Generation and characterization of specific antibodies directed to the binding domain of adhesive molecules.
Antibodies were selected in rabbits using as immunogens five synthetic peptides representing the putative cell-binding domains of von Willebrand factor (vWF), fibrinogen (Fn), fibronectin (Fn) and vitronectin. All peptide immunogens contained the sequence Arg-Gly-Asp, thought to be involved in ligand binding to platelet glycoprotein (GP) IIb-IIIa; two of them corresponded to two distinct regions in the alpha-chain of fibrinogen. All anti-peptide antibodies recognized the corresponding immunogen, and four of them, i.e. all except the anti-vitronectin antibody, reacted with the native macromolecule containing the cognate sequence. Each antibody was specific for the corresponding adhesive protein and did not cross-react with any of the others in spite of the presence of the common sequence Arg-Gly-Asp. Two murine monoclonal antibodies reacting with the vWF peptide and with native vWF were used to define the amino acid residues involved in the antigen-antibody interaction. These residues included the glycine and aspartic acid residues of the common Arg-Gly-Asp sequence, as well as other residues specific to the vWF sequence. The antibodies were potent inhibitors of vWF binding to GP IIb-IIIa. The present studies demonstrate that the putative cell binding domains of four adhesive proteins may be part of distinct antigenic epitopes with unique conformations in each of the four GP IIb-IIIa ligands. Moreover, they provide evidence that the Arg-Gly-Asp sequence is the only, or essential, GP IIb-IIIa binding site in the vWF molecule.
Berliner
S A
SA
Scripps Clinic and Research Foundation, La Jolla, CA 92037.
Roberts
J R
JR
Thorn
M L
ML
Ruggeri
Z M
ZM
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000276:immunology; D002448:Cell Adhesion / Q000276:immunology*; D015815:Cell Adhesion Molecules / Q000276:immunology*; D003429:Cross Reactions; D000939:Epitopes / Q000276:immunology*; D005340:Fibrinogen / Q000276:immunology; D005353:Fibronectins / Q000276:immunology; D006023:Glycoproteins / Q000276:immunology; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology*; D019096:Vitronectin; D014841:von Willebrand Factor / Q000276:immunology
D015815:Cell Adhesion Molecules; D000939:Epitopes; D005353:Fibronectins; D006023:Glycoproteins; D009842:Oligopeptides; D019096:Vitronectin; D014841:von Willebrand Factor; D005340:Fibrinogen