Sphk2 deficiency induces thrombocytopenia by aberrant S1P₂activation. (A) Effects of S1P₁ (W146, 10 mg/kg, left) or S1P₂ (JTE-013, 1.2 mg/kg) antagonism on Sphk2 deficiency-induced thrombocytopenia (24-hour platelet counts). (B-D) Platelet counts and MPV in litters from independent intercrosses of S1pr2^+/− in a wild-type background (B), S1pr2^+/− in a Sphk2-deficient background (C), and of Sphk2^+/− in a S1P₂-deficient background (D). Note that although S1P₂ deficiency does not itself affect platelet counts, it rescues Sphk2 deficiency-induced thrombocytopenia. (E) Transmission electron micrographs of bone marrow from Sphk2^+/+, Sphk2^−/−, and Sphk2^−/−:S1pr2^−/− mice. Although the majority of MKs from Sphk2^+/+ and Sphk2^−/−:S1pr2^−/− mice were singular and large, with a mature appearance and well-defined demarcation membrane systems (upper), MKs in Sphk2^−/− were highly heterogeneous, with clusters of immature MKs or mature MKs with limited DMS next to blood sinusoids (middle), low-contrast MK “ghosts” that appeared to be undergoing necrosis (bottom left, next to a normal MK) and platelet release within the bone marrow (bottom right). Representative images from n = 4 mice per genotype are shown. Scale bars, 2 μm. (F) Effect of a bolus injection of the Rho kinase inhibitor Y27632 (10mg/kg) on platelet counts in Sphk2^−/− and Sphk2^+/+ controls. Note a significant increase in platelet counts only in the knockout. (G) Sphingosine content of Sphk deficient platelets. Statistical analyses by 2-way analysis of variance (A,F) or the Mann-Whitney U test.