title: Activated protein C cofactor function of protein S: a novel role for a γ-carboxyglutamic acid residue.
abstract: Protein S has an important anticoagulant function by acting as a cofactor for activated protein C (APC). We recently reported that the EGF1 domain residue Asp95 is critical for APC cofactor function. In the present study, we examined whether additional interaction sites within the Gla domain of protein S might contribute to its APC cofactor function. We examined 4 residues, composing the previously reported "Face1" (N33S/P35T/E36A/Y39V) variant, as single point substitutions. Of these protein S variants, protein S E36A was found to be almost completely inactive using calibrated automated thrombography. In factor Va inactivation assays, protein S E36A had 89% reduced cofactor activity compared with wild-type protein S and was almost completely inactive in factor VIIIa inactivation; phospholipid binding was, however, normal. Glu36 lies outside the ω-loop that mediates Ca(2+)-dependent phospholipid binding. Using mass spectrometry, it was nevertheless confirmed that Glu36 is γ-carboxylated. Our finding that Gla36 is important for APC cofactor function, but not for phospholipid binding, defines a novel function (other than Ca(2+) coordination/phospholipid binding) for a Gla residue in vitamin K-dependent proteins. It also suggests that residues within the Gla and EGF1 domains of protein S act cooperatively for its APC cofactor function.
authors.affiliation: Centre for Haematology, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London, UK. j.ahnstrom@imperial.ac.uk
authors.forename: Josefin
authors.initials: J
authors.lastname: Ahnström
authors.forename: Helena M
authors.initials: HM
authors.lastname: Andersson
authors.forename: Kevin
authors.initials: K
authors.lastname: Canis
authors.forename: Eva
authors.initials: E
authors.lastname: Norstrøm
authors.forename: Yao
authors.initials: Y
authors.lastname: Yu
authors.forename: Björn
authors.initials: B
authors.lastname: Dahlbäck
authors.forename: Maria
authors.initials: M
authors.lastname: Panico
authors.forename: Howard R
authors.initials: HR
authors.lastname: Morris
authors.forename: James T B
authors.initials: JT
authors.lastname: Crawley
authors.forename: David A
authors.initials: DA
authors.lastname: Lane
mesh_terms: D015055:1-Carboxyglutamic Acid / Q000737:chemistry / Q000502:physiology*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000502:physiology; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D002478:Cells, Cultured; D015944:Factor VIIIa / Q000378:metabolism; D015943:Factor Va / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism / Q000502:physiology; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D011486:Protein C / Q000819:agonists / Q000378:metabolism* / Q000502:physiology; D054730:Protein Interaction Domains and Motifs / Q000235:genetics / Q000502:physiology; D017293:Protein S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
chemical_list: D050505:Mutant Proteins; D011486:Protein C; D017293:Protein S; D015055:1-Carboxyglutamic Acid; D015943:Factor Va; D015944:Factor VIIIa