Sialic acid incorporation decreases platelet activation in vitro and in vivo. (A) Platelet-rich plasma from mice was incubated with FITC-conjugated CMP-SA or FITC (Control) at a 40μM final concentration for the indicated time points. At the indicated times, platelets in plasma were activated with 25μM TRAP for 5 minutes (TRAP) or left untreated (Rest), and platelet-associated FITC fluorescence was measured by flow cytometry (n = 2). (B) Representative side and forward scatter plots of platelets isolated from mice 60 minutes after infusion of FITC-SA or FITC (Control) and activated with 25μM TRAP or left untreated (Resting; n = 3). (C) The ratio of side to forward and side scatter platelets isolated from mice injected with FITC-SA (■) and FITC (□, Control) was determined. Unstimulated platelets isolated at 0 minutes were set as 1, and platelet shapes of isolates at 30 and 60 minutes activated with 25μM TRAP were compared. Data are means ± SD (n = 3). n.s. indicates not significant. *P < .05. (D) P-selectin expression was determined after TRAP activation by flow cytometry using a PE-conjugated anti–mouse P-selectin mAb (n = 2). (E) Activation status of platelets isolated from peripheral (Peripheral) or blood shed from a tail wound (Shed blood) 60 minutes after infusion of FITC (Control) or FITC-SA as assessed by light scatter characteristics. In the left panel, platelets are identified by PE–anti-CD61 mAb binding. The FITC mean fluorescence associated with platelets is plotted in the middle panel (n = 3; **P < .01. The right panel shows the ratio of side versus forward scatter of platelets in shed blood obtained from control mice (Control) or mice injected with FITC-SA (FITC-SA; n = 3; **P < .01).