Studies on the interaction of platelet glycoprotein IIb-IIIa and glycoprotein IV with fibrinogen and thrombospondin: a new immunochemical approach.
We have designed a new binding assay based on crossed immunoelectrophoresis that allowed us to test for the relative capacities of platelet membrane glycoprotein IIb-IIIa (GP IIb-IIIa), and glycoprotein IV (GP IV) to bind purified Arg-Gly-Asp (RGD)-containing adhesive proteins. Preformed immune complexes were made by reacting a platelet lysate with murine monoclonal antibodies to GP IV (OKM5 and FA6-152) or to GP IIb-IIIa (AP-2). Upon two-dimensional electrophoretic separation in agarose gels and immunoprecipitation by a polyclonal antibody to mouse IgG, the immobilized complexes containing the desired antigen were further probed with purified 125I-labeled TSP or fibrinogen. Under these conditions, immobilized GP IV was found to specifically bind TSP, whereas it was unreactive with fibrinogen. By contrast, immobilized GP IIb-IIIa demonstrated fibrinogen binding capacity but did not demonstrate any reactivity toward TSP. These observations suggest that the overall structure of the adhesive protein may determine the accessibility of the RGD sequence to its binding site on GP IIb-IIIa.
Beiso
P
P
INSERM U 150, CNRS UA 334, Hôpital Lariboisière, Paris, France.
Pidard
D
D
Fournier
D
D
Dubernard
V
V
Legrand
C
C
D000595:Amino Acid Sequence; D001345:Autoradiography; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D007122:Immunoelectrophoresis; D007123:Immunoelectrophoresis, Two-Dimensional; D007163:Immunosorbent Techniques; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D019699:Thrombospondins
D008562:Membrane Glycoproteins; D010980:Platelet Membrane Glycoproteins; D019699:Thrombospondins; D005340:Fibrinogen