Activity of SRC tyrosine kinases is required for PMNL adhesion to activated platelets and for platelet-induced PMNL P-110 tyrosine phosphorylation.

HE-loaded PMNLs were preincubated for 1 minute at 37°C with different concentrations of (A) PP1 (■), PP2 (●), PP3 (▪); (B) tyrphostin AG 490 (■), piceatannol (○), PD98059 (▪) before the addition to PFA-fixed, BCECF-loaded thrombin-activated platelets. Mixed-cell suspensions were coincubated at 37°C and 1000 rpm stirring (standard conditions). The interaction was stopped at 2 minutes by the addition of 1 vol of 2% PFA, and samples were processed for FACS analysis. Data report the percentage of PMNLs displaying the platelet green fluorescent marker. Values are means ± SEM (n = 5). (C) PMNLs were preincubated for 1 minute at 37°C with different concentrations of PP1 or with 50 μM piceatannol or PD98059 before coincubation with PFA-fixed, thrombin-activated platelets. Coincubation in standard conditions was stopped at 2 minutes, and samples were processed for analysis of protein tyrosine phosphorylation. The figure shows the Western blot of samples from a representative of 3 experiments.