Platelet isolation

Platelet isolation was performed as previously described (29). Peripheral blood samples were drawn into acid-citrate-dextrose (ACD) and centrifuged at 200 x g for 20 min. Platelet-rich plasma (PRP) was obtained and recentrifuged (500 X g, 20 min) in the presence of 100 nM of prostaglandin E₁ (PGE₁; Cayman 1303) to pellet the platelets. Platelet pellets were resuspended in 25 ml of PSG (5 mM PIPES, 145 mM 35 NaCl, 4 mM KCl, 50 μM Na₂HPO₄, 1 mM MgCl₂·6H₂O, 5,5 mM glucose; pH 6,8) containing 100 nM PGE₁. The platelet suspension was centrifuged again at 500 X g for 20 min and the platelet pellet was resuspended in medium 199 (M199) containing 25 mM of HEPES to the concentration of 1 x 10⁹/mL. The platelet viability in dengue patients and healthy volunteers in the present study has been previously reported (26). The viability of platelets used for in vitro experiments was above 95% of live platelets.