Integrin activation and actin cytoskeletal reorganization in hESC-derived platelets. (A) Representative dot plots for platelets binding FITC-conjugated PAC-1 in the absence (left panel) or presence (middle panel) of 50 μM ADP. The right panel shows inhibition of PAC-1 binding by 10 μM tirofiban. Numbers on plots are the percentages of total cells within each quadrant. (B) Binding of FITC-conjugated PAC-1 to hESC-derived platelets was quantified in the absence and presence of ADP by flow cytometry. Some specimens were also incubated with tirofiban. Data depict means (± SD) from more than 3 independent experiments. (C) hESC-derived platelets spreading on fibrinogen-coated cover glass in the absence and presence of 50 μM ADP or 1.0 U/mL thrombin. Cells were fixed, permeabilized, and stained with rhodamine-phalloidin to label F-actin (red) and anti-CD41a antibody followed by Alexa 488–conjugated secondary antibody (green). αIIbβ3-dependent formation of stress fibers, lamellipodia, and filopodia was observed. Bar represents 10μm.