Localization of the binding regions of a murine monoclonal anti-factor VIII antibody and a human anti-factor VIII alloantibody, both of which inhibit factor VIII procoagulant activity, to amino acid residues threonine351-serine365 of the factor VIII heavy chain.
We have localized the binding region of a previously described monoclonal anti-factor VIII (FVIII) inhibitory antibody (C5) to amino acid residues Thr351-Ser365 of the thrombin-generated 54-kD fragment of the heavy chain of FVIII. Synthetic FVIII peptides were examined for the ability to competitively inhibit the binding of C5 to FVIII in an ELISA system. The synthetic FVIII peptide Thr351-Ser365 blocked C5 binding to FVIII in a dose-dependent manner in this system. Two other synthetic FVIII peptides, Asn340-Glu354 and Glu342-Asp356, which partially overlapped Thr351-Ser365, also blocked C5 binding to FVIII. Blocking of C5 binding with these peptides, however, required much greater concentrations (greater than 100 times stronger) than that required for Thr351-Ser365. The Thr351-Ser365 peptide also neutralized the FVIII inhibitory activity of C5 in plasma. A human FVIII inhibitor (anti-FVIII heavy chain alloantibody) was also partially neutralized by Thr351-Ser365. Thr351-Ser365 lies between a thrombin cleavage site (Arg372) and an activated protein C cleavage site (Arg336) and may be at or near a region of functional importance in the expression of FVIII procoagulant activity.
Foster
P A
PA
Department of Basic and Clinical Research, Scripps Clinic, La Jolla, California 92037.
Fulcher
C A
CA
Houghten
R A
RA
de Graaf Mahoney
S
S
Zimmerman
T S
TS
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000502:physiology*; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D001777:Blood Coagulation / Q000187:drug effects; D005169:Factor VIII / Q000037:antagonists & inhibitors* / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007518:Isoantibodies / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000276:immunology; D012694:Serine; D013912:Threonine
D000911:Antibodies, Monoclonal; D007518:Isoantibodies; D010455:Peptides; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; C047636:factor VIII receptor; D013912:Threonine; D012694:Serine; D005169:Factor VIII