title: Protein S Thr103Asn mutation associated with type II deficiency reproduced in vitro and functionally characterised.
abstract: Protein S functions as a cofactor to activated protein C (APC) in the degradation of FVa and FVIIIa. In protein S, the thrombin sensitive region (TSR) and the first EGF-like domain are important for expression of the APC cofactor activity. A naturally occurring Thr103Asn (T103N) mutation in the first EGF-like domain of protein S has been associated with functional (type II) protein S deficiency. To elucidate the functional consequences of the T103N mutation, recombinant protein S mutant was expressed in mammalian cells and functionally characterised. The expression level of protein S T103N from transiently transfected COS 1 cells was equal to that of wild type protein S. The mutant protein S and wild type protein S were also expressed in 293 cells after stable transfection, and the recombinant proteins purified. In APTT- and PT-based coagulation assays, the mutant protein demonstrated approximately 50% lower anticoagulant activity as compared to wild type protein S. The functional defect was further investigated in FVa- and FVIIIa-degradation assays. The functional defect of mutant protein S was attenuated at increasing concentrations of APC. The results demonstrate the region around residue 103 of protein S to be of functional importance, possibly through a direct interaction with APC.
authors.affiliation: Department of Laboratory Medicine, Lund University, University Hospital Malmö, Sweden.
authors.forename: T K
authors.initials: TK
authors.lastname: Giri
authors.forename: P
authors.initials: P
authors.lastname: García de Frutos
authors.forename: B
authors.initials: B
authors.lastname: Dahlbäck
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001780:Blood Coagulation Tests; D019556:COS Cells; D004305:Dose-Response Relationship, Drug; D005165:Factor V / Q000187:drug effects / Q000378:metabolism; D015944:Factor VIIIa / Q000187:drug effects / Q000378:metabolism; D015943:Factor Va / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D016297:Mutagenesis, Site-Directed; D010314:Partial Thromboplastin Time; D017354:Point Mutation; D011486:Protein C / Q000378:metabolism / Q000494:pharmacology; D017293:Protein S / Q000235:genetics* / Q000494:pharmacology*; D018455:Protein S Deficiency / Q000235:genetics*; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins / Q000235:genetics; D014162:Transfection
chemical_list: D011486:Protein C; D017293:Protein S; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins; C095381:factor V Leiden; D015943:Factor Va; D015944:Factor VIIIa; D005165:Factor V