Dissociation of activated protein C functions by elimination of protein S cofactor enhancement.

Hdl Handle:
http://hdl.handle.net/10147/82463
Title:
Dissociation of activated protein C functions by elimination of protein S cofactor enhancement.
Authors:
Harmon, Shona; Preston, Roger J S; Ainle, Fionnuala Ni; Johnson, Jennifer A; Cunningham, Moya S; Smith, Owen P; White, Barry; O'Donnell, James S
Affiliation:
Haemostasis Research Group, Institute of Molecular Medicine, St James's Hospital, Trinity College, Dublin 8, Ireland.
Citation:
Dissociation of activated protein C functions by elimination of protein S cofactor enhancement. 2008, 283 (45):30531-9 J. Biol. Chem.
Journal:
The Journal of biological chemistry
Issue Date:
7-Nov-2008
URI:
http://hdl.handle.net/10147/82463
DOI:
10.1074/jbc.M802338200
PubMed ID:
18779332
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.
Language:
en
MeSH:
Amino Acid Substitution; Antigens, CD; Binding Sites; Cell Line; Coenzymes; Endothelial Cells; Factor VIIIa; Factor Va; Humans; Mutagenesis, Site-Directed; Peptide Mapping; Protein C; Protein S; Receptor, PAR-1; Receptors, Cell Surface; Signal Transduction
ISSN:
0021-9258

Full metadata record

DC FieldValue Language
dc.contributor.authorHarmon, Shona-
dc.contributor.authorPreston, Roger J S-
dc.contributor.authorAinle, Fionnuala Ni-
dc.contributor.authorJohnson, Jennifer A-
dc.contributor.authorCunningham, Moya S-
dc.contributor.authorSmith, Owen P-
dc.contributor.authorWhite, Barry-
dc.contributor.authorO'Donnell, James S-
dc.date.accessioned2009-09-24T10:13:54Z-
dc.date.available2009-09-24T10:13:54Z-
dc.date.issued2008-11-07-
dc.identifier.citationDissociation of activated protein C functions by elimination of protein S cofactor enhancement. 2008, 283 (45):30531-9 J. Biol. Chem.en
dc.identifier.issn0021-9258-
dc.identifier.pmid18779332-
dc.identifier.doi10.1074/jbc.M802338200-
dc.identifier.urihttp://hdl.handle.net/10147/82463-
dc.description.abstractActivated 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.-
dc.language.isoenen
dc.subject.meshAmino Acid Substitution-
dc.subject.meshAntigens, CD-
dc.subject.meshBinding Sites-
dc.subject.meshCell Line-
dc.subject.meshCoenzymes-
dc.subject.meshEndothelial Cells-
dc.subject.meshFactor VIIIa-
dc.subject.meshFactor Va-
dc.subject.meshHumans-
dc.subject.meshMutagenesis, Site-Directed-
dc.subject.meshPeptide Mapping-
dc.subject.meshProtein C-
dc.subject.meshProtein S-
dc.subject.meshReceptor, PAR-1-
dc.subject.meshReceptors, Cell Surface-
dc.subject.meshSignal Transduction-
dc.titleDissociation of activated protein C functions by elimination of protein S cofactor enhancement.en
dc.contributor.departmentHaemostasis Research Group, Institute of Molecular Medicine, St James's Hospital, Trinity College, Dublin 8, Ireland.en
dc.identifier.journalThe Journal of biological chemistryen

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