Fig. 1. Mfsd2b is expressed in resting and activated platelets and is required for S1P release.

a Mfsd2b is expressed in resting and thrombin-activated platelets with comparable levels. Western blot analysis of Mfsd2b expression in resting and thrombin-activated platelets from WT and global knockout of Mfsd2b (KO). Mfsd2b expression is retained in thrombin-activated platelets at levels similar to resting cells. Note that PF4 is a soluble protein and it is strongly released from alpha-granules after activation with thrombin in both WT and KO platelets. WT wild-type, KO knockout. Tmem16F is a membrane protein of platelets. Experiments were repeated twice (n = 2). b, c Resting and thrombin-activated platelets export S1P in Mfsd2b-dependent manners. Resting and thrombin-activated platelets from WT and KO mice were incubated with [3-³H]-sphingosine in Tyrode H buffer containing 0.5% BSA for 1 h at 37 °C. The cell pellets and supernatants were collected for S1P determination. Scintillation quantification of S1P levels from supernatants and cell pellets expressed as percentage. Data are mean and SD. Experiments were repeated twice (n = 7–8). **P < 0.01, ***P < 0.001; ns not significant. P values were calculated using one-way ANOVA. d, e Time course transport assays for thrombin-activated platelets (platelets were activated before transport assays). Activated WT platelets readily release S1P in an Mfsd2b-dependent manner. Note that activated WT platelet cannot store S1P. Data are mean and SD. Experiments were repeated twice (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001. P values were calculated using one-way ANOVA. DPM disintegrations per minute.