A monoclonal antibody to von Willebrand factor (vWF) inhibits factor VIII binding. Localization of its antigenic determinant to a nonadecapeptide at the amino terminus of the mature vWF polypeptide.
vWF is a multimeric glycoprotein that serves as the major carrier in plasma of Factor VIII (FVIII). We have used an anti-human vWF MAb W5-6A to investigate the FVIII binding site on vWF. W5-6A inhibited FVIII binding to vWF-coated polystyrene tubes in a concentration-dependent manner with 90% inhibition of FVIII binding at a concentration of 10 micrograms/ml. The W5-6A epitope was identified by screening a vWF fragment library using the bacteriophage expression vector lambda gt11. DNA sequence analysis of 29 immunoreactive phage clones localized the W5-6A epitope to a nonadecapeptide spanning amino acid residues threonine 78 to threonine 96 at the amino-terminus of the mature vWF polypeptide. Purified beta-galactosidase/vWF fusion protein from one of these clones, vWF9, was incubated with radiolabeled W5-6A and caused near complete inhibition of W5-6A binding to vWF. Inhibitory activity was lost after vWF9 trypsinization or reduction and alkylation. These data indicate that (a) the antigenic determinant recognized by W5-6A localizes to a nonadecapeptide at the NH2 terminus of the mature vWF polypeptide, (b) disulfide bonds within vWF9 may be necessary to maintain the structure required for immunoreactivity with W5-6A, and (c) W5-6A recognizes an immunogenic region on vWF that may be at (or near) the major FVIII binding domain.
Bahou
W F
WF
Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor 48109.
Ginsburg
D
D
Sikkink
R
R
Litwiller
R
R
Fass
D N
DN
D000911:Antibodies, Monoclonal / Q000276:immunology*; D010582:Bacteriophage lambda / Q000235:genetics; D001667:Binding, Competitive; D015153:Blotting, Western; D004279:DNA, Viral / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D004926:Escherichia coli / Q000235:genetics; D005169:Factor VIII / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D010455:Peptides / Q000276:immunology; D014841:von Willebrand Factor / Q000276:immunology*
D000911:Antibodies, Monoclonal; D004279:DNA, Viral; D000939:Epitopes; D010455:Peptides; D014841:von Willebrand Factor; D005169:Factor VIII