Platelet responses to rhodocytin are impaired upon cytochalasin D treatment. (A) Whole-cell lysates from basal or agonist-stimulated platelets preincubated with various doses of cytochalasin D were separated by SDS-PAGE, and tyrosine phosphorylation was assessed using an antiphosphotyrosine antibody. (B) Whole-cell lysates were isolated from resting or platelets stimulated with (i) rhodocytin, (ii) CRP, or (iii) convulxin, treated with or without cytochalasin D. CLEC-2, PLCγ2, and Syk were immunoprecipitated and the samples separated by SDS-PAGE and immunoblotted for CLEC-2, PLCγ2, and Syk. Their tyrosine-phosphorylated states were assessed using an antiphosphotyrosine antibody. (C) Platelets pretreated with various concentrations of cytochalasin D were stimulated with (i) 30nM rhodocytin, (ii) 3 μg/mL CRP, and (iii) 1 μg/mL convulxin. Addition of agonist is indicated by an arrowhead. Representative aggregation traces are shown for control and cytochalasin D–treated platelets. (D) Dose responses to (i) rhodocytin, (ii) CRP, and (iii) convulxin. Platelets pretreated with DMSO or 10μM cytochalasin D are shown as mean and standard error of 3 independent experiments.