The cleaved form of FcγRIIa is detectable by immunoprecipitation and Western blot but not by flow cytometry. Washed human platelets were left untreated (NT) or treated with (final concentrations) 150 μM W7 or 10 mM NEM for 2 hours. Platelets were fixed and (A) lysed then analyzed for levels of GPVI and FcγRIIa by Western blot, or (B) stained with nonimmune mouse IgG (empty histogram in FcγRIIa panel), anti–FcγRIIa antibody IV.3, or anti–GPVI mAb, 1G5, and secondary FITC-labeled antibodies were then analyzed by fluorescence-activated cell sorting (FACS). The vertical line in each histogram indicates the mean fluorescence intensity in untreated platelets stained with each antibody. (C) Non-treated (NT) platelets or platelets treated with 150 μM W7 or 10 mM NEM for 2 hours were pelleted and lysed then mixed with 10 μg of either nonimmune mouse IgG (NIM) or IV.3. Immune complexes were captured and assessed by SDS-PAGE and Western blotting with anti–FcγRIIa tail antiserum or anti–FcγRIIa extracellular (EC) domain IgG.