The inhibitor IgG fraction used in this study revealed reactivity to sites within each FVIIIa subunit as judged by dot blots. Use of LC-MS/MS techniques coupled with IP resolved the multiple antibody-antigen interactions in this complex mixture, identifying an ensemble of epitopes that mapped to all A and C domains of FVIII. Indeed, the LC-MS approach proved far superior to the affinity-directed mass spectrometry approach employing MALDI-TOF that we used in an earlier study to map the epitope of anti-A2 domain monoclonal antibody [12
]. Only two of the 19 peptides identified by LC-MS were identified by MALDI, and this result emphasized the increased sensitivity and resolving power of the current method. Furthermore, unlike recent techniques using peptide arrays to map inhibitor epitopes [8
], the enhanced sensitivity of the LC-MS methods eliminated the requirement to affinity purify the IgG using immobilized FVIII.
Consistent with numerous reports (see Ref. [1
] for review), the majority of peptides we identified mapped to sequences within the FVIII A2 and C2 domains. The 10 peptides that mapped to the A2 domain (residues 373-714) represented 7 distinct, non-contiguous sequences and accounted for a significant fraction (29%, 100/341 residues) of this domain (). C2 domain (residues 2170-2332) showed a similar density of epitopes with three peptides representing 20% (33/163 residues) of this domain. We observed significantly less representation of A1, A3 and C1 domains in the epitope map. In most cases, epitopes were defined by relatively short peptide sequences of 12 residues or less, and in all cases sequences were no longer than 15 residues. Thus this approach provided a high level of definition for the residues comprising these antibody-binding sites.
FVIII structure indicating epitope sequences
A number of the peptide sequences identified have been shown by various biochemical or immunological methods to be important for specific FVIII functions, thus antibody binding these sites would be predicted to impair and/or eliminate that function. Importantly, two peptides comprising A2 residues 474-487 and 499-509 were identified that overlap the epitope for the inhibitory monoclonal antibody 413 (residues 484-509; [9
]). Inhibitors to this region block FXase activity in a non-competitive manner [16
], likely by preventing interaction between the A2 subunit of FVIIIa and the protease domain of FIXa [17
]. Furthermore, two apparent continuous epitopes represented by overlapping sequences 532-541 and 538-551, and 577-584 and 583-593 lie in close proximity to the 558-loop in the A2 subunit which comprises a FIXa-interactive site in FVIIIa [18
The three C2 peptide sequences identified fall within or overlap two larger segments, residues 2181-2243 [10
] and 2248-2312 [19
], to which sites for inhibitors have been mapped. The high-resolution structure of the C2 domain predicts both hydrophobic and electrostatic interactions in binding FVIII to membrane [20
]. We note that peptides 2216-2227 and 2240-2249 contain three of the four basic residues, Arg2220, Lys2227, and Lys2249 that contribute electrostatic interactions with phosphatidylserine. Furthermore, the former sequence includes Val2223, while the latter sequence lies in close proximity to residues Leu2250/Leu2251, which form two of the hydrophobic-interactive sites.
A final noteworthy sequence, residues 360-372, is contained within a1 segment separating A1 and A2 domains and overlaps with the inhibitory monoclonal antibody, C5 (residues 351-365 [21
]). It has been suggested anti-a1 inhibitors block the conformational change that yields active FVIIIa following activation [22
]. Furthermore, residues 337-372 form a FX-binding site [23
]. Thus antibody binding at this site could interfere with procofactor activation and/or the subsequent capacity for the cofactor to interact with substrate for FXase.
The neutralizing potential of antibodies binding to other sequences identified in the immuno-precipitate is not clear based upon failure to co-localize with known sites of function. However, we note that each sequence contains multiple residues that when mutated, yield a hemophilic phenotype as identified in the Hemophilia A database (http://hadb.org.uk
]). Indeed, one peptide comprising residues 200-208 shows point mutations at 6 of the 9 residues result in a hemophilia, suggesting a potential functional role for this site. Given the lack of functional information regarding these sequences, we speculate that these epitopes minimally represent sites for non-neutralizing antibodies, which could potentially facilitate clearance of the FVIII protein.
In conclusion, we employed immuno-precipitations coupled with the high resolving power and sensitivity of LC-MS techniques to obtain an epitope map for the IgG fraction prepared from a high-titer FVIII inhibitor plasma. While some limitations with this method exist, such as gaps in the sequence coverage and potential proteolytic destruction of the epitope, these concerns were minimized with use of more than one protease. Advantages of the current approach include a level of sensitivity that eliminates the need to enrich the antibody pool for anti-FVIII antibodies and results in fine-point mapping the epitope to a relatively short sequence length. Thus the present technique complements other established epitope mapping protocols, which in general represent more labor-intensive methodologies and suffer from their own particular limitations. Within this inhibitor plasma we identified a population of epitopes for neutralizing and likely non-neutralizing antibodies that map to all A and C domains.