Functional assay of the platelets derived from ES cells. Platelets were obtained from the culture medium on days 14 to 15, and functional assays were performed. (A) Unstimulated platelets (left panels) and stimulated platelets by PAR4 thrombin receptor-activating peptide AYPGFK without stirring (right panels) were analyzed by flow cytometry. In the stimulated platelets, particles with higher forward scatter are observed (indicated by an arrow), indicating aggregate formation. A microscopic photograph (original magnification, × 600) of the analyzed platelets is shown below each histogram. (B) Platelets were stimulated by AYPGFK (left) or ADP (right) in the presence of Alexa Fluor 488-labeled fibrinogen, and the fibrinogen binding was determined by flow cytometry (gray histograms). Simultaneous binding to unstimulated platelets and control binding in the presence of EDTA or the anti-CD41 inhibitory antibody MWReg30 are shown as open histograms. (C) Platelets were stimulated by AYPGFK and stained with anti-P-selectin antibody followed by FITC secondary antibody. Surface expression of P-selectin was analyzed by flow cytometry (gray histogram). The open histogram represents the control experiment with unstimulated platelets. (D) Platelets were allowed to adhere and spread on the immobilized fibrinogen (left) and BSA surface (right), stained with FITC-conjugated phalloidin, and observed under fluorescence microscopy. Original magnification, × 1000.