Human whole blood was collected in vacutainers containing sodium citrate buffer and centrifuged at 1000 rpm for 15 min to obtain PRP. Platelet-poor plasma (PPP), used as a blank control, was obtained by centrifuging at 3000 rpm for 15 min. A total of 450 μl of PRP or PPP was pipetted into an aggregometry cuvette, which was preheated to 37°C for 5 min. The light transmission was measured in a Chrono-log aggregometer (Helena Laboratories, Beaumont, Texas, USA) for 5 min after ADP (10 μM) or NS1 was added.
For the platelet aggregation enhancement assay, PRP was preincubated with BSA or NS1 (with or without the F(ab’)2 antibody fragment) at 37°C for 1 h. To investigate whether the NS1-induced enhancement of platelet aggregation was mediated by ADP receptors, PRP was preincubated with BSA or NS1 (with or without ADP receptors inhibitors, BMS-646786 (BM0020, Sigma-Aldrich) or Clopidogrel hydrogen sulfate (SML0004, Sigma-Aldrich)) at 37°C for 1 h. The light transmission was measured for 5 min after low-dose ADP (2.5 μM) was added.
For the platelet aggregation enhancement assay, PRP was preincubated with BSA or NS1 (with or without the F(ab’)2 antibody fragment) at 37°C for 1 h. To investigate whether the NS1-induced enhancement of platelet aggregation was mediated by ADP receptors, PRP was preincubated with BSA or NS1 (with or without ADP receptors inhibitors, BMS-646786 (BM0020, Sigma-Aldrich) or Clopidogrel hydrogen sulfate (SML0004, Sigma-Aldrich)) at 37°C for 1 h. The light transmission was measured for 5 min after low-dose ADP (2.5 μM) was added.