Summary
In a previous report, we detailed the fractionation of polyclonal human anti-Factor VIII :C into a component directed exclusively against the phospholipid-binding site on Factor VIII (PL-site antibody) and another directed at other sites (non-PL-site antibody). The location on the F.VIII molecule of its PL-binding site has now been studied by two different methods using this fractionated 125I-labelled anti-F.VIII: C Fab’.
The first method was modified from that of Weinstein et al. (Proc Natl Acad Sci USA 1981; 78: 5137-41), involving electrophoresis of F.VIII peptide-125I-Fab‘ A/F.VIII immunocomplexes in SDS-polyacrylamide gels. PL-site antibody reacted with F.VIII peptides of apparent Mr approximately 80 kDa and sometimes 160 kDa in plasma and concentrate, but not with larger peptides. Non-PL-site antibody, however, reacted with a range of peptides of apparent Mr 90 kDa to 280 kDa. In addition, when purified F.VIII containing heavy and light chains (HC + LC), and isolated LC peptides were analysed, PL-site antibody bound to LC peptides whereas non-PL-site antibody did not.
The second method used the antibody pools in immunoradiometric assays (IRMA’s) of purified F.VIII peptides. Both labels measured similar amounts of F.VIII: Ag in a sample of purified F.VIII containing both HC and LC; on assaying an HC preparation, however, PL-site label measured only 2% of F.VIII: Ag found by non-PL-site label, indicating that PL-binding sites are absent in HC preparations.
These results indicate that F.VIII binds to PL via its 80 kDa light chain.
Key words
Factor VIII - Phospholipid - Antibodies - Immunocomplexes
