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

Abstract

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.

Keywords: calcium; oxidants; phosphatidylserine exposure; sickle cell anaemia; thiols.

Figure 1

Effect of xanthine oxidase and hypoxanthine (XO/HO) mixtures on accumulation of reactive oxygen species (ROS) and phosphatidylserine (PS) exposure in red cells from patients with sickle cell anaemia (SCA). (A) Red cells were pre‐loaded with CM‐H₂DCF‐DA (100 μmol/l) to measure ROS levels [as median fluorescence intensity (MFI)] or treated with the same final concentration of dimethyl sulphoxide [DMSO] (control) before incubation with hypoxanthine (HO, 2 mmol/l) and xanthine oxidase (XO, 0–0·1 U/ml) mixtures for 30 min at 37°C (n = 2). (B) Red cells were permeabilised to Ca²⁺ with the ionophore bromo‐A23187 (6 μmol/l), and incubated in different free [Ca²⁺]_os, maintained using Ca²⁺ / 2 mmol/l EGTA mixtures, at 0·5% haematocrit for 30 min at 37°C in the absence (filled circles) or presence (open circles) of HO (2 mmol/l)/XO (0·015 U/ml) mixtures after which externalised PS was labelled with LA‐FITC, n = 8. PS exposure was normalised to that of control red cells at 1 μmol/l free [Ca²⁺]_o (31·1 ± 3·9% of total red cells). Symbols represent means ± SEM for red cells from n different individuals. *P < 0·05; **P < 0·005.

Figure 2

Prothrombinase activity in intact (A and B) or hypotonically lysed (C) red cells from patients with sickle cell anaemia. Red cells were treated as in Fig 1B with thrombin formation used as a measure of accessible phosphatidylserine (PS). (A) Thrombin formation in the absence (filled circles) or presence (open circles) of HO (2 mmol/l)/XO (0·015 U/ml) mixtures over a range of free extra cellular [Ca²⁺] ([Ca²⁺]_o; 0·1–10 μmol/l), with thrombin formation per min normalised to that of control red cells at 1 μmol/l free [Ca²⁺]_o. (B) Thrombin formation in the absence (control) or presence of HO (2 mmol/l)/XO (0·015 U/ml) mixtures or PMS (0·1 mmol/l), given as a percentage relative to the total value in lysed red cells at two different free [Ca²⁺]_os (0·1 and 1 μmol/l), indicative of prothrombin activity due to externalised PS present on only the outer bilayer of the red cell membrane. (C) Total thrombin formation at 0·1 and 1 μmol/l free [Ca²⁺]_o in the absence and presence of HO (2 mmol/l)/XO (0·015 U/ml) mixtures or PMS (0·1 mmol/l) as measured by thrombin formation (in arbitrary units, AU) of hypotonically lysed red cells to give prothrombin activity of total PS present on both the inner and outer bilayers of the red cell membrane. Symbols and histograms represent means ± SEM for red cells from 4 to 5 different individuals. *P < 0·05; **P < 0·005.

Figure 3

Effect of tert‐butyl hydroperoxide (tBHP) on accumulation of reactive oxygen species (ROS), phosphatidylserine (PS) exposure and membrane integrity in red cells from patients with sickle cell anaemia. (A) Red cells were pre‐loaded with CM‐H₂DCF‐DA (100 μmol/l) to measure ROS levels or treated with the same final concentration of dimethyl sulphoxide [DMSO] (control) before incubation with tBHP (0–1 mmol/l) at 0·5% haematocrit for 30 min at 37°C in HK‐HBS (n = 4). (B) Red cells were double labelled with CM‐H₂DCF to measure ROS levels (left ordinate) and with Alexa Fluor 647 anti‐Hb α chain (labelled anti‐Hb; right ordinate) to correlate ROS levels with labelling of intracellular haemoglobin (n = 3). (C) Red cells were double labelled with LA‐FITC to measure accessible PS and (left ordinate) and with Alexa Fluor 647 anti‐Hb α chain (labelled anti‐Hb; right ordinate) to correlate PS labelling with that of intracellular haemoglobin (n = 3). (D) Red cells were permeabilised to Ca²⁺ as in Fig 1B at 0·5% haematocrit for 30 min at 37°C in the absence (filled circles) or presence (open circles) of tBHP (0·1 mmol/l) after which externalised PS was labelled with LA‐FITC. PS exposure was normalised to that of control red cells at 1 μmol/l free [Ca²⁺]_o (29·3 ± 3·4% of total red cells, n = 7). Symbols represent means ± SEM for red cells from n different individuals. *P < 0·05; **P < 0·005.

Figure 4

Effect of thiol modification on phosphatidylserine (PS) exposure of red cells from sickle cell anaemia patients. Red cells were pre‐incubated at 4% haematocrit in the absence (solid circles) or presence (open circles) of N‐ethylmaleimide (NEM) and dithiothreitol (DTT), then permeabilised to Ca²⁺ as in Fig 1B for 30 min at 37°C, after which accessible PS was labelled with LA‐FITC. (A) Effect of NEM (1 mmol/l) on PS exposure (n = 8); (B) Effect of DTT (0·25 mmol/l) on PS exposure (n = 10). PS exposure was normalised to that of control red cells (in the absence of thiol modifiers) at 1 μmol/l free [Ca²⁺]_o (NEM control: 21·2 ± 4·7%, DTT control: 24·3 ± 4·6% of total red cells). Symbols represent means ± SEM for red cells from n different individuals. *P < 0·05; **P < 0·005.

Figure 5

Effect of hypochlorous acid (HOCl) on phosphatidylserine (PS) exposure of red cells from sickle cell anaemia patients. (A) Red cells were pre‐loaded with CM‐H₂DCF‐DA (100 μmol/l) or treated with the same final [DMSO] before incubation with HOCl (500 μmol/l) at 0·5% haematocrit for 30 min at 37°C in HK‐HBS (n = 3). (B) Red cells were permeabilised to Ca²⁺ as in Fig 1B at 0·5% haematocrit for 30 min at 37°C in the absence (full circles) or presence (open circles) of HOCl (500 μmol/l) after which accessible PS was labelled with LA‐FITC (n = 6). PS exposure was normalised to that of control red cells at 1 μmol/l free [Ca²⁺]_o (32 ± 5·4% of total red cells). Symbols represent means ± SEM for red cells from n different individuals.

Figure 6

Schematic digram of stimuli affecting phosphatidylserine (PS) distribution in red cells from patients with sickle cell anaemia. (A) (i) PS is usually confined to the inner leaflet of the lipid bilayer of red cells including sickle cells through high activity of the flippase and low activity of the scramblase, as externalisation is prothrombotic and increases phagocytosis; (ii) elevation of intracellular Ca²⁺ ([Ca²⁺]_i) via the deoxygenation‐induced cation conductance (or P_sickle) or via ionophore promotes PS exposure increasing the possibility of microvascular occlusion; (iii) the effect of oxidants either from within the sickle cell or from the circulation is uncertain. (B) (i) As before, entry of Ca²⁺ increases PS exposure; (ii) most oxidants (xanthine oxidase/hypoxanthine mixtures, nitrite, phenazine methosulphate) actually reduced Ca²⁺‐induced PS exposure by about 50% and would reduce thrombosis; (iii) the exception was thiol oxidation which markedly increased externalisation of PS. ●, known PS distribution; , PS distribution unknown.

Figure 7

Schematic diagram of the effect of tert‐butyl hydroperoxide (tBHP) on red cells from patients with sickle cell anaemia. (i) tBHP is thought to increase phosphatidylserine (PS) exposure but relatively high concentrations have been used, which may disrupt the integrity of the membrane; (ii) in the presence of high tBHP more PS was labelled, but the ability of anti‐Hb IgG to label intracellular Hb suggests that this was because loss of membrane integrity allowed labelling of both inner leaflet PS as well as externalised PS; (iii) by contrast, with lower tBHP concentrations that do not allow entry of anti‐Hb IgG, PS exposure was reduced by 50%, as for the other oxidants (in Fig 1b). ●, known PS distribution; , PS distribution unknown.