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Figure 4


Fig. 4. {alpha}IIbß3 integrin mediates DAB2 binding to platelets and the surface of CHO-{alpha}IIbß3-integrin cells. (A) Effect of the functionally blocking mAbs and the RGDS peptide on the interaction of DAB2 with platelets. Resting or TRAP-stimulated (10 µM) platelets (1.5x108) were incubated with the indicated mAbs (20 µg/ml) or RGDS peptide (100 µM) for 30 minutes. The percent of platelets with DAB2 binding was determined by flow cytomery after incubating the platelets with anti-DAB2 (H110) antibody (1:200 dilution) and FITC-conjugated donkey anti-rabbit secondary antibody. (B) Effects of functional blocking mAb and RGDS peptide on the binding of recombinant DAB2-PTB to platelet surface. TRAP-stimulated platelets (1.5x108) were incubated with the mAb or RGDS peptide at room temperature for 30 minutes. DAB2-PTB or control GST protein (100 µg/ml) was added into the assay mixture and incubated for 3 hours. DAB2-PTB binding was detected as described in A except that an anti-GST antibody was used. (C) Expression of {alpha}IIbß3 integrin on CHO-K1 and CHO-{alpha}IIbß3-integrin cells. The percent of cells with {alpha}IIbß3 integrin expression was determined by flow cytometry using 10E5 (20-30 µg/ml) and FITC-conjugated goat anti-mouse secondary antibody. (D) Binding of DAB2 to the surface of CHO-{alpha}IIbß3-integrin cells. DAB2-PTB or control GST protein (100 µg/ml) were added to the cultured CHO-K1 and CHO-{alpha}IIbß3-integrin cells for 2 hours. After incubation with FITC-conjugated anti-GST antibody (green) and counterstaining with DAPI (blue), confocal microscopy analysis was performed to visualize DAB2 binding. (E) Effects of function blocking mAbs and RGDS peptide on DAB2 interaction with CHO-{alpha}IIbß3-integrin. The CHO-{alpha}IIbß3-integrin cell suspension was incubated with the indicated mAbs or the RGDS peptide for 30 minutes. Then, DAB2-PTB or control GST protein (100 µg/ml) were added into the cell suspension and incubated for 3 hours. DAB2 binding was determined by flow cytometry using FITC-conjugated anti-GST antibody. The data represent the mean ± s.d. of three to six experiments. **P<0.001 compared with TRAP-stimulated platelets treated with mIgG (A and B), CHO-K1 cells (C) or CHO-{alpha}IIbß3-integrin cells pre-treated with mIgG (E).





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