Physical association of Src, Lyn, and tyrosine kinase activity with GPIb on VWF-botrocetin stimulation.

(A) After stimulation with 6 μg/mL botrocetin plus 10 μg/mL VWF for the indicated time periods (seconds), platelets were solubilized with lysis buffer (lanes 1-3) as described in “Materials and methods,” or with lysis buffer including 50 μg/mL calpeptin (lanes 4-6), or with lysis buffer including Complete (lanes 7-9). The lysates were immunoprecipitated with the anti-GPIb MoAb WGA-3. Precipitated proteins were then separated by SDS-PAGE and immunoblotted with anti-Src and anti-GPIb MoAb, respectively. (B) Platelets were preincubated for 10 minutes at 37°C with vehicle solutions (control) or 20 μg/mL jararaca GPIb-BP. After stimulation as described in panel A, the platelets were solubilized with a lysis buffer containing Complete, and subjected to immunoprecipitation with anti-GPIb MoAb. The immunoprecipitates were separated by SDS-PAGE and analyzed by immunoblotting with anti-Src, anti-Lyn, anti-Fyn, and anti-GPIb MoAbs as indicated at the left of each panel. (C) The precipitates with anti-GPIb MoAb were prepared as described in panel B and analyzed in an in vitro kinase assay using enolase as the exogenous substrate. The proteins were separated by 8% SDS-PAGE and quantified with a BAS 2000 phosphorimager. Equivalent precipitation of GPIb was confirmed by immunoblotting and is shown in the lower panel. HC represents the band presumably derived from the IgG heavy chain.