Quantitative lipidomics reveals SPC dysregulation at the trans-Golgi network in the Smpd1^−/−mouse strain. (A) Correlation of murine lipid concentrations between wild-type (WT) and acidic sphingomyelinase knockout (Smpd1^−/−) mutant mice in resting platelets (top panel) and after stimulation with CRP (1 µg/mL; bottom panel). (B) Regulated and nonregulated SLs in resting and activated platelets. Data from 3 independent experiments were combined. (C) SL metabolism in WT and Smpd1^−/− mice indicates a shift toward lysosphingomyelin. (D) ATP release in murine wild-type platelets pretreated with SPC or vehicle control for 2 minutes followed by a stimulation with CRP. Arithmetic means ± SEM (n = 6; left side) and characteristic graphs (right side) are shown. (E) Platelet light transmission aggregometry in murine wild-type platelets pretreated with SPC or vehicle control followed by a stimulation with CRP. Arithmetic means ± SEM (n = 6; left side) and characteristic graphs (right side) are shown. (F) Platelet secretion of dense (ATP release) and α (P-selectin) granules in Smpd1^+/+ and Smpd1^−/− platelets in the presence of SPC (20 µM) or vehicle control. (G) Platelet integrin α_IIbβ₃ activation (top panel) and aggregation (bottom panel) of Smpd1^+/+ and Smpd1^−/− platelets in the presence of SPC or vehicle control following stimulation with CRP. (H) Arithmetic means ± SEM (n = 6; top panel) and representative phase-contrast images (bottom panel) of platelet surface coverage after perfusion from blood from Smpd1^−/− and wild-type mice over a collagen-coated surface (200 µg/mL) for 5 minutes in the presence (gray bars) or absence (black bars) of SPC and at a shear rate of 1700^−sec. Scale bar equals 20 µm. *(P < .05) and **(P < .01) indicate statistically significant differences. LacCer, lactosylceramide; S1P, sphingosine-1-phosphate; Sa, sphinganine; Sa1P, sphinganine-1-phosphate; SEM, standard deviation of the mean; So, sphingosine.