Dissociation of activated protein C functions by elimination of protein S cofactor enhancement.
Harmon, Shona Preston, Roger J S Ainle, Fionnuala Ni Johnson, Jennifer A Cunningham, Moya S Smith, Owen P White, Barry O'Donnell, James S
Harmon, Shona
Preston, Roger J S
Ainle, Fionnuala Ni
Johnson, Jennifer A
Cunningham, Moya S
Smith, Owen P
White, Barry
O'Donnell, James S
Advisors
Editors
Other Contributors
Date
2008-11-07
Date Submitted
Keywords
Other Subjects
Subject 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
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
Planned Date
Start Date
Collaborators
Principal Investigators
Alternative Titles
Publisher
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
ISSN
0021-9258
eISSN
ISBN
DOI
10.1074/jbc.M802338200
PMID
18779332