Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC192

Poster Communications

Deoxygenation-induced phosphatidylserine exposure in human red blood cells from sickle cell patients

U. M. Cytlak1, D. C. Rees2, J. S. Gibson1

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


  • Figure 1. Normalised PS exposure in RBCs from HbSS SCD patients after 60min of deoxygenation in presence of channel inhibitors or calcium chelator (100μM), * p<0.02, + not significant (Student's t test).

The aminophospholipid phosphatidylserine (PS) is usually confined to the inner leaflet of red blood cells (RBCs). This is important because externalisation can result in thrombus formation and removal of RBCs. Externalised PS occurs in a small, but variable, percentage of RBCs from sickle cell disease (SCD) patients and may participate in the chronic anaemia and acute ischaemic complications, characteristic of SCD (1). Exposure increases on deoxygenation. Whilst this appears to be stimulated by elevated extracellular Ca2+, the mechanism remains unclear (2). It is hypothesised that the deoxygenation-induced cation channel (Psickle) of SCD patient RBCs is involved. Routine discarded blood samples were obtained from HbSS and HbSC SCD patients, using EDTA as anticoagulant. RBCs were washed and incubated in saline comprising (in mM): 4 KCl, 145 NaCl, 10 HEPES, 10 inosine, 0.15 MgCl2 with 1.1 CaCl2 unless otherwise indicated. Suspensions of RBCs (5% haematocrit, Hct) were deoxygenated (60min or less) using Eschweiler tonometers before diluting into test tubes pre-equilibrated with N2 (final Hct 0.5%) in the absence or presence of various potential inhibitors of PS exposure (10, 50 and 100μM). In some experiments, deoxygenated RBCs were preloaded with the Ca2+ chelator MAPTAM, before adding to test tubes. PS exposure was assessed by incubation with FITC-lactadherin (16nM, 105 RBCs, Haematologic Technologies Inc.) in the presence of vanadate (1mM) and analysed by flow cytometry (3). Deoxygenated RBCs with 1.1mM Ca2+o showed PS exposure of 3.8±0.3% (means±SEM, n=17), rising to 5.2±0.4 and 7.2±0.6% after 30 and 60min, respectively. In the absence of Ca2+o, PS exposure was reduced by 65.2±24.3% (n=10, p<0.02) after 60min of deoxygenation. Pre-incubation with MAPTAM inhibited PS exposure at 60min by 58.4±27.9%, n=4, p<0.02 (Figure 1). Partial inhibitors of Psickle DIDS, SITS and dipyridamole showed inhibition by 86.5±28.6, 91.9±25.4 and 50.4±25.0% (n=5, p<0.02), respectively. Inhibitors of the non-selective cation channel, EIPA, or of the peripheral benzodiazepine receptors, PK11195, had no effect. Data are shown for HbSS patients, but RBCs from HbSC genotype behaved similarly. Glutamate receptor agonists NMDA and homocysteine (HC) had little effect on PS exposure after 60min incubation in oxygenated RBCs being 2.5±0.6% (n=2) in controls and 2.3±0.2 and 2.6±0.3% with NMDA and HC (100µM), respectively. Findings show that Ca2+ was required for deoxygenation-induced PS exposure in sickle cells. The sickling shape change alone was insufficient. Intracellular Ca2+ chelation reduced the effect. Inhibitor data are consistent with Ca2+ entry via the Psickle pathway with no evidence for involvement of other putative RBC cation channels. In future work, the link between Ca2+i and PS exposure will be pursued.

Where applicable, experiments conform with Society ethical requirements