Evidence of increased ER stress pathways in ERp5-deficient platelets. (A) Proteomic analysis of resting platelet lysate from control (ERp5^fl/fl) and CKO (Pf4Cre⁺/Erp5^fl/fl) mice. Volcano plot for all proteins detected by tandem mass spectometry (MS/MS), with the log2 fold change (lfc) plotted on the x-axis vs the negative log10 (P) of a paired test statistic on the y-axis. Proteins in red are significantly increased (positive lfc value) or decreased (negative lfc value) in CKO compared with control mice (P < .05). Color scale represents negative log10 (P); P = .05 corresponds to a negative log10 P value of 1.3 (upper limit of grayed-out area). (B) Upregulated and downregulated protein pathways in the resting platelet lysate of CKO mice by gene ontology enrichment pathway analysis. (C) Western blot of resting platelet lysates from control (gray) and CKO (red) mice showing increased expression of PDI, ERp57, and ERp72 proteins in ERp5-deficient platelets. Top panels are representative blots. Bottom panels show the relative band density of protein, expressed as a ratio to the corresponding β-actin band, n = 4 to 7 per genotype. (D) Label-free quantification (LFQ) intensity plots for ER proteins GRP78, calreticulin, and ERp46 detected by MS/MS in the resting platelet lysates from control (gray) and CKO (red) mice, n = 4 to 5 per genotype. (E) Megakaryocyte expression of PDI and ERp57 in the bone marrow of control and CKO mice. Representative bone marrow histology sections, stained for Hoechst (nuclei, blue), GP1bβ (green), PDI, or ERp57 (red). Mean fluorescence intensity per megakaryocyte area. n = 28 to 35 megakaryocytes from 3 to 4 mice per genotype, mean ± SEM, Student t test. ∗P < .05, ∗∗P < .005, ∗∗∗P < .0005, ∗∗∗∗P < .0001.