De novo translation of αIIb protein is increased in human platelets during clinical sepsis. Platelets were isolated from septic patients or matched healthy donors. (A) Volcano plot showing significantly, differentially expressed (FDR <0.05) ribosomal protected mRNA reads (RPRs) in platelets from septic patients or healthy donors (n = 5/group). ITGA2B is labeled. (B) Magnification of IGV file demonstrating triplet periodicity for ITGA2B. Here, ribosomes read the mRNA in groups of 3 nucleotides (a triplet) at a time where each triplet codon represents an amino acid code. mRNAs undergoing active translation will have ribosome reads generally stacked on the first nucleotide of each codon across the mRNA. This is termed triplet periodicity. (C) Newly synthesized αIIb protein (arrow) in platelets from septic patients detected by radiolabel (³⁵S-methionine) and immunoprecipitation using an anti-αIIb antibody. An immunoglobulin G isotype-matched antibody was used as a control (representative of n = 3/group). (D) Confocal microscopy image of Duolink proximity ligation assay with puromycin demonstrating de novo translation of αIIb protein (green, arrows) in platelets from septic patients and healthy donors (n = 3/group). Anisomycin inhibits protein synthesis and was used here to demonstrate specificity of this system (eg, reduced staining for αIIb in the presence of anisomycin). (E) Quantification of percent positive cells for αIIb protein by CellProfiler, as described in “Methods.”