The Ca²⁺-activated protease, calpain, plays an important role in platelet function by cleaving a broad spectrum of proteins. However, we have shown that an increased activity of calpain is responsible of the enhanced aggregability of platelets from patients with type 2 diabetes and that PPAR-γ agonist therapy prevents 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.

2 Dimensional Differential in Gel electrophoresis (2D-DIGE) followed by mass spectrometry analysis of platelet lysates from patients with type 2 diabetes before and after treatment with the PPAR-γ agonist pioglitazone (8 weeks, 20 mg/kg/day) identified up to 50 different proteins altered. The SNARE-associated protein septin-5, the integrin-linked kinase (ILK) and the mitochondrial glycerol-3-phosphate dehydrogenase 2 (GPD2) were chosen for further characterization. Septin-5 and ILK were confirmed as direct calpain substrates in vitro while GPD2 which is a PPAR-regulated protein was not. 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 the potentiation of the thrombin-induced platelet α granule secretion. Interestingly, the platelet releasate obtained after calpain activation was able to stimulate endothelial cell proliferation. Moreover calpain stimulation potentiated the thrombin-induced increase in RANTES secretion and its subsequent cleavage to a variant possessing an enhanced chemotactic activity. The calpain-dependent cleavage of ILK led to its translocation onto the plasma membrane and its consequent activation. Indeed, the cleaved ILK was able to increase the phosphorylation of its substrate Akt, platelet adhesion and spreading. To further investigate the relevance of calpain activation in platelet function in vivo, diabetes was induced in mice. Diabetes induction resulted in the activation of calpain in murine platelets and a reduction in the levels of several calpain substrates including ILK and septin-5. Treatment of the animals with the calpain inhibitor A-705253 inhibited protein cleavage. Moreover, platelets from diabetic mice demonstrated an increased aggregation and thrombus formation in vivo which were restored by A-705253 treatment.

In conclusion, the increased calpain-dependent α granule secretion, platelet adhesion and spreading may account for the enhanced vascular proliferation and thrombosis formation in diabetes and calpain inhibition represent a promising anti-thrombotic therapy in patients with diabetes.