Extracellular S1P cannot rescue the defect of Sphk2^−/− MKs in PP fragmentation. (A) Representative autoradiogram of ³H-S1P in WT, Sphk1^−/−, and Sphk2^−/− MKs. (B) The number of PPs with or without fragmentation observed by DIC microscopy in vitro over 1 hour in the indicated groups. Data are pooled from 7 to 10 independent experiments from each group (n = 27-51 per group). (D) DIC microscopic analysis of PP shedding in the presence of 10 µM S1P. MKs were kept on a heated microincubator to keep the temperature at 37°C in serum-free medium and monitored using a DIC microscope system equipped with a ×40 oil objective lens with NA = 0.7. Arrow, platelets released from PP stems. WT MKs (upper row); Sphk2^−/− MKs (lower row). (D) Fold change of S1pr1, S1pr2, and S1pr4 mRNA by fetal liver–derived mature Sphk2^−/− MKs (white) compared with mature WT MKs (black). The mRNA levels were normalized to Gapdh. Representative of 3 independent experiments performed in triplicate. All scale bars represent 20 µm; time in minutes. All error bars represent SEM.