Binding of soluble fibrinogen to the cells on fibrinogen plate. (A) FXS-labeled fibrinogen was added to D3-adherent cells and the cells on the fibrinogen plate at the indicated concentrations and the binding was characterized by flow cytometry. The MFIs from the histograms for each concentration of FXS-labeled fibrinogen were plotted graphically. (B) FXS-labeled fibrinogen (200 μg/mL) was added either to the D3-detached cells (Detached), the cells on poly-L-lysine plate (broken line), the cells on fibrinogen plate, or D3-adherent cells (Plastic Plate) for 30 minutes at room temperature. Adherent cells were detached mechanically after the binding reaction and subjected to flow cytometry. In (C) and (D), D3-detached cells were incubated on the fibrinogen plate for the indicated times (0, 15, 30, and 45 minutes). The cell lysates were immunoprecipitated with (C) anti-FAK polyclonal antibody or (D) anti-Syk MoAb, and immunoblotting analysis was performed with antiphosphotyrosine MoAb (4G10). The blots were stripped and reprobed with indicated antibodies. +Soluble Fg. indicates the stimulation with 1 mg/mL soluble fibrinogen for 5 minutes. PL shows the result of plating on poly-L-lysine plate for 30 minutes. Ad+Soluble Fg. and De+Soluble Fg. are the results of 1 mg/mL soluble fibrinogen stimulation on D3-adherent cells and D3-detached cells, respectively. Results are representative of 3 experiments.