Influence of PI 3-kinase activation and calcium chelator on shear-induced tyrosine phosphorylation. Washed platelets (2.8 × 10⁸/mL) were preincubated with either wortmannin (100 nM) or vehicle (DMSO) for 15 minutes at 37°C, or EDTA (2 mM) for 5 minutes at room temperature. Platelets were submitted to high shear rates for 4.5 minutes at 37°C in the presence of 2B-rVWF (0.7 μg/mL), lysed, and separated on an 8% polyacrylamide gel. Immunoblotting was performed using antiphosphotyrosine MoAbs 4G10 and PY20 (top panel), stripped, and reprobed with anti–phospho-FAK polyclonal antibody (bottom panel). Results are representative of 3 experiments.