ABSTRACT
Whole blood (0.8 volumes) was collected, using 20G teflon catheters (Surflo, Terumo, Tokyo, Japan), from the marginal ear vein of male New Zealand White rabbits into 0.2 volumes of citrate 3.2% (w/v) (trisodium salt-dihydrate, Sigma Chemicals Co., St. Louis, MO). The citrated rabbit whole blood was added, in 50 μL aliquots, to each of the eight wells of a teflon micromixing device, followed by 25 μL of antagonist/vehicle. After a 30-sec temperature equilibration period, 25 μL of the agonist EC50/vehicle was added. A 10 μL sample was taken immediately and after 15 min incubation at 37°C for counting of the single, non-aggregated platelets (3-24 μm3) using the Baker Instruments Series 810 Platelet Analyzer (Baker Instruments, Allentown, PA). The results were expressed as the platelet aggregation index, i.e., 1-P15/P0, where P15 and P0 are the platelet count at 15 and 0 min, respectively. Therefore, an index of 0 signifies no platelet aggregation and, conversely, 1 is total platelet aggregation. The platelet count for male New Zealand White rabbits was approximately 250 × 103/μL, and there was minimal variation between experimental animals. The bioassay was performed immediately after blood collection.
