Vacuette sodium citrate 3.8

Venipuncture was performed with a 21-gauge needle, and after release of the tourniquet the first tube was obtained and always reserved for serological analyses. Next, for microparticle isolation blood was collected into 3 x 9 mL citrate tubes (Vacuette sodium citrate 3.8 %, Greiner Bio-One, Kremsmünster, Austria) which were gently mixed 5 times. Immediately after collection, blood cells were removed by centrifugation. The samples underwent a 2 step centrifugation procedure: 1800 g, 10 min, 21 °C, followed by a second centrifugation of the supernatant: 3000 g, 10 min, 21 °C, to obtain platelet poor plasma (PPP). The PPP was aliquoted, snap-frozen in liquid nitrogen, and stored at -80 °C until analysis [19 (link), 20 (link)].

Platelet-poor plasma (PPP) was isolated using a standardized protocol [17 (link)]. Briefly, venous blood was drawn into citrate tubes (Vacuette sodium citrate 3.8%, Greiner Bio-One, Kremsmünster, Austria). Immediately after blood collection, cells were removed by centrifugation (1800 g, 10 min, 21 °C) followed by centrifugation at 3000 g, 10 min, 21 °C to remove platelets. Samples were divided into 1000 μL aliquots, snap-frozen in liquid nitrogen, and stored at −80 °C.