Preparation of human platelets

1. Human blood was taken from drug-free volunteers on the day of the experiment using acidic citrate dextrose (ACD; 120 mmol/L sodium citrate, 110 mmol/L glucose, 80 mmol/L citric acid) as anticoagulant.
2. Platelet-rich plasma was obtained by centrifugation at 200g for 20 minutes.
3. Platelets were isolated by centrifugation at 1,000g for 10 minutes in the presence of prostacyclin (0.1 μg/mL) and resuspended in 25 mL of a modified Tyrodes-HEPES buffer (134 mmol/L NaCl, 0.34 mmol/L Na2HPO4 , 2.9 mmol/L KCl, 12 mmol/L NaHCO^₃ , 20 mmol/L HEPES, 5 mmol/L glucose, 1 mmol/L MgCl^₂ , pH 7.3) and 3 mL of ACD in the presence of prostacyclin (0.1 μg/mL).
4. Platelets were centrifuged at 1,000g for 10 minutes and resuspended at a concentration of 4 × 10⁸ cells/mL in Tyrodes-HEPES buffer containing EGTA (1 mmol/L) and indomethacin (10 μmol/L), unless indicated.
5. MgCl^₂ was omitted and 1 mmol/L EDTA was added to the Tyrodes-HEPES buffer where stimulation in the absence of MgCl^₂ was required.
6. Experiments were performed at 37°C in an aggregometer with continuous stirring (1,200 rpm).
7. The resulting platelet population was essentially free (<0.1%) of erythrocytes and peripheral mononuclear cells, and >99% pure, as assessed by flow cytometry (see below) for expression of the platelet specific antigen CD42b.
8. To ensure that the isolation procedure did not artificially stimulate platelets, their activation state was assessed before and after isolation by measuring P-selectin expression levels in whole blood and purified platelets by flow cytometry.