QUANTITATION OF MEMBRANE GLYCOPROTEIN-IIIA ON INTACT HUMAN-PLATELETS USING THE MONOCLONAL-ANTIBODY, AP-3

Abstract

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 Ca2+-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. 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. At saturation, 40,200 AP-3 molecules were bound/platelet, a value similar to that obtained for AP-2 or Tab. 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.