Platelets from patients with type 2 diabetes display hyperaggregability and increased thrombogenic potential. In platelets from patients with type 2 diabetes we found enhanced tyrosine nitration and inactivation of the sarcoplasmic endoplasmic reticulum Ca²⁺ ATPase (SERCA-2), elevated platelet [Ca²⁺]_i and the activation of the Ca²⁺ -activated protease m-calpain. The tyrosine nitration of SERCA-2 as well as the activation of m-calpain in vitro in platelets from healthy volunteers could be evoked in vitro by peroxynitrite and inhibited by 12 weeks therapy with the peroxisome proliferator-activated receptor-g (PPAR-g) agonist rosiglitazone. Thus while platelet endothelial cell adhesion molecule-1 (PECAM-1) and the sphingosine 1-phosphate 2 receptor were found to be degraded by calpain in diabetic platelets, these proteins were protected in PPARg-treated individuals. To determine the spectrum of proteins targeted by calpain in diabetic platelets we took a proteomic approach and isolated platelets from 6 diabetic subjects before and 8 weeks after therapy with pioglitazone. A total of 40 proteins were identified by differential in-gel electrophoresis followed by MS analysis in all of the subjects studied. According to function the majority of the proteins could be classified into the following groups; cytoskeletal proteins, signalling molecules, metabolism and vesicles/secretory trafficking. In order to differentiate between PPARg-regulated proteins and potential calpain targets the ionomycin-induced but calpastatin-sensitive cleavage of platelet proteins was determined. This procedure identified septin-5, integrin-linked kinase (ILK), focal adhesion kinase (FAK) and the proline rich tyrosine kinase (PYK2) as calpain substrates while the glycerol-2 phosphate dehydrogenase (a known PPARg-regulated gene) was unaffected by calpain activation. Indeed, thrombin-induced tyrosine phosphorylation was attenuated by calpain activation and over 40 altered phospho-proteins could be detected in platelets from control versus transgenic calpastatin mice that overexpress the endogenous calpain inhibitor (including ILK and ILK- linked protein kinase family protein). Fitting with the phosphorylation and degradation of the ILK platelet adhesion and clot formation were markedly altered by calpain activation. Moreover, as septin-5 constitutively inhibited a granule secretion, we found that calpain activation resulted in the enhanced release of angiogenic factors such as TGF-b from endothelial cells as well as of RANTES. These data indicate that the activation of calpain in diabetic platelets targets a spectrum of proteins involved in the regulation of focal adhesions and the secretory machinery. We propose that many of the platelet complications associated with the development of type 2 diabetes and eventually the accelerated development of cardiovascular disease may be at least partially addressed by platelet calpain inhibition.