Inhibition of human factor VIIIa by anti-A2 subunit antibodies.
Human inhibitory alloantibodies and autoantibodies to Factor VIII (FVIII) are usually directed toward the A2 and/or C2 domains of the FVIII molecule. Anti-C2 antibodies block the binding of FVIII to phospholipid, but the mechanism of action of anti-A2 antibodies is not known. We investigated the properties of a patient autoantibody, RC, and a monoclonal antibody, 413, that bind to the region which contains the epitopes of all anti-A2 alloantibodies or autoantibodies studied to date. mAb 413 and RC were noncompetitive inhibitors of a model intrinsic Factor X activation complex (intrinsic FXase) consisting of Factor IXa, activated FVIII (FVIIIa), and synthetic phospholipid vesicles, since they decreased the Vmax of intrinsic FXase by > 95% at saturating concentrations without altering the Km. This indicates that RC and mAb 413 either block the binding of FVIIIa to FIXa or phospholipid or interfere with the catalytic function of fully assembled intrinsic FXase, but they do not inhibit the binding of the substrate Factor X. mAb 413 did not inhibit the increase in fluorescence anisotropy that results from the binding of Factor VIIIa to fluorescein-5-maleimidyl-D-phenylalanyl-prolyl-arginyl-FIXa (Fl-M-FPR-FIXa) on phospholipid vesicles in the absence of Factor X, indicating it does not inhibit assembly of intrinsic FXase. Addition of Factor X to Fl-M-FPR-FIXa, FVIIIa, and phospholipid vesicles produced a further increase in fluorescence anisotropy and a decrease in fluorescence intensity. This effect was blocked completely by mAb 413. We conclude that anti-A2 antibodies inhibit FVIIIa function by blocking the conversion of intrinsic FXase/FX complex to the transition state, rather than by interfering with formation of the ground state Michaelis complex.
Lollar
P
P
Department of Medicine, Emory University, Atlanta, Georgia 30322.
Parker
E T
ET
Curtis
J E
JE
Helgerson
S L
SL
Hoyer
L W
LW
Scott
M E
ME
Scandella
D
D
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology*; D015944:Factor VIIIa / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005170:Factor X / Q000494:pharmacology; D005454:Fluorescence Polarization; D006801:Humans; D051379:Mice
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D015944:Factor VIIIa; D005170:Factor X