The Ca²⁺-activated protease, m-calpain, plays an important role in platelet function by cleaving a broad spectrum of proteins. We have recently demonstrated that an increased activity of m-calpain is responsible of the enhanced aggregability of platelets from patients with type 2 diabetes and that glitazone therapyprevents calpain activation. The aim of our study was to identify additional calpain substrates in platelets and their consequences on platelet signalling as well as on the interaction of platelet with other cells. To characterize new targets of m-calpain in platelets, we analyzed platelet lysates from patients with type 2 diabetes by 2 Dimensional Differential in Gel electrophoresis (2D-DIGE) followed by mass spectrometry before and after treatment with the PPARg agonist pioglitazone (8 weeks, 20 mg/kg/day). Up to 50 different proteins were altered in platelets from patients with type 2 diabetes and were reversed after pioglitazone treatment. Three proteins relevant for platelet activation were chosen for further characterization. In vitro stimulation of washed human platelets with the Ca²⁺ ionophore ionomycin in order to activate calpain led to the cleavage of the SNARE-associated protein septin-5 and the integrin-linked kinase (ILK). An effect inhibited by the calpain inhibitor calpeptin (10 mmol/L, 30 min). However, calpain failed to cleave the mitochondrial glycerol-3-phosphate dehydrogenase 2 (GPD2) suggesting that septin-5 and ILK are direct substrates of calpain while GPD2 is a PPAR-regulated protein. Further characterization of the effect of calpain on the function of the proteins identified showed that in resting platelets, septin-5 bound the SNARE protein syntaxin-4. Calpain activation led to the dissociation of septin-5 from syntaxin 4 and potentiation of the thrombin-induced platelet a granule secretion. Interestingly, the platelet releasate obtained after calpain activation was able to stimulate endothelial cell proliferation. Moreover, calpain stimulation increased RANTES secretion and its capacity to attract monocytes. On the other hand, the calpain-dependent cleavage of ILK led to its translocation onto the platelet membrane and its consequent activation. Indeed, the cleaved ILK was able to increase the phosphorylation of its substrate Akt. Functionally, ILK cleavage increased platelet adhesion and spreading. Thus, the calpain-dependent increase in a granule secretion, platelet adhesion and spreading may account for the increased vascular proliferation and inflammation as well as the enhanced thrombosis formation in diabetes.