INHIBITION OF HUMAN FACTOR VILLA BY ANTI-A2 SUBUNIT ANTIBODIES

Human inhibitory alloantibodies and autoantibodies to Factor VIII (FVI II) are usually directed toward the A2 and/or C2 domains of the FVIII molecule. Anti-C2 antibodies block the binding of FVIII to phospholipi d, but the mechanism of action of anti-A2 antibodies is not known. We investigated the properties of a patient autoantibody, RC, and a monoc lonal antibody, 413, that bind to the region which contains the epitop es of all anti-A2 alloantibodies or autoantibodies studied to date. mA b 413 and RC were noncompetitive inhibitors of a model intrinsic Facto r X activation complex (intrinsic FXase) consisting of Factor IXa, act ivated FVIII (FVIIIa), and synthetic phospholipid vesicles, since they decreased the V-max of intrinsic FXase by > 95% at saturating concent rations without altering the K-m. This indicates that RC and mAb 413 e ither block the binding of FVIIIa to FIXa or phospholipid or interfere with the catalytic function of fully assembled intrinsic FXase, but t hey 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 in-5-maleimidyl-D-phenylalanyl-prolyl-a rginyl-FIXa (FI-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 FI-M-FPR-FIXa, FVIIIa, and phospholipid vesic les produced a further increase in fluorescence anisotropy and a decre ase in fluorescence intensity. This effect was blocked completely by m Ab 413. We conclude that anti-A2 antibodies inhibit FVIIIa function by blocking the conversion of intrinsic FXase/FX complex to the transiti on state, rather than by interfering with formation of the ground stat e Michaelis complex.