Premium
Identification of a factor VIII peptide, residues 2315–2330, which neutralizes human factor VIII C2 inhibitor alloantibodies: requirement of Cys 2326 and Glu 2327 for maximum effect
Author(s) -
Nogami Keiji,
Shima Midori,
Nakai Hiroaki,
Tanaka Ichiro,
Suzuki Hiroshi,
Morichika Shogo,
Shibata Masaru,
Saenko Evgueni,
Scandella Dorothea,
Giddings John,
Yoshioka Akira
Publication year - 1999
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1046/j.1365-2141.1999.01673.x
Subject(s) - chemistry , peptide , epitope , neutralization , microbiology and biotechnology , phosphatidylserine , epitope mapping , recombinant dna , binding site , c2 domain , biochemistry , antibody , biology , phospholipid , immunology , gene , membrane
Factor VIII (FVIII) inhibitor alloantibodies react with combinations of the A2, C2 and A3‐C1 domains of the FVIII molecule. Some inhibitors block binding of FVIII to both von Willebrand factor (VWF) and phospholipid, and recognize a C2 domain epitope which overlaps both binding sites. In order to determine the essential binding regions for alloantibodies inhibitory for FVIII activity, we have performed inhibitor neutralization assays and competitive inhibition assays using 10 overlapping synthetic peptides spanning the carboxy‐terminal region of the C2 domain (residues 2288–2332). We found one peptide (2315–2330, L9) which neutralized the anti‐FVIII activity of four out of five different C2 alloantibodies by 50%, 39%, 47% and 57%, respectively. Neutralization of these alloantibodies by recombinant C2 domain (residues 2173–2332) was 68%, 50%, 59%, 86% and >95%, respectively. The inhibitor which was not neutralized by L9 peptide and reacted by immunoblotting with peptide 2218–2307, did not prevent binding of FVIII to VWF and only partially inhibited binding of FVIII to phosphatidylserine. Mutants of the L9 peptide were prepared in which each residue from 2315–2330 was sequentially substituted by glycine. Inhibitor neutralization experiments using these peptides demonstrated that Arg 2320 and Cys 2326 or Glu 2327 are important for the effect of L9 peptide, since their substitution by glycine reduced its neutralizing effect by 60% to >90%, suggesting that they are crucial for formation of the one of the C2 inhibitor epitopes.