The effect of S1P on PP fragmentation in vitro. (A) The number of MKs displaying PPF in the absence or presence of 10 µM S1P (230–590 MKs per experiment; three independent experiments with triplications). (B) The number of PPs with or without fragmentation observed by DIC microscopy in vitro over 1 h in the indicated groups. Data are pooled from 4–10 independent experiments for each group (n = 30–60 per group). (C) Representative dot plots show flow cytometric analyses of PP fragmentation. The first two panels show the gates for PPs. The CD41⁺CD61⁺ population was analyzed for the distribution of PPs according to FSC and SSC. MKs are G3; PPs with higher and lower FSC values are G2 and G1, respectively. The three representative microphotographs in the right show a representative brightfield image, as well as tubulin and CD41 stainings of fragments sorted using the gating strategy illustrated in the two plots. (D) Flow cytometric analyses of the PP fragmentation index in the presence or absence of various concentrations of S1P. The PP fragmentation index was calculated as described in Materials and methods. Data are representative of six independent experiments with triplication. (E) PP fragmentation by MKs exposed to shear stress. The efficiency of dynamic PP fragmentation was calculated as described in Materials and methods. Data are pooled from five independent experiments for each group. (F) Representative time-lapse video microscopy of PPs in the presence or absence of 5 µM S1P under shear stress (4 dynes/cm²). Arrows indicate direction of flow; arrowheads indicate PP shedding events. All error bars indicate SEM. Bars: (C) 10 µm; (F) 20 µm.