Oxidative stress and phosphatidylserine exposure in red cells from patients with sickle cell anaemia

Phosphatidylserine (PS) exposure increases as red cells age, and is an important signal for the removal of senescent cells from the circulation. PS exposure is elevated in red cells from sickle cell anaemia (SCA) patients and is thought to enhance haemolysis and vaso-occlusion. Although precise conditions leading to its externalisation are unclear, high intracellular Ca²⁺ has been implicated. Red cells from SCA patients are also exposed to an increased oxidative challenge, and we postulated that this stimulates PS exposure, through increased Ca²⁺ levels. We tested four different ways of generating oxidative stress: hypoxanthine and xanthine oxidase, phenazine methosulphate, nitrite and tert-butyl hydroperoxide, together with thiol modification with N-ethylmaleimide (NEM), dithiothreitol and hypochlorous acid (HOCl), in red cells permeabilised to Ca²⁺ using bromo-A23187. Unexpectedly, our findings showed that the four oxidants significantly reduced Ca²⁺ -induced PS exposure (by 40-60%) with no appreciable effect on Ca²⁺ affinity. By contrast, NEM markedly increased PS exposure (by about 400%) and slightly but significantly increased the affinity for Ca²⁺ . Dithiothreitol modestly reduced PS exposure (by 25%) and HOCl had no effect. These findings emphasise the importance of thiol modification for PS exposure in sickle cells but suggest that increased oxidant stress alone is not important.