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RED CELLS
From the Children's Hospital Oakland Research
Institute and the Lawrence Berkeley National Laboratory, CA.
Several transgenic murine models for sickle cell anemia have been
developed that closely reproduce the biochemical and physiological disorders in the human disease. A comprehensive characterization is
described of hematologic parameters of mature red blood cells, reticulocytes, and red cell precursors in the bone marrow and spleen of
a murine sickle cell model in which erythroid cells expressed
exclusively human Sickle cell disease (SCD) is characterized by
hemoglobin polymerization and sickling of the red blood cells (RBCs)
that, in turn, result in changes in the RBC plasma membrane. One of
these changes is the exposure of phosphatidylserine (PS) on the surface of a subpopulation of sickle RBCs.1,2 PS exposure on the RBC surface may lead to a prothrombotic state in patients with SCD3 and to the recognition and removal of RBCs by
macrophages.4-6 This PS-induced removal could contribute
to the markedly reduced RBC survival observed in sickle cell
anemia.7
In contrast to the anemic state in thalassemia that is largely due to
ineffective erythropoiesis (premature cell death of erythroblasts),
reduced RBC survival is thought to be the major contributor to anemia
in SCD. On the other hand, stress erythropoiesis in SCD may be
responsible for the membrane defects found in sickle erythrocytes and
reticulocytes. PS-exposing RBCs are found at all stages of the RBC life
in SCD, and a significant number of (young) reticulocytes expose PS on
their surface.8 Defects in erythroid cells can be expected
as soon as erythroblasts with sickle hemoglobin are exposed to hypoxic
conditions. Because hemoglobin synthesis begins in the early stages of
erythropoiesis,9 this situation could arise during
erythroid differentiation in hematopoietic organs. This defective
erythropoiesis could result in premature cell death and removal of
erythroblasts and in the appearance of abnormal reticulocytes in
the circulation. To further study this defective erythropoiesis, PS
exposure, and RBC survival, we used transgenic sickle mice developed by
Paszty et al10 as a model for the human disorder.
Several transgenic murine models for sickle cell anemia have been
developed that closely reproduce the biochemical and physiological disorders in the human disease (summarized in 11). Mice
used in this study express only human Mice
Hematologic characterization
Erythrocyte labeling and flow cytometric analysis Murine erythrocytes from tail vein or heart punctures were washed by suspension in HBSM (10 mM HEPES, 165 mM NaCl, pH 7.4) and centrifugation. PS exposure on the cell surfaces was determined by labeling with various fluorescent annexin V conjugates (see figure legends) in buffers containing 2 mM CaCl2 as described earlier.14 For flow cytometric analysis, RBCs were routinely suspended at 1 to 5 × 106 cells/mL in HBSM containing 2 mM CaCl2.Flow cytometric analysis of the reticulocyte fraction was performed by staining of the RNA-containing subset for 30 minutes at room temperature with 50 ng/mL thiazole orange (Aldrich, Milwaukee, WI) in HBSM containing 2 mM CaCl2. This dye was combined with labeling with biotin-annexin V (40 ng/mL) and 2 µg/mL phycoerythrin (PE)-conjugated streptavidin to identify the PS-exposing reticulocytes. Flow cytometry was carried out with a Becton Dickinson FACScalibur (4-color studies) or FACScan (Becton Dickinson, San Jose, CA), and data were analyzed using CellQuest software (Becton Dickinson). As described earlier,14 intact cells were selected by forward and side scatter parameters, and the percentage of positive intact cells was determined from the fluorescence signal in excess of that obtained with a negative (unlabeled) control for each sample. Electron microscopy Human sickle RBCs were obtained after informed consent from a patient with SCD (hemoglobin SS, 12.5% hemoglobin F) and washed in HBS (10 mM HEPES, 145 mM NaCl, pH 7.4). Murine sickle RBCs were washed in HBSM. Cells were suspended at 10% hematocrit in the appropriate HEPES buffers, subjected to deoxygenation under nitrogen gas for 3 minutes, and immediately fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer containing sucrose (pH 7.4, 350 mOsm). Control samples were fixed similarly without prior deoxygenation. Then the samples were washed in cacodylate buffer and fixed in 1% OsO4 in cacodylate buffer with sucrose, added just before use, for 30 minutes. After washing in buffer, fixed samples were dehydrated in ethanol. Cells were suspended in hexamethyldisilazane and dried on a glass coverslip. Then cells were sputter-coated with approximately 150 Å platinum and imaged in the secondary electron mode at 20 kV in an Amray 1000A scanning electron microscope (KLA Tencor, Bedford, MA).Survival studies RBC turnover was studied in sickle and normal mice using biotinylation of the entire RBC cohort at t = 0 and determination of the number of biotinylated cells by flow cytometry. This method provided direct measurement of the RBC replacement per day. Complete biotinylation of all RBCs was achieved by tail vein injection of 100 to 200 µL of 30 mg/mL sulfo-N-hydroxysuccinimide-biotin (sulfo-NHS-biotin; Pierce, Rockford, IL). For flow cytometric analysis, a few microliters blood was obtained by tail vein puncture, and 3 × 106 cells were labeled with 3 µg/mL PE-streptavidin in 0.25 mL HBSM. The small sample size was essential to allow for daily determinations without significant changes in circulating blood mass. Alternatively, to detect the number of PS-exposing cells in the biotinylated subpopulation, we added 75 ng/mL green fluorescence protein (GFP)- annexin V (a kind gift of Joel Ernst, University of California at San Francisco) in addition to PE-streptavidin in HBSM containing 2 mM CaCl2 and analyzed 20 000 of the labeled cells by dual-color flow cytometry.RBC morphology studies were conducted by comparing the number of surviving (biotinylated) discocytes and ISCs with the corresponding unlabeled population. The biotinylated fraction was labeled by incubating the samples with 2µg/mL Oregon green-conjugated streptavidin (Molecular Probes, Eugene, OR) in 0.25 mL HBSM so that the surviving cells could be identified in fluorescent micrographs taken on a model TMD inverted optical microscope (Nikon, Garden City, NY). Corresponding bright-field and fluorescent image fields were obtained by switching between green (546 nm)-filtered transillumination and epi-illumination, respectively, with a filter cube in the epi-light path incorporating a 480-nm excitation filter, a 520-nm emission filter, and a 510-nm dichroic mirror. Relative percentages of ISCs and biotinylated cells were determined by counting at least 600 cells per sample in the bright-field images and identifying biotinylated ISC and discocytes in the corresponding fluorescent images. Bone marrow analysis Precursors were harvested from murine femora and tibia by flushing out the bone marrow in HBSGM (10 mM HEPES, 165 mM NaCl, 5 mM glucose, pH 7.4) containing 1 U/mL heparin and 1 mM EDTA. Spleen cells were suspended by gentle pressing of the tissue, and all cells were filtered through a 70-µm cell strainer.To determine the prevalence of erythroblasts in bone marrow and spleen, we had to distinguish these cells from nonerythroid cells and from mature erythrocytes. We used multicolor flow cytometry combining the RNA dye thiazole orange, PE-conjugated Ter119 (Pharmingen, San Diego, CA), an erythroid marker, and CyChrome-conjugated CD45 (Pharmingen), a probe for early progenitors and white blood cell lineages. Bone marrow or spleen cells at 5 × 106 cells/mL were labeled with 0.32 µg/mL PE-Ter119 and 0.16 µg/mL CyChrome-CD45 in HBSGM for 30 minutes at room temperature, followed by resuspension of the cells in HBSGM containing 25 ng/mL thiazole orange less than 1 hour before analysis by flow cytometry. Intact cells were gated by selection of forward- and side-scatter parameters, and the intact Ter119-positive (erythroid) population was analyzed for the presence of RNA by comparing the thiazole orange fluorescence level to an unlabeled control. To determine PS exposure in the erythroblasts, we used allophycocyanin-conjugated streptavidin in combination with biotin-annexin V and PE-Ter119 and thiazole orange to distinguish the immature erythroid cell population. Annexin V has been commonly used to detect PS exposure in apoptotic cells.15 Bone marrow or spleen cells at 5 × 106 cells/mL were labeled with 0.32 µg/mL PE-Ter119 in HBSGM with 2 mM CaCl2 for 30 minutes at room temperature, followed by resuspension of the cells in HBSGM containing 25 ng/mL thiazole orange and 2 mM CaCl2. In addition, 7-amino actinomycin (7 AAD, 0.6 µg/mL) was added immediately before flow cytometric analysis to exclude dead cells from the population. Intact cells were defined by forward- and side-scatter patterns, and the intact, 7-AAD-negative population was further gated for erythroid (Ter119-positive) and RNA-containing (thiazole orange-positive) events. The percentage of annexin V-binding cells in the RNA-containing erythroblast selection was determined from the fluorescence signal in excess of that obtained with unlabeled control.
The morphology of human sickle cells and RBCs from our murine
sickle model, shown in Figure 1,
illustrates the similarities between human and murine sickle cells at
high and low partial oxygen pressures. Although the murine RBCs are
smaller than their human counterparts, the morphologic features are
indistinguishable. At oxygen tensions of approximately 160 mm Hg (room
air), most cells are discoid, but typical abnormal sickle RBC
morphology, including the presence of ISC, is also observed in both the
human and the murine samples. After deoxygenation, both human and
murine RBCs exhibit markedly distorted cell shapes because of sickle hemoglobin polymerization.
The osmotic deformability profile as determined by ektacytometry
(Figure 2) was shifted to a lower
osmolality range, and the maximal value of the deformability index
attained was significantly lower for murine sickle RBCs than for RBCs
from wild-type mice (Figure 2A). The shift to the left of the osmotic
deformability profile implied that murine sickle cells exhibited
decreased osmotic fragility compared to normal mouse RBC, whereas the
decreased maximal deformability index implied increased dynamic
rigidity of murine sickle RBCs. Similar osmotic deformability profiles were also seen with human sickle cells (Figure 2B). However, though the
osmotic deformability profiles were virtually identical between different mice (the overlay of RBC from 4 different mice is shown in
Figure 2A), individual differences were apparent between samples from
different sickle cell patients, as illustrated by the deformability profiles of 2 patients with SCD shown in Figure 2B. It is important to
note that these 2 patients were neither transfused nor treated with
hydroxyurea because such treatment would have affected the osmotic
deformability profile.
Using a prototype H3 Technicon hematology analyzer (Bayer
Diagnostics), we determined the reticulocyte count and the
distribution of cell volume and hemoglobin concentration of
reticulocytes and mature RBCs. Figure 3
shows typical volume and hemoglobin concentration histograms, and Table
1 indicates the mean values of various cell parameters derived for 40 control and 22 sickle mice, confirming the earlier reported preliminary data.10 Hematocrit levels
of sickle mice were significantly decreased compared to wild-type mice,
and the reticulocyte counts of sickle mice were increased 10-fold
compared to wild-type mice, which was confirmed by flow cytometry using
thiazole orange (data not shown). We did not find a difference in the
distribution of reticulocyte staining intensity between sickle and
normal mice. Volume histography (Figure 3A) showed that mature sickle
cells were smaller than their reticulocyte counterparts, whereas the
hemoglobin concentration histogram showed that the mature RBCs had
higher hemoglobin concentrations than reticulocytes. Furthermore, the
broad distribution of hemoglobin concentration of mature RBC implied
dehydration of sickle cells in the circulation.
Although enhanced erythropoietic activity in sickle mice was obvious
from the high reticulocyte count, additional evidence was obtained by
enumerating erythroblasts in the hematopoietic organs. In contrast to
humans, hematopoietic compensation for anemia in mice takes place
largely in the spleen rather than in the bone marrow. We found that the
sickle mouse spleens were grossly enlarged (1.2-3.0 g) compared with
spleens of normal mice (0.08 g) and contained on average 16-fold more
cells (n = 6) than normal spleens (n = 8). We used multicolor flow
cytometry to estimate the increase in erythropoietic activity in the
sickle mice by assessing the total number of erythroblasts (Table
2). Erythroid cells were defined as those
cells that could be labeled with Ter119, an erythroid marker that
labels murine glycophorin A.16 Erythroblasts were defined
as those cells that labeled with both Ter119 and thiazole orange, an
RNA stain. Because the spleen was analyzed in its entirety, the total
number of erythroid cells in this organ represented the total
spleen-derived erythroid cells. Bone marrow was harvested from femora
and tibia. Although this did not represent all bone marrow, these 2 sources accounted for most marrow-derived erythroblasts. The amount of
erythroid cells was slightly increased in the sickle bone marrow, with
a 1.6-fold increase in relative presence of erythroblasts over cells of
other lineages. On the other hand, the sickle spleens contained
on average 60-fold more immature erythroid cells than normal spleens.
Average total number of immature erythroid cells was 35-fold higher in
sickle cell mice, indicating a massive increase in erythropoiesis to
compensate for anemia. Despite this large increase in erythroid cells,
anemia persisted in these animals, implying that either peripheral RBC destruction was high in these animals or that not all erythroid cells
completed their maturation cycle, or both.
We determined the survival times of murine sickle cells in the
circulation by labeling the entire RBC cohort with biotin and monitoring the disappearance of the biotinylated cells using
streptavidin labeling at several time intervals after biotinylation.
Survival curves plotted in Figure 4 show
that normal mouse RBC removal was linear, as reported
earlier.17 The number of biotinylated cells decreased
approximately 2.5% per day, with 50% surviving at 20 days and
complete disappearance of all labeled cells by 42 days, indicating that
on average 2.5% of the senescent RBC mass was replaced per day. In
contrast, sickle RBCs (Figure 4, SS) showed a rapid exponential decline
in biotinylated cells. The exponential curve was fitted to the equation
A(t) = A0[1
Biotinylation of the cells, in conjunction with fluorescent
streptavidin labeling, provided a tag to identify the surviving fraction in flow cytometric and microscopic studies. We used
fluorescence microscopy to visualize the morphology of the biotinylated
cells (Figure 5) and to track the
surviving populations of ISC and non-ISC relative to the overall cell
count. Figure 6 shows the time-dependent reduction in the total number of fluorescent (biotinylated) cells as
determined by microscopic observation (solid line). These data are in
excellent agreement with the flow cytometry data shown in Figure 4. We
defined ISCs as cells at least twice as long as wide (typical ISCs are
indicated by arrows in Figure 5A). Total percentage of ISCs in the
blood of the sickle mice was constant at approximately 9% (Figure 6,
solid circles). Although the number of biotinylated ISCs decreased in
time (Figure 6, open circles), the rate of this decrease was slower
than the loss of all biotinylated RBCs (Figure 6, open squares). The
inset in Figure 6 depicts the same data differently to indicate that
the percentage of ISCs in the biotinylated RBC population increases
with time. These data indicate an enrichment of ISCs in the surviving
population of RBCs and suggest that the formation of ISCs is related to
the extent of time that a sickle cell spends in the circulation.
Human sickle cells show a subpopulation of RBCs that expose
PS.1,2 This membrane defect is typical for SCD. Similarly, we observed the presence of a subpopulation of PS-exposing RBCs in the
sickle mouse. In the human patient with SCD, the subpopulation of
PS-exposing RBCs can vary from a low of 0.2% to up to
10%.1,2 In contrast, the size of this subpopulation
varied less in the murine sickle cell population, with all sickle mice
showing a significant increase in PS-exposing cells (4.4% ± 1.9%,
n = 11) compared to control mice (0.4% ± 0.2%, n = 19; Figure
7). In addition, double labeling with the
RNA stain thiazole orange showed that reticulocytes bound annexin V in
sickle and in control mice, indicating that PS exposure can also be a
feature of immature circulating RBCs (Figure 6). Similar to the
situation in human sickle cells, the PS-exposing cells form a
heterogeneous population, with some cells entering the circulation
already with PS exposed on the surface while others acquire this defect
after entering the circulation.8
To determine a possible role of PS exposure in premature removal of the
cells from the circulation, we used double-label flow cytometric
analysis with fluorescent annexin V and streptavidin during survival
studies of biotinylated RBCs. Annexin V labeling indicated the PS
exposure on RBCs, and streptavidin labeling determined RBC survival as
shown in Figure 4. Although the total number of annexin V-binding
cells remained constant in time, the relative percentage of annexin
V-binding cells in the biotinylated fraction decreased more rapidly
with time than the total biotinylated fraction (Figure
8). This indicates that PS-exposing cells
are continuously formed in the sickle mouse circulation and are rapidly
removed from the circulation.
The observations that PS-exposing cells are present in reticulocytes
and that they are in fact less prevalent among the old fraction of
cells suggest that these cells are present as a subpopulation of the
erythroblasts of marrow, spleen, or both. In Figure
9, we show that such a subpopulation of
annexin V-binding, PS-exposing cells is indeed present in RBC
precursors of spleen and marrow. PS exposure is also recognized as an
early event in apoptosis,15 and we cannot exclude that a
fraction of the precursors expose PS as a result of the activation of
the apoptotic cascade during harvest
Several transgenic murine models for sickle cell anemia have been
developed that closely reproduce the biochemical and physiological disorders in the human disease (summarized in 11). In this
study, we describe characteristics of the transgenic sickle mice
developed by Paszty et al10 that express only human We find reduced mean corpuscular volume (MCV) and mean corpuscular
hemoglobin (MCH) values in the mature RBC population that are
distinctly different from those in the reticulocyte fraction. This
indicates that the RBC are rapidly remodeled after their release into
the circulation to reduce their volume by approximately 30% (from 50.4 to 37.5 fL). Previously, it was suggested that the low mean corpuscular
hemoglobin concentration (MCHC), in combination with the slight
imbalance in globin synthesis observed in these mice ( Access to early erythroblasts and RBC survival studies has enabled us
to examine the contributions to the anemia of reduced RBC survival or
ineffective erythropoiesis. Ineffective erythropoiesis is thought to
play a minor role in human sickle cell anemia. A recent report
describes ineffective erythropoiesis in SAD mice: transgenic mice that
express human As a comparison, we determined RBC survival in a well-established
strain of thalassemic mice. These Although erythropoiesis in the sickle mice may be mostly effective, reticulocytes show signs of defects during RBC maturation. In normal cells, PS is located exclusively in the inner lipid monolayer of the plasma membrane. In human SCD, a subpopulation of RBCs is found that exposes PS on its surface.1,2 Similarly, approximately 4% of the murine sickle cells show the same feature. Reticulocytes show a comparable percentage of PS-exposing RBCs, suggesting that these cells exposed PS before they entered the circulation. Indeed, when erythroblasts were analyzed, they also showed an increase in PS exposure. Interestingly, exposure of PS on the cell surfaces is a general feature of apoptosis,15 leading to recognition and removal by macrophages.4-6 PS exposure has been linked to ineffective erythropoiesis, or apoptosis of erythroid cells, in thalassemia.22 Although PS exposure is apparent in sickle erythroblasts, it does not seem to result in premature elimination of precursors and ineffective erythropoiesis. The machinery for recognition and removal of such cells may be simply overwhelmed by the highly increased turnout of defective erythroid cells. The RBC survival study using biotinylation allowed us to determine the number of surviving cells as a function of time and to characterize certain features of these cells, including morphologic changes and the presence of PS on the surfaces. Analysis of the number of ISCs surviving over time shows that there is an enrichment of these cells in the older cell fraction. This indicates that the formation of ISCs is correlated with the extent of time in the circulation, which is in line with the hypothesis that these cells are formed because of repeated sickling. Our data do not allow the conclusion that ISC are removed at a faster rate than other cells. However, because reductions in membrane deformability are thought to lead to the removal of RBCs from the circulation, we cannot exclude the possibility that the formation rate of ISCs is even faster than the removal rate. In time, the number of PS-exposing cells is constant (approximately 4%), but PS-exposing cells show a faster turnover than other cells, as indicated by the decreasing fraction of annexin V-positive cells in the biotinylated RBC population. In humans, PS-exposing cells from the dense fraction were not removed faster than the remainder of this population in 8 hours.23 However, the difference in removal rate may have gone undetected using their method. Our data suggest that PS-exposing cells are continuously formed, either before their release into the circulation or during their life in the circulation. PS-exposing cells are more rapidly removed from the circulation than other cells, which is in agreement with the reported role of PS exposure as a recognition signal and a trigger for removal by macrophages.4-6 Apoptosis could be the mechanism underlying PS exposure in precursors, but the mechanism that leads to PS exposure on enucleated, circulating sickle cells is thus far unclear. Although not addressed in our studies, it is possible that similar mechanisms responsible for ISC formation play a role in the generation of PS-exposing cells in older cell populations. It could be triggered by repeated sickling leading to alterations in the membrane that are of a similar nature, as observed in the apoptotic cascade. Hence, RBCs could enter the circulation with PS on the surface, or they could expose PS during their life. Because it is not possible to assess the formation rate of PS exposure in these 2 populations in vivo, the actual removal rate of these cells could be much higher than our data indicate. The high turnover of these cells suggests that the number of PS-exposing cells measured at a given time could reflect the steady state of a short-lived population of RBCs. The presence of the PS-exposing RBCs in sickle cell anemia could have important implications for the physiology of the disease. Recognition and removal4-6 will contribute to anemia. However, it is evident that the phagocyte system is unable to remove PS-exposing cells at the rate that they are formed. Therefore, remaining PS-exposing RBCs are continuously present in the circulation and could lead to an imbalance in hemostasis or altered cell-cell interactions. It is firmly established that PS-exposing surfaces propagate the proteolytic reactions that result in thrombin formation by promoting the assembly of coagulation factors on their surfaces.24,25 PS exposure also plays a role in the feedback inhibition of thrombin formation by activating the protein C pathway. Although these processes are essential for platelet functionality, they are not restricted to this cell type. In human sickle cell anemia, we found a correlation between the risk for stroke and the presence of PS-exposing cells3 and an increase in binding of PS-exposing cells to endothelial cells.26 In addition, significant alterations in other coagulative abnormalities, such as decreased activity of protein C and S and increased presence of anti-PS antibodies,27 may result from the presence of PS-exposing cells in the circulation. In summary, our data show that RBC survival is markedly reduced in sickle cell mice, which is in part compensated by a considerably enhanced erythropoiesis. Furthermore, we show that the PS-exposing RBCs in these mice may play a role in their premature removal from the circulation. The continuous presence of this subset of PS-exposing cells in the circulation could contribute to the hemostatic imbalance and may have a significant impact on the pathophysiology of SCD.
We thank Evelyn Clausnitzer for valuable help with the electron microscopy study and Joel Ernst for the kind gift of GFP-annexin V.
Submitted February 28, 2001; accepted May 2, 2001.
Supported by National Institutes of Health grants HL66355, DK32094, HL20985, and HL31579.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Frans A. Kuypers, Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr Way, Oakland, CA 94609; e-mail: fkuypers{at}chori.org.
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 E. A. Manci, C. A. Hillery, C. A. Bodian, Z. G. Zhang, G. A. Lutty, and B. S. Coller Pathology of Berkeley sickle cell mice: similarities and differences with human sickle cell disease Blood, February 15, 2006; 107(4): 1651 - 1658. [Abstract] [Full Text] [PDF]
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C. J. Wu, L. Krishnamurti, J. L. Kutok, M. Biernacki, S. Rogers, W. Zhang, J. H. Antin, and J. Ritz Evidence for ineffective erythropoiesis in severe sickle cell disease Blood, November 15, 2005; 106(10): 3639 - 3645. [Abstract] [Full Text] [PDF]
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L. De Franceschi, A. Rivera, M. D. Fleming, M. Honczarenko, L. L. Peters, P. Gascard, N. Mohandas, and C. Brugnara Evidence for a protective role of the Gardos channel against hemolysis in murine spherocytosis Blood, August 15, 2005; 106(4): 1454 - 1459. [Abstract] [Full Text] [PDF]
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L. S. Kean, E. A. Manci, J. Perry, C. Balkan, S. Coley, D. Holtzclaw, A. B. Adams, C. P. Larsen, L. L. Hsu, and D. R. Archer Chimerism and cure: hematologic and pathologic correction of murine sickle cell disease Blood, December 15, 2003; 102(13): 4582 - 4593. [Abstract] [Full Text] [PDF]
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Z. Yasin, S. Witting, M. B. Palascak, C. H. Joiner, D. L. Rucknagel, and R. S. Franco Phosphatidylserine externalization in sickle red blood cells: associations with cell age, density, and hemoglobin F Blood, July 1, 2003; 102(1): 365 - 370. [Abstract] [Full Text] [PDF]
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D. W. Emery, E. Yannaki, J. Tubb, T. Nishino, Q. Li, and G. Stamatoyannopoulos Development of virus vectors for gene therapy of beta chain hemoglobinopathies: flanking with a chromatin insulator reduces gamma -globin gene silencing in vivo Blood, August 28, 2002; 100(6): 2012 - 2019. [Abstract] [Full Text] [PDF]
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M. Walsh, R. J. Lutz, T. G. Cotter, and R. O'Connor Erythrocyte survival is promoted by plasma and suppressed by a Bak-derived BH3 peptide that interacts with membrane-associated Bcl-XL Blood, May 1, 2002; 99(9): 3439 - 3448. [Abstract] [Full Text] [PDF]
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| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
, 
, and 
S globin. Red cell
survival was dramatically decreased in these anemic animals, partially
compensated by considerable enhancement in erythropoietic
activity. As in humans, these murine sickle cells contain a
subpopulation of phosphatidylserine-exposing cells that may play a
role in their premature removal. Continuous in vivo generation of this
phosphatidylserine-exposing subset may have a significant impact on the
pathophysiology of sickle cell disease.
(Blood. 2001;98:1577-1584)
3/+ mice
have a heterozygous deletion of both 
(t/T)]e
, solid line), total percentage of ISCs in
the population as counted by microscopy (
), and percentage of ISCs
that are fluorescent (
). Inset shows the percentage of biotinylated
cells with ISC morphology.
ie, after release from their
natural environment. However, comparison of the average percentages of
annexin V-binding cells among sickle precursors (37% ± 17% in
bone marrow, 31% ± 10% in spleen; n = 5) to normal precursors
(18% ± 13% in bone marrow, n = 8; 16% ± 12% in spleen,
n = 5) suggests that the sickle precursor population either contains
a higher subpopulation of these PS-exposing cells or is more
susceptible to the induction of PS exposure.
