Epitope Mapping of a Human Inhibitor Plasma by Affinity-Directed Mass Spectrometry.

Abstract 3171

Poster Board III-111

Hemophilia A results from a defect or deficiency in the blood clotting protein, factor VIII. Treatment of patients with factor VIII results in the development of neutralizing antibodies in a significant fraction (∼30%) of patients with a severe hemophilia phenotype. We employed an affinity-directed mass spectrometry (MS) technique using immunoprecipitation coupled with protein fragmentation to map the epitopes recognized by the IgG fraction from a factor VIII inhibitor plasma. To accomplish this aim, antibody (IgG fraction) was purified from 10 ml of plasma obtained from a high titer (533 Bethesda Units/ml) inhibitor patient (obtained from George King BioMedical, Inc., GK1838-1156). This IgG fraction was immobilized onto Protein G agarose beads and then reacted with limited tryptic or chymotryptic digests of heavy chain and light chain purified from recombinant factor VIII. The factor VIII heavy and light chain digests showed peptide maps that covered approximately 20% and 40%, respectively, of the factor VIII sequence. The immunoprecipitated complexes were washed and directly applied to analysis by MALDI-TOF MS. Five peptides with mass values (m/z ratio) of 1105.6, 1291.7, 1309.7, 1362.7, and 1681.2 were identified. Database searches and direct sequencing showed these peaks corresponded to factor VIII peptides HYFIAAVER (residues 1697-1705), HNIFNPPIIAR (residues 2137-2147), SWYLTENIQR (residues 584-593), TRHYFIAAVER (1695-1705), and IRAEVEDNIMVTF (residues 1763-1775), respectively. This method of epitope identification does not discriminate neutralizing from non-neutralizing antibody binding. However, inspection of the sequence contained in these epitopes reveals that residues 1695-1705 occur near the amino terminal region of the A3 domain in close proximity to the thrombin cleavage site at Arg1689 and may contribute to facilitating factor VIII activation. Residues 2137-2147 (C1 domain) are of interest since residue Arg2150 contributes to binding von Willebrand factor. While no macromolecular interactions have been attributed to A2 domain residues 584-593 or the A3 domain residues 1763-1775, the former sequence appears on the same face of A2 as the 558-loop which is an important interactive site for factor IXa in the assembly of factor Xase. Thus, these observations suggest that several of the above sequences may possess functional attributes that, if bound by antibody, could lead to inhibition of factor VIII activity. To further test this hypothesis, synthetic peptides were employed in an attempt to reduce the potency of the inhibitor plasma. Two peptides, corresponding to residues 1697-1705 and 2137-2147 have been examined to date. Addition of either peptide (100 nM) to the inhibitor plasma reduced its titer by approximately 4-fold, suggesting these sequences represent sites in factor VIII that when bound by antibody, block cofactor function. Taken together, these results identify in part, the ensemble of epitopes recognized by the inhibitor antibody fraction derived from a single patient.