Human factor VIII inhibitor alloantibodies with a C2 epitope inhibit factor Xa-catalyzed factor VIII activation: a new anti-factor VIII inhibitory mechanism.
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.
Nogami
Keiji
K
Department of Pediatrics, Nara Medical University, Kashihara, Japan.
Shima
Midori
M
Nishiya
Katsumi
K
Sakurai
Yoshihiko
Y
Tanaka
Ichiro
I
Giddings
John C
JC
Saenko
Evgueni L
EL
Yoshioka
Akira
A
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
D000939:Epitopes; D007518:Isoantibodies; D010743:Phospholipids; D005169:Factor VIII; D015951:Factor Xa