(A) Kinetics of tyrosine phosphorylation of cortactin as a function of aggregation by comparison of unstirred and stirred control and thrombasthenic platelets. Platelets from control (control) or the Glanzmann thrombathenia patient of Fig 1A (patient) were activated with 0.05 U thrombin in nonstirring (unstirred) or stirring (stirred) conditions for 0, 30, and 120 seconds. Solubilized platelet proteins were analyzed for tyrosine phosphorylation by Western blotting. Aggregation rates are indicated. Arrows designate cortactin. (B) Effect of cytochalasin D on tyrosine phosphorylation of cortactin and 64 kD. Control platelets were activated with 0.1 U thrombin for 2 minutes with stirring after preincubation for 10 minutes at 37°C in the presence of buffer alone (thromb), 100 μmolL cytochalasin D (cytoD), or DMSO (DMSO). Unactivated platelets were incubated in parallel (control). Tyrosine-phosphorylated proteins were detected by Western blotting, and 130 kD, 100 to 105 kD, 77 to 80 kD, 64 kD, and src are indicated by arrows.