GW3965 inhibits tyrosine phosphorylation stimulated through GPVI. Platelets (8 × 108 cells/mL) pretreated with GW3965 (10-50μM) or DMSO (0.08% volume/volume) for 10 minutes and stimulated with collagen (50 μg/mL) for 90 seconds in the presence of ethyleneglycoltetraacetic acid (10μM), indomethacin (10μM), and apyrase (2 U/mL). For total tyrosine phosphorylation levels, lysates were immunoblotted with an antiphosphotyrosine antibody (Ai). For Syk phosphorylation levels (Bi), Syk was immunoprecipitated from lysates using an anti-Syk antibody and immunoblotted with an antiphosphotyrosine antibody. For phosphorylation of LAT (Bii) and PLC-γ2 (Biii), the phosphorylation of Tyr200 and Tyr1197, respectively, was examined using antiphospho-specific antibodies. Membranes were stripped and reprobed for glyceraldehyde-3-phosphate dehydrogenase (Ai,Bii,Biii) or Syk (Bi). Band intensities were quantified from 3 separate experiments (mean ± SEM) and normalized for protein loading. Bars represent the average percentage of tyrosine phosphorylation levels; 100% was defined as the activation achieved in DMSO (0.08% volume/volume)-treated platelets. *P < .05. **P < .01. MW indicates molecular weight; and IP, immunoprecipitation.
