The effect of S1P on PP fragmentation in vivo. (A) Percentage of PP fragmentation events observed by MP-IVM over 1 h in the indicated groups. n = 13–33 per group. Data are pooled from three to seven independent experiments. (B) Role of S1pr1 for PP shedding in vivo visualized by MP-IVM. Representative MP-IVM sequences show that WT MKs frequently shed PPs as shown in the first and the third rows. The inset shows a magnification of a shed PP particle. Asterisks show embedded platelet-like particles. Inhibition or loss of S1pr1 abolishes PP shedding (second and fourth rows). Arrowheads indicate intrasinusoidal PPs, and arrows show extrasinusoidal PPs in S1pr1^−/− chimaeras. The dashed lines highlight the sinusoids. Green or yellow indicates MKs and PPs; red indicates sinusoids. Bars, 20 µm. (C) Circulating reticulated (young) platelets in mice treated with W146, an S1pr1-specific antagonist, or vehicle as assessed by flow cytometry. n = 3 mice each group. (D) Circulating platelet counts in CD1 mice treated with W146 or vehicle. n = 4 mice each group. (E) The time point of fragmentation detected by MP-IVM in mice treated with W146 (<6 h) or vehicle. Data are pooled from three independent experiments each group. (F) Volumes of PP fragments in mice treated with W146 (within 6 h) or vehicle. Red lines indicate means. Data are pooled from three independent experiments each group. (G) Mean platelet volume in the indicated genotypes. n = 8 for WT; n = 6 for S1pr1^+/−; n = 15 for WT BM chimaeras; n = 9 for S1pr1^+/− BM chimaeras; n = 14 for S1pr1^−/− BM chimaeras. All values are mean and SEM.