Content uploaded by Leonid Livshits
Author content
All content in this area was uploaded by Leonid Livshits on Sep 15, 2023
Content may be subject to copyright.
Content uploaded by Ivana Pajić-Lijaković
Author content
All content in this area was uploaded by Ivana Pajić-Lijaković on Sep 15, 2023
Content may be subject to copyright.
Citation: Livshits, L.; Peretz, S.;
Bogdanova, A.; Zoabi, H.; Eitam, H.;
Barshtein, G.; Galindo, C.; Feldman,
Y.; Paji´c-Lijakovi´c, I.; Koren, A.; et al.
The Impact of Ca2+ on Intracellular
Distribution of Hemoglobin in
Human Erythrocytes. Cells 2023,12,
2280. https://doi.org/10.3390/
cells12182280
Academic Editors: Yubin Zhou
and Youjun Wang
Received: 3 August 2023
Revised: 2 September 2023
Accepted: 11 September 2023
Published: 15 September 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
cells
Article
The Impact of Ca2+ on Intracellular Distribution of Hemoglobin
in Human Erythrocytes
Leonid Livshits 1, 2, *, Sari Peretz 2,3,4, Anna Bogdanova 1,5 , Hiba Zoabi 3, Harel Eitam 3, Gregory Barshtein 6,
Cindy Galindo 7, Yuri Feldman 7, Ivanna Paji´c-Lijakovi´c 8, Ariel Koren 2, Max Gassmann 1,5
and Carina Levin 2,4
1Red Blood Cell Research Group, Vetsuisse Faculty, Institute of Veterinary Physiology, University of Zurich,
8057 Zürich, Switzerland; annab@access.uzh.ch (A.B.); maxg@access.uzh.ch (M.G.)
2Pediatric Hematology Unit, Emek Medical Center, Afula 1834111, Israel; sari_pe@clalit.org.il (S.P.);
koren_a@clalit.org.il (A.K.); levin_c@clalit.org.il (C.L.)
3Laboratory Division Unit, Emek Medical Center, Afula 1834111, Israel; hiba.zoabi@gmail.com (H.Z.);
eitam_ha@clalit.org.il (H.E.)
4The Bruce and Ruth Rapaport Faculty of Medicine, Technion–Israel Institute of Technology,
Haifa 3200003, Israel
5The Zurich Center for Integrative Human Physiology (ZIHP), 8057 Zürich, Switzerland
6Biochemistry Department, The Faculty of Medicine, The Hebrew University of Jerusalem,
Jerusalem 9112102, Israel; gregoryba@ekmd.huji.ac.il
7Institute of Applied Physics, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel;
cindy.galindohur@mail.huji.ac.il (C.G.); yurif@mail.huji.ac.il (Y.F.)
8Department of Chemical Engineering, University of Belgrade, 11000 Beograd, Serbia; iva@tmf.bg.ac.rs
*Correspondence: leonidlivshts@gmail.com
Abstract:
The membrane-bound hemoglobin (Hb) fraction impacts red blood cell (RBC) rheology
and metabolism. Therefore, Hb–RBC membrane interactions are precisely controlled. For instance,
the signaling function of membrane-bound deoxy-Hb and the structure of the docking sites in the
cytosolic domain of the anion exchanger 1 (AE-1) protein are well documented; however, much
less is known about the interaction of Hb variants with the erythrocyte’s membrane. Here, we
identified factors other than O
2
availability that control Hb abundance in the membrane-bound
fraction and the possible variant-specific binding selectivity of Hb to the membrane. We show that
depletion of extracellular Ca
2+
by chelators, or its omission from the extracellular medium, leads
to membrane-bound Hb release into the cytosol. The removal of extracellular Ca
2+
further triggers
the redistribution of HbA0 and HbA2 variants between the membrane and the cytosol in favor of
membrane-bound HbA2. Both effects are reversible and are no longer observed upon reintroduction
of Ca
2+
into the extracellular medium. Fluctuations of cytosolic Ca
2+
also impact the pre-membrane
Hb pool, resulting in the massive transfer of Hb to the cellular cytosol. We hypothesize that AE-1 is the
specific membrane target and discuss the physiological outcomes and possible clinical implications
of the Ca2+ regulation of the intracellular Hb distribution.
Keywords: hemoglobin distribution; red blood cells; hemoglobin A2; calcium
1. Introduction
Hemoglobin (Hb), by far the most abundant protein in red blood cells (RBCs), is
known primarily for its key function in blood gas exchange and transport. However, its
cellular activities are not limited to this function. Pre-membrane Hb and cytosolic Hb pools
have distinct roles in controlling the metabolic activity of glycolytic enzymes and nitric
oxide (NO) production and in the rheological properties of RBCs [
1
–
4
]. Interactions of
deoxy-Hb with the cytosolic domain of transmembrane band 3 protein controls the activity
of glycolytic enzymes [
3
,
5
]; deoxy-Hb catalyzes nitrite conversion to NO [
6
]. Mean cell
Hb concentration, its aggregation—as in the case of pathological variants such as HbS and
Cells 2023,12, 2280. https://doi.org/10.3390/cells12182280 https://www.mdpi.com/journal/cells
Cells 2023,12, 2280 2 of 19
HbC—and the thickness of the pre-membrane Hb layer affect membrane stability and RBC
rheology [7].
In the RBCs of healthy donors, 0.5–12% of the Hb forms a membrane-bound pool,
while the rest of the Hb remains in the cytosol [
2
]. The considerable variation in the extent of
the membrane-bound Hb fraction depends on a plethora of (patho)physiological conditions,
such as Hb O
2
saturation, oxidative stress, cellular Hb concentration, charge, stability of
Hb variants, and elevated Hb oxidation to methemoglobin, which are all reported to affect
Hb binding to the membrane [
8
–
13
]. The different methodologies used to isolate and detect
RBC membranes may also contribute to the variation (summarized in Ref. [9]).
Several types of Hb–membrane interactions are known. These include the following:
(a) electrostatic binding of deoxy-Hb to the cytoplasmic domain of band 3 anion trans-
port protein (anion exchanger (AE-1) protein) [
14
,
15
]; (b) covalent crosslinking with the
membrane components via disulfide bonds, and (c) adsorption to membrane lipids via
hydrophobic interactions [16,17].
To the best of our knowledge, no previous attempts have been made to determine
whether the distribution of Hb isoforms/variants between the membrane and the cytosol
in healthy individuals is random or if preference is given to specific variants over others.
In general, Hb in the RBCs of healthy adults is present in three main isoforms [
18
]. HbA0
makes up >96% of the Hb, whereas HbA2 typically does not exceed 2–3.5%, complemented
by the even less abundant remnants (~1%) of fetal Hb (HbF), which is the dominant Hb
isoform in the fetus. In contrast to HbF, HbA0 and HbA2 are ubiquitously distributed in
most of the circulating cells [
19
]. The Hb isoforms are not located uniformly within the
RBCs; due to its positive charge, HbA2 has a higher affinity for RBC membrane proteins
than the other Hb isoforms [
20
]. The potential regulatory or catalytic role of the HbA2
variant in RBC membranes has never been explored. It is suggested that HbA2 may be
involved in controlling RBC morphology by regulating the activity of the K–Cl co-transport
system or by tuning cell pH [
21
]. In addition to its involvement in oxygen transport, HbA2
0
s
signaling activity has been speculated but not confirmed [
19
,
21
]. Furthermore, if HbA2 is a
sensor that responds to stimulation via redistribution between the membrane-bound and
cytosolic pools, it is not clear what these stimuli are.
In the present study, we examined the distribution of HbA0 and A2 variants between
the cytosolic- and membrane-bound pools, focusing on the possible variant selectivity in
these responses to plasma-borne stressors. We identified the distinct roles of extracellular
and intracellular Ca
2+
as stimulants and monitored the potential outcome of Ca
2+
-induced
redistribution of Hb between the membrane-bound and cytosolic pools on the RBC’s mor-
phology and physiological parameters, such as membrane stability and met
abolic reado
uts.
2. Materials and Methods
2.1. Blood Samples
The remains of adults’ fresh blood samples sent for routine analysis to the central
laboratory at Emek Medical Center in K3EDTA-, citrate- or heparin sulfate-supplemented
tubes in the years 2021–2023 were chosen at random. The study was performed in accor-
dance with the Declaration of Helsinki and approved by the Emek Medical Center ethics
committee (EMC-0085-21).
2.2. Buffers and Chemicals
Commercial phosphate-buffered saline, either Ca2+/Mg2+-free (PBS) or with 0.9 mM
Ca
2+
and 0.5 mM Mg
2+
(DPBS), was purchased from Biological Industries (Haemek, Is-
rael) and Sartorius (Göttingen, Germany), respectively. Plasma-mimicking buffer (PMB)
contained the following: 140 mM of NaCl, 4 mM of KCl, 0.75 mM of MgSO
4
, 10 mM of
glucose, 0.015 mM of ZnCl
2
, 0.2 mM of glycine, 0.2 mM of sodium glutamate, 0.2 mM
of alanine, 0.1 mM of arginine, 0.6 mM of glutamine, and 20 mM of HEPES, adjusted to
pH 7.4 with imidazole and then supplemented with 0.01% bovine serum albumin (BSA).
When required, 2 mM of CaCl
2
or 5 mM of ethylenediaminetetraacetic acid (EDTA) was
Cells 2023,12, 2280 3 of 19
added. Citrate phosphate dextrose adenine solution (CPDA-1) was purchased from Ma-
copharma (Tourcoing, France). All other chemicals were bought from Sigma-Aldrich Israel
(Rehovot, Israel).
2.3. Hb Variant Analysis
Hb variants were detected by HPLC. The VARIANT
™
II
β
-thalassemia Short Program
(Bio-Rad, Hercules, CA, USA) method, which separates Hb variants by cation-exchange
chromatography using a salt gradient, was used, with the calibrators and controls provided
by the manufacturer with every batch. The analysis consisted of monitoring retention
times, area percentages, and concentrations of various peaks and windows for different Hb
variants: HbF (retention time of 1.1 min with 0.98–1.2 min window), HbA0 (2.5 min and
2.0–3.0 min), HbA2 (3.65 min and 3.57–3.75 min), and minor peaks, such as P2 (1.39 min
and 1.28–1.5 min) and P3 (1.7 min and 1.5–1.9 min).
2.4. RBC Membrane Preparation
To isolate the membrane fraction of RBCs, we adapted the protocol reported by
Ghashghaeinia et al. [
22
]. Briefly, a 150
µ
L aliquot of the blood sample was incubated
with 20 volumes of ice-cold HEPES-based hypoosmotic solution (20 mM of HEPES/NaOH,
1 m
M of PMSF, pH 7.4) for 10 min and then centrifuged (4
◦
C, 14,000
×
g, 20 min). This
procedure was repeated three times prior to the measurements of Hb content or Hb isoform
distribution. Hb concentrations in intact RBCs and RBC membranes were measured using
an MRC Spectro V-18 spectrophotometer (absorbance at 575 nm) and then evaluated in
accordance with a prior calibration with known concentrations of Hb. After the RBCs or
the membranes were isolated and washed, 5
µ
L of intact RBCs or 30
µ
L of RBC membranes
were diluted in 4 mL DDW and measured using the spectrophotometer. Hb isoform
distributions were examined as described in Section 2.3.
2.5. Morphological Characterization Using Cell Flow-Properties Analyzer (CFA)
To examine morphological changes, we used a computerized CFA [
23
–
25
]. Briefly,
5
0µ
L of RBC suspension (1% hematocrit, in the same medium used for pretreatment) was
placed into a flow chamber with an uncoated glass slide. The RBCs were allowed to adhere
to the slide surface for 10 min before capturing images. We captured at least 10 fields (more
than 1150 cells in total) for each sample. The CFA image analysis program can automatically
measure the major and minor cellular axes for individual cells. A major-to-minor axis ratio
of 1 reflects a round RBC. Cells with a ratio above 1.25 were removed from further analysis.
To compare RBC shapes in different examinations, we evaluated the projected area of each
cell by multiplying the major and minor axis values.
2.6. Glucose Consumption, Lactate Release, and K+Leakage Studies
After removal of the plasma and the buffy coat, the cells were incubated in PMB with
or without 2 mM of Ca
2+
, or with both 2 mM of Ca
2+
and 5 mM of EDTA (hematocrit
~20%) for 2 h. Then, the cells were centrifuged at 1700
×
gfor 5 min, the supernatant was
discarded, and the cells were resuspended in a fresh medium. The cells were quickly
mixed, and basal levels of extracellular K
+
, glucose, and lactate were detected using a
GEM
®
Premier
™
5000 blood gas analyzer (Werfen, Bedford, MA, USA). The cells were then
incubated for 4 h at 37
◦
C in a shaker, and the measurements of extracellular K
+
, glucose,
and lactate levels in PMB were repeated. Total Hb levels in the RBC suspension were also
assessed prior to the one-point test with the blood gas analyzer. Changes in K
+
, glucose,
and lactate concentrations in PMB representing glucose conversion to lactate, and K
+
loss
from RBCs over 4 h were then expressed in mmole per gram, Hb per hour.
Cells 2023,12, 2280 4 of 19
2.7. Labeling Experiments
Morphological changes, membrane potential, and intracellular Ca
2+
levels were investi-
gated via flow cytometry. The intracellular Ca
2+
dye Fluo-4 AM (1 mM stock, Thermo Fisher
Scientific, Waltham, MA, USA) and the voltage-sensitive dye bis (
1,3-dibuty
lbarbituric acid)
trimethine oxonol (DiBAC4(3); 0.2 mM stock, Molecular Probes, Eugene, OR, USA) were used.
Briefly, RBCs were washed free of the plasma and buffy coat, and 1
µ
L of packed cells was
resuspended in 1 mL of the desired buffer and incubated for 1 h at 37
◦
C. Then, 1
µ
L of
one of the dyes was added to 1 mL of the sample, and the cell suspensions were incubated
for another hour at 37
◦
C in the dark. Thereafter, fluorescence intensity was measured
in stained RBCs using a Navios EX flow cytometer (Beckman Coulter, Brea, CA, USA).
Measurements were repeated at least twice (>30,000 measured cells) and averaged for
each condition. All data were analyzed using Kaluza Analysis Software (Beckman Coulter,
https://www.beckman.co.il/flow-cytometry/software/kaluza, Version 2.1.00001.20653,
built on 8 March 2018).
2.8. Separation on a Percoll Density Gradient
Fractionation on a self-formed isotonic continuous Percoll density gradient was per-
formed as described [
26
]. Briefly, 1 mL of whole blood was gently layered on top of
13 mL
of a 90% isotonic Percoll mixture (consisting of nine parts of commercial Percoll (GE Health-
Care, Chicago, IL, USA) and one part of 10
×
concentrated PMB) supplemented with a final
0.1% BSA and 2 mM of CaCl
2
. RBCs were separated out via centrifugation at 1
8,514×g
for 60 min (minimal acceleration/deceleration) at 30
◦
C (Eppendorf Centrifuge 5810R,
F-34-6-38 rotor supplemented with specific adapters for 15 mL Falcon tubes).
2.9. Statistics
Data for the entire study were analyzed using GraphPad 5 software. The normality
of distribution of the values obtained in each experimental set was evaluated using the
Shapiro–Wilk test, and those with p> 0.05 were considered normally distributed. For those
parameters showing normal distribution, paired-matched values were compared using a
paired Student’s t-test. For the datasets that were not normally distributed, the Wilcoxon
signed-rank test was used. For all analyses, a two-tailed test with p< 0.05 was accepted as
statistically significant. For more details, see figure legends.
3. Results
3.1. Effect of Extracellular Constituents on Hb Distribution in the Membrane
Figure 1presents the membrane distribution of Hb isoforms in RBCs exposed to
routinely used anticoagulants and storage solution CPDA-1. In view of the minimal
cell fraction of HbF and its mostly homogeneous intracellular distribution (Table 1), we
concentrated on the HbA2-to-HbA0 isoform ratios in intact RBCs (which predominantly
correlate with the distribution of these Hb isoforms in the RBC cytosol) and in their
membrane compartment. Figure 1A,B present the HbA2/HbA0 ratios in RBCs collected in
K
3
EDTA- and heparin-supplemented tubes, respectively. Intriguingly, the HbA2 fraction
in the pre-membrane pool was significantly higher than those in intact RBCs and in the
cytosolic compartment. Furthermore, HbA2 abundance in the membrane was strongly
dependent on the type of supplemented anticoagulant (Figure 1C), with maximal values
found in RBCs collected into K
3
EDTA-supplemented tubes. Moreover, as demonstrated
in Figure 1D, RBC maintenance in the routine storage solution CPDA-1 led to an increase
in the membrane fraction of HbA2 compared to that in the cytosol or intact cells. The
pre-membrane HbA2/HbA0 ratio remained constant during prolonged storage of RBC
concentrates (i.e., for 3, 14, and 28 days at 4
◦
C). Intriguingly, 28 days of storage at 4
◦
C in
CPDA-1 medium caused a decrease in the total HbA2 fraction in intact RBCs. This finding
is in partial agreement with the findings of Hildrum et al. [
27
]; however, the mechanism
underlying this alteration is not clear and needs to be further investigated.
Cells 2023,12, 2280 5 of 19
Cells 2023, 12, x FOR PEER REVIEW 5 of 19
(A) (B)
(C) (D)
Figure 1. Effects of routinely used anticoagulants and CPDA-1 storage solution on Hb isoform dis-
tribution in RBCs. HbA2/HbA0 ratios in intact RBCs, and RBC cytosol and membrane of samples
collected into (A) EDTA-supplemented (n = 6) or (B) heparin-supplemented (n = 8) tubes are shown.
(C) HbA2/HbA0 membrane pools in RBCs collected in K
3
EDTA (n = 21), heparin (n = 16), and citrate
(n = 5) tubes from different individuals. The blood samples were kept at room temperature prior to
the tests. The total time period between the blood collection and the measurement did not exceed
four hours. RBCs were isolated from plasma and buffy coat via short 1700× g centrifugation. Imme-
diately after that, the measurement of Hb isoforms in intact RBCs and the isolation of RBC mem-
branes (as described above) were performed. Wilcoxon signed-rank test was used to test significance
for HbA2/HbA0 pools in the membranes of RBCs preserved in various anticoagulants; data are pre-
sented as median ± CI. Note the minimal differences (non-significant, NS) between intact RBCs’
HbA2/HbA0 ratios. (D) CPDA-1 study (n = 6), where cells were incubated at 4 °C for increasing
periods to a maximum of 28 days, and HbA2/HbA0 ratios for intact and membrane RBC fractions
were evaluated. The paired-matched data presented in panels (A,B,D) (means ± SD) were found to
be normally distributed and were compared using paired Student’s t-test. Distributions of Hb
isoforms (HbF, HbA0, and HbA2) corresponding to the data at current and next Figures are pro-
vided in the Supplementary Data document.
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
EDTA- Citrate-
0.001
Heparin-
<0.001 0.02
preserved blood
Intact Membrane Intact Membrane Intact Membrane
HbA2/ HbA0 ratio
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
RBC,
Membrane
RBC,
Intact
CPDA-1 storage
solution
0d 28d
NS
0.001
3d 14d 28d
HbA2/ HbA0 ratio
Figure 1.
Effects of routinely used anticoagulants and CPDA-1 storage solution on Hb isoform
distribution in RBCs. HbA2/HbA0 ratios in intact RBCs, and RBC cytosol and membrane of samples
collected into (
A
) EDTA-supplemented (n = 6) or (
B
) heparin-supplemented (n = 8) tubes are shown.
(
C
) HbA2/HbA0 membrane pools in RBCs collected in K
3
EDTA (n = 21), heparin (n = 16), and citrate
(n = 5) tubes from different individuals. The blood samples were kept at room temperature prior to
the tests. The total time period between the blood collection and the measurement did not exceed four
hours. RBCs were isolated from plasma and buffy coat via short 1700
×
gcentrifugation. Immediately
after that, the measurement of Hb isoforms in intact RBCs and the isolation of RBC membranes
(as described above) were performed. Wilcoxon signed-rank test was used to test significance
for HbA2/HbA0 pools in the membranes of RBCs preserved in various anticoagulants; data are
presented as median
±
CI. Note the minimal differences (non-significant, NS) between intact RBCs’
HbA2/HbA0 ratios. (
D
) CPDA-1 study (n = 6), where cells were incubated at 4
◦
C for increasing
periods to a maximum of 28 days, and HbA2/HbA0 ratios for intact and membrane RBC fractions
were evaluated. The paired-matched data presented in panels (
A
,
B
,
D
) (means
±
SD) were found
to be normally distributed and were compared using paired Student’s t-test. Distributions of Hb
isoforms (HbF, HbA0, and HbA2) corresponding to the data at current and next Figures are provided
in the Supplementary Data document.
Cells 2023,12, 2280 6 of 19
Table 1.
Distribution of Hb isoforms (HbF, HbA0, and HbA2) in intact RBCs and in their membrane
and cytosolic compartments. Blood samples collected into K
3
EDTA- or heparin-supplemented tubes
(n = 6 and 8, respectively) were kept at room temperature prior to the experimental manipulations.
The total time period between the blood collection and the measurement did not exceed four hours.
RBCs were isolated from plasma and buffy coat via short centrifugation at 1700
×
g. Immediately after
that, the measurement of Hb isoforms in intact RBCs was performed. Then, RBCs were lysed with ice-
cold HEPES-based hypoosmotic solution, and hemolysates were electrophoresed to evaluate fractions
of each Hb isoform in the RBC cytosol; the procedure was repeated three more times to obtain
membranes for determination of Hb isoform distribution. Percent of each Hb isoform out of total
Hb and HbA2/HbA0 ratios are shown. Data are presented as means
±
SD. Significance (presented
as superscript values) was determined compared to corresponding RBC membrane datasets using
paired Student’s t-test at p≤0.05; NS, non-significant.
EDTA-Supplemented Plasma
(n = 6)
Heparin-Supplemented Plasma
(n = 8)
Hb Isoforms RBC RBC
Intact Membrane Cytosol Intact Membrane Cytosol
HbF, % of
total Hb 0.30 ±0.09 0.04 0.17 ±0.14 0.27 ±0.08 NS 0.36 ±0.13 NS 0.38 ±0.16 0.36 ±0.12 NS
HbA2, % of
total Hb 3.02 ±0.28 0.002 6.42 ±1.46 2.98 ±0.26 0.003 2.65 ±0.52 <0.001 4.41 ±0.72 2.60 ±0.44 <0.001
HbA0, % of
total Hb 96.7 ±0.26 0.001 93.4 ±1.33 96.8 ±0.23 0.002 97.0 ±0.49 <0.001 95.2 ±0.67 97.0 ±0.40 <0.001
HbA2/HbA0 0.031 ±0.003 0.002 0.069 ±0.017 0.031 ±0.003 0.003 0.027 ±0.005 <0.001 0.046 ±0.008 0.027 ±0.005 <0.001
In the next set of experiments, we examined the size and content of the pre-membrane
Hb pool in RBCs from EDTA-preserved whole blood after the plasma had been substituted
with one of the routinely used isotonic buffers, DPBS or PMB (their chemical compositions
are detailed in Table 2). In a separate set of experiments, we assessed the possible tempera-
ture dependence of the HbA2/A0 distribution between the membrane and the cytosolic
pool. To do so, washed RBCs were resuspended in a Ca
2+
-free EDTA-containing medium
and incubated for two hours at either of the following temperatures: 4, 25, 37, or 42
◦
C.
No effect of temperature on the distribution of the Hb variant between the membrane
and the cytosol could be confirmed (Supplementary Figure S1). Based on these data, we
have chosen 37
◦
C as the optimal temperature for further investigation of the underlying
mechanism. The time dependence of the HbA2/HbA0 redistribution in the pre-membrane
pool was then examined. We observed at least a two-phase process of redistribution of
the HbA0 and HbA2 variants at the membrane (Figure 2A). First, a sharp decrease of the
pre-membrane HbA2/HbA0 was detected after 15 min incubation in either DPBS or PMB.
Then, the membrane fraction of HbA2 in the bulk pre-membrane Hb pool continued to
gradually decrease to a minimum at 2 h. There were no significant variations for the mem-
brane Hb ratios in DPBS- vs. PMB-treated RBCs, except after 1 h of maintenance. Longer
incubations, for 6–24 h, were associated with a modest partial recovery of the HbA2/HbA0
ratio in the pre-membrane fraction. Slight increases were observed at 6 and 24 h. Based
on these findings, 2 h was chosen as the optimal time point for further investigation of
the short-term mechanism. Numerically, the membrane-bound HbA2/HbA0 fractions in
samples maintained with DPBS or PMB were twice as small than in those suspended in
the EDTA-containing plasma after 2 h of incubation (Figure 2B). Intriguingly, the observed
alteration in the pre-membrane HbA2 fraction was associated with a significant rise in the
concentration of total Hb in the pre-membrane Hb pool in both DPBS and PMB (Figure 2C).
Cells 2023,12, 2280 7 of 19
Table 2. Chemical composition of the examined solutions.
Na+/K+/
Cl−,
mM
Phosphates,
mM
Mg2+/
Ca2+/Zn2+ ,
mM
Glucose,
mM
Amino
Acids
HEPES,
mM
Trisodium
Citrate,
mM
Citrate,
mM
Adenine,
mM
Albumin,
%pH
Plasma
(EDTA) Native Native Minor
(chelated) Native Native – – – Native Native 7–7.4
Plasma
(Citrate) Native Native Minor
(chelated) Native Native – – – Native Native 7–7.4
Plasma
(Heparin)
Native Native Native Native Native – – – Native Native 7–7.4
CPDA-1 16/0/0 16 0 161 – – 89.4 15.5 2 – 5.5
DPBS
146/4.1/141
1.5 0.5/0.9/0 – – – – – – – 7–7.4
PBS
146/4.1/141
1.5 0 – – – – – – – 7–7.4
PMB
140/4/144
–
0.75/2/0.015
10 Native 20 – – – 0.1 7–7.4
Cells 2023, 12, x FOR PEER REVIEW 7 of 19
(A)
(B) (C)
Figure 2. Changes in Hb concentration and isoform distribution in the membranes of RBCs incu-
bated in cell-maintenance solutions DPBS and PMB. For time–response studies (A), RBCs from the
same individuals (n = 3) collected in EDTA tubes were exposed to the media for 24 h with sampling
at different time points. The HbA2/HbA0 ratio in intact RBCs was 0.032 ± 0.001. The matched com-
parison for (B) HbA2/HbA0 in membrane pools (n = 6) and (C) Hb concentration (n = 6) in intact
RBCs and RBC membranes exposed to EDTA plasma, DPBS, and PMB (2 h, 37 °C) are shown. Re-
sults are presented as means ± SD. Significance for each presented set was determined using paired
Student’s t-test at p ≤ 0.05; NS, not significant. Mean HbA2/HbA0 ratio in intact RBCs was 0.028 ±
0.003, while bulk Hb concentration (±SD) in intact RBCs made up 19.4 ± 0.76 mM.
Table 2. Chemical composition of the examined solutions.
pH
Al-
bu-
min,
%
Ade-
nine,
mM
Cit-
rate,
mM
Triso-
dium Cit-
rate, mM
HEPE
S,
mM
Ami
no
Ac-
ids
Glu-
cose,
mM
Mg
2+
/Ca
2+
/Z
n
2+
, mM
Phos-
phates,
mM
Na
+
/K
+
/
Cl
−
, mM
7–7.4
Na-
tive
Native – – –
Na-
tive
Native
Minor
(chelated)
Native Native
Plasma
(EDTA)
7–7.4
Na-
tive
Native – – –
Na-
tive
Native
Minor
(chelated)
Native Native
Plasma
(Citrate)
7–7.4
Na-
tive
Native – – –
Na-
tive
Native Native Native Native
Plasma
(Heparin)
5.5 – 2 15.5 89.4 – – 161 0 16 16/0/0 CPDA-1
7–7.4 – – – – – – – 0.5/0.9/0 1.5
146/4.1/1
41
DPBS
Figure 2.
Changes in Hb concentration and isoform distribution in the membranes of RBCs incubated
in cell-maintenance solutions DPBS and PMB. For time–response studies (
A
), RBCs from the same
individuals (n = 3) collected in EDTA tubes were exposed to the media for 24 h with sampling
at different time points. The HbA2/HbA0 ratio in intact RBCs was 0.032
±
0.001. The matched
comparison for (
B
) HbA2/HbA0 in membrane pools (n = 6) and (
C
) Hb concentration (n = 6) in
intact RBCs and RBC membranes exposed to EDTA plasma, DPBS, and PMB (2 h, 37
◦
C) are shown.
Results are presented as means
±
SD. Significance for each presented set was determined using
paired Student’s t-test at p
≤
0.05; NS, not significant. Mean HbA2/HbA0 ratio in intact RBCs was
0.028 ±0.003, while bulk Hb concentration (±SD) in intact RBCs made up 19.4 ±0.76 mM.
Cells 2023,12, 2280 8 of 19
3.2. The Key Role of Extracellular Ca2+ in HbA2 Enrichment of the Membrane-Bound Hb Pool
Analysis of the experimental conditions supporting the enrichment of the pre-membrane
Hb pool with HbA2 summarized in Tables 2and 3suggested that extracellular divalent
cations play an important role in it, while plasma proteins are not involved in the preferential
HbA2 binding at the membrane. To verify this assumption, the cells were incubated
in Ca
2+
/Mg
2+
-free PBS with specific supplementation of the chloride salts of either Fe
3+
(
0.1 m
M), Zn
2+
(0.02 mM), Ca
2+
(2 mM), Cu
2+
(1 mM), or Mg
2+
(1 mM) at near-physiological
concentrations (Figure 3). The presence of Ca
2+
, but not of the other cations tested, resulted
in the observed significant reduction in the HbA2/HbA0 ratio in the membrane-bound
pool. Moreover, the selective preferential recruitment of HbA2 to the membrane showed
dose dependence with the extracellular Ca
2+
concentrations within the 0–2 mM range in
both PBS and PMB (Figure 4).
Table 3.
Effect of content and activity of plasma proteins on HbA2/HbA0 ratio. RBCs were incubated
in autologous plasma preheated at 56
◦
C for 30 min or in PMB supplemented with 5% BSA for 2 h at
37
◦
C. Data are presented as means
±
SD. Significance was determined using paired Student’s t-test
at p≤0.05; NS, non-significant.
HbA2/HbA0 Ratio
Number Intact Membrane
EDTA-plasma 4 0.031 ±0.003 0.101 ±0.017
EDTA-plasma, heated at 56 ◦C0.099 ±0.014 NS
EDTA-plasma 7 0.023 ±0.002 0.072 ±0.012 <0.001
PMB 0.040 ±0.006
PMB, supplemented with 5% BSA 0.040 ±0.008 NS
Cells 2023, 12, x FOR PEER REVIEW 8 of 19
7–7.4
0 1.5
146/4.1/1
41
PBS
7–7.4 0.1 – – – 20
Na-
tive
10 0.75/2/0.015 –
140/4/14
4
PMB
3.2. The Key Role of Extracellular Ca2+ in HbA2 Enrichment of the Membrane-Bound Hb Pool
Analysis of the experimental conditions supporting the enrichment of the pre-mem-
brane Hb pool with HbA2 summarized in Tables 2 and 3 suggested that extracellular di-
valent cations play an important role in it, while plasma proteins are not involved in the
preferential HbA2 binding at the membrane. To verify this assumption, the cells were in-
cubated in Ca2+/Mg2+-free PBS with specific supplementation of the chloride salts of either
Fe3+ (0.1 mM), Zn2+ (0.02 mM), Ca2+ (2 mM), Cu2+ (1 mM), or Mg2+ (1 mM) at near-physio-
logical concentrations (Figure 3). The presence of Ca2+, but not of the other cations tested,
resulted in the observed significant reduction in the HbA2/HbA0 ratio in the membrane-
bound pool. Moreover, the selective preferential recruitment of HbA2 to the membrane
showed dose dependence with the extracellular Ca2+ concentrations within the 0–2 mM
range in both PBS and PMB (Figure 4).
Table 3. Effect of content and activity of plasma proteins on HbA2/HbA0 ratio. RBCs were incubated
in autologous plasma preheated at 56 °C for 30 min or in PMB supplemented with 5% BSA for 2 h
at 37 °C. Data are presented as means ± SD. Significance was determined using paired Student’s t-
test at p ≤ 0.05; NS, non-significant.
HbA2/HbA0 Ratio
Number Intact Membrane
EDTA-plasma 4 0.031 ± 0.003 0.101 ± 0.017
EDTA-plasma, heated at 56 °C 0.099 ± 0.014 NS
EDTA-plasma 7 0.023 ± 0.002 0.072 ± 0.012 <0.001
PMB 0.040 ± 0.006
PMB, supplemented with 5% BSA 0.040 ± 0.008 NS
Figure 3. Ca2+, but not other bi-/trivalent cations, modifies membrane Hb isoform distribution. Cells
from the same individuals were exposed to bi-/trivalent cation-free DPBS (i.e., commercially pro-
duced buffer with negligible contents of these electrolytes) supplemented with either Zn2+, Ca2+,
Mg2+, Cu2+, or Fe3+ at near-physiological concentrations for 2 h at 37 °C prior to membrane isolation.
Data are presented as means ± SD. Significance was determined for each presented set using paired
Student’s t-test at p ≤ 0.05; NS, not significant. Mean HbA2/HbA0 ratio (± SD) in intact RBC was
0.030 ± 0.002.
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
NS
0.002
+0.02mM
Zn2+
+2mM
Ca2+
+0.1mM
Cu2+
NS NS
NS NS
+0.1mM
Fe3+
PBSPlasma-EDTA +1mM
Mg2+
HbA2/ HbA0 ratio
in RBC membrane
Figure 3.
Ca
2+
, but not other bi-/trivalent cations, modifies membrane Hb isoform distribution.
Cells from the same individuals were exposed to bi-/trivalent cation-free DPBS (i.e., commercially
produced buffer with negligible contents of these electrolytes) supplemented with either Zn
2+
, Ca
2+
,
Mg
2+
, Cu
2+
, or Fe
3+
at near-physiological concentrations for 2 h at 37
◦
C prior to membrane isolation.
Data are presented as means
±
SD. Significance was determined for each presented set using paired
Student’s t-test at p
≤
0.05; NS, not significant. Mean HbA2/HbA0 ratio (
±
SD) in intact RBC was
0.030 ±0.002.
Cells 2023,12, 2280 9 of 19
Cells 2023, 12, x FOR PEER REVIEW 9 of 19
Figure 4. Dose response to extracellular Ca2+ of Hb isoform ratio in RBC membrane. Erythrocytes
were exposed to increasing concentrations of Ca2+ pre-added to Ca2+-free DPBS (n = 6) or PMB (n =
4). Data are presented as means ± SD. Significance compared to the corresponding ‘0 mM Ca2+’ set
of DPBS or PMB was determined using paired Student’s t-test at p ≤ 0.05; NS, not significant. Non-
paired Student’s t-test was used to determine the significance of HbA2/HbA0 membrane pools in
DPBS- vs. PMB-exposed RBCs for the same Ca2+ concentrations. Except for the significance shown
for the ‘2 mM Ca2+’ sets (p = 0.008), no substantial differences for supplemented Ca2+ concentrations
were noted for independent measurements of maintenance in DPBS vs. PMB. Mean HbA2/HbA0
ratios (±SD) in intact RBCs exposed to DPBS and PMB were 0.029 ± 0.003 and 0.032 ± 0.003, respec-
tively.
3.3. Possible Interrelated Effect of Hypocalcemia on Hb Distribution and RBC Structural and
Metabolic Features
The following sets of experiments were designed to explore (a) whether the chelation
of extracellular Ca2+ triggers Hb pre-membrane redistribution and (b) if the recovery of
physiological concentrations of Ca2+ will rescue the native HbA2 distribution. For this set
of experiments, we collected RBCs into heparin-supplemented tubes, thus keeping the
native extracellular Ca2+ concentration unchanged. EDTA (final concentration of 5 mM)
was then added to the heparinized whole blood samples (Figure 5, upper panel). We
found that chelation of extracellular Ca2+ leads to a significant increase in the membrane
HbA2 fraction. Moreover, when Ca2+ was supplemented after EDTA removal (by the cy-
cles of washes with heparinized plasma) and the cells were incubated for an additional 2
h in the presence of extracellular Ca2+, we found a complete recovery of HbA2 distribution
to that in the initial (pre-EDTA supplemented) plasma. Total Hb content in the membranes
mirrored these changes: Ca2+ isolation decreased the abundance of Hb in the membrane-
bound pool, and the replenishing of Ca2+ led to the recovery of the membrane-bound Hb
fraction (Figure 5, boom panel). These observations were supported by experiments per-
formed using PBS or PMB as an exterior milieu instead of blood plasma. Thus, no specific
blood plasma constituents (except Ca2+) are required to regulate the abundance of Hb in
the pre-membrane pool or its variant-specific distribution.
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
+2mM
Ca2+
+0.5mM
Ca2+
+0.125mM
Ca2+
0.008
0.003
vs
0mM
0.006
vs
0mM
0.001
vs
0mM
0.004
vs
0mM
0.01
vs
0mM 0.005
vs
0mM
0.005
vs
0mM
0.005
vs
0mM
0.007
vs
0mM
0.05
vs
0mM
0mM
Ca2+
+0.25mM
Ca2+
+1mM
Ca2+
PMB
PBS
HbA2/ HbA0 ratio
in RBC membrane
Figure 4.
Dose response to extracellular Ca
2+
of Hb isoform ratio in RBC membrane. Erythrocytes
were exposed to increasing concentrations of Ca
2+
pre-added to Ca
2+
-free DPBS (n = 6) or PMB
(
n=4
). Data are presented as means
±
SD. Significance compared to the corresponding ‘0 mM Ca
2+
’
set of DPBS or PMB was determined using paired Student’s t-test at p
≤
0.05; NS, not significant. Non-
paired Student’s t-test was used to determine the significance of HbA2/HbA0 membrane pools in
DPBS- vs. PMB-exposed RBCs for the same Ca
2+
concentrations. Except for the significance shown for
the ‘2 mM Ca
2+
’ sets (p= 0.008), no substantial differences for supplemented Ca
2+
concentrations were
noted for independent measurements of maintenance in DPBS vs. PMB. Mean HbA2/HbA0 ratios
(
±
SD) in intact RBCs exposed to DPBS and PMB were 0.029
±
0.003 and 0.032
±
0.003, respectively.
3.3. Possible Interrelated Effect of Hypocalcemia on Hb Distribution and RBC Structural and
Metabolic Features
The following sets of experiments were designed to explore (a) whether the chelation
of extracellular Ca
2+
triggers Hb pre-membrane redistribution and (b) if the recovery of
physiological concentrations of Ca
2+
will rescue the native HbA2 distribution. For this
set of experiments, we collected RBCs into heparin-supplemented tubes, thus keeping the
native extracellular Ca
2+
concentration unchanged. EDTA (final concentration of 5 mM)
was then added to the heparinized whole blood samples (Figure 5, upper panel). We found
that chelation of extracellular Ca
2+
leads to a significant increase in the membrane HbA2
fraction. Moreover, when Ca
2+
was supplemented after EDTA removal (by the cycles of
washes with heparinized plasma) and the cells were incubated for an additional 2 h in the
presence of extracellular Ca
2+
, we found a complete recovery of HbA2 distribution to that in
the initial (pre-EDTA supplemented) plasma. Total Hb content in the membranes mirrored
these changes: Ca
2+
isolation decreased the abundance of Hb in the membrane-bound
pool, and the replenishing of Ca
2+
led to the recovery of the membrane-bound Hb fraction
(Figure 5, bottom panel). These observations were supported by experiments performed
using PBS or PMB as an exterior milieu instead of blood plasma. Thus, no specific blood
plasma constituents (except Ca
2+
) are required to regulate the abundance of Hb in the
pre-membrane pool or its variant-specific distribution.
Because of the previously reported contribution of membrane-associated Hb to cellular
stability and metabolic processes, we further tested whether the 2 h of hypocalcemia (by
incubating the cells in a Ca
2+
-free medium or in the presence of EDTA) and the correspond-
ing membrane Hb redistribution would have an effect on these parameters. Examination
of CFA images indicated a barely noticeable impact on the shape and morphology of RBCs
(Figure 6A,B). In addition, a slight effect on RBC hydration (as assessed through separation
on a Percoll density gradient) was found. In parallel, we revealed a significant increase in
membrane permeability (estimated with elevated K
+
leakage) under Ca
2+
-free or chelated
conditions and complete membrane recovery with Ca2+ reconstitution (Table 4).
Cells 2023,12, 2280 10 of 19
Cells 2023, 12, x FOR PEER REVIEW 10 of 19
Figure 5. Involvement of additional extracellular factors in changes in membrane Hb concentration
and isoform distribution under normo- and hypo-calcemic conditions. The erythrocytes were incu-
bated in heparin-preserved plasma or 2 mM Ca
2+
-supplemented PBS or PMB (indicated by various
colors) with or without 5 mM EDTA for 2 h at 37 °C. Erythrocytes were then quickly washed and
incubated for an additional 2 h with EDTA-free plasma or buffers. The corresponding comparisons
for the membrane-bound HbA2 pool (upper panel) and Hb concentration (boom panel) are shown.
Wilcoxon signed-rank test was used for statistical analysis; the data are presented as median ± CI.
NS, not significant. Median HbA2/HbA0 ratio and Hb concentration in intact RBCs were 0.028 ±
0.003 and 20.7 ± 1.06 mM, respectively.
Because of the previously reported contribution of membrane-associated Hb to cel-
lular stability and metabolic processes, we further tested whether the 2 h of hypocalcemia
(by incubating the cells in a Ca
2+
-free medium or in the presence of EDTA) and the corre-
sponding membrane Hb redistribution would have an effect on these parameters. Exam-
ination of CFA images indicated a barely noticeable impact on the shape and morphology
of RBCs (Figure 6A,B). In addition, a slight effect on RBC hydration (as assessed through
separation on a Percoll density gradient) was found. In parallel, we revealed a significant
increase in membrane permeability (estimated with elevated K
+
leakage) under Ca
2+
-free
or chelated conditions and complete membrane recovery with Ca
2+
reconstitution (Table
4).
Figure 5.
Involvement of additional extracellular factors in changes in membrane Hb concentration
and isoform distribution under normo- and hypo-calcemic conditions. The erythrocytes were incu-
bated in heparin-preserved plasma or 2 mM Ca
2+
-supplemented PBS or PMB (indicated by various
colors) with or without 5 mM EDTA for 2 h at 37
◦
C. Erythrocytes were then quickly washed and
incubated for an additional 2 h with EDTA-free plasma or buffers. The corresponding comparisons
for the membrane-bound HbA2 pool (
upper
panel) and Hb concentration (
bottom
panel) are shown.
Wilcoxon signed-rank test was used for statistical analysis; the data are presented as median
±
CI. NS,
not significant. Median HbA2/HbA0 ratio and Hb concentration in intact RBCs were 0
.028 ±0.00
3
and 20.7 ±1.06 mM, respectively.
Table 4.
Effect of hypocalcemia on RBC membrane permeability and metabolic properties. Ery-
throcytes were preincubated in PMB with or without 2 mM Ca
2+
or in Ca
2+
-supplemented PMB
with or without 5 mM EDTA for 2 h at 37
◦
C. In the subsequent experiment, RBCs exposed to
Ca
2+
-supplemented PMB with 5 mM EDTA were quickly washed with Ca
2+
-supplemented PMB and
then incubated for another 2 h at 37
◦
C. All samples were then washed and supplemented with fresh
buffer. Medium K
+
, glucose, and lactate contents were immediately determined via GEM
®
Premier
™
5000 blood gas analyzer (0 h). Samples were then incubated for 4 h with gentle shaking at 37
◦
C and
remeasured. The 4 h vs. 0 h difference was normalized to the total Hb concentration of the sample
(tHb), which was measured at each time point. Significance was determined using paired Student’s
t-test at p≤0.05; NS, non-significant.
Number K+Release, mM
Normalized to tHb
Glucose Consumption,
mM Normalized to tHb
Lactate Efflux, mM
Normalized to tHb
PMB
PMB + 2 mM Ca2+ 10 0.061 ±0.017
0.082 ±0.035 0.03
1.866 ±0.796
1.814 ±0.649 NS
0.184 ±0.049
0.185 ±0.038 NS
PMB + 2 mM Ca2+ 8 0.098 ±0.036 1.614 ±0.517 0.171 ±0.021
PMB + 2 mM Ca2+ + 5 mM EDTA 0.188 ±0.036 <0.001 0.745 ±0.806 0.02 0.098 ±0.032 <0.001
PMB + 2 mM Ca
2+
+ 5 mM EDTA ->
PMB + 2 mM Ca2+ 0.099 ±0.017 1.410 ±0.657 0.137 ±0.019 0.01
Cells 2023,12, 2280 11 of 19
Cells 2023, 12, x FOR PEER REVIEW 11 of 19
(A)
(B)
Figure 6. Extracellular Ca
2+
has a minimal influence on RBC morphology and heterogeneity. The
erythrocytes were preincubated in PMB with or without 2 mM Ca
2+
or in Ca
2+
-supplemented PMB
with or without 5 mM EDTA for 2 h at 37 °C. In the subsequent experiment, RBCs exposed to Ca
2+
-
supplemented PMB with 5 mM EDTA were quickly washed with Ca
2+
-PMB and then incubated for
another 2 h at 37 °C. In panels (A,B), pixeled projected areas were evaluated as described in Section
2.5. The datasets for 5 samples are presented as means ± SD, and no significant differences between
the experimental groups were found. In corresponding studies, only a minimal effect of the treat-
ments was observed on RBC heterogeneity (tested by RBC separation on a Percoll density gradient).
Representative images for each experimental set are shown.
350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800 825 850 875 900 925 950 975
0
2
4
6
8
10
12
14
16
18
20
PMB+2mM Ca2+
Pixel intensity
PMB
RBC, % of total
350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800 825 850 875 900 925 950 975
0
2
4
6
8
10
12
14
16
18
20 PMB+2mM Ca2+
Pixel intensity
PMB+2mM Ca2++5mM EDTA
PMB+2mM Ca2++5mM EDTA ->
PMB+2mM Ca2+
RBC, % of total
Figure 6.
Extracellular Ca
2+
has a minimal influence on RBC morphology and heterogeneity. The
erythrocytes were preincubated in PMB with or without 2 mM Ca
2+
or in Ca
2+
-supplemented PMB
with or without 5 mM EDTA for 2 h at 37
◦
C. In the subsequent experiment, RBCs exposed to
Ca
2+
-supplemented PMB with 5 mM EDTA were quickly washed with Ca
2+
-PMB and then incubated
for another 2 h at 37
◦
C. In panels (
A
,
B
), pixeled projected areas were evaluated as described in
Section 2.5. The datasets for 5 samples are presented as means
±
SD, and no significant differences
between the experimental groups were found. In corresponding studies, only a minimal effect of
the treatments was observed on RBC heterogeneity (tested by RBC separation on a Percoll density
gradient). Representative images for each experimental set are shown.
Cells 2023,12, 2280 12 of 19
No specific effect of the absence of Ca
2+
on metabolic properties (glucose consumption
or lactate release) was observed in the Ca
2+
-added vs. lacking RBCs (Table 4). In contrast,
we observed significant inhibition of both processes with EDTA-mediated chelation and
their recovery with Ca
2+
replenishment. The observed differences in Ca
2+
-free vs. chelating
media may reflect the requirement for additional tri-/bivalent cations for the physiological
regulation of metabolic processes.
3.4. Possible Intracellular Mechanism(s) Governing Ca2+ -Mediated Hb Translocation
We then set out to clarify the possible intracellular mechanism(s) underlying the
observed phenomenon of Ca
2+
-mediated Hb translocation. First, we tested whether the
observed Hb redistribution is activated by Ca
2+
uptake into the cells. We measured the
size and isoform composition of the pre-membrane Hb pool after RBC incubation with
increasing concentrations of Ca
2+
in the presence of the Ca
2+
ionophore A23187 (at 1
0µM
final concentration) (Figure 7). The uptake of Ca
2+
in the cells was confirmed by the
simultaneous monitoring of intracellular Ca
2+
change via flow cytometry using Fluo-4
fluorescence (Table 5). We found a significantly higher fraction of HbA2 in the RBC
membrane Hb pool when the cells were incubated in the presence of 0.125 or 0.5 mM
extracellular Ca
2+
along with A23187. The less pronounced action of the higher (2 mM)
Ca
2+
concentration in the medium could be explained by the shedding of A23187 due to
vesiculation caused by acute Ca2+ overload.
Cells 2023, 12, x FOR PEER REVIEW 13 of 19
Figure 7. Effect of increasing concentrations of Ca
2+
on Hb isoform distribution in the membranes of
RBCs in the presence of Ca
2+
ionophore A23187 (final 10 µM). Data are presented as average ± SD.
Significance of values for RBCs exposed to increasing Ca
2+
levels vs. those for ‘Ca
2+
-free PMB’ was
evaluated. In addition, data for Ca
2+
-exposed RBCs in the presence vs. absence of the ionophore
were compared.
Tabl e 5. Effect of increasing Ca
2+
concentration in the external milieu on intracellular Ca
2+
content in
the presence or absence of Ca
2+
ionophore A23187. Measurements were performed with the intra-
cellular Ca
2+
dye, Fluo-4 AM, and a flow cytometry approach. Results (normalized to ‘0 mM Ca
2+
’
test value) are presented as means ± SD. Significance for each test was determined vs. ‘0 mM Ca
2+
’
using paired Student’s t-test at p ≤ 0.05; NS, non-significant.
Number Fluo-4, Normalized to PMB Result
PMB 5 1
PMB + 0.125 mM Ca
2+
1.136 ± 0.085
0.02
PMB + 0.5 mM Ca
2+
1.178 ± 0.091
0.01
PMB + 2 mM Ca
2+
1.174 ± 0.098
0.02
PMB + 10 µM A23187 0.946 ± 0.088
NS
PMB + 10 µM A23187 + 0.125 mM Ca
2+
0.878 ± 0.105
NS
PMB + 10 µM A23187 + 0.5 mM Ca
2+
5.223 ± 0.427
<0.001
PMB + 10 µM A23187 + 2 mM Ca
2+
6.63 ± 0.731
<0.001
We have tested if Ca
2+
supplementation results in a change in the transmembrane
potential which, in turn, could trigger Hb trafficking to the membrane to balance the mem-
brane charge. We took two approaches to test this hypothesis: (a) using the specific volt-
age-sensitive fluorescent dye DiBAC4(3) [28,29] and (b) by varying KCl concentrations in
the extracellular buffer in the presence of the K
+
ionophore valinomycin [30]. Specifically,
DiBAC4(3) enters depolarized cells with the corresponding binding of intracellular pro-
teins and elevation in fluorescence [31]. Using the first approach, we found small but sig-
nificant Ca
2+
-dose-dependent changes in the transmembrane potential (Figure 8A). Using
the second approach, the transmembrane potential was affected by maintaining the RBCs
in media with increasing concentrations of KCl (Figure 8B). KCl substituted the equivalent
fractions of NaCl in the presence of valinomycin (1 µM final concentration). In accordance
with the Nernst equation, the transmembrane potential in the exposed RBCs increases
with gradually increasing concentrations of KCl to a maximum of + 10 mV in 150 mM of
KCl medium [29]. Intriguingly, we observed reduced and minimally varying membrane
HbA2/HbA0 values in all spectra of the examined NaCl/KCl concentrations, raising
doubts as to the role of transmembrane potential in regulating Hb membrane distribution.
Figure 7.
Effect of increasing concentrations of Ca
2+
on Hb isoform distribution in the membranes of
RBCs in the presence of Ca
2+
ionophore A23187 (final 10
µ
M). Data are presented as av
erage ±SD
.
Significance of values for RBCs exposed to increasing Ca
2+
levels vs. those for ‘Ca
2+
-free PMB’ was
evaluated. In addition, data for Ca
2+
-exposed RBCs in the presence vs. absence of the ionophore
were compared.
We have tested if Ca
2+
supplementation results in a change in the transmembrane
potential which, in turn, could trigger Hb trafficking to the membrane to balance the
membrane charge. We took two approaches to test this hypothesis: (a) using the specific
voltage-sensitive fluorescent dye DiBAC4(3) [
28
,
29
] and (b) by varying KCl concentrations
in the extracellular buffer in the presence of the K
+
ionophore valinomycin [
30
]. Specifically,
DiBAC4(3) enters depolarized cells with the corresponding binding of intracellular proteins
and elevation in fluorescence [
31
]. Using the first approach, we found small but significant
Ca
2+
-dose-dependent changes in the transmembrane potential (Figure 8A). Using the
second approach, the transmembrane potential was affected by maintaining the RBCs in
media with increasing concentrations of KCl (Figure 8B). KCl substituted the equivalent
fractions of NaCl in the presence of valinomycin (1
µ
M final concentration). In accordance
with the Nernst equation, the transmembrane potential in the exposed RBCs increases
with gradually increasing concentrations of KCl to a maximum of + 10 mV in 150 mM of
Cells 2023,12, 2280 13 of 19
KCl medium [
29
]. Intriguingly, we observed reduced and minimally varying membrane
HbA2/HbA0 values in all spectra of the examined NaCl/KCl concentrations, raising
doubts as to the role of transmembrane potential in regulating Hb membrane distribution.
Table 5.
Effect of increasing Ca
2+
concentration in the external milieu on intracellular Ca
2+
content
in the presence or absence of Ca
2+
ionophore A23187. Measurements were performed with the
intracellular Ca
2+
dye, Fluo-4 AM, and a flow cytometry approach. Results (normalized to ‘0 mM
Ca2+’ test value) are presented as means ±SD. Significance for each test was determined vs. ‘0 mM
Ca2+’ using paired Student’s t-test at p≤0.05; NS, non-significant.
Number Fluo-4, Normalized to PMB Result
PMB 5 1
PMB + 0.125 mM Ca2+ 1.136 ±0.085 0.02
PMB + 0.5 mM Ca2+ 1.178 ±0.091 0.01
PMB + 2 mM Ca2+ 1.174 ±0.098 0.02
PMB + 10 µM A23187 0.946 ±0.088 NS
PMB + 10
µ
M A23187 + 0.125 mM Ca
2+ 0.878 ±0.105 NS
PMB + 10 µM A23187 + 0.5 mM Ca2+ 5.223 ±0.427 <0.001
PMB + 10 µM A23187 + 2 mM Ca2+ 6.63 ±0.731 <0.001
Cells 2023, 12, x FOR PEER REVIEW 14 of 19
(A) (B)
Figure 8. Possible influence of transmembrane potential. (A) Effect of increasing Ca2+ concentration
on transmembrane potential was determined using means of the voltage-sensitive dye DiBAC4(3)
and a flow cytometry approach as detailed in Section 2.7. Data were normalized to the ‘0 mM Ca2+’
value, and significance relative to this value is shown. (B) Membrane HbA2/HbA0 ratios in RBCs
exposed to increasing fractions of KCl (0, 50, 100, and 150 mM) replacing the equivalent fractions of
NaCl, in the presence of the K+ ionophore valinomycin (final 1 µM). Data are presented as means ±
SD; minimal differences (all NS, p > 0.05) between all matching tests were examined using paired
Student’s t-test. Mean HbA2/HbA0 ratio (±SD) in intact RBCs in these experiments was 0.031 ± 0.001.
3.5. AE-1 as a Membrane Target for Ca2+-Induced Hb Binding
Previous studies have reported that AE-1 is a primary target membrane molecule for
Hb binding to the membrane [8,14,15]; we tested whether alterations in this exchanger’s
structure or activity would affect Hb distribution. We treated the cells with a well-known
AE-1 inhibitor, DIDS (50 µM, 30 min in 37 °C), or with 2 mM of ZnCl2 (1 h, 25 °C), which,
at this supraphysiological concentration, causes mild AE-1 clustering [32]. Intriguingly,
we found that AE-1 clustering in the presence of Ca2+ returned the HbA2 membrane frac-
tion to the corresponding values of calcium-unexposed RBCs (Figure 9). In contrast, inhi-
bition of AE-1-mediated transport with DIDS minimally affected the Hb variant ratio.
These results suggest the possible involvement of AE-1 in the mechanism of calcium-me-
diated Hb trafficking.
Figure 9. Possible involvement of AE-1 in the observed mechanism. Cells collected in heparin tubes
were washed of plasma and treated in 2 mM of Ca2+-PMB with either 5 mM of EDTA, 50 µM of DIDS
or 2 mM of ZnCl2. Wilcoxon signed-rank test was used for statistical analysis; data for 8 subjects are
0.80
0.85
0.90
0.95
1.00
+2mM
Ca2+
+1mM
Ca2+
+0.5mM
Ca2+
+0.25mM
Ca2+
+0.125mM
Ca2+
0.02
0.04
0.03
NS
NS
PMB
Transmembrane potential,
normalized to PMB (0 mM Ca
2+
) data
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
All NS
NaCl
150 mM
KCl
150 mM
NaCl 100 mM
+KCl 50 mM
NaCl 50 mM
+KCl 100 mM
HbA2/ HbA0 ratio
in RBC membrane
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
PMB+2mM Ca
NS
<0.001
+EDTA +50μM DIDS +2mM ZnCl2
<0.001
HbA2/ HbA0 ratio
in RBC membrane
Figure 8.
Possible influence of transmembrane potential. (
A
) Effect of increasing Ca
2+
concentration
on transmembrane potential was determined using means of the voltage-sensitive dye DiBAC4(3)
and a flow cytometry approach as detailed in Section 2.7. Data were normalized to the ‘0 mM
Ca
2+
’ value, and significance relative to this value is shown. (
B
) Membrane HbA2/HbA0 ratios
in RBCs exposed to increasing fractions of KCl (0, 50, 100, and 150 mM) replacing the equivalent
fractions of NaCl, in the presence of the K
+
ionophore valinomycin (final 1
µ
M). Data are presented
as means
±
SD; minimal differences (all NS, p> 0.05) between all matching tests were examined
using paired Student’s t-test. Mean HbA2/HbA0 ratio (
±
SD) in intact RBCs in these experiments
was 0.031 ±0.001.
3.5. AE-1 as a Membrane Target for Ca2+-Induced Hb Binding
Previous studies have reported that AE-1 is a primary target membrane molecule for
Hb binding to the membrane [
8
,
14
,
15
]; we tested whether alterations in this exchanger’s
structure or activity would affect Hb distribution. We treated the cells with a well-known
AE-1 inhibitor, DIDS (50
µ
M, 30 min in 37
◦
C), or with 2 mM of ZnCl
2
(1 h, 25
◦
C), which,
Cells 2023,12, 2280 14 of 19
at this supraphysiological concentration, causes mild AE-1 clustering [
32
]. Intriguingly, we
found that AE-1 clustering in the presence of Ca2+ returned the HbA2 membrane fraction
to the corresponding values of calcium-unexposed RBCs (Figure 9). In contrast, inhibition
of AE-1-mediated transport with DIDS minimally affected the Hb variant ratio. These
results suggest the possible involvement of AE-1 in the mechanism of calcium-mediated
Hb trafficking.
Cells 2023, 12, x FOR PEER REVIEW 14 of 19
(A) (B)
Figure 8. Possible influence of transmembrane potential. (A) Effect of increasing Ca2+ concentration
on transmembrane potential was determined using means of the voltage-sensitive dye DiBAC4(3)
and a flow cytometry approach as detailed in Section 2.7. Data were normalized to the ‘0 mM Ca2+’
value, and significance relative to this value is shown. (B) Membrane HbA2/HbA0 ratios in RBCs
exposed to increasing fractions of KCl (0, 50, 100, and 150 mM) replacing the equivalent fractions of
NaCl, in the presence of the K+ ionophore valinomycin (final 1 µM). Data are presented as means ±
SD; minimal differences (all NS, p > 0.05) between all matching tests were examined using paired
Student’s t-test. Mean HbA2/HbA0 ratio (±SD) in intact RBCs in these experiments was 0.031 ± 0.001.
3.5. AE-1 as a Membrane Target for Ca2+-Induced Hb Binding
Previous studies have reported that AE-1 is a primary target membrane molecule for
Hb binding to the membrane [8,14,15]; we tested whether alterations in this exchanger’s
structure or activity would affect Hb distribution. We treated the cells with a well-known
AE-1 inhibitor, DIDS (50 µM, 30 min in 37 °C), or with 2 mM of ZnCl2 (1 h, 25 °C), which,
at this supraphysiological concentration, causes mild AE-1 clustering [32]. Intriguingly,
we found that AE-1 clustering in the presence of Ca2+ returned the HbA2 membrane frac-
tion to the corresponding values of calcium-unexposed RBCs (Figure 9). In contrast, inhi-
bition of AE-1-mediated transport with DIDS minimally affected the Hb variant ratio.
These results suggest the possible involvement of AE-1 in the mechanism of calcium-me-
diated Hb trafficking.
Figure 9. Possible involvement of AE-1 in the observed mechanism. Cells collected in heparin tubes
were washed of plasma and treated in 2 mM of Ca2+-PMB with either 5 mM of EDTA, 50 µM of DIDS
or 2 mM of ZnCl2. Wilcoxon signed-rank test was used for statistical analysis; data for 8 subjects are
0.80
0.85
0.90
0.95
1.00
+2mM
Ca2+
+1mM
Ca2+
+0.5mM
Ca2+
+0.25mM
Ca2+
+0.125mM
Ca2+
0.02
0.04
0.03
NS
NS
PMB
Transmembrane potential,
normalized t o PMB (0mM Ca
2+
) data
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
All NS
NaCl
150 mM
KCl
150 mM
NaCl 100 mM
+KCl 50 mM
NaCl 50 mM
+KCl 100 mM
HbA2/ HbA0 ratio
in RBC membrane
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
PMB+2mM Ca
NS
<0.001
+EDTA +50μM DIDS +2mM ZnCl2
<0.001
HbA2/ HbA0 ratio
in RBC membrane
Figure 9.
Possible involvement of AE-1 in the observed mechanism. Cells collected in heparin tubes
were washed of plasma and treated in 2 mM of Ca
2+
-PMB with either 5 mM of EDTA, 50
µ
M of DIDS
or 2 mM of ZnCl
2
. Wilcoxon signed-rank test was used for statistical analysis; data for 8 subjects are
presented as median
±
CI. Significance vs. control ‘PMB + 2 mM Ca
2+
’ dataset is presented, where
NS relates to p> 0.05. Median HbA2/HbA0 ratio (±CI) in intact RBCs was 0.029 ±0.001.
4. Discussion
We examined the regulation of the intracellular distribution of Hb between the mem-
brane and the cytosol, namely, the size of the pre-membrane Hb pool and its isoform
composition. Our main finding was the unique and distinct roles of intracellular and
extracellular Ca
2+
in the control of pre-membrane Hb pool size and composition (Figure 10).
Reversibility of the effects indicated that the pre-membrane Hb pool in the RBCs of healthy
donors is largely populated by non-covalently-bound Hb, both A0 and A2 isoforms, and
not with covalently-bound Hb, as reported previously (see, for instance, Ref. [
8
]). Redis-
tribution of HbA2 and HbA0 variants in response to chelation of extracellular Ca
2+
was a
biphasic process, involving Hb release from the membrane through acute (minute range)–
intensive and then prolonged–gradual stages. Extracellular Ca
2+
supported a higher level
of Hb in the pre-membrane pool with lower HbA2 abundance, and an increase in cytosolic
Ca
2+
induced Hb translocation to the cytosolic compartment. Selectivity for HbA2 control
in the pre-membrane pool may be associated with its higher binding affinity [20].
We also assumed a correlative effect of changes in the extracellular Ca
2+
level on
the RBCs’ physiological changes. We found that Ca
2+
removal, with the corresponding
Hb rearrangement, specifically affects RBC membrane stability and cellular metabolism.
These findings may be crucial for understanding the molecular mechanisms controlling
Ca
2+
-mediated Hb presence in the membrane, mainly because of the possible involvement
of membrane proteins in building the cytoskeleton or directly regulating its stability (dis-
cussed in Section 4.1). However, a deeper exploration of the mechanisms governing the
involvement of the membrane Hb in physiological processes was outside the scope of the
current work and warrants future research.
Cells 2023,12, 2280 15 of 19
Cells 2023, 12, x FOR PEER REVIEW 15 of 19
presented as median ± CI. Significance vs. control ‘PMB + 2 mM Ca
2+
’ dataset is presented, where
NS relates to p > 0.05. Median HbA2/HbA0 ratio (±CI) in intact RBCs was 0.029 ± 0.001.
4. Discussion
We examined the regulation of the intracellular distribution of Hb between the mem-
brane and the cytosol, namely, the size of the pre-membrane Hb pool and its isoform com-
position. Our main finding was the unique and distinct roles of intracellular and extracel-
lular Ca
2+
in the control of pre-membrane Hb pool size and composition (Figure 10). Re-
versibility of the effects indicated that the pre-membrane Hb pool in the RBCs of healthy
donors is largely populated by non-covalently-bound Hb, both A0 and A2 isoforms, and
not with covalently-bound Hb, as reported previously (see, for instance, Ref. [8]). Redis-
tribution of HbA2 and HbA0 variants in response to chelation of extracellular Ca
2+
was a
biphasic process, involving Hb release from the membrane through acute (minute range)–
intensive and then prolonged–gradual stages. Extracellular Ca
2+
supported a higher level
of Hb in the pre-membrane pool with lower HbA2 abundance, and an increase in cytosolic
Ca
2+
induced Hb translocation to the cytosolic compartment. Selectivity for HbA2 control
in the pre-membrane pool may be associated with its higher binding affinity [20].
Figure 10. The proposed model.
We also assumed a correlative effect of changes in the extracellular Ca
2+
level on the
RBCs’ physiological changes. We found that Ca
2+
removal, with the corresponding Hb re-
arrangement, specifically affects RBC membrane stability and cellular metabolism. These
findings may be crucial for understanding the molecular mechanisms controlling Ca
2+
-
mediated Hb presence in the membrane, mainly because of the possible involvement of
membrane proteins in building the cytoskeleton or directly regulating its stability (dis-
cussed in Section 4.1). However, a deeper exploration of the mechanisms governing the
involvement of the membrane Hb in physiological processes was outside the scope of the
current work and warrants future research.
4.1. Possible Membrane Targets for Ca
2+
-Regulated Presence of Hb
The most intriguing question that arises from our data is how Ca
2+
regulates Hb’s
presence in the membrane. Our experiments with A23187 showed that Ca
2+
directly
Figure 10. The proposed model.
4.1. Possible Membrane Targets for Ca2+-Regulated Presence of Hb
The most intriguing question that arises from our data is how Ca
2+
regulates Hb’s
presence in the membrane. Our experiments with A23187 showed that Ca
2+
directly
decreases the protein’s translocation to the membrane. Whether this process is controlled
by the direct interaction of Hb and Ca
2+
(as proposed in [
33
,
34
]), or if there is some essential
involvement of other cytosolic molecules, is still an open question, warranting further
investigation. However, the increase in Hb with elevated Ca
2+
implies that the process is
initiated via stimulation of an extracellular target. Band 3 protein (AE-1) may be viewed as
an ideal candidate, supported by evidence from the literature as well as from the current
study. First, AE-1 is a known target for Hb–membrane binding [
8
,
14
,
15
]. For example,
competition between Hb and metabolic enzymes for attachment to AE-1 is one of the
central regulators of glucose metabolism in RBC [
3
,
5
]. Second, HbA2 has a more positive
charge than the other Hb variants, providing increased affinity for membrane AE-1 [
19
].
Finally, AE-1 itself is a Ca
2+
-binding protein [
35
–
37
]. Taken together, we cannot exclude the
role of AE-1 in these processes, but further studies are needed to confirm the hypothesis.
There may be other membrane components regulated by intracellular Ca
2+
that can
impact the presence of Hb on the membrane. Hb has been reported to interact with
spectrin [
38
], mainly as part of a high-molecular-weight cytoskeletal complex that also
contains ankyrin and band 4.2 [
8
]. These membrane proteins may be direct targets of Ca
2+
,
or at least affected by Ca
2+
-activated proteins [
39
–
41
]. Ca
2+
activation of the calmodulin
4.1R has been found to reduce the latter’s affinity for its cytoskeletal targets, thus affecting
the spectrin/actin network and cell shape [
42
]. Moreover, the possible involvement of
intracellular rearrangement of Na
+
and K
+
in Hb redistribution due to functional alteration
of the Na
+
/K
+
pump should also be considered [
43
]. The specific interactions of Hb
and Ca
2+
with anionic phosphatidylserine [
44
,
45
] may indicate that the more positive
HbA2 variant shares binding sites for Ca
2+
in stabilizing the membrane’s phospholipid
distribution. Therefore, the identification of which Ca
2+
-initiated molecular cascades are
primarily involved in Hb reorganization in the membrane is one of our paramount tasks.
Cells 2023,12, 2280 16 of 19
4.2. Suggestions for Laboratory and Clinical Practice
The current results may have significant clinical importance. One possible conse-
quence might be to reconsider the strategy for choosing specific chelators for routine
blood collection, the maintenance of RBCs prior to laboratory examination, and better
interpretation of the clinical analytical data. Specifically, EDTA is a frequently used metal
chelator for blood sample anticoagulation [
46
]. Due to its higher Ca
2+
ion binding constant,
EDTA could be used at lower concentrations than, for example, citric acid [
47
] (at the
same time explaining the limited effect of citrate on Hb distribution (Figure 1C)). However,
EDTA chelating activity is not limited to the buffer’s cations; although EDTA does not
cross the intact membrane of erythrocytes, it may bind to the external membrane surface
and remove ~90% of the membrane-bound Ca
2+
[
48
]. As a result, some cellular features
initiated by Ca
2+
-binding to the RBC surface or its uptake are immediately altered with
EDTA supplementation. For example, the appearance of hundreds of thin extrusions on
the RBC membrane was observed by Pinteric et al. [
49
] after EDTA supplementation to an
RBC sample. It is important to note that these membrane abnormalities were completely
recovered by Ca
2+
supplementation, meaning that these RBC modifications are due to the
specific removal of Ca2+ and not to the Ca2+-independent side effects of the chelator. This
phenomenon may provide a partial explanation for the observed increase in membrane
permeability observed in the current study (Table 4).
The proposed mechanism is essential for understanding and preventing numerous
post-transfusion events. Routinely used storage solutions are Ca
2+
-free; therefore, massive
transfusion of Ca
2+
-free RBC suspension (especially in severe cases) can cause an acute
decrease in the normal level of Ca
2+
in patients. Because of the dose–response effect of
Ca
2+
on Hb cellular distribution, the subsequent outcome on Hb-associated RBC features
may appear, even with relatively small fluctuations of plasma Ca
2+
in recipients immedi-
ately after blood transfusions. Several post-transfusion vascular complications have been
reported [
50
], and the possible influence of the proposed mechanisms on the pathogene-
sis of these events should therefore be further investigated. This is also relevant for the
advanced attempts to incorporate EDTA-based chelating therapies (for example, the Trial
to Assess Chelation Therapy (TACT) projects) that require detailed information on the
possible changes in RBCs to prevent side-effect complications [51].
5. Conclusions
Novel mechanisms for the Ca
2+
regulation of Hb localization in RBCs are presented.
We show that the total content and variant distribution of Hb are strongly associated with
the presence of Ca
2+
in the extracellular milieu, and any fluctuation in Ca
2+
level may
significantly affect the membrane fraction of Hb. Fluctuations of cytosolic Ca
2+
also impact
the pre-membrane Hb pool resulting in massive transfer of Hb to the cellular cytosol. We
also suggest an interrelationship between the revealed mechanisms and Ca
2+
-mediated
changes in RBC structural and metabolic properties. A detailed delineation of the causes
and consequences of this interaction awaits further clarification.
Supplementary Materials:
The following supporting information can be downloaded at https://www.
mdpi.com/article/10.3390/cells12182280/s1, Figure S1. Temperature-dependence of the HbA2/HbA0
ratios in pre-membrane Hb pool. Table S1. The numerical data related to Figure 1C,D. Table S2. The
numerical data related to Figure 2A. Table S3. The numerical data related to Figure 2B,C. Table S4.
The numerical data related to Figure 3. Table S5. The numerical data related to Figure 4. Table S6.
The numerical data related to Figure 5. Table S7. The numerical data related to Figure 7. Table S8.
The numerical data related to Figure 8B. Table S9. The numerical data related to Figure 9.
Cells 2023,12, 2280 17 of 19
Author Contributions:
Conceptualization, L.L., A.B., Y.F., I.P.-L., A.K., M.G. and C.L.; methodology,
L.L., A.B., G.B., I.P.-L. and C.L.; formal analysis, L.L., A.B. and G.B.; investigation, L.L., S.P., H.Z., G.B.
and C.G.; data curation, L.L., S.P., A.K. and C.L.; writing—original draft preparation, L.L. and A.B.;
writing—review and editing, A.K., M.G. and C.L.; visualization, L.L.; supervision, A.K., H.E., M.G.
and C.L.; project administration, L.L., A.B., H.E., A.K. and C.L.; funding acquisition, A.B., M.G. and
C.L. All authors have read and agreed to the published version of the manuscript.
Funding:
This project is partially funded by the Fondation Botnar as well as by the Baugarten Stiftung,
Susanne & RenéBraginsky Stiftung, and Ernst Göhner Stiftung.
Institutional Review Board Statement:
The study was conducted in accordance with the Declaration
of Helsinki and approved by the Emek Medical Center ethics committee (EMC-0085-21).
Informed Consent Statement:
In view of the specificity of the study’s protocol, informed consent
was not obtained from the subjects whose blood samples were investigated.
Data Availability Statement:
Data are contained within the article. The data presented in this study
are available upon request from the corresponding author.
Acknowledgments:
Special thanks to Eitan Fibach (Hadassah Medical Center, Jerusalem, Israel) for
the highly useful discussions, Mia Levite (Hadassah Medical Center, Jerusalem, Israel) for her kind
gift of the DIBAC4(3) sample, and the personnel of the Pediatric Hematology Unit and the Laboratory
Division at Emek Medical Center for their incredible technical help.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Barvitenko, N.; Adragna, N.; Weber, R. Erythrocyte Signal Transduction Pathways, Their Oxygenation Dependence and Functional
Significance. Cell. Physiol. Biochem. 2005,15, 1–18. [CrossRef] [PubMed]
2.
Kosmachevskaya, O.V.; Topunov, A.F. Alternate and Additional Functions of Erythrocyte Hemoglobin. Biochemistry
2018
,83,
1575–1593. [CrossRef] [PubMed]
3.
Issaian, A.; Hay, A.; Dzieciatkowska, M.; Roberti, D.; Perrotta, S.; Darula, Z.; Redzic, J.; Busch, M.P.; Page, G.P.; Rogers, S.C.; et al.
The Interactome of the N-Terminus of Band 3 Regulates Red Blood Cell Metabolism and Storage Quality. Haematologica
2021
,106,
2971–2985. [CrossRef] [PubMed]
4.
Patel, R.P. Biochemical Aspects of the Reaction of Hemoglobin and NO: Implications for Hb-Based Blood Substitutes. Free Radic.
Biol. Med. 2000,28, 1518–1525. [CrossRef] [PubMed]
5.
Puchulu-Campanella, E.; Chu, H.; Anstee, D.J.; Galan, J.A.; Tao, W.A.; Low, P.S. Identification of the Components of a Glycolytic
Enzyme Metabolon on the Human Red Blood Cell Membrane. J. Biol. Chem. 2013,288, 848–858. [CrossRef]
6. Gell, D.A. Structure and Function of Haemoglobins. Blood Cells Mol. Dis. 2018,70, 13–42. [CrossRef]
7. McMahon, T.J. Red Blood Cell Deformability, Vasoactive Mediators, and Adhesion. Front. Physiol. 2019,10, 1417. [CrossRef]
8.
Welbourn, E.M.; Wilson, M.T.; Yusof, A.; Metodiev, M.v.; Cooper, C.E. The Mechanism of Formation, Structure and Physiological
Relevance of Covalent Hemoglobin Attachment to the Erythrocyte Membrane. Free Radic. Biol. Med.
2017
,103, 95–106. [CrossRef]
9.
Kosmachevskaya, O.v.; Nasybullina, E.I.; Blindar, V.N.; Topunov, A.F. Binding of Erythrocyte Hemoglobin to the Membrane to
Realize Signal-Regulatory Function (Review). Appl. Biochem. Microbiol. 2019,55, 83–98. [CrossRef]
10.
Morabito, R.; Romano, O.; la Spada, G.; Marino, A. H
2
O
2
-Induced Oxidative Stress Affects SO
4=
Transport in Human Erythrocytes.
PLoS ONE 2016,11, e0146485. [CrossRef]
11.
Arashiki, N.; Kimata, N.; Manno, S.; Mohandas, N.; Takakuwa, Y. Membrane Peroxidation and Methemoglobin Formation Are
Both Necessary for Band 3 Clustering: Mechanistic Insights into Human Erythrocyte Senescence. Biochemistry
2013
,52, 5760–5769.
[CrossRef] [PubMed]
12.
Signorini, C.; Ferrali, M.; Ciccoli, L.; Sugherini, L.; Magnani, A.; Comporti, M. Iron Release, Membrane Protein Oxidation and
Erythrocyte Ageing. FEBS Lett. 1995,362, 165–170. [CrossRef] [PubMed]
13.
Remigante, A.; Spinelli, S.; Basile, N.; Caruso, D.; Falliti, G.; Dossena, S.; Marino, A.; Morabito, R. Oxidation Stress as a Mechanism
of Aging in Human Erythrocytes: Protective Effect of Quercetin. Int. J. Mol. Sci. 2022,23, 7781. [CrossRef]
14.
Rauenbuehler, P.B.; Cordes, K.A.; Salhany, J.M. Identification of the Hemoglobin Binding Sites on the Inner Surface of the
Erythrocyte Membrane. Biochim. Biophys. Acta (BBA)-Biomembr. 1982,692, 361–370. [CrossRef]
15.
Sega, M.F.; Chu, H.; Christian, J.A.; Low, P.S. Fluorescence Assay of the Interaction between Hemoglobin and the Cytoplasmic
Domain of Erythrocyte Membrane Band 3. Blood Cells Mol. Dis. 2015,55, 266–271. [CrossRef]
16.
Datta, P.; Chakrabarty, S.; Chakrabarty, A.; Chakrabarti, A. Membrane Interactions of Hemoglobin Variants, HbA, HbE, HbF and
Globin Subunits of HbA: Effects of Aminophospholipids and Cholesterol. Biochim. Biophys. Acta (BBA)-Biomembr.
2008
,1778, 1–9.
[CrossRef] [PubMed]
17.
Shaklai, N.; Ranney, H.R. Interaction of Hemoglobin with Membrane Lipids: A Source of Pathological Phenomena. Isr. J. Med. Sci.
1978,14, 1152–1156.
Cells 2023,12, 2280 18 of 19
18.
Giambona, A.; Passarello, C.; Renda, D.; Maggio, A. The Significance of the Hemoglobin A2 Value in Screening for
Hemoglobinopathies. Clin. Biochem. 2009,42, 1786–1796. [CrossRef]
19.
Steinberg, M.H.; Rodgers, G.P. HbA2: Biology, Clinical Relevance and a Possible Target for Ameliorating Sickle Cell Disease. Br. J.
Haematol. 2015,170, 781–786. [CrossRef]
20.
Fischer, S.; Nagel, R.L.; Bookchin, R.M.; Roth, E.F.; Tellez-Nagel, I. The Binding of Hemoglobin to Membranes of Normal and
Sickle Erythrocytes. Biochim. Biophys. Acta (BBA)-Biomembr. 1975,375, 422–433. [CrossRef]
21.
Wong, P. A Hypothesis on the Role of the Electrical Charge of Haemoglobin in Regulating the Erythrocyte Shape. Med. Hypotheses
2004,62, 124–129. [CrossRef] [PubMed]
22.
Ghashghaeinia, M.; Cluitmans, J.C.; Toulany, M.; Saki, M.; Köberle, M.; Lang, E.; Dreischer, P.; Biedermann, T.; Duszenko, M.;
Lang, F.; et al. Age Sensitivity of NF
κ
B Abundance and Programmed Cell Death in Erythrocytes Induced by NF
κ
B Inhibitors.
Cell. Physiol. Biochem. 2013,32, 801–813. [CrossRef] [PubMed]
23.
Barshtein, G.; Gural, A.; Zelig, O.; Arbell, D.; Yedgar, S. Unit-to-Unit Variability in the Deformability of Red Blood Cells. Transfus.
Apher. Sci. 2020,59, 102876. [CrossRef] [PubMed]
24.
Barshtein, G.; Rasmusen, T.L.; Zelig, O.; Arbell, D.; Yedgar, S. Inter-donor Variability in Deformability of Red Blood Cells in Blood
Units. Transfus. Med. 2020,30, 492–496. [CrossRef]
25.
Barshtein, G.; Pries, A.R.; Goldschmidt, N.; Zukerman, A.; Orbach, A.; Zelig, O.; Arbell, D.; Yedgar, S. Deformability of Transfused
Red Blood Cells Is a Potent Determinant of Transfusion-Induced Change in Recipient’s Blood Flow. Microcirculation
2016
,23,
479–486. [CrossRef]
26.
Peretz, S.; Livshits, L.; Pretorius, E.; Makhro, A.; Bogdanova, A.; Gassmann, M.; Koren, A.; Levin, C. The Protective Effect of the
Spleen in Sickle Cell Patients. A Comparative Study between Patients with Asplenia/Hyposplenism and Hypersplenism. Front.
Physiol. 2022,13, 796837. [CrossRef]
27.
Hildrum, J.M.; Fjeld, B.; Risahagen, S.M.; Bernatek, B.J.; Klingenberg, O. Assessment of Hemoglobin A2 Stability at Room
Temperature during 24 or 25 Days as Measured by High Pressure Liquid Chromatography and Capillary Electrophoresis. Int. J.
Lab. Hematol. 2021,43, e266–e270. [CrossRef]
28.
Balach, M.M.; Casale, C.H.; Campetelli, A.N. Erythrocyte Plasma Membrane Potential: Past and Current Methods for Its
Measurement. Biophys. Rev. 2019,11, 995–1005. [CrossRef]
29.
Moersdorf, D.; Egee, S.; Hahn, C.; Hanf, B.; Ellory, C.; Thomas, S.; Bernhardt, I. Transmembrane Potential of Red Blood Cells
Under Low Ionic Strength Conditions. Cell. Physiol. Biochem. 2013,31, 875–882. [CrossRef]
30.
Hoffman, J.F.; Laris, P.C. Determination of Membrane Potentials in Human and Amphiuma Red Blood Cells by Means of a
Fluorescent Probe. J. Physiol. 1974,239, 519–552. [CrossRef]
31.
Epps, D.E.; Wolfe, M.L.; Groppi, V. Characterization of the Steady-State and Dynamic Fluorescence Properties of the Potential-
Sensitive Dye Bis-(1,3-Dibutylbarbituric Acid)Trimethine Oxonol (Dibac
4
(3)) in Model Systems and Cells. Chem. Phys. Lipids
1994
,
69, 137–150. [CrossRef] [PubMed]
32.
Koshkaryev, A.; Livshits, L.; Pajic-Lijakovic, I.; Gural, A.; Barshtein, G.; Yedgar, S. Non-Oxidative Band-3 Clustering Agents Cause
the Externalization of Phosphatidylserine on Erythrocyte Surfaces by a Calcium-Independent Mechanism. Biochim. Biophys. Acta
(BBA)-Biomembr. 2020,1862, 183231. [CrossRef] [PubMed]
33.
Makhro, A.; Hänggi, P.; Goede, J.S.; Wang, J.; Brüggemann, A.; Gassmann, M.; Schmugge, M.; Kaestner, L.; Speer, O.;
Bogd
anova, A
. N-Methyl-d-Aspartate Receptors in Human Erythroid Precursor Cells and in Circulating Red Blood Cells
Contribute to the Intracellular Calcium Regulation. Am. J. Physiol.-Cell Physiol. 2013,305, C1123–C1138. [CrossRef] [PubMed]
34.
Makhro, A.; Haider, T.; Wang, J.; Bogdanov, N.; Steffen, P.; Wagner, C.; Meyer, T.; Gassmann, M.; Hecksteden, A.; Kaestner, L.;
et al. Comparing the Impact of an Acute Exercise Bout on Plasma Amino Acid Composition, Intraerythrocytic Ca2+ Handling,
and Red Cell Function in Athletes and Untrained Subjects. Cell Calcium 2016,60, 235–244. [CrossRef] [PubMed]
35.
Low, P.S.; Allen, D.P.; Zioncheck, T.F.; Chari, P.; Willardson, B.M.; Geahlen, R.L.; Harrison, M.L. Tyrosine Phosphorylation of Band
3 Inhibits Peripheral Protein Binding. J. Biol. Chem. 1987,262, 4592–4596. [CrossRef]
36.
Harrison, M.L.; Rathinavelu, P.; Arese, P.; Geahlen, R.L.; Low, P.S. Role of Band 3 Tyrosine Phosphorylation in the Regulation of
Erythrocyte Glycolysis. J. Biol. Chem. 1991,266, 4106–4111. [CrossRef]
37.
Passing, R.; Schubert, D. The Binding of Ca
2⊕
to Solubilized Band 3 Protein of the Human Erythrocyte Membrane. Hoppe Seylers
Z. Physiol. Chem. 1983,364, 873–878. [CrossRef]
38.
Datta, P.; Chakrabarty, S.B.; Chakrabarty, A.; Chakrabarti, A. Interaction of Erythroid Spectrin with Hemoglobin Variants:
Implications in β-Thalassemia. Blood Cells Mol. Dis. 2003,30, 248–253. [CrossRef]
39.
Wallis, C.J.; Babitch, J.A.; Wenegieme, E.F. Divalent Cation Binding to Erythrocyte Spectrin. Biochemistry
1993
,32, 5045–5050.
[CrossRef]
40. Hall, T.G.; Bennett, V. Regulatory Domains of Erythrocyte Ankyrin. J. Biol. Chem. 1987,262, 10537–10545. [CrossRef]
41.
Korsgren, C.; Peters, L.L.; Lux, S.E. Protein 4.2 Binds to the Carboxyl-Terminal EF-Hands of Erythroid
α
-Spectrin in a Calcium-
and Calmodulin-Dependent Manner. J. Biol. Chem. 2010,285, 4757–4770. [CrossRef] [PubMed]
42.
Nunomura, W. Regulation of Protein 4.1R Interactions with Membrane Proteins by Ca2+ and Calmodulin. Front. Biosci.
2006
,11,
1522–1539. [CrossRef] [PubMed]
43.
Slatinskaya, O.; Maksimov, G. In the Erythrocyte Hemoglobin Conformation and Distribution during RBC Volume Changes.
Russ. J. Biol. Phys. Chemisrty 2022,7, 297–302. [CrossRef]
Cells 2023,12, 2280 19 of 19
44.
Shviro, Y.; Zilber, I.; Shaklai, N. The Interaction of Hemoglobin with Phosphatidylserine Vesicles. Biochim. Biophys. Acta
(BBA)-Biomembr. 1982,687, 63–70. [CrossRef]
45.
Bucki, R.; Bachelot-Loza, C.; Zachowski, A.; Giraud, F.; Sulpice, J.-C. Calcium Induces Phospholipid Redistribution and
Microvesicle Release in Human Erythrocyte Membranes by Independent Pathways. Biochemistry
1998
,37, 15383–15391. [CrossRef]
46.
Sears, M.E. Chelation: Harnessing and Enhancing Heavy Metal Detoxification—A Review. Sci. World J.
2013
,2013, 219840.
[CrossRef]
47.
Keowmaneechai, E.; McClements, D.J. Influence of EDTA and Citrate on Physicochemical Properties of Whey Protein-Stabilized
Oil-in-Water Emulsions Containing CaCl2.J. Agric. Food Chem. 2002,50, 7145–7153. [CrossRef]
48. Harrison, D.G.; Long, C. The Calcium Content of Human Erythrocytes. J. Physiol. 1968,199, 367–381. [CrossRef]
49.
Pinteric, L.; Manery, J.; Chaudry, I.; Madapallimattam, G. The Effect of EDTA, Cations, and Various Buffers on the Morphology of
Erythrocyte Membranes: An Electron-Microscopic Study. Blood 1975,45, 709–724. [CrossRef]
50.
Hall, C.; Nagengast, A.K.; Knapp, C.; Behrens, B.; Dewey, E.N.; Goodman, A.; Bommiasamy, A.; Schreiber, M. Massive
Transfusions and Severe Hypocalcemia: An Opportunity for Monitoring and Supplementation Guidelines. Transfusion 2021,61,
S188–S194. [CrossRef]
51.
Diaz, D.; Fonseca, V.; Aude, Y.W.; Lamas, G.A. Chelation Therapy to Prevent Diabetes-Associated Cardiovascular Events. Curr.
Opin. Endocrinol. Diabetes Obes. 2018,25, 258–266. [CrossRef] [PubMed]
Disclaimer/Publisher’s Note:
The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.
