S1P regulates PPF. (A) The percentage of MKs displaying PPF. PPF is expressed as the percentage of MKs carrying PPs (8,000-10,000 MKs per experiment, five independent experiments with triplications). (B) The percentage of MKs displaying PPF in S1pr1^+/+ or S1pr1^−/− MKs transduced with lenti-GpIbα-S1pr1 or empty control vectors (3,000-8,000 MKs per experiment, two independent experiments with triplications). (C) Representative electron micrographs of WT and S1pr1^−/− MKs in BM. Arrowheads indicate the DMS. Red color highlights the DMS. N, nucleus. (D) The percentage of MKs with polarized PPF in the presence or absence of S1P and the S1pr1-specific inhibitor VPC23019 (VPC). n = 127–265 MKs per group. Data are pooled from three to five independent experiments. (E) Y10/L8057 cells were incubated with 10 µM S1P or vehicle for 2 min. The activities of Rac-GTP were quantified by pull-down assay (n = 5 independent experiments). (F) Y10/L8057 cells were incubated with 1 µM S1pr1 agonist, SEW2871, or vehicle for 5 min. The activities of Rac-GTP were quantified by pull-down assay (n = 3 independent experiments). (G) The percentage of MKs displaying PPF in the presence or absence of 50 µM NSC23766, a Rac GTPase inhibitor (4,000-7,000 MKs per experiment; three independent experiments with triplications). (H) Representative MP-IVM images of MKs with YFP⁺ or EGFP⁺ PPs. Green indicates MKs and PPs; red indicates sinusoids. Arrowheads indicate extravascular YFP⁺ or EGFP⁺ PPs; arrows indicate interstitial YFP⁺ or EGFP⁺ PPs. The inset shows magnification for the dotted box. The arrow in the inset indicates the connection between extravascular YFP⁺ PPs. Bars: (C) 2 µm; (H) 20 µm. (I) MKs displaying intrasinusoidal PPF in vivo presented as a percentage of all MKs carrying PPs (20–30 MKs per group; five independent experiments for WT; three independent experiments for other genotypes). (J) The lateral (x-y) speed of PP elongation was measured in naive (nontransplanted) S1pr1^+/+CD41-YFP^ki/+ mice, S1pr1^+/−CD41-YFP^ki/+ mice, and S1pr1^+/+CD41-YFP^ki/+ mice treated with W146. Red lines indicate means. Data are pooled from three mice each group. All error bars indicate SEM.