Ligands of ITAM-containing receptors, FcγRIIa or GPVI, induce shedding of these receptors from platelets. Washed human platelets were resuspended in Tyrode buffer and either not treated (NT) or treated with FcγRIIa ligands, VM58 or 14A2 (2 μg/mL, final concentration), or with the GPVI ligand, convulxin (Cvx; 0.5 μg/mL, final concentration) for 1 hour, in the presence (+) or absence (−) of EDTA (10 mM, final concentration). Platelets were isolated by centrifugation and lysed in Triton X-100 buffer as described in “Proteolysis of platelet receptors.” Immunoblots of (A) full-length GPVI (top) and FcγRIIa (bottom) detected using anti-GPVI mAb (6B-12) or anti-FcγRIIa tail antiserum, respectively; (B) full-length and cleaved GPV in platelet lysates detected by using anti-GPV tail IgG; and (C) soluble GPIbα present in supernatant fractions detected by using anti-glycocalicin IgG. (D) Timecourse analysis of levels of platelet proteins in response to treatment by 14A2, WM23, TRAP, VWF/botrocetin, or convulxin, using antibodies directed against the cytoplasmic tail of GPVI and FcγRIIa. Blots were visualized using HRP-conjugated secondary antibodies and ECL. 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.