2.2 Platelet aggregometry
Aggregation in whole blood was examined utilizing a 4-channel Model 700
impedance aggregometer (Chrono-Log, Havertown, Pennsylvania, USA)
according to the manufacturer’s specifications, and the experimental
protocol applied was as described by us previously
(Nooney et al. , 2015). In brief,
tests were performed at 37°C and a stirring speed of 900 rpm. Blood
samples were diluted two-fold with normal saline (final volume 1 mL) and
pre-warmed for 5 min at 37°C. Aggregation was induced with ADP (final
concentration of 2.5 µM). The NO donor sodium nitroprusside (SNP, 10 µM)
was added to samples 1 min before ADP. Aggregation was monitored
continually for 7 min, and responses were recorded for electrical
impedance in ohms. Results obtained from aggregometry assays with
respect to responses to SNP were evaluated as a percentage of the extent
of maximal aggregation in the presence and absence of SNP, and reported
as percent inhibition of aggregation. For in vitro experiments examining
effects of hydrogen sulphide (H2S) on anti-aggregatory
responses to SNP , NAC and the H2S donor NaHS were added
15 minutes and 2 minutes, respectively, prior to ADP. Inhibitors of
H2S formation [aminooxyacetic acid (AOAA, 0.5 mM) and
D,L-propargylglycine (PAG, 3.3 mM)] were pre-incubated with blood
samples for 15 minutes prior to ADP.