Serotonin exacerbates platelet activation to DENV via 5HT₂ receptors and downstream pathways. (A) Whole blood from WT mice was isolated and stimulated ex vivo with DENV2, in the presence of serotonin (50 nM, 200 nM, 1 μM or 5 μM) for 15 minutes. Cells were then fixed and stained for platelet marker CD41 and activation marker CD62P and analyzed by flow cytometry (n = 4). The flow cytometry gating strategy is presented in supplemental Figure 2A. DENV raised the activation levels of platelets, which was significantly further increased in the presence of serotonin. (B) WT mice were infected with DENV2 (1 × 10⁶ PFU I) and treated with 150 mg/kg of serotonin or vehicle daily. Serotonin treatment worsened thrombocytopenia during DENV infection compared with vehicle-treated mice, at 3 days postinfection. (C) DENV infection resulted in increased platelet activation, which was further increased when mice were also treated with exogenous serotonin (n = 7-13 per group). (D) Representative peripheral blood smears, day 3 postinfection, show platelet aggregation (dashed lines) was more visually apparent after exogenous serotonin treatment compared with DENV infection. Some individual platelets are indicated by arrows. Scale bar = 25 μm. (E) Whole blood from WT mice was isolated and stimulated with DENV2, MCs or MC+DENV2 in the presence of vehicle or ketanserin (100 nM or 500 nM) for 15 minutes. Cells were then analyzed by flow cytometry. Coculture with DENV or MCs raised the activation levels of platelets but was significantly further enhanced in the presence of MC+DENV2. This was abolished with ketanserin treatment (n = 4). (F) Whole blood from healthy human volunteers was isolated and stimulated with DENV2, serotonin, or DENV2+serotonin for 15 minutes. Cells were then fixed and stained for platelet markers CD41 and CD62P and analyzed by flow cytometry. DENV raised the activation levels of platelets. DENV in the presence of serotonin significantly enhanced platelet activation over control and DENV groups (n = 4). (G) Human whole blood was stimulated with DENV2, MCs or DENV2+MCs in the presence of vehicle or ketanserin (100 nM or 500 nM) for 15 minutes before assessment of platelet activation. Ketanserin treatment abolished the significant increase in platelet activation that occurred in the DENV2+MC treated group compared with controls (n = 4). (H) Mouse blood was incubated with 25 nM treprostinil for 10 minutes before stimulation with DENV and 5 μM serotonin for 15 minutes, followed by assessment of platelet activation. Treprostinil prevented DENV-induced platelet activation, which was overcome in the presence of serotonin (n = 4). (I-K) WT mice (n = 5 per group) were treated with either fluoxetine or vehicle for 2 weeks and subsequently infected with DENV2 (1 × 10⁶ PFU intraperitoneally). (I) Fluoxetine pretreatment prevented thrombocytopenia after DENV2 infection. (J) There was reduced platelet activation when mice were treated with fluoxetine prior to DENV2 infection. (K) Representative peripheral blood smears, day 3 postinfection, show decreased platelet aggregation (dashed lines) when mice were treated with fluoxetine. Some individual platelets are indicated by arrows. Scale bar = 25 μm. Error bars represent the standard error of the mean. P values were determined by 1-way ANOVA or by Student’s unpaired t test for panels in vivo experiments. ns, not significant; *P < .05; **P < .01; ***P < .001; ****P < .0001.