Fig. 4: The p38 MAPK/NF-κB pathway mediates E protein-induced platelet activation.

a Washed human platelets isolated from healthy donors were incubated with or without the E protein (2 μg/ml) at 37 °C, and then platelet RNA was isolated for RNA-Seq (n = 3). Heat map of significantly differentially expressed platelet transcripts from human platelets treated with or without the E protein. b Gene Ontology (GO) analysis identified enriched pathways (Log₂FC ≥ 1, adjusted P < 0.05). c–e Human platelets were preincubated with the E protein (2 μg/ml) or the S protein (4 μg/ml) for 5 min at 37 °C and stimulated with or without ADP (10 μM). Western blot analysis showed that the E protein potentiated the phosphorylation of p38 and NF-κB in platelets (n = 5). Washed human platelets were pretreated with 10 μM SB203580 (a p38 inhibitor) or dimethyl sulfoxide (DMSO) for 10 min at 37 °C, followed by incubation with the E protein (0.5, 1, 2 μg/ml) for 5 min as a pretreatment step. f Potentiated platelet aggregation induced by the E protein combined with ADP 10 μM was suppressed by SB203580 (n = 4). g and h Enhanced P-selectin exposure and fibrinogen (Fg) binding of platelets induced by the E protein were suppressed by SB203580 (n = 4). i SB203580 inhibited the E protein promoting human platelet spreading on Fg (n = 4). Pretreated platelets were allowed to spread on a Fg-coated surface at 37 °C for 1 h. Data was shown with quantification analysis of the areas of spreading platelets (pixel numbers). j SB203580 attenuated accelerating clot retraction induced by the E protein in response to thrombin (n = 4). Data were presented with quantification analysis of clot retraction at 20, 40, and 60 min. k–m Western blot analysis of phosphorylation of p38 and NF-κB in platelets. SB203580 (10 μM) suppressed the elevated phosphorylated levels of p38 and NF-κB promoted by the E protein (2 μg/ml) in response to ADP (10 μM) (n = 4). The molecular weight markers are shown (c, k). Data were analyzed by 1-way ANOVA with Tukey multiple-comparisons test (d–i, l, m) or 2-way ANOVA with Tukey multiple-comparisons test (j). Data are presented as mean ± SEM. Source data are provided as a Source Data file.