Proceedings of The Physiological Society

University College Dublin (2009) Proc Physiol Soc 15, PC179

Poster Communications

Ca2+ and phosphatidylserine exposure in red blood cells from patients with sickle cell disease

E. Weiss1, D. Rees2, J. S. Gibson1

1. Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom. 2. Molecular Haematology, King's College London School of Medicine, London, United Kingdom.


Patients with sickle cell disease (SCD) have HbS (rather than normal HbA) in their red blood cells (RBCs). How HbS results in the symptoms of SCD remains incompletely understood. Occlusion of small blood vessels is implicated and involves exposure of phosphatidylserine (PS) on the outer leaflet of the RBC membrane (Kuypers, 2008). This phospholipid is normally confined to the inner leaflet through the action of an aminophospholipid translocase. On exposure, it makes RBCs stickier. We have investigated the extent to which calcium entry through the deoxygenation-induced pathway, Psickle (Lew and Bookchin, 2005), contributes to PS exposure. Blood samples were obtained with ethical approval from consenting volunteers homozygous for HbSS. RBCs were washed into low (LK) or high potassium (HK) saline, comprising (in mM) NaCl 140, KCl 4, glucose 5, HEPES 10 for LK saline, and NaCl 55, KCl 90, glucose 5 and HEPES 10 for HK saline, all pH 7.4 at 37oC. They were incubated at different extracellular [calcium]s for up to 18h. RBCs were subsequently treated with vanadate (1mM), harvested, and labelled with FITC-annexin (Becton Dickinson, BD). Percentage of RBCs with PS exposed on their external membrane was then measured by flow cytometry. Data are given as means±SEM for blood samples from n patients. After 18h, the percentage of RBCs expressing PS when incubated under oxygenated LK conditions was 5.3±1.4, 5.7±1.0, 5.7±1.1 and 6.2±0.9 (n = 4) at 0.5, 1.1, 2 and 5mM [calcium], respectively. At the same [calcium]s, when deoxygenated, values increased to 11.1±2.3, 11.1±1.4, 13.3±0.9 and 18.2±1.7. When deoxygenated at 5mM Ca2+ in LK saline, 10.3±4.4% (n = 3) RBCs exposed PS, falling to 5.4±1.7% in HK saline (compared with 5.3±0.8% in oxygenated conditions). The deoxygenation-induced increase in PS exposure was abolished by loading cells with the calcium chelator, MAPT-AM. In deoxygenated LK saline with 5mM calcium, the number of RBCs with PS exposure fell by about 15% when incubated with either dipyridamole (100μM) or clotrimazole (10μM). These results agree with previous findings that PS exposure increases upon deoxygenation. They also show that the effect is dependent on extracellular calcium and is prevented by its chelation intracellularly. PS exposure is largely prevented by removing the gradient for loss of potassium efflux by suspension in HK saline. There is modest inhibition by dipyridamole (a partial Psickle inhibitor) and clotrimazole (which inhibits the Gardos channel). We conclude that PS exposure is induced by calcium entry following deoxygenation-induced Psickle activation, and that intracellular calcium has its main effect via cell shrinkage due to solute loss following activation of the Gardos channel.

Where applicable, experiments conform with Society ethical requirements