Platelet and CHO-P cell adhesion to PMNL induces CD18-dependent activation of LYN and HCK.

PMNLs were coincubated with PFA-fixed and -activated platelets (A, C) or CHO-P cells (B, D) for different times (A, B) or 2 minutes (C, D) in standard conditions. Where indicated (C, D), PMNLs were preincubated with the anti-CD18 antibody IB4 (20 μg/mL) for 10 minutes at 4°C, and platelets or CHO-P cells were preincubated with the anti–P-selectin antibody, WAPS 12.2 (20 μg/mL), for 10 minutes at room temperature. Samples were lysed by the addition of RIPA or Triton X-100–containing buffer. LYN and HCK were immunoprecipitated, and immune complexes were in part subjected to in vitro kinase assay and in part analyzed by Western blotting with biotinylated anti-LYN and anti-HCK antibodies. The figure shows autoradiograms of phosphorylated kinases (ka) and Western blots (Wb) from a representative of 3 different experiments.