Preexposure to NS1 amplifies platelet activation by suboptimal concentrations of procoagulant agonists. Platelets were stimulated with NS1 (0, 0.5, or 5 µg/mL) produced in SF9 cells for 2.5 hours and restimulated with thrombin (0.05 U/mL), PAF (40 nM), oxLDL (5 µg/mL), epinephrine (10 µM), or ATP (2mM) for 30 minutes. (A) The percentage of platelets expressing surface P-selectin (CD62P); and the concentrations of MIF (B) and TXB₂ (C) in platelet supernatants were evaluated. (D) Platelets were pretreated for 30 minutes with aspirin (100 μM) or vehicle, stimulated for 2.5 hours with 5 µg/mL of NS1 produced in SF9 cells, and restimulated for 30 minutes with thrombin (0.05 U/mL) or PAF (40 nM). The percentage of platelets expressing surface P-selectin was evaluated. Bars represent mean ± standard error of the mean of 5 independent experiments. *P < .05 compared with unstimulated platelets; #P < .05 compared with procoagulant agonists or NS1 alone.