Inhibition of platelet Dll4 reduces hepatocyte Notch1 activation and TPO production. (A) Desialylated platelets were pretreated with Dll4 antibody (1 μg/mL) for 5 minutes and then incubated with hepatocytes for 30 or 60 minutes, followed by measuring Notch1 activation (cleaved Notch1 expression), HES5 expression, phosphorylation of JAK2/STAT3, and TPO level by western blot (mean ± SD; n = 3; 2-way ANOVA). The expression of cleaved Notch1, HES5, and TPO was quantified as a ratio relative to β-actin level, and the phosphorylation level of JAK2 and STAT3 was quantified as a ratio relative to respective total level. (B) Normal platelets (Plts), desialylated platelets (Desia-Plts), or desialylated platelets pretreated with Dll4 antibody (Desia-Plts-Dll4) (all at a total number of 2.5 × 10⁸) were infused into wild-type mice, and at indicated time points, liver was isolated to measure hepatic TPO mRNA by quantitative PCR assay (mean ± SD; n = 3; 2-way ANOVA) (compared with Plts or Desia-Plts-Dll4 group, ∗∗∗P < .001). (C) Schematic outline of the role of Notch1 in the regulation of hepatic TPO production. PCR, polymerase chain reaction.