Expression of GPIIb-IIIa in the patient's platelets.

(A) Cell-surface expression was assessed using monoclonal antibodies (10 μg/mL) directed against the GPIIb-IIIa complex (6E1), GPIIb (SZ22), and GPIIIa (AP3) subunits. Bound IgG was assessed by flow cytometry using FITC-conjugated goat anti–mouse antibody. Results are given as the percentage (mean + range from 4 separate experiments) of the binding of the same antibodies to control platelets, assigned as 100%. N.M.'s platelets expressed about 20% GPIIb-IIIa compared with normal platelets. Surface expression of GPIb was similar on both N.M.'s and control platelets as shown by the binding of SZ1. (B) Total platelet GPIIb-IIIa was evaluated by immunoblotting. SDS-soluble platelet proteins (12 μg) from both control and N.M.'s platelets were subjected to electrophoresis on a 7% polyacrylamide gel under reducing conditions, transferred to PVDF membrane, and incubated with 20 μg/mL rabbit polyclonal antibodies specific for GPIIb and GPIIIa. Bound antibodies were detected using alkaline phosphatase–conjugated goat anti–rabbit IgG, followed by color development using the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrates.