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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.

The effectiveness of flow cytometry in the detection of drug-dependent, platelet-reactive antibodies was investigated. In studies of seven sera known to contain quinine- or quinidine-dependent, platelet-reactive antibodies, flow cytometry was 5 to 10 times more sensitive in detecting drug-dependent antibodies (DDAbs) than the 51Cr release assay, antigen-capture enzyme-linked immunosorbent assay (ELISA), and indirect immunofluorescence microscopic assay. With flow cytometry, DDAbs could be detected at drug concentrations as low as 0.1 microM, or less than one-tenth the level required with other methods. Antigen-capture ELISA was not as sensitive as flow cytometry in DDAb detection, but it did allow identification of the DDAbs' target molecules. With this assay, five of the seven DDAbs recognized both the glycoprotein Ib/IX (GPIb/IX) and glycoprotein IIb/IIIa (GPIIb/IIIa) complexes, while the remaining two sera reacted only with GPIb/IX. Of 44 consecutive patients who developed thrombocytopenia while taking quinidine, DDAbs were detected by flow cytometry in 11 (25%), more than twice the number detected by other methods. In one patient who developed thrombocytopenia while taking trimethoprim/sulfamethoxazole, DDAbs could be detected only by flow cytometry. It can be concluded that flow cytometry is highly sensitive in detecting DDAbs and allows their detection at pharmacologic concentrations of the drug. Most quinidine-dependent antibodies recognize at least two different glycoprotein complexes in the platelet membrane.

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Glycoprotein (GP) IIb-IIIa serves as the platelet fibrinogen receptor. Studies of the tertiary structure of GPIIIa have shown that the protein has a large loop structure of at least 325 amino acids in length. To further characterize this loop structure, intact platelets were digested with alpha-chymotrypsin. Digestion products were examined using the anti-GPIIIa monoclonal antibodies (MoAbs) AP3, D3GP3, and C5GP3, as well as the human alloantibody, anti-PLA1. AP3 recognized GPIIIa digestion products of 109, 95, and 68 Kd. D3GP3 and C5GP3 recognized an additional band of 51 Kd. Time course digestions demonstrated that the 51-Kd fragment was generated by proteolysis of the 68-Kd peptide. Sequence analysis of the reduced 51-Kd peptide showed that this fragment began at amino acid 422. The nonreduced 51-Kd peptide was reactive with antibodies directed against the first 13 amino acids of GPIIIa, demonstrating the presence of a covalently attached N-terminal peptide. These data suggest that: (1) the minimum length of the loop structure is at least 384 amino acids; (2) the AP3 epitope is formed at least in part by a determinant contained within residues 348 to 421; and (3) the D3GP3 and C5GP3 epitopes are contained within amino acids 422 to 692 of GPIIIa, a region that may be flexible and involved in conformational changes that occur after ligand binding.