The Arg-Gly-Asp (RGD) recognition site of platelet glycoprotein IIb-IIIa on nonactivated platelets is accessible to high-affinity macromolecules.
We have characterized a murine IgG monoclonal antibody, OP-G2, specific for platelet glycoprotein (GP) IIb-IIIa (alpha IIb beta 3). OP-G2 Fab fragments inhibit fibrinogen-mediated platelet aggregation and competitively inhibit adenosine diphosphate-induced binding of 125I-fibrinogen to washed platelets. OP-G2 binding to GPIIb-IIIa is specifically inhibited by RGD-containing peptides but not the fibrinogen gamma-chain carboxy-terminal peptide, and OP-G2 Fab fragments, like RGD-containing peptides, alter the conformation of GPIIb-IIIa resulting in the expression of a ligand-induced binding site (LIBS) recognized by PMI-1. OP-G2 fails to bind to the recombinant Cam variant of GPIIb-IIIa (alpha III beta 3Cam) wherein an Asp119 to Tyr119 substitution in GPIIIa abrogates the ability to recognize RGD. These data indicate that OP-G2 recognizes an epitope at or in very close proximity to the RGD recognition site of GPIIb-IIIa and that, in every aspect tested, OP-G2 behaves like a macromolecular RGD ligand. Interestingly, two-color flow cytometry shows that OP-G2 IgG can bind to nonactivated platelets. Quantitative binding assays indicate that nonactivated platelets bind approximately 50,000 125I-OP-G2 molecules/platelet. Furthermore, the affinity of OP-G2 for platelets activated with thrombin is roughly fivefold higher (nonactivated, kd = 24.8 nmol/L; activated, kd = 4.9 nmol/L). These results suggest that the RGD recognition site of GPIIb-IIIa is available to macromolecules that contain RGD even on nonactivated platelets, provided that the affinity of the ligand is adequate.
Tomiyama
Y
Y
Blood Center of Southeastern Wisconsin, Milwaukee 53233.
Tsubakio
T
T
Piotrowicz
R S
RS
Kurata
Y
Y
Loftus
J C
JC
Kunicki
T J
TJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism*; D000939:Epitopes / Q000032:analysis; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D009842:Oligopeptides; D010980:Platelet Membrane Glycoproteins; C047981:arginyl-glycyl-aspartic acid; D005340:Fibrinogen