Time dependence of tyrosine phosphorylation of proteins from platelets activated by alboaggregin A in the presence of Fab fragments of anti-GPVI Abs.

Washed platelets (700 μL) were stirred at 1100 rpm at 37°C with or without Fab fragments of GPVI Abs. After the addition of 0.5 μg/mL alboaggregin A, aliquots were removed at the times indicated and dissolved in SDS buffer containing inhibitors. (A) After separation by SDS-PAGE (7%-17% acrylamide gradient) and transfer to PVDF membranes, the proteins were incubated with the antiphosphotyrosine mAb 4G10 before detection by using a peroxidase-linked second Ab and chemiluminescence. (B-D, upper bands) Lysates of resting platelets and platelets activated with 0.5 μg/mL alboaggregin A. Aliquots were immunoprecipitated with antibodies against (B) Fcγ, (C) LAT, or (D) PLCγ2. After SDS-PAGE and Western blotting, the proteins were detected with 4G10 antiphosphotyrosine antibody. (B-D, lower bands) The membranes were then stripped and treated with anti-Fcγ, anti-LAT, or anti-PLCγ2 Abs.