Fig. 3. DAB2 binds to platelets and megakaryocytic differentiating K562 cells. (A) DAB2 binds to the surface of activated platelets. Washed platelets (1.5x10⁸) were either untreated (R, resting platelets) or were stimulated with thrombin (Th, 0.1 U/ml), TPA (1 µg/ml), collagen (Col, 10 µg/ml), U46619 (U, 1 µM) or TRAP (10 µM) for 10 minutes. Platelets were analyzed by flow cytometry after incubation with anti-DAB2 (1:100 dilution) and FITC-conjugated goat anti-mouse secondary antibody. The percent of platelets with specific DAB2 binding is presented. (B) The PTB domain of DAB2 is crucial for the interaction with platelets. Resting or TRAP-stimulated (10 µM) platelets (1.5x10⁸/well) were added for 3 hours to 24-well plates pre-coated with DAB2-PTB, DAB-M or control GST proteins (100 µg/ml). Platelet adhesion was then quantified by Crystal Violet assay. (C) TPA-treated (T, 10 ng/ml) or vehicle control ethanol-treated (E, 0.01%) K562 cells (5x10⁵ cells/well) were added to plates pre-coated with DAB2-PTB or control GST protein (100 µg/ml). Cell adhesion was quantified by Crystal Violet assay. (D,E) TPA- and ethanol-treated K562 cells (5x10⁵) were incubated with DAB2-PTB for 3 hours. Binding of DAB2 was analyzed by incubating K562 cells with FITC-conjugated anti-GST antibody, counterstained with 4',6-diamidino-2-phenylindole (DAPI) and observed by confocal microscopy (D). The percent of K562 cells with DAB2-PTB binding was determined by flow cytometry (E). The data represent the mean ± s.d. of three to six experiments. **P<0.001 when compared with thrombin-treated platelets (A), TRAP-stimulated platelet adhesion to GST (B) and ethanol-treated K562 cell adhesion (C), or binding to DAB2-PTB (E).