GPIb-vWF interaction induces tyrosine phosphorylation of PLCγ2, independently of integrin αIIbβ3 signaling.

Human platelets were preincubated with a vehicle solution as the control (A-C, left panels), 1 mM RGDS peptide plus 1 mM EGTA (A, right panel), 20 μg/mL jararaca GPIb-BP (B, right panel), or 10 μM PP1 (C, right panel) for 5 minutes and then stimulated with 6 μg/mL botrocetin plus 10 μg/mL vWF for the indicated time periods. After cells were solubilized, PLCγ2 was isolated by immunoprecipitation with the corresponding Ab. Precipitated proteins were then separated by SDS-PAGE and analyzed by immunoblotting with 4G10 plus PY20. The membranes were reprobed by immunoblotting with anti-PLCγ2. The data are representative of 3 experiments. The numbers below the panels show time in seconds. pY indicates phosphotyrosine.