A1 domain of VWF interaction with GPIb selectively activates Src and stimulates Src association with GPIb.

(A) After stimulation with 6 μg/mL botrocetin plus 20 μg/mL A1 domain of VWF for the indicated time periods (seconds), platelets were solubilized with lysis buffer containing Complete. The lysates were divided into 2 equal volumes, which were subjected to either immunoprecipitation with anti-Src or anti-Lyn MoAbs, respectively. In vitro kinase assays were performed using enolase as exogenous substrate. The proteins were separated by 8% SDS-PAGE and quantified with a BAS 2000 phosphorimager. Equivalent precipitation of Src or Lyn was confirmed by immunoblotting as shown in the lower panel. The arrows represent the bands presumably derived from IgG heavy chain. (B) The level of enolase phosphorylation in anti-Src (♦) and anti-Lyn (▪) immunoprecipitates shown in panel A was quantified by densitometry, and represented as a fold-increase over the control (0 seconds). The data are expressed as mean ± SEM of 3 separate experiments. (C) The samples were prepared as described in panel A and the subsequent lysates immunoprecipitated with anti-GPIb MoAb. The immunoprecipitates were separated by SDS-PAGE and analyzed by immunoblotting with anti-Src and anti-GPIb MoAbs, respectively.