Quantitation of membrane glycoprotein IIIa on intact human platelets using the monoclonal antibody, AP-3.
A murine monoclonal antibody specific for glycoprotein (GP)IIIa was prepared by immunization with a GPIIb- and GPIIIa-enriched Triton X-114 extract of platelet membranes. This antibody, designated AP-3, was shown by indirect immunoprecipitation to react solely with GPIIIa derived from either P1A1-positive or -negative individuals. The epitope on GPIIIa recognized by AP-3 is expressed on dissociated GPIIIa as well as on Ca+2-dependent complexes of GPIIb and GPIIIa, as shown by crossed immunoelectrophoresis in the presence or absence of EDTA. A previously described monoclonal antibody specific for the GPIIb/IIIa complex (AP-2) inhibited platelet aggregation induced by ADP, thrombin, collagen, or arachidonic acid (Pidard et al, J Biol Chem 258:12582-12586, 1983). In contrast, AP-3 had no effect on aggregation induced by any of these reagents, a finding similar to that previously reported for the GPIIb-specific monoclonal antibody, Tab (McEver et al, J Clin Invest 66:1311-1318, 1980). At saturation, 40,200 AP-3 molecules were bound per platelet, a value similar to that obtained for AP-2 or Tab. Thus, data derived using AP-3 indicate that significant amounts of free GPIIIa are not present, thereby supporting the hypothesis that GPIIb and GPIIIa exist complexed in a 1:1 stoichiometry in the plasma membrane of intact, nonactivated platelets.
Newman
P J
PJ
Allen
R W
RW
Kahn
R A
RA
Kunicki
T J
TJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D001792:Blood Platelets / Q000276:immunology* / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis* / Q000276:immunology; D006801:Humans; D007519:Isoantigens / Q000276:immunology; D008565:Membrane Proteins / Q000032:analysis* / Q000276:immunology; D051379:Mice; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins
D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D007519:Isoantigens; D008565:Membrane Proteins; D010980:Platelet Membrane Glycoproteins