Multiplate 5.0 analyzer

For the multiple channel electrical impedance platelet aggregometry (Multiplate 5.0 Analyzer, Diapharma Group Inc), heparinized blood samples were kept at room temperature and analyzed within 40 minutes of blood collection (range, 29–40 minutes) for 12 minutes. Before each analysis, the test cells were warmed to 37°C within the aggregometer and an automated pipette was used to decrease preanalytical variability. Heparinized blood samples (300 µL) were diluted with 300 µL of diluent and incubated for 3 minutes as recommended by the manufacturer (Multiplate 5.0 Analyzer, Diapharma Group Inc). Adenosine diphosphate (ADP; Diapharma Group Inc, West Chester, Ohio) and arachidonic acid (AA; Diapharma Group Inc) were used as platelet agonists and area under the curve (AUC) for ADP (AUC_ADP) and for AA (AUC_AA) were recorded. Note that the AUC value is an arbitrary unit. As recommended by the manufacturer (Multiplate 5.0 Analyzer, Diapharma Group Inc), the final concentration of ADP was 6.5 µM and the final concentration of AA was 0.5 mM. To serve as a control and to evaluate for spontaneous platelet aggregation, an identical volume of saline was used in place of the agonists (AUC_saline). The reagents were reconstituted and stored according to the manufacturer's recommendations (Diapharma Group Inc) in 60 µL aliquots.

Whole venous blood was used to measure platelet aggregation, diluted 1:1 in isotonic sodium chloride with 3 mM sodium citrate. A Multiplate 5.0 Analyzer from diaPharma was used for platelet aggregation studies. Aggregation was induced by ADP (1 μM) after preincubation with NO donors (all at 1 μM final concentration) for five minutes. All these procedures were carried out at 37 °C.