Characterization of new calpain substrates in human and murine platelets. (A) Representative differential in-gel electrophoresis of platelet proteins from the same diabetic patient treated with placebo or pioglitazone (red spots indicate proteins down-regulated by pioglitazone; green spots, proteins that were up-regulated). (B) Representative blots showing the effect of Ca²⁺ (5mM) and ionomycin (Io; 1μM)–induced μ- and m-calpain activation on the levels of septin-5, ILK, and GPD2 in washed human platelets. (C) Levels of septin-5 and ILK in washed platelets from healthy donors (CTL) and patients with type 2 diabetes treated with placebo or pioglitazone (PPAR) (data shown from same patients). (D) Effect of μ- and m-calpain activation on septin-5 and ILK in platelets from μ-cal^fl/lfl and PF4-μ-cal^−/− mice. Experiments were performed in the absence or in the presence of calpeptin (Cpt, 10μM). Arrow indicates the product after μ-calpain activation; arrowhead, product after m-calpain activation. Blots are representative of 5-6 additional experiments and graphs summarize data obtained in 8 subjects/animals per group. *P < .05; **P < .001; ***P < .001 versus healthy donors or untreated platelets; ^###P < .001 versus placebo.