Immunoblot and immunoprecipitation of solubilized, surface-biotinylated platelets. (A) Left panel, immunoblot of patient and control platelet lysates using the GPIIb-specific MoAb, PMI-1.32,33 This pattern is essentially identical to that in Fig 1 except that because the control sample was diluted 20-fold, the bands in the patient's sample are more intense than the bands in the control sample. Right panel, immunoprecipitation of lysates using the GPIIb-specific MoAb, Tab34 and immunoblotting with PMI-1. Samples were electrophoresed under reduced conditions and arrows mark the positions of pro-GPIIb, mature GPIIb, and the GPIIb fragment. These data indicate that Tab recognizes all forms of the mutant GPIIb. (B) Patient and normal control surface-biotinylated platelet lysates were immunoprecipitated with the GPIIb/IIIa complex-specific MoAb, 10E5,26 the GPIIb-specific MoAb, Tab, and the GPIIIa-specific MoAb, 7H2.29 Immunoprecipitates were electrophoresed under nonreduced conditions and blotted onto PVDF membranes. The membranes were treated with HRP-streptavidin and the bands developed. Arrows mark the positions of GPIIb and GPIIIa. Because only surface-labeled molecules are detected by the avidin reagent, the failure to identify the patient's abnormal GPIIb bands indicates that these were not present on the surface of the patient's platelets. (C) Experiment conducted as in (B) except immunoprecipitates were reduced before electrophoresis.