Identification of the Ca(2+) entry pathway involved in deoxygenation-induced phosphatidylserine exposure in red blood cells from patients with sickle cell disease

Phosphatidylserine (PS) exposure in red blood cells (RBCs) from sickle cell disease (SCD) patients is increased compared to levels in normal individuals and may participate in the anaemic and ischaemic complications of SCD. Exposure is increased by deoxygenation and occurs with elevation of intracellular Ca(2+) to low micromolar levels. The Ca(2+) entry step has not been defined but a role for the deoxygenation-induced pathway, P(sickle), is postulated. Partial P(sickle) inhibitors 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonic acid (SITS), 4,4′-dithiocyano-2,2′-stilbene-disulphonic acid (DIDS) and dipyridamole inhibited deoxygenation-induced PS exposure (DIDS IC(50), 118 nM). Inhibitors and activators of other pathways (including these stimulated by depolarisation, benzodiazepines, glutamate and stretch) were without effect. Zn(2+) and Gd(3+) stimulated PS exposure to high levels. In the case of Zn(2+), this effect was independent of oxygen (and hence HbS polymerisation and RBC sickling) but required extracellular Ca(2+). The effect was completely abolished when Zn(2+) (100 μM) was added to RBCs suspended in autologous plasma, implying a requirement of high levels of free Zn(2+).