Protein-tyrosine phosphorylation is required for platelet/PMN adhesion. (A) PMN were preincubated for 2 minutes at 37°C with different concentrations of genistein or an equivalent amount of DMSO before addition of PFA-fixed thrombin-activated platelets. In parallel, HE-loaded PMN were preincubated for 2 minutes at 37°C with different concentrations of genistein or an equivalent amount of DMSO before addition of PFA-fixed BCECF-loaded thrombin-activated platelets. Coincubation in standard conditions was stopped at 2 minutes and samples processed for the evaluation of protein-tyrosine phosphorylation and cell-cell adhesion. The figure shows the Western blot of samples from a representative experiment, and the graph reports the optical density of the tyrosine-phosphorylated P∼110 in parallel with the number of platelets bound by 100 PMN. Values, reported as percentage of control, are means ± SEM, n = 3. (B) HE-loaded PMN were preincubated for 2 minutes at 37°C with erbstatin A (10 μg/mL; black triangles), genistein (10 μg/mL; black squares), or DMSO (control; white squares) before addition of PFA-fixed BCECF-loaded thrombin-activated platelets and incubation in standard conditions. The reaction was stopped at different times and samples processed for fluorescence-activated cell sorter (FACS) analysis. Data report PLT/100 PMN and are expressed as percentage of the peak level (at 1 minute) of the DMSO-treated samples. At this time, 65 ± 8% of PMN bound 361 ± 150 platelets (means ± SEM, n = 3).