title: Cofactor activity in factor VIIIa of the blood clotting pathway is stabilized by an interdomain bond between His281 and Ser524 formed in factor VIII.
abstract: The factor VIII (FVIII) crystal structure suggests a possible bonding interaction of His(281) (A1 domain) with Ser(524) (A2 domain), although the resolution of the structure (∼4 Å) does not firmly establish this bonding. To establish that side chains of these residues participate in an interdomain bond, we prepared and examined the functional properties of a residue swap variant (H281S/S524H) where His(281) and Ser(524) residues were exchanged with one another and a disulfide-bridged variant (H281C/S524C) where the two residues were replaced with Cys. The latter variant showed efficient disulfide bonding of the A1 and A2 domains. The swap variant showed WT-like FVIII and FVIIIa stability, which were markedly reduced for H281A and S524A variants in an earlier study. The disulfide-bridged variant showed ∼20% increased FVIII stability, and FVIIIa did not decay during the time course measured. This variant also yielded 35% increased thrombin peak values compared with WT in a plasma-based thrombin generation assay. Binding analyses of H281S-A1/A3C1C2 dimer with S524H-A2 subunit yielded a near WT-like affinity value, whereas combining the variant dimer or A2 subunit with the WT complement yielded ∼5- and ∼10-fold reductions, respectively, in affinity. Other functional properties including thrombin generation potential, FIXa binding affinity, Km for FX of FXase complexes, thrombin activation efficiency, and down-regulation by activated protein C showed similar results for the two variants compared with WT FVIII. These results indicate that the side chains of His(281) and Ser(524) are in close proximity and contribute to a bonding interaction in FVIII that is retained in FVIIIa.
authors.affiliation: From the Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642.
authors.forename: Hironao
authors.initials: H
authors.lastname: Wakabayashi
authors.affiliation: From the Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642.
authors.forename: Morgan
authors.initials: M
authors.lastname: Monaghan
authors.affiliation: From the Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642 philip_fay@urmc.rochester.edu.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Fay
mesh_terms: D001777:Blood Coagulation; D015949:Factor IXa / Q000378:metabolism; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015944:Factor VIIIa / Q000378:metabolism*; D006639:Histidine; D006801:Humans; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D012694:Serine; D013917:Thrombin / Q000378:metabolism
chemical_list: D012694:Serine; D006639:Histidine; D015944:Factor VIIIa; D005169:Factor VIII; D015949:Factor IXa; D013917:Thrombin
keywords: Blood Coagulation Factors; Factor VIII; Mutagenesis; Protein Stability; Protein Structure