Carbon Monoxide–Sensitive Apoptotic Death of Erythrocytes

Carbon Monoxide–Sensitive Apoptotic Death of ErythrocytesElisabeth Lang1,†, Syed M. Qadri1,†, Kashif Jilani1, Christine Zelenak1, Adrian Lupescu1, Erwin Schleicher2 and Florian Lang1*1Department of Physiology, University of Tübingen, Tübingen, Germany and 2Department of Internal Medicine, University of Tübingen,

Tübingen, Germany

(Received 19 April 2012; Accepted 19 June 2012)

Abstract: Carbon monoxide (CO) intoxication severely interferes with the oxygen-transporting function of haemoglobin. Beyondthat, CO participates in the regulation of apoptosis. CO could be generated from CO-releasing molecules (CORM), such as thetricarbonyl-dichlororuthenium (II) dimer (CORM-2), which is presently considered for the treatment of vascular dysfunction,inflammation, tissue ischaemia and organ rejection. CORM-2 is at least partially effective by modifying gene expression andmitochondrial potential. Erythrocytes lack nuclei and mitochondria but may undergo suicidal cell death or eryptosis, characterizedby cell shrinkage and phospholipid scrambling of the cell membrane. Eryptosis is triggered by the increase in cytosolic Ca2+

activity ([Ca2+]i). The present study explored whether CORM-2 influences eryptosis. To this end, [Ca2+]i was estimated fromFluo-3-fluorescence, cell volume from forward scatter, phospholipid scrambling from annexin-V-binding and haemolysis fromhaemoglobin release. CO-binding haemoglobin (COHb) was estimated utilizing a blood gas analyser. As a result, exposure oferythrocytes for 24 hr to CORM-2 (� 5 lM) significantly increased COHb, [Ca2+]i, forward scatter, annexin-V-binding andhaemolysis. Annexin-V-binding was significantly blunted by 100% oxygen and was virtually abolished in the nominal absenceof Ca2+. In conclusion, CORM-2 stimulates cell membrane scrambling of erythrocytes, an effect largely due to Ca2+ entry andpartially reversed by O2.

Carbon monoxide (CO) intoxication is a common cause ofmorbidity and mortality affecting approximately 50,000 indi-viduals annually in the United States [1, 2]. Frequent causesinclude car exhaust, malfunctioning heating systems andinhaled smoke [1, 3]. CO tightly binds to haemoglobin withan affinity 210-fold of that for oxygen [4]. In heavy smokers,the percentage of CO-binding haemoglobin (COHb) mayexceed 20% [1]. The negative effect of CO poisoning onoxygen transport is compounded by an effect of CO on the O2

equilibrium curve with the impairment of O2 binding at highand O2 release at low O2 partial pressure [5]. Effects of COcould partially be prevented by hyperbaric O2 [6].CO is generated in mammalian cells by haem oxygenase [7]

and participates in the regulation of diverse cellular functions[8–22], including apoptosis [23–34].CO could be generated from CO-releasing molecules

(CORM), such as tricarbonyl-dichlororuthenium (II) dimer(CORM-2) [12]. CORM are presently considered for the treat-ment of vascular dysfunction, inflammation, tissue ischaemiaand organ rejection [12]. CORM-2 counteracts apoptosis bystimulating Bcl-2 expression and blunting caspase-3 activation[28].Similar to apoptosis of nucleated cells, suicidal death of

erythrocytes or eryptosis involves cell shrinkage and cellmembrane scrambling with phosphatidylserine exposure at thecell surface [35]. Eryptosis is triggered by Ca2+ entry after theactivation of Ca2+-permeable cation channels [36–44]. Ca2+

activates Ca2+-sensitive K+ channels [45, 46], resulting in K+

exit, hyperpolarization and subsequent Cl� exit followed bycellular water loss and thus cell shrinkage [47]. Ca2+ furtherstimulates phospholipid scrambling of the cell membrane withphosphatidylserine exposure at the cell surface [44, 48–50].Ca2+ sensitivity of erythrocyte membrane scrambling is increasedby ceramide [51, 52], which is generated by a sphingomyelin-ase, an enzyme stimulated by platelet-activating factor [53].Erythrocyte cell membrane scrambling could further resultfrom ATP depletion [54] and activation of caspases [35].Eryptosis may be modulated by kinases such as p38 MAPK[55], CK1 [56] and JAK3 [57].The present study explored whether CORM-2 influences eryp-

tosis. To this end, human erythrocytes were exposed to CORM-2and CO-binding haemoglobin, cytosolic Ca2+ concentration, cellvolume, phosphatidylserine exposure and haemolysis determined.

Materials and Methods

Erythrocytes, solutions and chemicals. Leucocyte-depletederythrocytes (aged 1–2 weeks) were kindly provided by the bloodbank of the University of Tübingen. Viability of erythrocytes maydepend on the donor and the storage time, thus causing someinterindividual variability. To avoid any bias potentially introduced bythe use of different erythrocyte batches, comparison was always madewithin a given erythrocyte batch. The study was approved by theethics committee of the University of Tübingen (184/2003 V).Erythrocytes were incubated in vitro at a haematocrit of 0.4% in

Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO4, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 glucose,1 CaCl2, pH 7.4 at 37°C for 24 hr. Where indicated, CORM-2 [trica-rbonyldichlororuthenium(II) dimer] and ruthenium chloride (both fromSigma, Freiburg, Germany) were added at the indicated concentrations.

Author for correspondence: Florian Lang, Physiologisches Institut,der Universität Tübingen, Gmelinstr. 5, D-72076 Tübingen (fax+ 49 7071 29 5618, e-mail [email protected]).†These authors contributed equally and thus share the first authorship.

© 2012 The AuthorsBasic & Clinical Pharmacology & Toxicology © 2012 Nordic Pharmacological Society

Basic & Clinical Pharmacology & Toxicology Doi: 10.1111/j.1742-7843.2012.00915.x

Gas bottles containing 100% O2 were purchased from Westfalen(Münster, Germany). In Ca2+-free Ringer solution, 1 mM CaCl2 wassubstituted by 1 mM glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraace-tic acid (EGTA, Sigma, Freiburg, Germany).To study the effect of CO2 exposure on CO-triggered eryptosis,

erythrocytes (4% final haematocrit) were incubated in 10 ml Ringersolution (containing 25 mM NaHCO3 and 1 mM HCl) in the presenceand absence of 10 lM CORM-2. The samples were incubated simulta-neously for 24 hr in two different incubators equilibrated with CO2

concentrations of 0.04% and 5%, respectively.

Exposure of erythrocytes to 100% oxygen. Erythrocytes (finalhaematocrit 0.4%) were incubated in 8 ml Ringer solution in thepresence and absence of 10 lM CORM-2. At time-points 1, 3, 6 and12 hr of incubation, the samples were bubbled with 100% O2 for30 min. To ensure maximum exposure of erythrocytes to 100% O2,the tubes were tightly closed and incubated for the indicated timeperiods.

Measurement of carboxyhaemoglobin. For the determination ofcarboxyhaemoglobin, 200 ll of erythrocytes was incubated for 24 hr(final haematocrit 2%) in the presence and absence of 10 lM CORM-2. Where indicated, erythrocytes were exposed to 100% O2. Thesamples were centrifuged (3 min. at 400 9 g, room temperature), andthe erythrocyte pellet was resuspended in 200 ll Ringer solution (finalhaematocrit 50%) for the determination of carboxyhaemoglobin usinga blood gas analyzer (Radiometer ABL 700, West Sussex, UK).

FACS analysis of annexin-V-binding and forward scatter. Afterincubation under the respective experimental conditions, 50 ll cellsuspension was washed in Ringer solution containing 5 mM CaCl2 andthen stained for 20 min. with annexin-V-FITC (1:200 dilution;ImmunoTools, Friesoythe, Germany) under protection from light [58]. Inthe following, the forward scatter (FSC) of the cells was determined andannexin-V fluorescence intensity was measured in FL-1 with anexcitation wavelength of 488 nm and an emission wavelength of530 nm on a FACS calibur (BD, Heidelberg, Germany).

Measurement of intracellular Ca2+. After incubation in the respectiveexperimental solution, 50 ll suspension erythrocytes was washed inRinger solution and then loaded with Fluo-3/AM (Biotium, Hayward,USA) in Ringer solution containing 5 mM CaCl2 and 2 lM Fluo-3/AM. The cells were incubated at 37°C for 20 min. and washed twicein Ringer solution containing 5 mM CaCl2. The Fluo-3-/AM-loadederythrocytes were resuspended in 200 ll Ringer solution. Then, Ca2+-dependent fluorescence intensity was measured in fluorescencechannel FL-1 in FACS analysis.

Measurement of haemolysis. For the determination of haemolysis,the samples were centrifuged (3 min. at 400 9 g, room temperature)and the supernatants were harvested. As a measure of haemolysis,the haemoglobin (Hb) concentration of the supernatant wasdetermined photometrically at 405 nm. The absorption of thesupernatant of erythrocytes lysed in distilled water was defined as100% haemolysis.

Confocal microscopy and immunofluorescence. For the visualizationof eryptotic erythrocytes, 4 ll of erythrocytes, incubated in therespective experimental solutions, was stained with FITC-conjugatedannexin-V (1:100 dilution; ImmunoTools, Friesoythe, Germany) in200 ll Ringer solution containing 5 mM CaCl2. Then, the

erythrocytes were washed twice and finally resuspended in 50 ll ofRinger solution containing 5 mM CaCl2. 20 ll was mounted withProlong Gold antifade reagent (Invitrogen, Darmstadt, Germany) ontoa glass slide and covered with a coverslip, and images were subsequentlytaken on a Zeiss LSM 5 EXCITER confocal laser scanning microscope(Carl Zeiss MicroImaging, Oberkochen, Germany) with a waterimmersion Plan-Neofluar 63/1.3 NA DIC.

Statistics. Data are expressed as arithmetic mean ± SEM. Statisticalanalysis was made using t test or paired ANOVA with Tukey’s test aspost-test, as indicated in the figure legends. n denotes the number ofdifferent erythrocyte specimens studied. The batches of erythrocytesdiffered moderately in their susceptibility to eryptosis. Thus, thecontrol values were not identical in all series of experiments. To avoidany bias potentially introduced by the use of different erythrocytebatches, comparison was always made within a given erythrocytebatch.

Results

Carbon monoxide (CO) tightly binds to haemoglobin formingCOHb. A 24-hr exposure to the CO-releasing molecule(CORM) tricarbonyl-dichlororuthenium (II) dimer (CORM-2)indeed resulted in a significant increase in COHb (fig. 1). Theincrease could be significantly blunted by increasing the oxy-gen concentration from 21% to 100% (fig. 1).In order to estimate the effect of CORM-2 on cytosolic Ca2+

concentration, Fluo 3 fluorescence was determined in FACSanalysis. As illustrated in fig. 2A,B, CORM-2 exposure wasfollowed by an increase in Fluo-3 fluorescence, reflecting anincrease in cytosolic Ca2+ concentration. Upon a 24-hr expo-sure to Ringer either in the absence or in the presence ofCORM-2 (1–10 lM), cytosolic Ca2+ concentration was higher

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Fig. 1. Effect of CORM-2 on CO haemoglobin. Arithmetic means ±SEM (n = 4) of the percentage of haemoglobin-binding CO in ery-throcytes exposed for 24 hr to Ringer solution without (white bar)or with (black bar) 10 lM CORM-2 and with 10 lM CORM-2 inthe presence of 100% O2 (grey bars). ***(p < 0.001) significantdifference from the respective value in the absence of CORM-2;###(p < 0.001) significant difference from the respective value at21% O2 (ANOVA).


2 ELISABETH LANG ET AL.

in the presence than in the absence of CORM-2, an effectreaching statistical significance at � 5 lM CORM-2.An increase in cytosolic Ca2+ concentration was expected to

activate Ca2+-sensitive K+ channels, which should be followedby the exit of KCl and cell shrinkage. Accordingly, forwardscatter was determined to estimate the cell volume. As shownin fig. 3A,B, exposure of erythrocytes for 24 hr to Ringersolution containing CORM-2 (1–10 lM) increased forwardscatter, an effect reaching statistical significance at 5 lMCORM-2. Thus, CORM-2 exposure resulted in cell swellinginstead of the expected cell shrinkage.An increase in cytosolic Ca2+ concentration was further

expected to stimulate cell membrane scrambling with phosphati-dylserine exposure at the cell surface. Erythrocytes exposingphosphatidylserine at the cell surface were identified by thedetermination of annexin-V binding. As a first step, the fluores-cent annexin-V binding was visualized by confocal imaging. Asillustrated in fig. 4, a 24-hr exposure to 10 lM CORM-2 wasindeed followed by the appearance of an increased number ofannexin-V-positive erythrocytes. In a second step, annexin-Vbinding was quantified by FACS analysis. As shown in fig. 5A,B, the percentage of annexin-V-binding erythrocytes was higherafter the exposure of erythrocytes for 24 hr to Ringer solutioncontaining CORM-2 than after the exposure of erythrocytes for24 hr to Ringer solution without CORM-2. The difference in thepercentage of annexin-V-binding erythrocytes between the pres-ence and absence of CORM-2 reached a statistical significance at� 5 lM CORM-2. Further experiments were performed with

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Fig. 2. Effect of CORM-2 on erythrocyte cytosolic Ca2+ concentration. (A) Original histogram of Fluo-3 fluorescence in erythrocytes after theexposure for 24 hr to Ringer solution without (-, black line) and with (+, red line) the presence of 10 lM CORM-2. (B) Arithmetic means ± SEM(n = 16) of the geo means (geometric mean of the histogram in arbitrary units) of Fluo-3 fluorescence in erythrocytes exposed for 24 hr to Ringersolution without (white bar) or with (black bars) 1–10 lM CORM-2. ***(p < 0.001) indicates significant difference from the respective value inthe absence of CORM-2 (ANOVA).

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Fig. 3. Effect of CORM-2 on erythrocyte forward scatter. (A) Original histogram of forward scatter of erythrocytes after the exposure for 24 hr toRinger solution without (-, black line) and with (+, red line) the presence of 10 lM CORM-2. (B) Arithmetic means ± SEM (n = 16) of the eryth-rocyte forward scatter after the incubation for 24 hr in Ringer solution without (white bar) or with (black bars) 1–10 lM CORM-2***(p < 0.001) indicates significant difference from the absence of CORM-2 (ANOVA).

10 µM0 µM CORM-2

Fig. 4. Confocal images of PS-exposing erythrocytes with or withoutCORM-2 treatment. Confocal microscopy of FITC-dependent fluores-cence (upper panels) and light microscopy (lower panels) of humanerythrocytes stained with FITC-conjugated annexin-V after 24-hr incu-bation in Ringer solution without (left panels) and with (right panels)10 lM CORM-2.


CORM-2-INDUCED CELL MEMBRANE SCRAMBLING 3

ruthenium chloride, a substance related to CORM-2 but notreleasing CO. As a result, the percentage of annexin-V-bindingerythrocytes after the exposure of erythrocytes for 24 hr toRinger solution was 1.0 ± 0.1 (n = 4) in the absence of ruthe-nium chloride and 1.3 ± 0.1 (n = 4) or 1.1 ± 0.1 (n = 4) in thepresence of 10 or 20 lM ruthenium chloride, respectively.To explore whether CORM-2 triggers haemolysis,

haemoglobin release was determined after the exposure ofthe erythrocytes to Ringer solution for 24 hr without or with1–10 lM CORM-2. As shown in fig. 5B, the exposure toCORM-2 was followed by haemolysis, an effect reaching

statistical significance at 10 lM CORM-2. The percentage ofhaemolysed erythrocytes remained, however, more than onemagnitude smaller than the percentage of phosphatidylserine-exposing erythrocytes (fig. 5B).Additional experiments explored whether the observed

increase in cytosolic Ca2+ activity accounted for the stimula-tion of cell membrane scrambling after the CORM-2 exposure.Erythrocytes were thus treated with CORM-2 in the presenceand nominal absence of extracellular Ca2+. As illustrated infig. 6, the effect of CORM-2 on annexin-V binding was virtu-ally abolished in the nominal absence of Ca2+.As the treatment of the erythrocytes with 100% O2 almost

fully reversed the effect of CORM-2 on CO haemoglobin(fig. 1), a further series of experiments explored whether theCORM-2-induced cell membrane scrambling and Ca2+ entrycould be partially reversed by increasing partial oxygen pres-sure. To this end, erythrocytes were treated with CORM-2 at21% and 100% O2. As illustrated in fig. 7A, the effect ofCORM-2 on annexin-V binding was significantly blunted byan increase in O2 from 21% to 100%. Similarly, enhancedFluo 3 fluorescence triggered by CORM-2 treatment was sig-nificantly reduced upon exposure to 100% O2 (fig. 7B).A further series of experiments explored whether CO-trig-

gered suicidal death of erythrocytes is sensitive to extracellularCO2 concentrations. To this end, erythrocytes were incubatedat 0.04% or 5% CO2 with or without CORM-2 (10 lM). As aresult, CORM-2 increased significantly (p < 0.001) the per-centage of phosphatidylserine-exposing erythrocytes at both0.04% CO2 (from 2.2 ± 0.5 to 70.7 ± 2.8, n = 4) and 5%CO2 (from 1.6 ± 0.1 to 42.7 ± 1.7, n = 4). Accordingly, theeffect of CORM-2 was significantly (p < 0.001) higher at0.04% CO2 than at 5% CO2.

Discussion

The present study discloses a novel effect of the CO donor tri-carbonyl-dichlororuthenium (II) dimer CORM-2, that is, trig-gering of Ca2+ entry, cell swelling and cell membranescrambling in human erythrocytes. As the related substanceruthenium chloride did not exert comparable effects, thepresent observations strongly suggest that the effects wereattributable to the release of CO. The exposure to CORM-2

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Fig. 5. Effect of CORM-2 on phosphatidylserine exposure and erythrocyte membrane integrity. (A) Original histogram of annexin-V binding oferythrocytes after the exposure for 24 hr to Ringer solution without (-, black line) and with (+, red line) the presence of 10 lM CORM-2. (B)Arithmetic means ± SEM (n = 16) of erythrocyte annexin-V binding after incubation for 24 hr in Ringer solution without (white bar) or with(black bars) the presence of 1–10 lM CORM-2. For comparison, arithmetic means ± SEM (n = 4) of the percentage of haemolysis is shown asgrey bars. **(p < 001), ***(p < 0.001) significant difference from the absence of CORM-2 (ANOVA).

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Fig. 6. Effect of Ca2+ withdrawal on CORM-2-induced phosphatidyl-serine exposure. Arithmetic means ± SEM (n = 4) of the percentageof annexin-V-binding erythrocytes after a 24-hr treatment with Ringersolution without (white bar) or with (black bars) 10 lM CORM-2 inthe presence (left bars, +Ca2+) and absence (right bars, -Ca2+) ofcalcium. ***(p < 0.001) indicates significant difference from theabsence of CORM-2 (ANOVA), and ###(p < 0.001) indicates significantdifference from the respective values in the presence of Ca2+.



was followed by a moderate increase in CO haemoglobin. Theincrease in CO haemoglobin remained, however, below 5%,that is, much lower than the >20% CO haemoglobin observedin heavy smokers [1]. Thus, the CO concentration required forthe observed effects was well in the range of those encoun-tered in vivo. The effect of CORM-2 is reversed by 100% O2

and slightly blunted upon an increase in CO2 concentrationfrom 0.04% to 5%.The Ca2+ entry into erythrocytes is accomplished by Ca2+-

permeable unselective cation channels presumably involvingTRPC6 [39]. CORM-2 has previously been shown to activateunselective cation channels in endothelial cells [59].An increase in erythrocyte Ca2+ concentration was expected

to activate Ca2+-sensitive K+ channels [45, 46], which shouldresult in the exit of K+, hyperpolarization of the cell mem-brane, exit of Cl� and exit of osmotically obliged water [47].In contrast to this expectation, exposure of erythrocytes toCORM-2 increased the forward scatter reflecting cell swelling.Thus, CORM-2 possibly inhibits the Ca2+-sensitive K+ chan-nels or activates channels or carriers increasing cell volume.As a matter of fact, CORM-2 modifies the activity of a varietyof channels [27, 60–70] including the large Ca2+-activated K+

channel BK [62].The increase in Ca2+ concentration after CORM-2 exposure

largely accounts for the stimulation of cell membrane

scrambling with subsequent annexin-V binding at theerythrocyte surface. The effect of cytosolic Ca2+ concentrationon erythrocyte membrane scrambling has been shown before[48, 50]. The effect of CORM-2 on cell membrane scramblingis, however, not fully abolished in the nominal absence ofextracellular Ca2+, an observation pointing to some additionalmechanism.Eryptosis is observed in several clinical conditions, such as

iron deficiency [71], phosphate depletion [72], haemolyticuraemic syndrome [73], sepsis [74], sickle cell disease [75],malaria [75–79], Wilson’s disease [80], tumours in APC genemutations [81] and possibly metabolic syndrome [82]. Erypto-sis is further stimulated by a wide variety of xenobiotics andother small molecules [55,57,83–92]. At least in theory, thoseclinical disorders or chemicals could enhance the sensitivity tothe scrambling effect of CORM-2.Erythrocyte phospholipid scrambling fosters the clearance

of affected erythrocytes from circulating blood and may thuslead to the development of anaemia [35]. The anaemia iscounteracted by compensatory stimulation of erythrocyte for-mation, apparent from an increase in the reticulocyte number[40,93]. Thus, overt anaemia develops only if those compensa-tory mechanisms fail or are overridden.Erythrocyte phospholipid scrambling is further expected to

impede microcirculation resulting from the adherence of phos-phatidylserine-exposing erythrocytes to the vascular wall [94–98]. Moreover, phosphatidylserine-exposing erythrocytes couldstimulate blood clotting [94,99,100]. Beyond that, CORM-2may stimulate coagulation and inhibit fibrinolysis [101–105].On the other hand, CO inhibits platelet aggregation [14] andmodifies the function of endothelial cells [13] and may thuscounteract thrombus formation [16].CO is known to interact with superoxide formation by ery-

throcytes [106]. Under hypoxia, erythrocytes produce reactiveoxygen species (ROS), which in turn triggers vascular inflam-mation in lung tissue [106]. Accordingly, perfusion with eryth-rocyte-free perfusate prevents the hypoxia-induced inflammation[106]. The erythrocyte superoxide formation is triggered byhaemoglobin autoxidation, which is in turn inhibited by CO ornitrite [106].In conclusion, the present observations disclose that

CORM-2 stimulates cell membrane scrambling. The effect isat least partially due to Ca2+ entry and partially reversed bythe increase in O2 concentration. As excessive eryptosis mayresult in anaemia and impairment of microcirculation, CO-induced eryptosis may contribute to the pathophysiology ofCO intoxication.

AcknowledgementsThe authors acknowledge the meticulous preparation of the

manuscript by Sari Rübe and Lejla Subasic. The authors thankPaola M. Tripodi and Luisa Rosaclerio for their valuable tech-nical support. This study was supported by the Deutsche Fors-chungsgemeinschaft, Nr. La 315/4-3 and La 315/6-1 and theBundesministerium für Bildung, Wissenschaft, Forschung undTechnologie (Center for Interdisciplinary Clinical Research).

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Fig. 7. Effect of oxygen pressure on CORM-2-induced phosphatidyl-serine exposure and enhanced cytosolic Ca2+ concentration. (A) Arith-metic means ± SEM (n = 4) of the percentage of annexin-V-bindingerythrocytes after a 24-hr treatment with Ringer solution without(white bar) or with (black bars) 10 lM CORM-2 in the presence of21% O2 (left bars) or of 100% O2 (right bars). ***(p < 0.001) indi-cates significant difference from the absence of CORM-2 (ANOVA), and#(p < 0.05) indicates significant difference from the respective valuesat 21% O2. (B) Arithmetic means ± SEM (n = 4) of the geo means(geometric mean of the histogram in arbitrary units) of Fluo-3 fluores-cence in erythrocytes incubated in Ringer solution without (white bar)or with (black bars) 10 lM CORM-2 in the presence of 21% O2

(left bars) or of 100% O2 (right bars). ***(p < 0.001) indicatessignificant difference from the absence of CORM-2 (ANOVA), and###(p < 0.001) indicates significant difference (p < 0.001) from therespective values at 21% O2.



Confl ict of InterestThe authors state that they have no conflict of interest to

disclose.

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Carbon Monoxide–Sensitive Apoptotic Death of Erythrocytes