The effect of Src family tyrosine kinase inhibition on platelet aggregation caused by the simultaneous stimulation of G_i and G_z pathways. (A) Platelets were preincubated at 37°C with DMSO (vehicle) or varying doses of PP2 for10 minutes prior to the stimulation of the platelets. Platelets were stimulated with 100 nM 2-MeSADP plus 100 μM MRS 2179 (G_i signaling), 10 μM epinephrine (G_z signaling), or a combination of both agonists, as noted. MRS2179 (100 μM) was added 1 minute prior to platelet stimulation. (B) Washed human platelets were preincubated at 37°C with DMSO (vehicle) or with 10 μM PP1, 10 μM PP2, or 10 μM PP3 for 10 minutes prior to the stimulation of the platelets. Platelets were stimulated with 100 nM 2-MeSADP, 10 μM epinephrine, or a combination of both agonists, as noted. MRS2179 (100 μM) was added 1 minute prior to platelet stimulation. ADP (10 μM) was added for simultaneous stimulation of G_q and G_i pathways. All aggregation tracings were performed in the presence of 1 mg/mL fibrinogen. Tracings are representative of results obtained from 3 donors and 3 separate experiments. (C) Washed, aspirin-treated platelets (2 × 10⁸ platelets/mL) were stimulated as noted in “Materials and methods.” Chemiluminescence was measured on membranes that were probed with an anti–phospho-Src⁴¹⁶ antibody for measurement of Src kinase activity. The intensity of the bands was first calculated as a ratio of phosphorylated-to-total Src in each lane. Then the ratios from 3 independent experiments were normalized to the control, taken as 1, and expressed as fold increase in other lanes (mean ± SEM). The blot shown is representative of 3 independent experiments.