Background While G6PD deficiency is one of the major causes of acute hemolytic anemia, the membrane changes leading to red cell lysis have not been extensively studied. lysis and vesiculation rates. In control RBCs we observed only transient AE1 phosphorylation. Conclusions/Significance Collectively, our findings indicate that prolonged tyrosine phosphorylation produces extensive membrane destabilization leading to the loss of vesicles which contain hemichromes. The proposed mechanism of hemolysis may be applied to other hemolytic diseases characterized by the accumulation of hemoglobin denaturation products. Introduction G6PD TP53 deficiency affects more than 400 million people worldwide, with a prevalence varying from 10 to 25% in most areas where malaria is usually endemic. This genetic defect provides partial protection against malaria, but may lead to severe hemolytic episodes after the administration of some drugs (anti-malarials, anti-inflammatories, vitamin K, etc.), the ingestion of fava beans (favism) or contamination [1]C[3]. Typically the appearance of the first symptoms occurs 24C48 hours after the intake of pro-oxidant drugs or fava beans. While the molecular biology of G6PD deficiency has been extensively studied [2], the molecular mechanisms leading to the hemolytic problems are still unclear. G6PD deficient red cells (G? RBCs) display a failure of the protective response to oxidant stress, which leads to irreversible oxidation of glutathione [1], [2], [4]C[6]. The accumulation of large hemichrome aggregates (Heinz bodies) is usually an additional hallmark of the hemolytic problems in G? individuals [7]. Some membrane alterations have been described in G? RBCs, such as the oxidation and clustering of membrane proteins, the binding of hemichromes to the internal face of the membrane, the destabilization of the membrane and the release of micro-vesicles [8]C[10]. Oddly enough, increased hemichrome formation has been observed in G? RBCs infected by malaria parasites [11]. The data available on membrane modifications are in any case insufficient to formulate a clear hypothesis Imipenem IC50 as to the mechanisms of membrane destabilization and G? RBC destruction. The dearth of information concerning the mechanisms of red cell lysis represents a practical drawback which impedes both any prediction about the hemolytic activity of drugs and the understanding of the large individual susceptibility even in presence of the same G6PD mutation [1]. The authors, as well as others have shown that band 3 red cell membrane protein (AE1) displays a noticeable tendency to become tyrosine phosphorylated in G- RBCs after CSH group oxidation or GSH depletion by 1-chloro-2,4-dinitrobenzene (CDNB) or diamide [12], [13]. We have also exhibited that Syk tyrosine kinase strongly increases its affinity to oxidized AE1 and induces its selective phosphorylation [13]. Hyper-phosphorylated AE1 showed a manifest tendency to cluster, indicating a change in its interactions with the cytoskeletal network. Furthermore, abnormal AE1 tyrosine phosphorylation has been observed in a number of red cell disorders [14]. In the present study we have exhibited that following CSH group oxidation induced by diamide (CSH group oxidant) and divicine, an oxygen reactive compound held responsible for favism [15], AE1 becomes increasingly and irreversibly phosphorylated in G? RBCs. Syk kinase inhibition largely prevents red cell membrane lysis and vesiculation, strongly suggesting a functional role of AE1 tyrosine phosphorylation in the red cell membrane destabilization. Results Short and long term effects of oxidants in G6PD deficient red cells Previous work has described how oxidant treatments induce more intense AE1 tyrosine phosphorylation in G? RBCs than in control RBCs [13], [14], [16]. In the present study, we analyzed AE1 phosphorylation and a series of additional parameters for longer time exposure Imipenem IC50 with diamide, an -SH group oxidant reagent, or with divicine [15], a Imipenem IC50 compound extracted from fava beans considered responsible for severe hemolytic crises in G? deficient subjects [5], [15]. The long term effects of oxidants in the G? RBCs were not easily predictable as, although the G? RBC samples used in our experiments had low G6PD levels (Mediterranean variant 563 C > T with.