Platelet aggregation study
Whole blood was collected from antecubital vein and mixed with 3.8 percent sodium citrate as an anticoagulant and centrifuged at 250 g for 15 minutes at room temperature. Platelet-rich plasma (PRP) obtained from supernatant suspension was pooled together(5). Aggregation was determined by Born’s technique(6) using aggregometer with Adr and ADP as platelet agonists. Platelet aggregation was recorded as an increase in the percentage of light transmission and was evaluated by comparing the amplitudes of the aggregation curves. Each experiment was repeated twice.
Platelet aggregation was also detected by using microplate reader as previously described(7,8) with some modification(9). Reading of optical density (OD) at 595 nm was taken using kinetic mode of
microplate reader (Synergy HT, Biotex, USA) at 1 minute intervals for 8 minutes. The decrease in OD of PRP after being mixed with each agonist was considered to indicate platelet aggregation. Percentage of aggregation was then calculated from the following formula: Mean values for each point were plotted against time.
Percentage of aggregation = (A-B)/(A-C) x 100 A = OD PRP plus normal saline
B = OD PRP plus agonist
C = OD PFP plus normal saline