title: Mutations associated with hemophilia A in the 558-565 loop of the factor VIIIa A2 subunit alter the catalytic activity of the factor Xase complex.
abstract: The 558-565 loop region in the A2 subunit of factor (F) VIIIa forms a direct interface with FIXa. We have expressed and purified B-domainless FVIII (FVIII(WT)) and B-domainless FVIII containing the hemophilia A-associated mutations Ser558Phe, Val559Ala, Asp560Ala, Gln565Arg, and the activated protein C cleavage site mutant Arg562Ala. Titration of FVIIIa in FXa generation assays showed that the mutant and wild-type proteins had similar functional affinities for FIXa (dissociation constant [K(d)] values approximately 5 nM-20 nM and approximately 100 nM-250 nM in the presence and absence of phospholipid, respectively). The catalytic activities of the factor Xase complex composed of the hemophilia A-associated FVIII species were markedly reduced both in the presence and absence of phospholipid. FVIII(WT) and FVIII(Arg562Ala) showed catalytic rate constant (k(cat)) values of approximately 60 minute(-1) in the presence of phospholipid, whereas the hemophilia A-associated mutants showed k(cat) values ranging from 3.3 minute(-1) to 7.5 minute(-1). In the absence of phospholipid, all k(cat) values were reduced but FVIII(WT) and FVIII(Arg562Ala) retained higher activities as compared with the hemophilic mutant FVIII forms. Fluorescence anisotropy experiments using fluorescein-modified FIXa confirmed that all FVIII forms interacted with FIXa. However, the presence of factor X yielded minimal increases in anisotropy observed with the mutant factor VIII forms, consistent with their reduced activity. These results show that residues within the 558-565 loop are critical in modulating FIXa enzymatic activity but do not contribute significantly to the affinity of FVIIIa for FIXa.
authors.affiliation: Department of Biochemistry and Biophysics and the Department of Medicine, University of Rochester Medical Center, Rochester, NY 14642, USA.
authors.forename: P Vincent
authors.initials: PV
authors.lastname: Jenkins
authors.forename: Jan
authors.initials: J
authors.lastname: Freas
authors.forename: Kyla M
authors.initials: KM
authors.lastname: Schmidt
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhou
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Fay
mesh_terms: D001483:Base Sequence; D002384:Catalysis; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015949:Factor IXa / Q000378:metabolism; D015944:Factor VIIIa / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005454:Fluorescence Polarization; D006467:Hemophilia A / Q000097:blood / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D009154:Mutation; D009363:Neoplasm Proteins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits
chemical_list: D009363:Neoplasm Proteins; D021122:Protein Subunits; D015944:Factor VIIIa; D015949:Factor IXa; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant