Factor VIII procoagulant protein interacts with phospholipid vesicles via its 80 kDa light chain.
In a previous report, we detailed 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.
Kemball-Cook
G
G
National Institute for Biological Standards and Control, Potters Bar, Herts, UK.
Edwards
S J
SJ
Sewerin
K
K
Andersson
L O
LO
Barrowcliffe
T W
TW
D000936:Antigen-Antibody Complex / Q000032:analysis; D000941:Antigens / Q000032:analysis; D001345:Autoradiography; D002021:Buffers; D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D005404:Fixatives; D005782:Gels; D006801:Humans; D008970:Molecular Weight; D010743:Phospholipids / Q000378:metabolism*; D011863:Radioimmunoassay; D014841:von Willebrand Factor / Q000032:analysis
D000936:Antigen-Antibody Complex; D000941:Antigens; D002021:Buffers; D005404:Fixatives; D005782:Gels; D010743:Phospholipids; D014841:von Willebrand Factor; D005169:Factor VIII