Quantitative Assessment of Platelet Aggregation

The aggregability of platelets can be assessed by quantitative determination of ATP exocytosis [21 (link)]. WB samples containing low, physiological or high levels of albumin were centrifuged at 150 g for 12 min in order to obtain platelet rich plasma (PRP) samples. Platelet aggregation was induced by addition of collagen (2 μg mL^-1, final concentration) to 500 μL of the PRP samples. After two minutes, the PRP samples were centrifuged at 1,500 g for two minutes and proteins in the supernatant were precipitated with 0.4 M perchloric acid. After centrifugation at 12,000 g 100 μL of the supernatant were neutralized by addition of 10–12 μL of 2 M K₂CO₃ at 4°C. The supernatant obtained after centrifugation was used for HPLC analysis (injection volume: 40 μL). Separation of adenine nucleotides was performed on a Hypersil ODS column (5 μm, 250 × 4 mm I.D., equipped with a precolumn; Thermo Electron Corp. Runcorn, Cheshire, UK) using a L-2200 autosampler, two L-2130 HTA pumps, and a L-2450 diode array detector (all VWR International, West Chester, PA, USA) as previously described [22 (link)]. Detector signals (absorbance at 254 nm) were recorded and the program EZchrom Elite (VWR) was used for data acquisition and analysis.