Stimulation of platelets with vWF plus botrocetin induces tyrosine phosphorylation of FcR γ-chain.

(A) Human platelets were preincubated for 10 minutes at 37°C with a vehicle solution (lanes 1-4, 9), 1 mM RGDS plus 1 mM EGTA (lanes 5-7), or 20 μg/mL jararaca GPIb-BP (lane 8). After stimulation with 6 μg/mL botrocetin plus 10 μg/mL vWF (lanes 1-8) or 50 μg/mL collagen (lane 9) for the indicated time periods, the reactions were terminated by the addition of lysis buffer. The platelet lysates were then precipitated with GST-Syk-SH2. (B) After preincubation with 0.25% dimethyl sulfoxide (DMSO) as control or 10 μM PP1 for 5 minutes at 37°C, the human platelets were activated by the addition of 6 μg/mL botrocetin plus 10 μg/mL vWF for the indicated time frames. The cells were processed for precipitation with GST-Syk-SH2 as described in the legend for A. For A and B, precipitated proteins were then separated by SDS-PAGE (15% polyacrylamide) and immunoblotted with anti-FcR γ-chain (i) and 4G10 plus PY20 (ii). The numbers below the panels show time in seconds. The data are representative of 3 experiments. The open arrowheads indicate the proteins of 13, 11 kd. The closed arrowheads indicate the proteins of 8.5, 7 kd. wc indicates whole cell lysates.