Effect of genistein on protein tyrosine phosphorylation and FAK phosphorylation. Washed platelets (2.8 × 108/mL) were preincubated without (–) or with genistein (G; 100 μM-1000 μM) or vehicle (DMSO) for 30 minutes at 37°C, prior to exposure to 2B-rVWF (0.7 μg/mL) for 4.5 minutes at 37°C under high shear rates. Buffer indicates samples without 2B-rVWF. Total cell lysates were analyzed by immunoblotting with antiphosphotyrosine antibodies (4G10 and PY20) upon protein separation on a 8% polyacrylamide gel (A). Membranes were then stripped and reblotted with anti–phospho-FAK polyclonal antibody (B). DSP are indicated on the bottom part in the corresponding samples. In separate experiments, platelets were preincubated with genistein at the indicated concentration, exposed to shear, and fixed with PFA. An aliquot was deposited in microtiter plates for microscopic observation of aggregates. Images were recorded following sedimentation in the wells. Bar represents 50 μm (C). Results are representative of 3 experiments.
