Upper panel: inhibition of Ca²⁺-ionomycin–induced prothrombinase activity in control erythrocytes by compound R5421. Cells were incubated with increasing concentrations of R5421 for 10 minutes, followed by ionomycin (5 μmol/L) in the presence of Ca²⁺ (1 mmol/L) for 60 minutes. Subsequently, samples were analyzed for prothrombinase activity as described in Materials and Methods. Insert: structural formula of compound R5421. Lower panel: tyrosine phosphorylation in control erythrocytes incubated for 15 minutes with Ca²⁺-ionomycin in the absence (lane 1) and presence (lane 2) of R5421 (0.1 mmol/L). Lanes 3 to 6 represent phosphorylation patterns of white ghosts incubated with four different concentrations of ATP (0.05 mmol/L ATP, lane 3; 0.5 mmol/L ATP, lane 4; 1.0 mmol/L ATP, lane 5; and 2.0 mmol/L ATP, lane 6), all in the presence of R5421 (0.1 mmol/L). Contrary to the incubations with intact cells, the experiments with ghosts were performed in the absence of Ca²⁺ to ensure sufficient levels of phosphorylation (see text).