Immunogold-surface replica study of ADP-induced ligand binding and fibrinogen receptor clustering in human platelets.
Platelet cohesion requires the binding of fibrinogen to its receptor, a heterodimer consisting of the plasma-membrane glycoproteins GPIIb and GPIIIa. Although the GPIIb-IIIa complex is present on the surface of unstimulated platelets, it binds fibrinogen only after platelet activation. We have used an immunogold-surface replica technique to study the distribution of GPIIb-IIIa and bound fibrinogen over broad expanses of surface membranes in unstimulated and ADP-activated human platelets. We found that the gold prove was monodispersed over the surface of unstimulated platelets, although the cell surface lacked immunoreactive fibrinogen. To ascertain whether the receptors clustered prior to ligand binding or as a consequence thereof, we studied the surface distribution of GPIIb-IIIa after stimulation with ADP, which causes activation of the fibrinogen receptor function of GPIIb-IIIa without inducing the secretion of fibrinogen. In the absence of added fibrinogen, the unoccupied, yet binding-competent receptors on ADP-stimulated platelets were monodispersed. The addition of fibrinogen caused the GPIIb-IIIa molecules to cluster on the cell surface. Clustering was also induced by the addition of the GPIIb-IIIa binding domains of fibrinogen--namely, the tetrapeptide Arg-Gly-Asp-Ser on the alpha-chain or the gamma-chain decapeptide gamma 402-411. These results show that receptor occupancy causes clustering of GPIIb-IIIa in activated platelets.
Department of Pathology, University of California, San Francisco 94143.
W M
WM
Isenberg
R P
RP
McEver
D R
DR
Phillips
M A
MA
Shuman
D F
DF
Bainton
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D001792:Blood Platelets / Q000378:metabolism*; D006046:Gold; D007120:Immunochemistry / Q000379:methods; D008024:Ligands / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000097:blood; D008565:Membrane Proteins / Q000097:blood; D008854:Microscopy, Electron; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011940:Receptor Aggregation; D013268:Stimulation, Chemical
D008024:Ligands; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010980:Platelet Membrane Glycoproteins; D000244:Adenosine Diphosphate; D006046:Gold