DENV-induced thrombocytopenia is MC-dependent. (A) Platelet counts were not reduced in MC-deficient (Sash) mice infected with DENV2 (1 × 10⁶ PFU via intraperitoneal injection) at 24 and 72 hours postinfection (n = 8-12 per group). (B) No significant differences in platelet activation were observed between uninfected and DENV2-infected Sash mice, determined by staining blood isolated 24 and 72 hours postinfection for platelet marker CD41 and activation marker CD62P, followed by analysis by flow cytometry (n = 4-5 per group). Flow cytometry gating strategy is presented in supplemental Figure 2A. (C) Platelets were labeled in mice through injection of an antibody against platelet GP1b. Spleens of DENV2-infected and control Sash mice were isolated 3 days postinfection, and cells were stained for CD11b and F4/80 and analyzed by flow cytometry. No significant differences in the numbers of Platelet⁺ Macrophages (CD11b⁺F4/80⁺GP1b⁺) were observed between uninfected and DENV-infected Sash mice (n = 4). Flow cytometry gating strategy is presented in supplemental Figure 2B. (D) Sash mice that were reconstituted with MCs (Sash-R) were infected with DENV2. A significant reduction in circulating platelets was observed in Sash-R mice infected with DENV compared with uninfected controls at 24 and 72 hours postinfection (n = 7-8 per group). (E) Increased numbers of activated platelets (CD41⁺CD62P⁺) were detected in the circulation of DENV-infected Sash-R mice compared with uninfected controls at 24 and 72 hours postinfection (n = 3-4 per group). (F) Increased numbers of Platelet⁺ Macrophages were detected in the spleens of DENV-infected Sash-R mice compared with uninfected control spleens (n = 3-4 per group). (G) Representative eosin and methylene blue staining of peripheral blood smears from WT, Sash and Sash-R mice, day 3 postinfection showing aggregates in infected WT and Sash-R mice, but not Sash mice. Some individual platelets are indicated by arrows. Platelet aggregates are surrounded by dashed lines. Scale bar, 25 μm. Whole blood from (H) WT or (I) Sash mice was isolated and stimulated with DENV2, or cocultured with MCs or MCs+DENV2 for 15 minutes. Cells were then fixed and stained for CD41 and CD62P and analyzed by flow cytometry (n = 6-7 per group). For panels D-E, coculture with DENV or MCs raised the activation levels of platelets, but was significantly enhanced with coculture with MCs+DENV2. (J) Ketotifen treatment (6 mg/kg) of DENV-infected mice significantly increased platelet counts compared with vehicle treatment (24 hours postinfection). (K) Ketotifen also reduced platelet activation compared with controls receiving vehicle treatment (n = 4-5 per group). Error bars represent the standard error of the mean. P values were determined by Student’s unpaired t test. ns, not significant; *P < .05; **P < .01; ***P < .001; ****P < .0001.