Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1785-1792
Targeted Inactivation of Murine Band 3 (AE1) Gene Produces a
Hypercoagulable State Causing Widespread Thrombosis In Vivo
By
Hani Hassoun,
Ying Wang,
John Vassiliadis,
Mohini Lutchman,
Jiri Palek,
Leo Aish,
Irene S. Aish,
Shih-Chun Liu, and
Athar H. Chishti
From the Department of Biomedical Research, St Elizabeth's Medical
Center, Tufts University School of Medicine, Boston, MA.
 |
ABSTRACT |
Only 5% to 10% of band 3 null mice survive the neonatal period. To
determine the cause of death, 3 adult and 11 newborn band 3 null mice
were submitted for histopathologic examination. All but 1 pup showed
evidence of thrombosis including: (1) large thrombotic lesions in the
heart, which were partially organized, calcified in some fields, and
endothelialized, indicating a process that developed premortem (3 of 3 adults and 6 of 11 pups). (2) Subcapsular necrotic areas in the liver
suggestive of premortem ischemic events caused by arteriolar occlusions
(8 of 11 pups). (3) Large vein thrombi (4 of 11 pups). To investigate
the etiology of this hypercoagulable state, we have used the Russell's
viper venom test (RVV) to show that red blood cells (RBCs) from band 3 null mice significantly shorten the RVV clotting time of normal plasma
in a dose-dependent fashion, whereas RBCs from normal mice have no
effect, suggesting that the membrane of band 3 null RBCs provides a
suitable surface for activation of the prothrombinase complex. Using
flow cytometry, we have examined the phosphatidylserine (PS)-specific
binding of fluorescein isothiocyanate (FITC)-annexin V to normal and
band 3 null RBCs. A subpopulation of cells (3% to 5% of RBCs) with increased FITC-annexin V binding was detected in band 3 null RBCs as
compared with normal RBCs. Furthermore, the entire cell population of
band 3 null RBCs shows a measurable increase in the mean fluorescence intensity, suggesting that band 3 null RBCs may have increased PS
exposure on the outer membrane leaflet. These findings are further
supported by direct fluorescence microscopy of normal and band 3 null
RBCs labeled with FITC-annexin V. Based on these observations, we
postulate that the high mortality of band 3 null mice may be related to
a hypercoagulable state, which appears to originate from changes in the
phospholipid composition of the membrane leading to PS exposure on the
outer leaflet.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
BAND 3 IS ONE OF the most abundant
integral proteins of the red blood cell (RBC) membrane. With 
1.2 × 106 copies per RBC, band 3 constitutes 25% to
30% of the total protein mass in the purified membrane (for review,
see Tanner1 and Low2). The principal role of
band 3 is to function as an anion exchange protein (AE1) and to provide
mechanical stability to the plasma membrane. The mature protein,
consisting of 911 amino acids,3,4 is composed of two
structural domains, which can be readily cleaved by trypsin and
chymotrypsin, retaining their distinct functions. The
52-kD membrane spanning domain mediates the anion
transport function of band 3 thus allowing efflux of HCO3
from the cell in exchange for
Cl-.5 The amino terminal 43-kD cytoplasmic
domain harbors association sites for several cytoplasmic and
membrane-associated proteins. These proteins include (1) several
glycolytic enzymes whose activity is modulated by their binding to band
36-8; (2) hemichromes that are produced by oxidation and
denaturation of hemoglobin and whose binding to band 3 leads to
clustering of band 3 in the membrane9-11; (3) ankyrin whose
binding to band 3 is believed to anchor the erythrocyte membrane
skeleton to the lipid bilayer12-15; and (4) protein 4.2 and
protein 4.1 whose associations with the cytoplasmic domain of band 3 have functional implications for the stability of the membrane.
We and others have recently developed murine animal models in which the
expression of erythroid band 3 has been eliminated via targeted
mutagenesis.16,17 Although no fetal loss occurs among band
3 null mice, only 5% to 10% of the mice survive the neonatal period.
The cause of death is currently unknown. Adult band 3 null mice, which
survive the critical neonatal period, display severe hemolytic anemia
with characteristic spherocytic RBCs. The band 3 null RBCs have an
increased osmotic fragility indicating a low surface area-to-volume
ratio due to a marked loss of membrane surface. The loss of membrane is
presumed to occur through the shedding of vesicles, an observation
substantiated by electron micrographs of band 3 null RBCs, which
display vesicles and rod-like membrane structures expanding from the
cell surface. These findings are also corroborated by observations made
of RBCs from cattle with a naturally occurring homozygous nonsense
mutation of the band 3 gene, which exhibit similar
properties.18 To investigate the cause of high mortality,
we have performed a detailed histopathologic analysis of band 3 null
mice. Our results show a striking propensity for thrombosis in newborn
and adult band 3 null mice. Mechanistically, the complete loss of band
3 appears to disrupt the phospholipid asymmetry of the red
blood cell membrane, thereby generating a membrane
surface suitable for the activation of the coagulation cascade.
| |
MATERIALS AND METHODS |
Collection and isolation of RBCs.
Whole blood (200 to 300 µL) was collected from control and band 3 null adult mice in heparin-coated glass tubes by retroocular puncture
and immediately washed once with phosphate-buffered saline (PBS). After
centrifugation, the cells were collected and resuspended in 300 µL of
PBS. The cells were then centrifuged at 1,000g for 5 minutes in
a pipette tip sealed at the pointed end. After centrifugation, the
buffy coat was removed by clipping the open end of the tip, discarding
a generous top layer, and RBCs were collected from the bottom by
clipping the sealed pointed end. The isolated RBCs were washed twice in
PBS. An aliquot of the cells was then used for complete blood count
(CBC) and Wright-Giemsa staining. At this stage, the RBC samples are
platelet- and white cell-depleted.
Hematologic analysis of adult band 3 null mice.
Previously we reported hematologic measurements on blood collected from
2-day old pups.16 Here we report hematologic measurements on adult band 3 null mice (129/SvJ strain), which suffer from thrombotic lesions. It is worth noting that because of accelerated in
vitro clotting, the blood from band 3 null mice has to be collected in
the presence of an excess of citrate-phosphate-dextrose buffer anticoagulant (30% vol/vol). The following values were corrected for
the dilution factor. RBC count: wild-type (WT), 8.0 ± 0.5 × 106/µL; band 3 null, 2.6 ± 0.4 × 106/µL. Hemoglobin: WT, 11.9 ± 0.4 g/dL; band 3 null,
3.8 ± 0.3 g/dL. Hematocrit: WT, 42.2% ± 1.5%; band 3 null,
15.9% ± 1.6%. Reticulocytes: WT, 2.3% ± 0.3%; band 3 null,
56.0% ± 10%. Platelets: WT, 471 × 103/µL;
band 3 null, 539 × 103/µL.
Histopathologic evaluation.
Adult band 3 null mice were killed and major organs including heart,
liver, spleen, kidneys, brain, lungs, stomach, colon, small intestine,
and esophagus were dissected and fixed in buffered formalin. Newborn
pups were killed and fixed in buffered formalin. Sections of organs
were examined by light microscopy after staining with hematoxylin and
eosin.
Russell's viper venom (RVV) test.
The RVV test (American Diagnostica Inc, Greenwich, CT) is a one-stage
prothrombinase assay in which the conversion of factor X to its active
form, factor Xa, is catalyzed by RVV in the presence of calcium and
phospholipids. We have adapted this assay, as described previously,19,20 to determine the effect of membrane
phospholipids from normal and band 3 null RBCs on the clotting time of
normal plasma. Aliquots of citrated platelet-poor plasma (150 µL)
were incubated at 37°C with 20 µL of PBS containing predetermined
numbers of normal or band 3 null RBCs. After 2 minutes of incubation, 150 µL of RVV test solution containing RVV and calcium were added. The time required for the formation of a fibrin clot was measured with
a fibrometer.
Measurement of phosphatidylserine (PS)-mediated binding of
fluorescein isothiocyanate (FITC)-annexin V to RBCs by flow-cytometry.
Annexins specifically bind to acidic phospholipids, particularly PS.
Flow-cytometric analysis of FITC-annexin V binding to the surface of
platelets, platelet-derived microvesicles,21 cells
undergoing apoptosis,22 and pathologic
RBCs23-25 has been used to quantify PS exposure on the
outer membrane leaflet. We have used this assay to detect PS exposure
on the surface of normal and band 3 null RBCs using FITC-annexin V
(Annexin-V-Fluos; Boehringer Mannheim, Indianapolis, IN). RBCs were
collected as described above and immediately processed for labeling
with FITC-annexin V. Aliquots of washed RBCs (2 × 106
cells) were incubated with FITC-annexin V in 200 µL of a buffer containing 10 mmol/L HEPES/NaOH, pH 7.4, 140 mmol/L NaCl, and 5 mmol/L
CaCl2 (buffer A). After incubation for 15 minutes at room
temperature, the RBCs were aspirated into a flow cytometer (Coulter
EPICS XL model Y44299; Coulter, Fullerton, CA) for
analysis (excitation wavelength 488 nm, emission wavelength 545 nm). It is important to point out that membrane vesicles and ghosts were "gated out" using parameters that allow detection of erythrocytes exclusively; the size of membrane vesicles is below the detection level
and the forward light scatter of erythrocyte ghosts is beyond the
window under examination. This conclusion was reached by studying ghosts and vesicles in independent experiments (data not shown).
Fluorescence microscopy analysis of FITC-annexin V binding to RBCs.
RBCs were collected, washed twice in the labeling buffer A, and
incubated with FITC-annexin V as described above. The cells were
collected by centrifugation, placed between siliconized slide and
coverslip, and examined by fluorescence microscopy.
| |
RESULTS |
Band 3 null mice display widespread thrombosis suggesting an underlying
hypercoagulable state.
Full-length band 3 (AE1) is believed to be expressed only in RBCs,
while a truncated form of the AE1 protein is found in
kidneys.1 Our gene targeting construct was engineered to
selectively inactivate the transcription of the erythroid band 3 gene.16 To investigate the cause of excessive mortality
among band 3 null mice, 11 newborn pups and 3 adult mice that survived
the neonatal period were killed and submitted for histopathologic
examination of major organs including heart, liver, spleen, kidneys,
brain, lungs, stomach, colon, small intestine, and esophagus.
Thrombotic lesions around the atrioventricular valves and within the
atrial or ventricular chambers were detected in all 3 adult mice and 6 of 11 newborn pups (Fig 1A through C). The
thrombotic lesions are large, partially organized, calcified in some
fields, and endothelialized, indicating a subacute process that
developed premortem. Large vein thrombi were detected in four pups (Fig
1D and E). In addition, the liver of 8 band 3 null pups showed
subcapsular necrotic areas (Fig 1F), suggesting premortem acute
ischemic events caused by small arterial occlusions. All but 1 of the
band 3 null mice examined showed evidence of thrombotic lesions. The
wide-spread occurrence of thrombotic lesions strongly suggests an
underlying hypercoagulable state in band 3 null mice. The considerable
size of these thrombi and their critical location in organs such as the
heart suggest that thromboembolic events may constitute the major cause
of death in band 3 null mice.
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| Fig 1.
Histopathologic analysis of band 3 null mice. Band 3 null
pups and organs from band 3 null adult mice were fixed in buffered
formalin for histopathologic examination. Tissues were stained with
hematoxylin and eosin. (A and B) (original magnification × 20) show
characteristic thrombi in the atrial and ventricular cavities. Similar
lesions were observed in nine of 12 mice examined. A higher
magnification of one thrombus in (C) (original magnification × 60)
clearly shows endothelial lining of the organized thrombus, indicating
premortem development of the lesion. (D) (original magnification × 40) shows two thrombi in a medium-size vessel in the lung parenchyma.
(E) (original magnification × 40) shows a large organized thrombus in
the portal vein in one adult band 3 null mouse. (F) (original
magnification × 40) shows a liver section from a band 3 null pup
revealing subcapsular hepatocellular necrosis suggesting an ischemic
event due to a small arterial occlusion. M, myocardium; T, thrombus;
RA, right atrium; RV, right ventricle; LA, lung alveoli; H,
hepatocytes; N, necrosis.
|
|
An increased exposure of PS is found on the surface of band 3 null
RBCs.
Because band 3 is the most abundant integral protein of the RBC
membrane and because it has been ascribed a flippase
activity,26-28 we speculated that the hypercoagulable state
of band 3 null mice might be related to an altered phospholipid (PL)
asymmetry in the membrane of RBCs. In normal human RBCs, the major
phospholipids of the membrane are asymmetrically distributed between
the two bilayers (for review, see Zwaal and Schroit29 and
Williamson and Schlegel30). The cholinephospholipids
(sphingomyelin [Sph] and phosphatidylcholine [PC]) are
predominantly found in the outer lipid leaflet, whereas the
aminophospholipids (phosphatidylserine [PS] and
phophatidylethanolamine [PE]) are mainly confined to the inner lipid
leaflet. This asymmetric distribution of the phospholipids is the
result of a dynamic equilibrium whereby phospholipids are constantly
exchanged between the two lipid bilayers by active transport
mechanisms. An altered phospholipid asymmetry on the membrane leading
to the exposure of PS on the outer leaflet is known to provide a
suitable surface for activation of the coagulation cascade and may
potentially contribute to the hypercoagulable state of band 3 null
mice. We have investigated this possibility using the following
complementary approaches.
The RVV test was used to determine the effect of RBCs from normal and
band 3 null adult mice on the clotting time of normal plasma. The
effect of normal and band 3 null RBCs on the time required for fibrin
formation is shown in Fig 2. RBCs from
normal adult mice have no effect on the RVV clotting time even at high concentrations, whereas RBCs from adult band 3 null mice significantly shorten the RVV clotting time in a dose-dependent and saturable fashion. In agreement with previously reported
results,19,20 these findings suggest that the phospholipid
organization in normal erythrocytes does not provide a suitable
catalytic surface for activation of the prothrombinase complex. In
contrast, the membrane of band 3 null RBCs provides a procoagulant
surface that enhances the activation of the prothrombinase complex
through the exposure of PS on its outer lipid leaflet.
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| Fig 2.
RVV test. Aliquots (150 µL) of citrated platelet-poor
plasma were mixed with 20 µL of PBS containing increasing numbers of
normal ( ) or band 3 null ( ) RBCs from adult mice. After
incubation at 37°C for 2 minutes, 150 µL of RVV test solution
containing RVV and calcium was added. The time required for the
formation of a fibrin clot was measured with a fibrometer. Note that
RBCs from normal mice have no effect on the RVV clotting time, whereas
RBCs from band 3 null mice significantly shorten the RVV clotting time.
The values shown are the mean of four independent experiments ± standard deviation (SD).
|
|
To further demonstrate surface exposure of PS on band 3 null RBCs, we
examined the specific binding of FITC-annexin V to RBCs using flow
cytometry. Binding of FITC-annexin V to the surface of platelets and
platelet-derived microvesicles has been previously used to document PS
exposure upon stimulation with various agonists.21 The same
approach has also been used to detect PS exposure on the surface of
cells undergoing apoptosis22 and has allowed the
identification of a subpopulation of RBCs with increased surface exposure of PS in patients with sickle cell anemia.23-25
Using flow cytometry, the possibility of abnormal PS exposure on the surface of RBCs from adult band 3 null mice was examined. As shown in
Fig 3A, the background fluorescence of
normal RBCs incubated with FITC-annexin V is attributed to the
nonspecific binding of FITC-annexin V to the surface of the cells. Gate
D defines the fluorescence intensity exceeding the level of nonspecific
binding and is arbitrarily set to encompass less than 0.5% of normal
RBCs. Flow cytometric analysis of band 3 null erythrocytes (Fig 3C) showed a subpopulation of cells (3.8% of the total cell population in
the experiment shown; range, 3% to 5% in four independent
experiments) exhibiting an increased FITC-annexin V binding as
reflected by a fluorescence intensity that is above the defined
background level (ie, included within gate D). Importantly, when only
the cell population included in gate F (ie, entire cell population excluding cells within gate D) is considered, the band 3 null erythrocytes show a 1.2-fold increase in fluorescence intensity when
compared with normal erythrocytes. This experiment, which results are
summarized in a tabular form in Fig 3, is representative of four
independent experiments that yielded similar findings.
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| Fig 3.
Flow cytometric analysis of FITC-annexin V binding to
RBCs. RBCs from normal (+/+) and band 3 null ( /) mice were
examined at the time of collection (0 hour, [A] and [C]) and after
36 hours (B and D) of incubation in PBS. The cells were washed three
times in PBS before each measurement. (A) Shows the nonspecific
background fluorescence of normal RBCs shortly after collection. Gate D
defines the fluorescence intensity exceeding the level of nonspecific
binding and is arbitrarily set to encompass less than 0.5% of normal
RBCs. (C) Shows a subpopulation of band 3 null RBCs included within
gate D (3.8% of the total population in the experiment shown; range,
3% to 5% in four independent experiments with increased FITC-annexin
V binding. (B) Shows that normal RBCs show no increase in FITC-annexin
V binding when examined 36 hours after collection. The subpopulation of
erythrocytes included in gate D remains below 1%. In contrast, the
percentage of band 3 null RBCs included in gate D increases to 15.7%
at 36 hours (in the experiment shown; range, 15% to 20% in four
independent experiments. Importantly, the entire population of band 3 null RBCs shows an increase in FITC-annexin V binding as reflected by
the doubling of the mean fluorescence of the cell population included
within gate F (entire cell population excluding cells in gate D),
whereas no significant change is detected in normal cells. This
experiment, which results are summarized in a tabular form, is
representative of four independent experiments that yielded similar
findings.
|
|
There remains the possibility that some RBC ghosts and vesicles may be
present among the RBCs and conceivably contribute to the procoagulant
activity of the RBC population detected by the RVV test. However, it is
important to mention that in the flow cytometry studies, vesicles and
ghosts have been "gated out" using parameters that focus
exclusively on erythrocytes. The size of membrane vesicles is below the
level of detection, and the forward light scatter of erythrocyte ghosts
is above the window under examination. We have confirmed this fact by
studying purified ghosts and vesicles in independent experiments (data
not shown).
It is pertinent to mention here that RBCs isolated from band 3 null
mice contain a higher percentage of reticulocytes than their normal
counterparts (56.0% ± 10% v 2.3% ± 0.3%). We have examined the effect of elevated reticulocytosis on the RVV clotting time, as well as on annexin binding as assessed by flow cytometry. The
results are as follows: (1) RBCs isolated from normal pups contain 30%
to 40% reticulocytes and yet show no propensity to shorten the RVV
clotting time and no increase in the annexin V binding as measured by
flow cytometry. Therefore, the properties of neonatal RBCs, as assessed
by these two methods, are identical to those of adult normal mice that
contain only 2% to 3% of reticulocytes. (2) Normal mice were treated
with subcutaneous injections of phenylhydrazine for 5 days to boost the
percentage of reticulocytes to 50%. Again, RBCs from
phenylhydrazine-treated mice had no effect on the RVV clotting time.
These data strongly suggest that an increased reticulocytosis cannot
account for the abnormal findings observed in the band 3 null RBC
population.
These data, together with the results of the RVV test, suggest that
band 3 null RBCs, when examined shortly after collection, contain a
subpopulation of cells with increased PS exposure on the outer membrane
leaflet. Furthermore, the entire cell population exhibits an increased
affinity for FITC-annexin V binding, thus reflecting that band 3 null
erythrocytes as a whole exhibit more PS on their outer lipid leaflet.
Based on these results, we postulate that the altered phospholipid
asymmetry in the plasma membrane may be responsible for the
procoagulant activity of band 3 null RBCs in vivo.
Finally, fluorescence microscopy was used to visualize FITC-annexin V
binding to normal and band 3 null RBCs. As shown in Fig 4C, normal RBCs do not show detectable
binding of FITC-annexin V, whereas a subpopulation of band 3 null RBCs
shows a conspicuous membrane-associated fluorescence indicating an
abnormal exposure of PS on the cell surface (Fig 4D).
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| Fig 4.
Fluorescence microscopy analysis of FITC-annexin V
binding to RBCs. RBCs were examined after 36 hours of incubation in PBS
to facilitate the detection of abnormal cells due to progressive loss
of phospholipid asymmetry. Washed RBCs were incubated with FITC-annexin
V at room temperature. After two washings in PBS, RBCs were collected
by centrifugation at 1,000g and placed between siliconized
slide and coverslip. Normal and band 3 null RBCs were examined by phase
contrast microscopy (A and B) and fluorescence microscopy (C and D).
Note that normal RBCs do not bind FITC-annexin, whereas some band 3 null RBCs show a conspicuous membrane-associated fluorescence
reflecting FITCannexin V binding.
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Band 3 null red blood cells undergo a progressive loss of
phospholipid asymmetry.
The exposure of PS on the outer lipid leaflet serves as a recognition
signal for capture and clearance of cells by macrophages. Therefore,
RBCs that have lost the normal phospholipid asymmetry are likely to be
rapidly cleared from circulation in vivo. We hypothesized that as cells
circulate in vivo, the randomization of phospholipids may slowly
progress over time, thus triggering macrophage recognition and
clearance. Because the macrophage clearance mechanism is absent in
vitro, we anticipated that band 3 null RBCs may show a progressive loss
of phospholipid asymmetry when compared with normal cells. We tested
this hypothesis by analyzing in vitro the binding of FITC-annexin V to
normal and band 3 null erythrocytes after incubation of the cells on
ice, for 36 hours, in PBS. As shown in Fig 3A and B, normal RBCs show a
steady level of fluorescence when examined over a period of 36 hours.
The subpopulation of erythrocytes showing a level of fluorescence
intensity exceeding the level of nonspecific binding (included in gate
D) remains below 1% over the course of 36 hours of incubation on ice.
In contrast, the percentage of band 3 null RBCs showing a fluorescence intensity above background increases steadily from 3.8% shortly after
collection (0 hour) to 15.7% at 36 hours (in the experiment shown;
range, 15% to 20% in four independent experiments) (Fig 3C and D).
Importantly, the entire population of band 3 null cells included within
gate F shows a nearly twofold increase in the mean fluorescence level,
whereas no significant increase is observed in normal cells. This
experiment, which results are summarized in a tabular form in Fig 3, is
representative of four independent experiments that yielded similar
findings. In addition, we have examined the adenosine triphosphate
(ATP) content of band 3 null RBCs and have excluded the
possibility that the loss of phospholipid asymmetry in these cells is
simply due to depletion of intracellular ATP (data not shown). This
conclusion is further buttressed by the observation of a similar
increase in annexin binding to band 3 null RBCs occuring even when the
cells are incubated in CPDA (citrate/phosphate/dextrose/adenine). In
summary, these observations support our hypothesis that RBCs from band
3 null mice undergo a progressive loss of membrane phospholipid
asymmetry in vitro.
| |
DISCUSSION |
Membrane anionic phospholipids play a crucial role at several levels of
the coagulation pathway. These phospholipids provide an essential
attachment surface for coagulation factors, thereby promoting their
assembly into potent enzymatic complexes and enhancing their catalytic
activity. These coagulation factors include the tissue factor/factor
VII complex of the extrinsic pathway,31,32 as well as the
tenase and prothrombinase complexes of the intrinsic pathway.33 PS has been shown to be the most effective
phospholipid in supporting this procoagulant function, and the presence
of a negatively charged PS on the cell surface produces a million fold
increase in the rate of thrombin formation. Although the proteins
responsible for the phospholipid translocation across the membrane have
not yet been identified, several studies have shown that the
phospholipid asymmetry in the membrane is regulated by three major
protein activities: (1) The ATP-dependent aminophospholipid translocase
activity is characterized by its ability to transport PS and PE from
the outer to the inner leaflet (flip)34-36; (2) an
ATP-dependent floppase activity allows the transport of both
aminophospholipids and cholinephospholipids from the inner to the outer
leaflets (flop)35,37,38; and (3) a scramblase activity is
known to disrupt the lipid asymmetry within minutes in the presence of
increased cytoplasmic calcium.39,40
The results reported here suggest that the phospholipid asymmetry is
altered in the membrane of band 3 null RBCs. These abnormal erythrocytes show an excessive exposure of PS on the surface as assessed by prothrombinase and FITC-annexin V binding assays. We
postulate that the altered phospholipid distribution may play a role in
the development of a hypercoagulable state in the band 3 null mice.
Indeed, loss of phospholipid asymmetry has previously been implicated
in the thrombogenic potential of pathologic RBCs. Sickle erythrocytes
are known to accelerate clotting in vitro,19 and the
deoxygenation of RBCs from sickle cell patients results in a reduced
transbilayer movement of PS41 leading to loss of
phospholipid asymmetry. Furthermore, a subpopulation (2% to 3%) of
sickle cell RBCs exhibits a substantial increase in PS exposure when
assessed by flow cytometry analysis of fluorescent annexin V
binding.24,25 It has been suggested that even a small number of abnormal sickle cells with altered phospholipid asymmetry could induce a substantial thrombogenic effect and may contribute to
the hypercoagulable state observed in sickle cell anemia.25 Similarly, the loss of phospholipid asymmetry has been implicated in
the increased incidence of thrombotic events observed in patients with
thalassemia.42,43 RBCs from thalassemic patients examined by the prothrombinase assay display a procoagulant activity indicating an excessive exposure of PS on the outer leaflet.42
In addition to its modulating effect on the coagulation pathway, the
loss of phospholipid asymmetry in the membrane has two other major
consequences: (1) it results in recognition and clearance of altered
RBCs by macrophages.44 This phagocytosis can be completely inhibited by artificial PS vesicles, suggesting that the presence of PS
on the surface of RBCs may represent the sole recognition signal for
attachment and clearance of these cells by macrophages.45 Incorporation of PS in the outer leaflet exceeding 1 to 2 mol% was
determined to be the threshold level beyond which abnormal RBCs are
recognized and cleared from circulation in vivo.46,47 A
similar mechanism for the clearance of apoptotic cells has also been
demonstrated.22,48 (2) The loss of phospholipid asymmetry has been correlated with vesiculation of the plasma membrane. The
influx of calcium in platelets and RBCs induces shedding of vesicles
from the plasma membrane.21,49-51 This event is synchronous with the development of a procoagulant surface due to the loss of
phospholipid asymmetry in the membrane of remnant
platelets.49 Loss of phospholipid asymmetry and
vesiculation are also recognized synchronous events of cells undergoing
apoptosis. Furthermore, the translocation of PS to the outer leaflet
precedes and facilitates membrane fusion.49,50 Because
blebbing and shedding of microvesicles entails fusion of apposing
segments of plasma membrane, it has been proposed that membrane fusion
and vesiculation are two consequences of the loss of phospholipid
asymmetry in the membrane.52-54
Here, we propose a model that provides an explanation for the
hypercoagulable state and the RBC phenotype of band 3 null mice. The
complete loss of band 3 produces changes in the phospholipid composition of the membrane leading to an accelerated loss of phospholipid asymmetry in circulating RBCs. The consequent exposure of
PS on the cell surface results in the activation of the prothrombinase complex by providing a procoagulant anionic phospholipid surface, thereby generating a hypercoagulable state. The loss of membrane asymmetry could also contribute to the shedding of membrane vesicles thereby leading to the spherocytic shape of band 3 null erythrocytes. Because these membrane vesicles are also likely to retain the altered
phospholipid distribution, membrane vesiculation may accentuate the
procoagulant activity in vivo. Finally, the PS exposure on the surface
of band 3 null erythrocytes is likely to trigger their clearance by
macrophages, accounting for the shortened life span of these cells in
vivo.
The proposed model, which ascribes to band 3 a role in the maintenance
of the membrane phospholipid asymmetry, is consistent with the recently
reported flippase function of band 3.26-28 The evidence for
the flippase activity of band 3 is based on several observations. The
translocation of anionic amphiphiles
(1-palmitoyl-sn-glycero-3-phosphomethanol and
5-aminonaphtalene-2-sulfonic acid) is hindered by several established
inhibitors of band 3-mediated anion exchange including stilbene
disulfonates (H2 DIDS or DIDS).26 The translocation of
these lipids was accelerated after external proteolysis of band 3 with
papain, which cleaves band 3 and affects its anion transport
function.26 A similar role of band 3 has also been described for the flip of a long chain amphiphilic anion,
5-(N-decyl)aminonaphtalene-2-sulfonate (DENSA).27
Likewise, the translocation of several mono- and divalent anionic
fluorescent phospholipids was modulated by selected inhibitors of the
anion exchange function of band 3 and by treatment with
papain.28 However, it is worth noting that the proposed model may be more complex in view of the recently published data reporting thrombosis as a major complication in two murine models of
hereditary spherocytosis in which the primary genetic defects involved
and 
spectrin.55
This model is also consistent with several studies that suggest an
interaction between membrane proteins and surrounding phospholipids. Nuclear magnetic resonance measurements have provided evidence for at
least two phospholipid domains in the presence of membrane proteins;
one resembles normal phospholipid bilayer, whereas the other domain
appears to be motionally restricted. These studies are consistent with
phospholipids tightly bound to the membrane proteins and undergoing
rotational diffusion. It is intriguing to note that glycophorin A,
which is missing in the membrane of band 3 null RBCs,56
preferentially associates with the anionic phospholipids PS and
PI.57-60
In summary, we have provided evidence that band 3 null mice have a
hypercoagulable state, which is likely to be responsible for the high
neonatal mortality. The data presented are consistent with the notion
that band 3, either directly or in association with other proteins,
modulates the phospholipid distribution in the membrane of
erythrocytes. However, there remains the possibility that altered
phospholipid asymmetry in the membrane of other cells (ie, platelets
and endothelial cells), where small amounts of the AE1 transcript have
been detected, may also contribute to the overall hypercoagulable state
of band 3 null mice.
| |
FOOTNOTES |
Submitted February 6, 1998;
accepted April 28, 1998.
Supported by Grants No.HL 51445 and CA 66263 (to A.H.C.) and KO8
HL02720 (to H.H.) from the National Institutes of Health. A.H.C. is an
established investigator of the American Heart Association.
Address reprint requests to Athar H. Chishti, PhD,
ACH4, St Elizabeth's Medical Center, 736 Cambridge St, Boston, MA
02135.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Donna-Marie Mironchuk for the artwork.
| |
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Abstract
Introduction
Abstract
