The antibody domain controlling reactions between platelet membranes and drug-dependent (dd) antibodies from patients with thrombocytopenia induced by cinchona alkaloids was studied using F(ab')2, Fab, and Fc fragments made from purified ddIgG. By direct binding radioimmunoassay (RIA) measurements, 20,000 to 50,000 antibody molecules bound per platelet equivalent of purified platelet membranes at apparent saturation with three different antibodies. F(ab'`) and Fab fragments bound to platelet membranes drug dependently but Fc fragments did not.The ability of dd-IgG fragments to compete with intact IgG was quantitatively measured by RUI and by complement fixation. F(ab'`) and Fab competed with intact IgG at an 8:1 and > 50:1 molar ratio, respectively, in RIA, and at a 1.6-3:1 and 44-75:1 ratio, respectively, by complement fixation assays. Fc did not compete with IgG in either assay. We conclude that the Fab domain supports attachment of dd antibody to the platelet surface.ntroduction Drug-induced antibodies that cause thrombocytopenia attach to platelet membranes in a high affinity reaction only when the sensitizing drug or one of its analogues is present in solution (1-4). In vitro binding of the antibody to platelet membranes has been measured over drug concentrations of lo-7 to> 10-2 M (2-4), while in vivo binding, as evidenced by decreases in circulating platelets, has been observed at drug concentrations as low as l0-9 M (5, 6). The highest concentrations of drug used in antibody binding experiments, 10-310-18 M, supported maximal attachment ofantibody to platelets even when antibody concentrations were in the range of I0-8-10-9 M (3, 4) and cell membrane receptors were in the range of 10-8 M. Thus, under conditions of a fixed ratio of antibody concentration to platelet concentration, antibody binding to platelets in vitro is saturable with increasing concentrations of free drug and remains so as drug is increased to at least 105-fold greater than the concentration of either antibody or platelet binding sites.A preliminary report of this work was presented at the 26th Annual

Immunoglobulins (Igs) and serum albumin were eluted from normal platelets and platelets from patients with idiopathic thrombocytopenic purpura (ITP) with a quantitative acid elution procedure followed by solid-phase radioimmunoassay (SPRIA). Acid elution was shown to release a reproducible fraction of platelet-associated Igs, and the amounts released per platelet were independent of the platelet concentration over a wide range of concentrations. This procedure is suitable for sensitive, reproducible, and specific quantitation of large numbers of samples. Washed platelets from 13 normal donors contained the following components (expressed in femtograms per platelet, mean +/- 2 SEM): IgG, 1.40 +/- 0.26; IgA, 0.72 +/- 0.36; IgM 0.078 +/- 0.036; albumin 7.7 +/- 1.5. Immunoglobulins and albumin eluted from the platelets of ten ITP patients (two in remission), expressed as femtograms per platelet, mean (range), were: IgG 104 (0.3 to 750); IgA 90 (0.9 to 715); IgM 162 (1.2 to 1,300); and albumin 34 (6.8 to 199). All platelet-associated Igs from thrombocytopenic ITP patients were found to be elevated twofold to 2,300-fold with one Ig class occasionally elevated 50-fold to 100-fold higher than the others. A similar group of ten thrombocytopenic ITP patients was found to have twofold to 26-fold elevations of platelet- associated albumin. This demonstration of increases in multiple classes of Igs as well as serum albumin associated with platelets from ITP patients suggests that some nonimmune process may be contributing to the phenomenon of increased platelet-associated proteins in ITP.