title: Functional characterization of factor V-Ile359Thr: a novel mutation associated with thrombosis.
abstract: A missense mutation, FV-Ile359Thr (FV Liverpool), associated with thrombosis has recently been described. This mutation creates an additional potential N-linked glycosylation site (Asn-X-Ser/Thr) in factor V (FV) at Asn357 that could interfere with secretion and/or protein interactions. To investigate the molecular pathology of FV-Ile359Thr, the mutation was created by site-directed mutagenesis and expressed together with other mutations that could help explain the phenotype (FV-Arg306Gln/Ile359Thr/Arg679Gln, FV-Ile359Thr/Arg506Gln/Arg679Gln, and FV-Asn357Gln/Ile359Thr). The FV-Ile359Thr was secreted normally and had full procoagulant activity. Western blot analysis showed that FV-Ile359Thr migrated more slowly, while the FV-Asn357Gln/Ile359Thr was indistinguishable from FV-wild type (FV-WT), indicating that FV-Ile359Thr was expressed with an additional carbohydrate chain. Activated protein C (APC)-mediated inactivation in an FVa degradation assay showed that the Ile359Thr mutation significantly reduced the cleavage at Arg306 both in the presence and absence of protein S, whereas the cleavage at Arg506 was unaffected. When tested in an FVIIIa degradation assay, the FV-Ile359Thr variant exhibited equally low APC cofactor activity as FV Leiden (FVArg506Gln). In conclusion, the Ile359Thr mutation appears to affect anticoagulation by 2 mechanisms, impeding the APC-mediated down-regulation of the FVa molecule and additionally being a poor APC cofactor for the down-regulation of FVIIIa. These findings explain the association of the FV-Ile359Thr mutation with thrombosis.
authors.affiliation: Department of Laboratory Medicine, Division of Clinical Chemistry, Lund University, The Wallenberg Laboratory, University Hospital, Malmö, Sweden.
authors.forename: Mårten
authors.initials: M
authors.lastname: Steen
authors.forename: Eva A
authors.initials: EA
authors.lastname: Norstrøm
authors.forename: Ann-Louise
authors.initials: AL
authors.lastname: Tholander
authors.forename: Paula H B
authors.initials: PH
authors.lastname: Bolton-Maggs
authors.forename: Andrew
authors.initials: A
authors.lastname: Mumford
authors.forename: John H
authors.initials: JH
authors.lastname: McVey
authors.forename: Edward G D
authors.initials: EG
authors.lastname: Tuddenham
authors.forename: Björn
authors.initials: B
authors.lastname: Dahlbäck
mesh_terms: D019943:Amino Acid Substitution; D001777:Blood Coagulation; D005165:Factor V / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D006031:Glycosylation; D006801:Humans; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010641:Phenotype; D011486:Protein C / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017293:Protein S / Q000378:metabolism; D013927:Thrombosis / Q000209:etiology / Q000235:genetics*
chemical_list: D011486:Protein C; D017293:Protein S; D005165:Factor V