Carbonylation of platelet proteins occurs as consequence of oxidative stress and thrombin activation, and is stimulated by ageing and type 2 diabetes

Abstract

Background: In normal ageing, as well as in diabetes mellitus, blood platelets are exposed to increasing amounts of reactive oxygen species. Because occurrence of carbonyl groups is the first step in oxidative damage of proteins, we questioned the formation of carbonylated proteins (i) in vitro: platelets exposed to oxidant hydrogen peroxide (H2O2) and agonist thrombin (in the presence/absence of antioxidants), and (ii) in vivo: during ageing progression, and in type 2 diabetes.

Methods: Platelets were isolated from the blood of experimental animals (rats and hamsters) and humans (healthy, young and elderly), as well as from type 2 diabetics (matures and elderly). 2,4-Dinitrophenyl derivatization of the amino acid side chains was used to quantify protein carbonyls (spectrophotometry) and to immunodetect carbonylated protein bands (Western blotting).

Results: In animal models and humans, H2O2 produced dose-dependent increases in carbonylation of platelet proteins (vs. basal condition). Thrombin activation stimulated protein carbonyl formation in a process quenched by antioxidant catalase, suggesting that carbonylation was induced by the oxidative stress generated by activated platelets. Progression of ageing caused increased carbonylation of platelet proteins (vs. young age); enriched in carbonylated proteins, platelets of elderly subjects were less sensitive to H2O2. Type 2 diabetes additionally enhanced carbonylation of human platelet proteins (vs. the levels at young and elderly healthy subjects). In all experiments, protein carbonyl concentrations were correlated with changes in intensity of 2,4-dinitrophenyl-hydrazine-reactive protein bands on immunoblots.

Conclusions: The results suggest that exogenous oxidative stress, thrombin activation, progression of ageing and type 2 diabetes lead to protein carbonyls formation in platelets, and this modification can be attenuated by antioxidant enzymes.