Translation-initiation signaling by plasma GFHs in human platelets. (A) Human washed platelets were resuspended in SB for the indicated times, and whole-platelet lysates were immunoblotted with antibodies as indicated. p, phosphorylated; t, total. (B) Platelets maintained in SB for 120 minutes, as in panel A, were pelleted by gentle centrifugation and resuspended in autologous platelet-poor plasma for 5 minutes, then pelleted, lysed, and subject to immunoblotting with the indicated antibodies. (C) Platelets were harvested directly from platelet-rich plasma (Fresh), or otherwise resuspended in SB for 120 minutes, then treated as in panel B for 5 minutes with platelet-poor plasma (PPP) or each indicated GFH, then pelleted for immunoblotting with the indicated antibodies. Vascular endothelial growth factor (VEGF), 100 pg/mL; insulin-like growth factor-1 (IGF-1), 200 ng/mL; insulin, 500 pM; EGF, 2 ng/mL; high mobility group box protein 1 (HMGB1), 2 ng/mL; epinephrine (Epi), 500 pM; serotonin (5-hydroxytryptamine [5-HT]), 1 nM. Fold change in phosphorylated (phospho) to total proteins from immunoblots as measured by densitometric analysis are shown to the right ± standard error of the mean, normalized to Fresh (1) and Starved (0). All were significantly increased over starved levels (P < .05) except for the following: IGF-1: p-4E-BP1; Epi: p-ERK, pAKT, p-eIF4E, p-4E-BP1; 5-HT: p-ERK, p-AKT, and p-4E-BP1; n = 5.