Phosphatidylserine Exposure in Human Red Blood Cells Depending on Cell Age

Background: Eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phosphatidylserine-translocation, is triggered by fever and inflammation. Signaling includes increased cytosolic Ca²⁺-activity ([Ca²⁺]_i), caspase activation, and ceramide. Inflammation is associated with increased plasma concentration of C-reactive protein (CRP). The present study explored whether CRP triggers eryptosis. Methods: Phosphatidylserine abundance at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, ceramide abundance and caspase-3-activity utilizing FITC-conjugated antibodies. Moreover, blood was drawn from patients with acute appendicitis (9♀,11♂) and healthy volunteers (10♀,10♂) for determination of CRP, blood count and phosphatidylserine. Results: A 48h CRP treatment significantly increased the percentage of annexin-V-binding cells (≥5µg/ml), [Ca²⁺]_i (≥5µg/ml), ceramide (20µg/ml) and caspase-activity (20µg/ml). Annexin-V-binding was significantly blunted by caspase inhibitor zVAD (10µM). The percentage of phosphatidylserine-exposing erythrocytes in freshly drawn blood was significantly higher in appendicitis patients (1.83±0.21%) than healthy volunteers (0.81±0.09%), and significantly higher following a 24h incubation of erythrocytes from healthy volunteers to patient plasma than to plasma from healthy volunteers. The percentage of phosphatidylserine-exposing erythrocytes correlated with CRP plasma concentration. Conclusion: C-reactive protein triggers eryptosis, an effect at least partially due to increase of [Ca²⁺]_i, increase of ceramide abundance and caspase activation.

“…Moreover, eryptosis is enhanced in elderly individuals [64]. It is sensitive to erythrocyte age [65] and enhanced eryptosis is observed following erythrocyte storage [66, 67]. …”

Section: Introductionmentioning

confidence: 99%

Stimulation of Erythrocyte Cell Membrane Scrambling by C-Reactive Protein

Abed

Thiel

Towhid

et al. 2017

“…The increase of the intracellular Ca 2+ content as well as the external presentation of PS after stimulation of RBCs by LPA or PMA has been investigated by many research groups [3,7,8,[15][16][17][18][19][20][21][22][23]. These investigations were mainly carried out to investigate physiological parameters affecting the PS exposure.…”

Section: Introductionmentioning

confidence: 99%

Measurements of Intracellular Ca2+ Content and Phosphatidylserine Exposure in Human Red Blood Cells: Methodological Issues

Wesseling

Wagner-Britz²,

Boukhdoud³

et al. 2016

Self Cite

Background/Aims: The increase of the intracellular Ca²⁺ content as well as the exposure of phosphatidylserine (PS) on the outer cell membrane surface after activation of red blood cells (RBCs) by lysophosphatidic acid (LPA) has been investigated by a variety of research groups. Carrying out experiments, which we described in several previous publications, we observed some discrepancies when comparing data obtained by different investigators within our research group and also between batches of LPA. In addition, we found differences comparing the results of double and single labelling experiments (for Ca²⁺ and PS). Furthermore, the results of PS exposure depended on the fluorescent dye used (annexin V-FITC versus annexin V alexa fluor® 647). Therefore, it seems necessary to investigate these methodological approaches in more detail to be able to quantify results and to compare data obtained by different research groups. Methods: The intracellular Ca²⁺ content and the PS exposure of RBCs separated from whole blood have been investigated after treatment with LPA (2.5 µM) obtained from three different companies (Sigma-Aldrich, Cayman Chemical Company, and Santa Cruz Biotechnology Inc.). Fluo-4 and x-rhod-1 have been used to detect intracellular Ca²⁺ content, annexin V alexa fluor® 647 and annexin V-FITC have been used for PS exposure measurements. Both parameters (Ca²⁺ content, PS exposure) were studied using flow cytometry and fluorescence microscopy. Results: The percentage of RBCs showing increased intracellular Ca²⁺ content as well as PS exposure changes significantly between different LPA manufacturers as well as on the condition of mixing of LPA with the RBC suspension. Furthermore, the percentage of RBCs showing PS exposure is reduced in double labelling compared to single labelling experiments and depends also on the fluorescent dye used. Finally, data on Ca²⁺ content are slightly affected whereas PS exposure data are not affected significantly by the measuring method (flow cytometry, fluorescence microscopy). Conclusion: The LPA batch used and the mixing procedure of LPA and the RBC suspension has to be taken into consideration when comparing results of intracellular Ca²⁺ content and PS exposure of RBCs after LPA activation. In addition, one should consider that the results of single and double labelling experiments might be different depending on the fluorescent dyes used.

“…The preparation of RBCs for microscopy experiments were the same as for flow cytometry measurements. Erythrocytes were monitored using an inverted fluorescence microscope (Eclipse TE2000-E, Nikon, Tokyo, Japan) as described before [45, 46]. The diluted erythrocyte samples (approximately 0.025% haematocrit) were placed on a cover slip in a dark room at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Trifluoperazine-Induced Suicidal Erythrocyte Death and S-Nitrosylation Inhibition, Reversed by the Nitric Oxide Donor Sodium Nitroprusside

Ghashghaeinia

Wesseling

Ramos

et al. 2017

Self Cite

Background and Purpose: The high potency antipsychotic drug trifluoperazine (10-[3-(4-methyl-1-piperazinyl)-propyl]-2-(trifluoromethyl)-(10)H-phenothiazine dihydrochloride; TFP) may either counteract or promote suicidal cell death or apoptosis. Similar to apoptosis, erythrocytes may enter eryptosis, characterized by phosphatidylserine exposure at the cell surface and cell shrinkage. Eryptosis can be stimulated by an increase in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and inhibited by nitric oxide (NO). We explored whether TFP treatment of erythrocytes induces phosphatidylserine exposure, cell shrinkage, and calcium influx, whether it impairs S-nitrosylation and whether these effects are inhibited by NO. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, and protein nitrosylation from fluorescence switch of the Bodipy-TMR/Sypro Ruby signal. Results: Exposure of human erythrocytes to TFP significantly enhanced the percentage of annexin-V-binding cells, raised [Ca²⁺]_i, and decreased S-nitrosylation. The effect of TFP on annexin-V-binding was not affected by removal of extracellular Ca²⁺ alone, but was significantly inhibited by pre-treatment with sodium nitroprusside (SNP), an effect significantly augmented by additional removal of extracellular Ca²⁺. A 3 hours treatment with 0.1 µM Ca²⁺ ionophore ionomycin triggered annexin-V-binding and cell shrinkage, effects fully reversed by removal of extracellular Ca²⁺. Conclusions: TFP induces eryptosis and decreases protein S-nitrosylation, effects blunted by nitroprusside. The effect of nitroprusside is attenuated in the presence of extracellular Ca²⁺.