Anti-idiotypic antibodies against an antibody to the platelet glycoprotein (GP) IIb-IIIa complex mimic GP IIb-IIIa by recognizing fibrinogen.
Binding of the adhesive ligand fibrinogen and the monoclonal antibody PAC1 to platelet glycoprotein (GP) IIb-IIIa is dependent on cell activation and inhibited by Arg-Gly-Asp (RGD)-containing peptides. Previously, we identified a sequence in a hypervariable region of PAC1 (mu-CDR3) that mimics the activity of the antibody. Here we examine whether monoclonal antibodies to this idiotypic determinant in PAC1 can mimic GP IIb-IIIa by binding to fibrinogen. Mice were immunized with a peptide derived from the mu-CDR3 of PAC1. Four antibodies were obtained that recognized fibrinogen as well as a recombinant form of the variable region of PAC1. However, they did not bind to other RGD-containing proteins, including von Willebrand factor, fibronectin, and vitronectin. Several studies suggested that these anti-PAC1 peptide antibodies were specific for GP IIb-IIIa recognition sites in fibrinogen. Three such sites have been proposed: two RGD-containing regions in the A alpha chain, and the COOH terminus of the gamma chain (gamma 400-411). Two of the antibodies inhibited fibrinogen binding to activated platelets, and all four antibodies bound to the fibrinogen A alpha chain on immunoblots. Antibody binding to immobilized fibrinogen was partially inhibited by monoclonal antibodies specific for the two A alpha chain RGD regions. However, the anti-PAC1 peptide antibodies also bound to plasmin-derived fibrinogen fragments X and D100, which contain gamma 400-411 but lack one or both A alpha RGD regions. This binding was inhibited by an antibody specific for gamma 400-411. When fragment D100 was converted to D80, which lacks gamma 400-411, antibody binding was reduced significantly (p less than 0.01). Electron microscopy of fibrinogen-antibody complexes confirmed that each antibody could bind to sites on the A alpha and gamma chains. These studies demonstrate that certain anti-PAC1 peptide antibodies mimic GP IIb-IIIa by binding to platelet recognition sites in fibrinogen. Furthermore, they suggest that the gamma 400-411 region of fibrinogen may exist in a conformation similar to that of an A alpha RGD region of the molecule.
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104.
C S
CS
Abrams
Z M
ZM
Ruggeri
R
R
Taub
J A
JA
Hoxie
C
C
Nagaswami
J W
JW
Weisel
S J
SJ
Shattil
D000595:Amino Acid Sequence; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D005340:Fibrinogen / Q000276:immunology* / Q000378:metabolism; D005353:Fibronectins / Q000276:immunology; D006023:Glycoproteins / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D010980:Platelet Membrane Glycoproteins / Q000276:immunology* / Q000378:metabolism; D011487:Protein Conformation; D019096:Vitronectin; D014841:von Willebrand Factor / Q000276:immunology
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D005353:Fibronectins; D006023:Glycoproteins; D007135:Immunoglobulin Variable Region; D010980:Platelet Membrane Glycoproteins; D019096:Vitronectin; D014841:von Willebrand Factor; D005340:Fibrinogen