Fig 1: Evaluation of platelet activation during the platelet separation process. The activation of platelets was evaluated by the expression of (A) CD63 and (B) CD62P on the platelet surface using flow cytometry. The expression of CD63 and CD62P was measured in whole blood immediately after collection (indicated as “WB T0”) and in platelet-rich plasma (PRP) 4 hours after platelet isolation (indicated as “PRP T4”). In addition, 4 hours after platelet separation, platelet activation was induced by incubation with E. coli (107/ml, indicated as “(PRP T4 + Ca2+) + E. coli” after re-calcification. Data are presented as mean of median fluorescence intensity (MFI) ± standard deviation (SD) (n = 9). (A, B) have the same annotation as shown in (B). In addition, platelet activation was evaluated by its soluble activation marker (C) ß-thromboglobulin (ßTG) and thrombin generation by (D) thrombin-antithrombin complex (TAT) in plasma from whole blood after blood sampling (WB + Ca2+) and whole blood after platelet-depletion [(B - Plt) + Ca2+], or after incubation with E. coli for 15 minutes, indicated as “(WB + Ca2+) + E. coli” and “(B - Plt + Ca2+) + E. coli”, respectively. Data are represented as mean ± standard deviation (SD) (n = 6). (C, D) have the same annotation as shown in (D). Platelet activation was further evaluated with scanning electron microscopy, as presented in an image representative of 6-9 samples collected (E–G). Platelets were collected in plasma-rich plasma (PRP) from whole blood anticoagulated with 3.2% (w/v) citrate (E), lepirudin (50 µg/ml) supplemented with 0.7% (w/v) citrate (F), and GPRP (8 mg/ml) supplemented with 0.7% (w/v) citrate (G). Thrombin was used as a platelet activator. The scale bar at the lower right represents 2 µm. ns, non significant.