Carol A. Fulcher

Localization of the binding regions of a murine monoclonal anti-factor VIII antibody and a human anti-factor VIII alloantibody, both of which inhibit factor VIII procoagulant activity, to amino acid residues threonine351-serine365 of the factor VIII heavy chain.

We have localized the binding region of a previously described monoclonal anti-factor VIII (FVIII) inhibitory antibody (C5) to amino acid residues Thr351-Ser365 of the thrombin-generated 54-kD fragment of the heavy chain of FVIII. Synthetic FVIII peptides were examined for the ability to competitively inhibit the binding of C5 to FVIII in an ELISA system. The synthetic FVIII peptide Thr351-Ser365 blocked C5 binding to FVIII in a dose-dependent manner in this system. Two other synthetic FVIII peptides, Asn340-Glu354 and Glu342-Asp356, which partially overlapped Thr351-Ser365, also blocked C5 binding to FVIII. Blocking of C5 binding with these peptides, however, required much greater concentrations (greater than 100 times stronger) than that required for Thr351-Ser365. The Thr351-Ser365 peptide also neutralized the FVIII inhibitory activity of C5 in plasma. A human FVIII inhibitor (anti-FVIII heavy chain alloantibody) was also partially neutralized by Thr351-Ser365. Thr351-Ser365 lies between a thrombin cleavage site (Arg372) and an activated protein C cleavage site (Arg336) and may be at or near a region of functional importance in the expression of FVIII procoagulant activity.

Immunochemistry of factor VIII:C inhibitor antibodies

Factor VIII:C (FVIII:C) inhibitors are pathologic circulating antibodies that reduce FVIII:C activity. They can arise either as alloantibodies in some congenital hemophiliacs, or as autoantibodies in nonhemophilic patients with acquired inhibitors. The reason for development of such antibodies is not known, but their basic biochemical and clinical characteristics have been reviewed extensively in the past several years. This article surveys recent immunochemical studies to characterize the FVIII:C molecule and antibodies against it. For instance, epitope (antigenic site) mapping investigations conducted to date suggest that binding sites for most inhibitory antibodies can be localized to limited regions of the FVIII:C molecule. Further progress in the understanding of immune responses against FVIII:C is likely to provide a sound basis for the design of new therapeutic approaches to patients suffering the sequelae of FVIII:C inhibitor antibodies.

A murine monoclonal anti-factor VIII inhibitory antibody and two human factor VIII inhibitors bind to different areas within a twenty amino acid segment of the acidic region of factor VIII heavy chain.

The factor VIII (FVIII) binding regions of the monoclonal anti-FVIII inhibitory antibody C5 and a human FVIII inhibitor antibody have previously been reported to be contained within amino acid residues 351-365 of FVIII. Localization of the binding regions of these two antibodies was based on their reactivity with four synthetic FVIII peptides. Nineteen synthetic FVIII peptides spanning the entire acidic region of the FVIII heavy chain have now been evaluated for the ability to inhibit the binding of C5 to FVIII in an ELISA assay. The smallest peptide tested that inhibited C5 binding to FVIII consisted of residues 351-361. Those peptides that were able to inhibit C5 binding in the ELISA assay were also able to neutralize the FVIII inhibitory activity of C5 in plasma. The FVIII inhibitory activity of two human FVIII inhibitor antibodies was also partially neutralized by peptides from this region. Evaluation of the pattern of peptides reactive with the three antibodies indicates that the binding regions of these antibodies are in very close proximity to each other, but are not identical. Their respective binding regions are contained within residues 351-361 (C5), 354-362 (inhibitor 1), and 342-354 (inhibitor 2). These results suggest that this 20 amino acid segment of the acidic region of the heavy chain of FVIII may be functionally important in the expression of FVIII procoagulant activity.