title: Human factor VIII inhibitor alloantibodies with a C2 epitope inhibit factor Xa-catalyzed factor VIII activation: a new anti-factor VIII inhibitory mechanism.
abstract: Factor VIII (FVIII) inhibitor alloantibodies react with the A2, C2, or A3-CI domains of FVIII and inactivate FVIII activity. We recently demonstrated that an anti-C2 monoclonal antibody with a Val2248-Gly2285 epitope, inhibited factor Xa (FXa)-catalyzed FVIII activation, and that a FXa binding site for FVIII was located within residues Thr2253-Gln2270. In this study, we investigated whether anti-C2 alloantibodies inhibit FXa-catalyzed FVIII activation. Anti-C2 alloantibodies from four patients inhibited FVIII activation by FXa in one-stage clotting assay. Furthermore, analysis by SDS-PAGE showed that all alloantibodies inhibited FVIII proteolytic cleavage by FXa independently of phospholipid. To confirm direct inhibition of FVIII and FXa interaction, we examined the effect of alloantibodies on FVIII binding to anhydro-FXa, a catalytically inactive FXa, in ELISA. All alloantibodies and C2-affinity purified F(ab)'2 preparations inhibited FVIII binding to anhydro-FXa dose-dependently. Our results revealed a new inhibitory mechanism of FVIII, mediated by inhibition of FXa in the presence of anti-C2 alloantibodies.
authors.affiliation: Department of Pediatrics, Nara Medical University, Kashihara, Japan.
authors.forename: Keiji
authors.initials: K
authors.lastname: Nogami
authors.forename: Midori
authors.initials: M
authors.lastname: Shima
authors.forename: Katsumi
authors.initials: K
authors.lastname: Nishiya
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Sakurai
authors.forename: Ichiro
authors.initials: I
authors.lastname: Tanaka
authors.forename: John C
authors.initials: JC
authors.lastname: Giddings
authors.forename: Evgueni L
authors.initials: EL
authors.lastname: Saenko
authors.forename: Akira
authors.initials: A
authors.lastname: Yoshioka
mesh_terms: D000918:Antibody Specificity; D004789:Enzyme Activation / Q000276:immunology; D000939:Epitopes / Q000276:immunology*; D005169:Factor VIII / Q000276:immunology* / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism*; D006467:Hemophilia A / Q000276:immunology; D006801:Humans; D007518:Isoantibodies / Q000276:immunology*; D010743:Phospholipids / Q000494:pharmacology; D011485:Protein Binding / Q000276:immunology; D017434:Protein Structure, Tertiary
chemical_list: D000939:Epitopes; D007518:Isoantibodies; D010743:Phospholipids; D005169:Factor VIII; D015951:Factor Xa