunningham
authors.forename: Owen P
authors.initials: OP
authors.lastname: Smith
authors.forename: Barry
authors.initials: B
authors.lastname: White
authors.forename: James S
authors.initials: JS
authors.lastname: O'Donnell
mesh_terms: D019943:Amino Acid Substitution; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D003067:Coenzymes / Q000235:genetics / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D000074283:Endothelial Protein C Receptor; D015944:Factor VIIIa / Q000235:genetics / Q000378:metabolism; D015943:Factor Va / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping / Q000379:methods; D011486:Protein C / Q000235:genetics / Q000378:metabolism*; D017293:Protein S / Q000235:genetics / Q000378:metabolism*; D044463:Receptor, PAR-1 / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D015703:Antigens, CD; D003067:Coenzymes; D000074283:Endothelial Protein C Receptor; C478370:PROCR protein, human; D011486:Protein C; D017293:Protein S; D044463:Receptor, PAR-1; D011956:Receptors, Cell Surface; D015943:Factor Va; D015944:Factor VIIIa
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