Figure 4. Platelet activation and apoptosis mediated by DENV2 in vitro.

Platelet rich plasma (PRP) was incubated with different MOI of DENV2. (A) P-selectin expression and (B) PAC-1 binding on platelet, and (C) platelet-derived MPs from supernatant were measured by flow cytometry. Data are the mean ± SEM of percent fluorescence from three different experiments. The DENV2 treatment increased both parameters in a concentration-dependent manner, ***p < 0.0001 (one way ANOVA). The above expressions were significantly inhibited by the platelet activation inhibitor PGI2 (50 and 100 ng/mL) ***p < 0.0001 (Student’s t-test). The DENV2 treatment has increased the (D) phosphorylation of ERK and (E) activation of caspase 9 (activation generates 37 kDa cleaved band) and cyclophilin D in platelets in a concentration-dependent manner, measured by immunoblotting. The densitometry data and uncut immunoblots of pERK/ERK, caspase 9 and cyclophilin D are mentioned in Suppl. Fig. 4(A–H). (F) DENV2 also increased PS expression in a concentration-dependent manner, **p < 0.001 and ***p < 0.0001. Further, both concentrations of PGI2 inhibited the PS expression significantly, **p < 0.002 and ***p < 0.0003. (G) The qPCR data show the higher number of DENV2 genome copy in platelet pellets when PRP was treated with higher MOI of DENV2, ***p < 0.0001, NS = non-significant. (H) P-selectin expression on platelets was measured following the treatment of PRP with different MOI of Japanese Encephalitis Virus (JEV) and DENV2. DENV2 activated platelets in a concentration-dependent manner, p < 0.001, while JEV did not show significant effect. The data for PAC1 binding, PS expression on platelets and platelet-MPs generation by DENV and JEV are mentioned in Suppl. Fig. 5.