Hemavet 950 analyzer

Mice (strain C57BL/6) were bled under anesthesia from the retro-orbital plexus, and ∼1 ml of blood was collected using heparin (20 U/ml in TBS) as the anticoagulation reagent. Blood was then centrifuged at 100 × g for 7 min to obtain platelet-rich plasma, which we termed the “crude fraction.” The platelet-rich plasma was centrifuged at 700 × g to concentrate the platelets in the top layer (termed the “purified fraction”). This procedure was repeated once to obtain the “highly purified fraction.” Eventually the platelet pellet was resuspended in 1 ml of Tyrode's buffer containing PGl₂ and apyrase, after which it underwent a centrifugation step (Fig. 1A) (“ultra-purified fraction”). For protein correlation profiling, because greater sample amounts are required, we mixed blood from different mice and then performed the extensive purification described above. For each sample 30 × 10⁶ platelets were resuspended in lysis buffer (2% SDS, 100 mm Tris, pH 7.5, 100 mm DTT), boiled at 95 °C, and further processed using the filter-aided sample preparation method (11 (link)). In brief, SDS was exchanged to urea on a 30-kDa filter. Peptides were eluted after digestion with trypsin and subjected to a StageTip-based (14 (link)) strong anion exchange fractionation (12 (link)). Platelet counts were determined using a Hemavet950 analyzer (Drew Scientific, Waterbury, CT).