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.