Erythrocytes, in the days preceding their destruction, experience a marked oxidative burden exerted by the denaturation of hemoglobin and production of ROS. They react to oxidative stress activating a complex phosphorylation response. To gain insight on its physiological significance, we used three different models of oxidative stress "in vitro". 1) Treatment with diamide, a relatively specific sulfhydryl groups reagent, that exerts a reversible oxidative effect in erythrocytes; 2) treatment with hydrogen peroxide (H2O2) that is physiologically produced in erythrocytes and is rapidly metabolized by catalase to water and oxygen; 3) treatment with phenylhydrazine (Phz) that mimics the oxidative damage exerted by denatured hemoglobin.

We observed that diamide caused an increased tyrosine phosphorylation in a concentration dependent way and band 3 appears to be the major protein subjected to tyrosine phosphorylation. We also observed that Phz does not exert a direct oxidant effect on membrane proteins but specifically oxidizes and denatures hemoglobin that forms hemichromes inducing an indirect oxidative stress mediated by ROS, while H2O2 exerted a measurable oxidative action on erythrocytes basically at very high and at non-physiological concentrations. We also investigated the involvement of PKA and PKC using Src kinase inhibitors. The enhancement of protein tyrosine phosphorylation is likely due to Syk kinase while the protein serine phosphorylation response was due to PKC and may be the result of calcium leak through partially destabilized membranes. The present findings constitute a valuable background revealing new insights on a new pathway finalized to regulate the lifespan of circulating erythrocytes.