Role of calcium in phosphatidylserine externalisation in red blood cells from sickle cell patients

Abstract

Phosphatidylserine exposure occurs in red blood cells (RBCs) from sickle cell disease (SCD) patients and is increased by deoxygenation. The mechanisms responsible remain unclear. RBCs from SCD patients also have elevated cation permeability, and, in particular, a deoxygenation-induced cation conductance which mediates Ca(2+) entry, providing an obvious link with phosphatidylserine exposure. The role of Ca(2+) was investigated using FITC-labelled annexin. Results confirmed high phosphatidylserine exposure in RBCs from SCD patients increasing upon deoxygenation. When deoxygenated, phosphatidylserine exposure was further elevated as extracellular [Ca(2+)] was increased. This effect was inhibited by dipyridamole, intracellular Ca(2+) chelation, and Gardos channel inhibition. Phosphatidylserine exposure was reduced in high K(+) saline. Ca(2+) levels required to elicit phosphatidylserine exposure were in the low micromolar range. Findings are consistent with Ca(2+) entry through the deoxygenation-induced pathway (P(sickle)), activating the Gardos channel. [Ca(2+)] required for phosphatidylserine scrambling are in the range achievable in vivo.

Figure 1

Effect of oxygen tension and extracellular Ca²⁺ on phosphatidylserine (PS) exposure in red blood cells (RBCs) from sickle cell patients. RBCs were incubated for 18 hours at four extracellular [Ca²⁺]'s (0.5, 1.1, 2.0 and 5.0 mM) after which they were labelled with FITC-annexin (as described in Section 2). Histograms representing mean percentage of positive RBCs ± S.E.M. for 5 different patients. *P < .01 deoxy compare to oxy; ⁺ P < .05 cf 0.5 mM Ca²⁺ deoxy; ^# P < .01 cf 0.5 mM Ca²⁺ deoxy.

Figure 2

Effect of inhibitors on phosphatidylserine (PS) exposure in red blood cells (RBCs) from sickle cell patients. RBCs were incubated under deoxygenated conditions (1% O₂) for 3 hours (5 mM extracellular [Ca²⁺]) after which they were labelled with FITC-annexin. Four conditions (all with 0.5% DMSO) are shown: MAPTAM-treated RBCs (loaded with 5 μM MAPTAM prior to deoxygenation), clotrimazole (10 μM), dipyridamole (50 μM), and DIDS (50 μM). Results are presented as percentage PS exposing RBCs relative to control RBCs exposed to 0.5% DMSO only. Histograms represent means ± S.E.M. (n = 3). *P < .01 and ^# P < .0001 cf DMSO controls.

Figure 3

Effect of extracellular K⁺ on phosphatidylserine (PS) exposure in red blood cells (RBCs) from sickle cell patients. RBCs were incubated for 18 hours with extracellular [Ca²⁺] of 5 mM under oxygenated (20% O₂) or deoxygenated (1% O₂) conditions in either low K⁺-containing (extracellular [K⁺] of 5 mM) saline or high K⁺-containing (90 mM [K⁺]) saline. Histograms represent means ± S.E.M. (n = 3). *P < .001 compare to LK oxy; ^#N.S. cf. LK oxy.

Figure 4

Effect of manipulation of intracellular Ca²⁺ on phosphatidylserine (PS) exposure in red blood cells (RBCs) from sickle cell patients. RBCs were first treated with vanadate (1 mM) to inhibit the plasma membrane Ca²⁺ pump and also the aminophospholipid translocase (flippase) before addition of bromo-A23187 (1.2 μM, 1% haematocrit) and requisite extracellular [Ca²⁺]s for 30 min. They were then treated with Co²⁺ (0.4 mM) before labelling with FITC-annexin. Intracellular [Ca²⁺] is calculated from extracellular [Ca²⁺] × r ², where r ² was taken as 2.05 [36]. Results presented are from a single experiment representative of 5 others.

Figure 5

Ca²⁺ loading of red blood cells (RBCs) from sickle cell patients. RBCs were loaded with the Ca²⁺ fluorophore fluo-4 (see Methods). They were then incubated for 30 min in the absence (left—thin line) or presence (right—thick line) of bromo-A23187 at an extracellular [Ca²⁺] of 1 μM. Results are presented as histogram of fluorescence of a single experiment representative of 3.

Figure 6

Effect of K⁺ and calmodulin inhibition on Ca²⁺-induced exposure of phosphatidylserine (PS) in red blood cells (RBCs) from sickle cell patients. Experimental details were as described in the legend to Figure 4, except that in (a) where incubation was carried out in either high K⁺-(HK, K⁺ = 90 mM) or low K⁺-containing saline (LK, 4 mM), and, in (b) where HK saline was used in the absence or presence of W-7 (100 μM). Results are presented as single experiments representative of 3 others.