Ligand-mediated activation of FcγRIIa leads to activation of both a metalloproteinase and a calpain-like protease in platelets. Washed human platelets were resuspended in Tyrode buffer and either not treated (NT) or treated with W7 (150 μM, final concentration), NEM (2 mM), or 14A2 (2 μg/mL) for 1 hour. Some samples also contained EDTA (10 mM), GM6001 (100 μM), E64d (10-100 μM), GRGDSP peptide (RGD; 1 mM), or methanol (MeOH) or DMSO vehicle controls. Levels of (A) GPVI and (B) FcγRIIa in platelet lysates were assessed by Western blotting with anti-GPVI (6B-12) or anti–FcγRIIa tail antiserum. Blots were visualized using HRP-conjugated secondary antibodies and ECL. (C) Platelets were treated with 14A2 in the presence of either membrane-permeable (E64d) or membrane-impermeable (leupeptin) inhibitors of calpain, or GM6001 (100 μM, final concentration). Levels of full-length FcγRIIa and full-length and cytoplasmic tail remnant of GPVI were assessed by Western blotting with anti–FcγRIIa tail antiserum or anti–GPVI tail IgG. 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.