Generation of Singlet Oxygen Induces Phospholipid Scrambling in Human Erythrocytes

Abstract

Maintenance of phospholipid asymmetry of the plasma membrane is essential for cells to prevent phagocytic removal or acceleration of coagulation. Photodynamic treatment (PDT), which relies on the generation of reactive oxygen species to achieve inactivation of pathogens, might be a promising approach in the future for decontamination of red blood cell concentrates. To investigate whether PDT affects phospholipid asymmetry, erythrocytes were illuminated in the presence of 1,9-dimethyl-methylene blue (DMMB) as photosensitizer and subsequently labeled with FITC-labeled annexin V. This treatment resulted in about 10% annexin V positive cells, indicating exposure of phosphatidylserine (PS). Treatment of erythrocytes with N-ethylmaleimide (NEM) prior to illumination, to inhibit inward translocation of PS via the aminophospholipid translocase, resulted in enhanced PS exposure, while treatment with H(2)O(2) (previously shown to inhibit phospholipid scrambling) greatly diminished PS exposure, indicating the induction of phospholipid scrambling by PDT. Only erythrocytes illuminated in the presence of DMMB showed translocation of NBD-phosphatidylcholine (NBD-PC), confirming scrambling induction. Double label experiments indicated that PS exposure does not occur without concurrent scrambling activity. Induction of scrambling was only moderately affected by Ca(2+) depletion of the cells. In contrast, scavengers of singlet oxygen were found to prevent phospholipid scrambling induced by PDT. The results of this study show that phospholipid scrambling is induced in human erythrocytes by exposure to singlet oxygen.

Low-Level Mercury Can Enhance Procoagulant Activity of Erythrocytes: A New Contributing Factor for Mercury-Related Thrombotic Disease

“…We measured phospholipid translocation according to the method described by Hilarius et al (2004). Briefly, erythrocytes (5 × 10 7 cells/mL) were incubated with Hg 2+ and then loaded with 0.5 μM 1-Palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phospho-l-serine (C 6 -NBD-PS; Avanti Polar Lipids, Alabaster, AL, USA) for the flippase activity assay or 1-oleoyl-2-[6-[(7-nitro-2–1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phosphocholine (C 6 -NBD-PC; Avanti Polar Lipids) for the scramblase activity assay.…”

Erythrocyte Aging: A More than Superficial Resemblance to Apoptosis?

“…In erythrocyte pathologies such as sickle cell anemia, thalassemia and spherocytosis, a decrease in erythrocyte survival is associated with an increase in phosphatidylserine exposure [13]. Treatments leading to an increased calcium influx and oxidation, that induce more or less presumed aspects of erythrocyte aging and decreased erythrocyte survival, also lead to inhibition of flipase and/or activation of scramblase, both leading to loss of phospholipid asymmetry [15]. In general, all processes that lead to increased destruction are associated with an increased exposure of phosphatidylserine at the erythrocyte surface.…”

Effect of N‐ethylmaleimide, chymotrypsin, and H2O2 on the viscoelasticity of human erythrocytes: Experimental measurement and theoretical analysis

“…As noted earlier, since the effects of each of these chemicals on the molecular structures are by no means specific or exclusive, detailed biochemical analyses (which is beyond the scope of this paper) will be required to further dissect the effects of each chemical [44,45] to tell a more complete story.…”