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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
html.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.


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