Agonist-induced activation of human platelets results in tyrosine phosphorylation of FcγRIIa, Syk, and PLCγ2 in a ligand binding–dependent manner. Washed human platelets stirring in an aggregometer cuvette were stimulated with TRAP in the presence or absence of 2 mM RGD peptide or 10 μg/mL IV.3 Fab, as indicated. After detergent lysis, proteins were immunoprecipitated using antibodies specific for FcγRIIa, Syk, or PLCγ2, and immunoblots containing immunoprecipitated proteins stained either for antigen (Ag) or phosphotyrosine (PY). Note that FcγRIIa, Syk, and PLCγ2 each become tyrosine phosphorylated after platelet activation, and that preventing ligand binding with RGD peptide (left half of the figure) largely inhibits tyrosine phosphorylation of each of these signaling components, demonstrating that ligand binding to the integrin αIIbβ3 activates the FcγRIIa→Syk→PLCγ2 signaling pathway. Also note that preincubation of platelets with IV.3 (right half of the figure) prevents FcγRIIa, Syk, and PLCγ2 activation in response to TRAP. Data shown are representative of 3 separate experiments performed using 2 different platelet donors.