Inhibition of ITAM-associated signaling molecules or cytoplasmic calpain blocks loss of FcγRIIa from platelets. Washed human platelets were resuspended in Tyrode buffer and either not treated (NT) or treated with 2 μg/mL (final concentration) of either VM58 or 14A2, or 0.1 μg/mL convulxin (Cvx) for 1 hour. Some samples also contained PP2 (10μM), piceatannol (30 μg/mL), or staurosporine (10 μM) as indicated. Levels of (A) GPVI fragment in supernatants or (B) FcγRIIa in platelet lysates was assessed by SDS-PAGE and Western blotting using anti–GPVI mAb (6B-12) or anti–FcγRIIa tail antiserum, respectively. PL indicates platelet lysate, containing full-length GPVI for reference. Vertical lines have been inserted to indicate a repositioned gel lane. All lanes within each figure came from the same experiment and the same gel/Western blot.