Mapping of natural anti-factor VIII antibodies in plasma pools from healthy donors: use of rationally designed synthetic peptides.

Inhibitor antibodies of blood coagulation factor VIII (FVIII) impair FVIII replacement therapy, constituting a serious complication in haemophilic patients. anti-FVIII antibodies may also develop in a variety of disease-associated autoimmunity. Mapping of human FVIII inhibitors in haemophilia A or autoantibody origin have delineated three major clusters of B-cell inhibitory epitopes (domain A2, A3 and C2). Inhibitory and non-inhibitory FVIII antibodies have also been described in plasma of healthy donors and pools of immunoglobulins. The purpose of this study was to use synthetic FVIII-peptides to more closely define regions of the molecule targeted by natural anti-FVIII antibodies. Predictive algorithms were used for defining the positions of potential continuous epitopes. To investigate the presence of peptide-reactive antibodies in normal plasma pools of healthy donors, a plasma fraction (Cohn fraction II+III) containing all IgG subclasses was purified by affinity chromatography on peptide-Sepharose columns. The results of ELISAs and Western blotting experiments (with the selected peptides and well-defined recombinant FVIII thrombin fragments) confirmed the reaction specificities of the affinity-purified human antibodies. For each IgG preparation, the isotopic subclass was also determined. In the clotting assay, several IgG preparations showed neutralising activity in a dose-dependent manner. Our observations support the recent hypothesis that FVIII inhibitors in haemophilia A and autoimmune disease may originate from the proliferation of natural FVIII-specific B-cell clones.