From the Institut d'Hématologie et
d'Immunologie, Université Louis Pasteur, Faculté de
Médecine, 4 rue Kirschleger, Strasbourg 67085, France and the
§ Unité 143 INSERM, Hôpital de Bicêtre, Le
Kremlin-Bicêtre 94275, France
Received for publication, August 30, 2000, and in revised form, October 31, 2000
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ABSTRACT |
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The phosphatidylserine transmembrane
redistribution at the cell surface is one of the early characteristics
of cells undergoing apoptosis and also occurs in cells fulfilling a
more specialized function, such as the
phosphatidylserine-dependent procoagulant response of platelets
after appropriate activation. Although an increase in cytoplasmic
Ca2+ is essential to trigger the remodeling of the
plasma membrane, little is known about intracellular signals leading to
phosphatidylserine externalization. Here, the role of store-operated
Ca2+ entry on phosphatidylserine exposure was investigated
in human erythroleukemia HEL cells, a pluripotent lineage with
megakaryoblastic properties. Ca2+ entry inhibitors
(SKF-96365, LaCl3, and miconazole) inhibited store-operated
Ca2+ entry in A23187- or thapsigargin-stimulated
cells and reduced the degree of phosphatidylserine externalization
concomitantly, providing evidence for a close link between the two
processes. In cells pretreated with cytochalasin D, an agent that
disrupts the microfilament network of the cytoskeleton, store-operated Ca2+ entry and phosphatidylserine externalization at the
cell surface were inhibited. In a context where most of the key actors
remain to be identified, these results provide evidence for the
implication of both store-operated Ca2+ entry and
cytoskeleton architectural organization in the regulation of
phosphatidylserine transbilayer migration.
Changes in the cytoplasmic free Ca2+ concentration
([Ca2+]i)1
constitute one of the main pathways by which information is transferred from extracellular signals into various intracellular compartments (1,
2). The induction of receptor-mediated cytosolic Ca2+
signals involves two interdependent and closely coupled events: (i) a
rapid and transient release of Ca2+ stored in the
endoplasmic reticulum, (ii) followed by slowly developing extracellular
Ca2+ entry (1, 3, 4).
In many cell types, including human platelets, depletion of the
intracellular Ca2+ stores induces entry of Ca2+
across the plasma membrane (5), referred to as capacitative or
store-operated Ca2+ entry (SOCE) (6). Cell stimulation by
Ca2+-mobilizing agents triggers the migration of
phosphatidylserine (PS) to the exoplasmic leaflet (7), an
aminophospholipid sequestered in the inner leaflet of the plasma
membrane of nonstimulated cells (8), followed by the shedding of small
vesicles released from the plasma membrane (9, 10). These
microparticles contain surface proteins and cytoplasmic components of
the original cell. Thus, shedding of PS-expressing microvesicles
appears to be closely associated with cell surface externalization of
PS (11). The exposure of PS is a key step for the hemostatic response
(11) and becomes a recognition signal for the phagocytosis of apoptotic cells (12). It has been demonstrated that exposure of PS in erythrocytes and platelets (13) as well as during apoptosis (14) is
dependent on extracellular Ca2+ and that a continuously
elevated [Ca2+]i level may be necessary to
sustain the externalization process (15). Furthermore, Ca2+
influx across the plasma membrane and maximal exposure of
aminophospholipids seem necessary for membrane microparticle formation
in platelets (10). In a previous study (16), we have shown that an
alteration of SOCE is correlated with a defective exposure of PS in
cells isolated from a patient with an extremely rare inherited disorder of PS externalization, the Scott syndrome. Nevertheless, little information regarding the upstream and downstream intracellular signals
leading to PS externalization or phospholipid scrambling has been
provided. Although several hypotheses have been proposed regarding
direct or indirect coupling mechanisms of the endoplasmic reticulum and
the plasma membrane (17, 18), little is known about the intracellular
signals governing SOCE or the Ca2+ channels mediating this
particular form of Ca2+ entry. Recently, Rosado et
al. (19) suggested that SOCE is mediated by actin cytoskeleton in
human platelets.
To examine the significance of SOCE on PS externalization, we have
combined the detection of PS exposure and the measurement of
[Ca2+]i in human erythroleukemia (HEL) cells, a
megakaryoblastic cell line expressing specific receptors found in
platelets (20, 21). We report here that SOCE regulates PS
externalization, and cytoskeletal elements modulate both SOCE and PS
transmembrane migration. The present study opens a new field of
investigation of possible relationships between Ca2+
signaling and the remodeling of the plasma membrane.
Materials--
RPMI 1640 and fetal calf serum were from
Life Technologies (Paisley, UK), and other cell culture reagents were
from BioWhittaker (Walkersville, MD). Ca2+ ionophore A23187
and SKF-96365
(1-[ Cell Culture--
HEL cells constitute a continuous
megakaryoblastic cell line expressing markers such as thrombopoietin,
thrombin receptors, and glycoprotein IIb/IIIa, with some of them being
found in platelets (24-26). HEL were seeded at 1 × 105 cells/ml and cultured in RPMI 1640 (free
Ca2+ concentration = 0.4 mM) supplemented
with L-glutamine (2 mM), 1% (v/v) nonessential
amino acids, sodium pyruvate (1 mM), gentamicin (20 µg/ml), and 10% (v/v) heat-inactivated fetal calf serum, at 37 °C
in humidified 5% CO2 atmosphere. All experiments were
performed with maximal concentrations of inhibitors at which no
cytotoxicity was observed.
Assessment of PS Exposure by Prothrombinase Functional
Assay--
Cell suspensions were centrifuged at 600 × g for 5 min, washed once in Hanks' balanced salt solution,
and resuspended at the concentration of 1 × 105
cells/ml. Cells were preincubated at 37 °C for 15 min with 3 and 10 µM SKF-96365 or 10 µM miconazole and for 30 min with 10 µM CytD. Cells were then stimulated with 2 µM ionophore A23187 for 10 min at 37 °C, and
centrifuged at 12,000 × g for 2 min to separate the
microparticle-containing supernatant from the corresponding cells.
Procoagulant PS exposure in stimulated cells and derived microparticles
was detected, in the presence of 1 mM CaCl2,
using a human prothrombinase assay in which this phospholipid is the rate-limiting parameter promoting the activation of prothrombin by
factor Xa in the presence of factor Va (27). Thrombin generated by the
assembled prothrombinase complex was measured using a chromogenic assay, as described elsewhere (23).
Measurements of [Ca2+]i by Flow
Cytometry--
HEL cells were used at 106 cells/ml. They
were loaded with 3 µM Fluo-3 for 30 min at room
temperature and then washed twice in RPMI 1640 medium supplemented with
CaCl2 (final Ca2+ concentration ~ 1 mM) and resuspended at the same concentration (106 cells/ml, 500 µl/analysis). To study
Ca2+ release and Ca2+ entry separately, the
experiments were performed in the presence of EGTA (1 mM),
able to chelate the totality of Ca2+ in RPMI 1640 medium.
Cells were then stimulated by the different agents, and
CaCl2 was added to restore an extracellular
Ca2+ concentration of ~1 mM, allowing
capacitative Ca2+ entry. Fluo-3 fluorescence was monitored
using a FACScan Becton-Dickinson flow cytometer and the CELLQuest
software. A baseline value was obtained for each sample by fluorescence
measurement for 30 s before addition of pharmacologic agents.
Collection was immediately resumed, then terminated after additional 5 min (~50,000 events). Fluo-3 fluorescence was expressed in arbitrary
fluorescence intensity units and plotted as FL-1 versus
time. To convert these values into absolute
[Ca2+]i, calibration was performed at the end of
each experiment. [Ca2+]i was calculated using the
equation: [Ca2+]i = Kd[(F Flow Cytometry Analysis--
PS probing was achieved using
annexin VFITC (140 nM final concentration).
Incubation at room temperature was allowed to proceed for 10 min before
data acquisition. In some cases, cells were activated in the presence
of 1 mM CaCl2 with 2 µM
Ca2+ ionophore A23187 for 10 min at 37 °C before
addition of annexin VFITC. In another set of experiments,
cell-derived microparticles were separated from activated cells by
centrifugation at 400 × g for 5 min at room
temperature. Under this condition, 100% of cells were eliminated as
well as the larger cell fragments. Annexin VFITC wad added
to the microparticle-containing supernatant. Samples were analyzed
using the FACScan flow cytometer. The forward light scatter and
fluorescence channels were set at logarithmic gains. The forward light
scatter setting was E-01. The identification of cell and microparticle
populations was performed as previously already described (29).
Statistical Analysis--
Results were expressed as mean ± S.E. of at least four separate experiments performed at different
culture stages and/or passages, with no obvious difference attributable
to the culture conditions. Paired Student's t test was used
for the statistical analysis. A p value of <0.05 was
considered significant.
Changes of [Ca2+]i--
To investigate the
changes of [Ca2+]i, HEL cells were loaded with
the Ca2+-binding fluorophore Fluo-3, and emission
fluorescence shifts were monitored by flow cytometry. As shown in Fig.
1, stimulation by the
Ca2+-ionophore A23187 evoked Ca2+ signal with
two characteristic phases: a rapid rise in
[Ca2+]i within a few seconds, followed by a
sustained phase of elevated [Ca2+]i lasting
several minutes, which resembles Ca2+ entry. To determine
whether this sustained Ca2+ phase reflected
Ca2+ entry, cells were treated with SKF-96365, an agent
known to block store-operated Ca2+ channels (30) and
other Ca2+ channels. SKF-96365 by itself significantly
increased (p < 0.05) basal fluorescence intensity,
[Ca2+]i basal values being 43 ± 5 nM, 87 ± 10, and 103 ± 27 nM in the
absence and in the presence of 3 and 10 µM SKF-96365, respectively. At 1 µM, SKF-96365 did not significantly
modify Ca2+ response induced by calcium ionophore A23187
(Table I). As illustrated in Fig. 1,
SKF-96365 treatment reduced the amplitude of the
[Ca2+]i sustained phase in HEL cells stimulated
with A23187 in the presence of external Ca2+ in the medium
(~1 mM). SKF-96365 was responsible for a
dose-dependent inhibition of Ca2+ entry between
3 and 10 µM, with reductions of ~13 and 20%,
respectively (Table I). Above 10 µM SKF-96365,
cytotoxicity precluded further observations (not shown).
In platelets, A23187 has been reported to induce Ca2+
release from intracellular stores, and after sustained depletion,
voltage-independent Ca2+ influx across the plasma membrane
becomes activated (7). To confirm which is the pathway affected by
SKF-96365 in HEL cells, the same experiments were repeated in the
presence of LaCl3, an inhibitor of divalent cationic
channels (31), and miconazole, an imidazole compound able to inhibit
SOCE (32). No significant difference was detected in the basal
fluorescence intensity after LaCl3 treatment
([Ca2+]i basal values were 43 ± 6 and
48 ± 12 nM, in the absence and in the presence of
LaCl3, respectively). However, miconazole increased
significantly (p < 0.05) the basal fluorescence
intensity ([Ca2+]i basal value was 76 ± 10 nM). As illustrated in Table I, LaCl3 (30 µM) or miconazole (10 µM) treatment reduced
Ca2+ entry by ~20 and 15%, respectively. Collectively,
these data suggest that the sustained phase of elevated
[Ca2+]i induced by A23187 in HEL cells is due to
SOCE activation.
Effect of Ca2+ Entry Inhibitors on PS
Externalization--
In the absence of stimulation (Fig.
2A), HEL cells and the
corresponding supernatants showed a basal PS-dependent
prothrombinase activity of 0.01 ± 0.005 and 0.02 ± 0.008 NIH units of thrombin generated per min/ml/2 × 105
cells, respectively. Following treatment with 3 and 10 µM
SKF-96365, these activities increased ~2-fold, testifying to a weak
ability of this agent to perturb PS asymmetric distribution by itself. After Ca2+ ionophore treatment, prothrombinase activity was
~12-fold and ~3.5-fold enhanced in cells and corresponding
supernatants, respectively. SKF-96365 significantly (p < 0.05) affected the development of prothrombinase activity induced by
Ca2+ ionophore with an inhibition of ~40 and 48% in the
presence of 3 and 10 µM SKF-96365, respectively. The same
behavior was observed in the corresponding supernatants.
Prothrombinase activity measurements were also performed in the
presence of LaCl3 and miconazole. LaCl3
interfered in this assay, probably because it was able to form a
coordinate complex with PS (33), making the latter inaccessible for the
assembly of the coagulation factors at the cell surface. As shown in
Fig. 2B, miconazole also decreased the development of
prothrombinase activity induced by A23187 in HEL cells; however, the
inhibition (34%, p < 0.05) was lower than that
observed with 10 µM SKF-96365. Prothrombinase activity
was reduced accordingly in the corresponding supernatants.
To establish whether microparticles from HEL cells released after
A23187 treatment bear exposed PS at their surface, cells and/or
corresponding supernatants were incubated with annexin VFITC, a widely used probe for PS (Fig.
3). Unstimulated HEL cells presented a
basal level of fluorescence concerning ~75% of the cells (Fig.
3B, upper left region), testifying to the
probable weak binding of annexin VFITC to various membrane
components, including phospholipids other than PS, because interaction
may be favored in the presence of Ca2+ at concentration as
high as 1 mM (34). The remaining ~25% of events in the
upper right region of Fig. 3B likely represents spontaneously activated and/or apoptotic cells expressing a proportion of PS at their surface, with the corresponding PS-positive
(i.e. annexin VFITC-positive) microparticle
population in the lower right region of Fig. 3E.
In agreement with prothrombinase assay (Figs. 2A, 2B, and 4C), A23187
induced extensive PS exposure in cells (Fig. 3C), as shown
by the marked shift of ~99% of the cells to the upper right
region, i.e. that of the highest fluorescence
intensities. This was indeed also the case for microparticle shedding
(Fig. 3F), as evidenced by the large proportion of annexin
VFITC-positive events in the lower right region
(91 ± 2%). Hence, microparticles virtually stemming from
activated cells bear accessible PS and are therefore
procoagulant.
TG-evoked Ca2+ Influx--
To establish whether the
Ca2+ entry inhibitors affect Ca2+ release from
intracellular stores or SOCE, HEL cells were initially stimulated with
TG (1 µM), an inhibitor of reticulum endoplasmic Ca2+-ATPases, in the presence of EGTA (1 mM) in
the extracellular medium (Fig. 4A). Under these conditions,
the addition of 1 µM TG to Fluo-3-loaded cells evoked a
transient and slow elevation in [Ca2+]i due to
the release of Ca2+ from internal stores. Subsequent
reintroduction of 1 mM CaCl2 in the external
medium induced a sustained increase in [Ca2+]i,
indicative of SOCE. At 1 µM, SKF-96365 had no effect on
CaCl2-evoked response in TG-exposed cells (not shown).
Relative to controls, the magnitude of SOCE upon CaCl2
addition was significantly reduced (~20%) in 3 or 10 µM SKF-96365-treated cells after stimulation with 1 µM TG (Fig. 4, A and B).
As shown above with SKF-96365, both LaCl3 and miconazole
did not affect Ca2+ release from intracellular stores
(Table II). After CaCl2
reintroduction, these inhibitors were able to significantly reduce SOCE
(~19 and 12%, respectively).
Disruption of the Actin Cytoskeleton Inhibits SOCE and PS Exposure
in HEL Cells--
HEL cells were preincubated with 10 µM
CytD, a widely used membrane-permeant inhibitor of actin
polymerization, which binds to the barbed end of actin filaments (35).
Under these conditions, basal values of [Ca2+]i
were 50 ± 5 and 133 ± 34 nM, in the absence and
in the presence of CytD, respectively, but this difference was not statistically significant. CytD reduced A23187-induced Ca2+
signal by ~20% (Fig. 5A).
As illustrated in Fig. 5B, in the presence of EGTA, CytD
reduced TG-evoked Ca2+ signal (by 32%, p < 0.05), indicating that Ca2+ stores are affected by the
inhibition of actin polymerization. In addition, CytD reduced
CaCl2-evoked response in TG-exposed cells (by 21%,
p < 0.01).
Fig. 5C shows the effect of CytD on PS externalization at
the HEL cell membrane surface. CytD by itself did not significantly modify prothrombinase activity. However, CytD treatment reduced A23187-induced PS exposure by 38% (p < 0.05),
suggesting that PS transmembrane movements are, at least in part,
modulated by actin polymerization. The decrease of prothrombinase
activity in the corresponding supernatants was weaker.
The present study provides evidence that PS externalization is
related to Ca2+ entry in HEL cells, and more specifically
to a particular way of Ca2+ entry, referred to as SOCE. In
addition, we show that both events, SOCE and PS exposure, are
regulated, at least in part, by actin cytoskeleton.
Like platelets and many other nonexcitable cells (5, 36), HEL cells
undergo SOCE, as observed here after CaCl2-induced response
in TG-treated cells. Three common inhibitors of SOCE, SKF-96365,
LaCl3, and miconazole (30-32, 37), partially inhibited capacitative Ca2+ entry in HEL cells without affecting
Ca2+ release from intracellular stores. At the actual
concentrations used, miconazole inhibited less efficiently A23187- and
TG-induced Ca2+ entry compared with SKF-96365 and
LaCl3. However, in several other studies (37, 38), it has
been observed that the inhibitory effect of these compounds differs in
magnitude depending on the cell type and concentration.
Little is known about the intracellular signals leading to PS
externalization. Our results from the present and previous (16) studies
strongly suggest that an alteration of SOCE is correlated with a
reduced PS exposure. Indeed, Ca2+ entry inhibitors,
SKF-96365 and miconazole, which inhibit store-operated Ca2+
channels, are also able to significantly reduce PS exposure. Ca2+ entry inhibitors also reduced prothrombinase activity
in the supernatants, suggesting a Ca2+ dependence for
microvesicle release. These inhibitors were deliberately used at low
concentration to prevent artificial perturbation of the lipid
organization in the plasma membrane, because this may happen in the
presence of membrane-binding molecules (8).
After stimulation, HEL cells and microparticles were both strongly
labeled by annexin VFITC, confirming that a major fraction
of microparticles bore exposed PS (91 ± 2%). Similar results
were obtained in platelets (10, 39, 40), red blood cells (41),
monocytes (29), and endothelial cells (42). This suggests that most if
not all shed vesicles express PS at their surface and may therefore
disseminate membrane-associated procoagulant activity.
The actin microfilaments of the cytoskeleton form a complex network,
providing the structural basis for simultaneous interactions between
multiple cellular structures. For example, the actin-based cytoskeleton
is involved in the control of ion channel activity across the plasma
membrane of different cell types (43, 44). In the present study, CytD
treatment inhibited A23187-induced Ca2+ responses and both
Ca2+ release and entry evoked by TG. Similar observations
have been reported in astrocytes after agonist stimulation, suggesting
that disruption of microfilaments inhibits
receptor/Gi-protein interaction (45). However, here we can
exclude this hypothesis, because HEL cells were stimulated by two
agents, A23187 and TG, involving distinct signaling pathways. It is
likely that the cytoskeleton regulates Ca2+ signaling at
different stages of Ca2+ release and SOCE. In addition, the
results concerning the effect of CytD on Ca2+ release
and/or Ca2+ entry are conflicting. Ribeiro et
al. (46) and Patterson et al. (47) have observed that
CytD did not block TG-induced Ca2+ entry in NIH 3T3 or
smooth muscle cells, respectively. Other authors have observed that
CytD did not alter the agonist-induced Ca2+ release in
vascular endothelial cells, but Ca2+ entry was reduced
(48). Moreover, in human platelets, Ca2+ stores released by
TG were not affected by CytD, but SOCE was greatly reduced by
cytoskeletal depolymerization (19). These discrepancies are probably
related to a wide variability in the cytoskeletal intrinsic properties
as a function of the cell type. Thus, the fact that cytoskeleton
disruption attenuates A23187- and TG-induced Ca2+ release
and entry in HEL cells indicates that cytoskeletal integrity or closely
associated processes are essential for a normal coupling between
Ca2+ stores and SOCE.
The role of cytoskeletal reorganization in PS exposure and
microparticle formation has been emphasized by several groups who studied the relationships between both events in platelets. Inhibition of actin polymerization by CytD affected the shedding of membrane vesicles but had no effect on the redistribution of PS between the two
leaflets of the platelet plasma membrane (49). Other authors (50-53)
suggest that platelet cytoskeletal proteins are involved in the
regulation of membrane lipid asymmetry. However, in the present study,
we have observed that the diminution of Ca2+ influx across
plasma membrane induced by CytD results in a significant reduction of
PS exposure with almost no effect on microparticle shedding. Here, the
partial inhibition suggests that the cytoskeleton plays a modulatory
role rather than a mandatory one in this particular Ca2+
signaling pathway regulating PS exposure at the HEL cell surface. Recently, it has been observed that the actin-cytoskeleton is also able
to modulate the uptake of membrane-derived microparticles by cells
(54). These findings, when added to the results of the present study,
suggest that cytoskeletal elements actively participate in the
intercellular traffic of membrane proteins and, more generally, in the
transcellular exchange of biological information.
HEL cells express platelet-specific membrane glycoproteins (26) and
possess a strong ability of PS externalization following stimulation.
Hence, such cells represent a useful model to investigate platelet
properties. Among the latter, PS-dependent procoagulant response has an essential physiologic significance as demonstrated by
the bleeding tendency in subjects with Scott syndrome, a genetic defect
of PS transmembrane migration (55, 56). This study provides new
insights into the control of PS transbilayer movements by both SOCE and
cytoskeleton organization, and opens a new field of investigation of
possible relationships with Ca2+-dependent
scramblase (57), and ATP-binding cassette (ABC) transporters such as multidrug resistance P-glycoproteins (58), MRP1 (59), or ABC1
(60), suspected to act as lipid translocases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride) were obtained from Calbiochem (La Jolla, CA).
Fluo-3/acetoxymethyl ester (Fluo-3) was from Molecular Probes (Eugene,
OR) and thapsigargin (TG) from Alexis Corp. (San Diego, CA).
Miconazole, cytochalasin D (CytD) and LaCl3 were products
from Sigma Chemical Co. (St. Louis, MO). Human blood coagulation
factors Xa and prothrombin, annexin V and annexin VFITC,
were, respectively, the same as those previously used in our laboratory
(22, 23). Factor V was purchased from Diagnostica Stago
(Asnières, France) and chromogenic substrate
H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline-dihydrochloride (Chromozyme TH) from Roche Diagnostics (Mannheim, Germany).
Fmin)/(Fmax
F)], where Kd is the dissociation
constant of the Ca2+·Fluo-3 complex (400 nM)
(28). Fmax represents the maximum fluorescence (obtained by treating cells with 10 µM A23187), and
Fmin corresponds to the minimum fluorescence
(obtained for ionophore-treated cells in the presence of 1 mM EGTA). F is the actual sample fluorescence. Fluorescence intensities were expressed as the increase in fluorescence with respect to baseline fluorescence intensity before stimulation.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of SKF-96365 on Ca2+
ionophore-induced response. Representative traces of
Ca2+ signal induced by 2 µM Ca2+
ionophore A23187 in the presence of Ca2+ in the external
medium (1 mM), in the absence or in the presence of
SKF-96365 (10 µM). Note that SKF-96365 reduced the
amplitude of the sustained elevation of [Ca2+]i
seen in A23187-stimulated cells. Solid lines represent the
averaged and smoothed data as a function of time.
A23187-induced Ca2+ response in HEL cells
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Fig. 2.
Extent of PS exposure revealed by
prothrombinase activity measured in HEL cells and corresponding
supernatants. Cells were preincubated with SKF-96365 (3 and 10 µM, A) or miconazole (10 µM,
B) for 15 min at 37 °C and were either nonstimulated or
stimulated by 2 µM A23187 for 10 min at 37 °C, in the
presence of 1 mM external CaCl2. Data are
expressed as mean ± S.E. of five to six independent experiments
(each with duplicate samples). *, p < 0.05.
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Fig. 3.
Assessment of PS exposure in nonstimulated
and stimulated HEL cells and derived microparticles by flow
cytometry. PS externalization was revealed through the binding of
140 nM annexin VFITC. When stimulated, cells
were incubated with 2 µM A23187 for 10 min at 37 °C,
in the presence of 1 mM external CaCl2. Derived
microparticles were separated from activated cells by centrifugation as
described under "Experimental Procedures." Top panels,
HEL cells and derived microparticles; bottom panels,
HEL-derived microparticles; A, D, unstimulated;
B, E, unstimulated + annexin VFITC;
C, F, stimulated + annexin VFITC.
Dot plots are representations of forward light scatter
versus annexin VFITC fluorescence signals. Cell
(upper region) and microparticle (lower region)
populations were distinguished according to size reflected by forward
light scatter signals. Fluorescence thresholds were set on the basis of
respective autofluorescence signals, i.e. in the absence of
annexin VFITC.
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[in a new window]
Fig. 4.
Effect of SKF-96365 on TG-evoked
Ca2+ response. A, representative traces
showing the effect of SKF-96365 (10 µM) on
Ca2+ signal induced by 1 µM TG in the
presence of EGTA, and the subsequent reintroduction of 1 mM
CaCl2 in the external medium. Solid lines
represent the averaged and smoothed data as a function of time. It has
to be emphasized that SKF-96365 reduced the amplitude of the sustained
elevation of [Ca2+]i observed in TG-stimulated
cells. B, histograms showing the effect of SKF-96365 (3 and
10 µM) on the increase in Fluo-3 fluorescence evoked by
TG in the presence of EGTA, and the subsequent addition of 1 mM CaCl2 in the external medium. Results
are expressed as the mean of fluorescence ± S.E. of five
experiments. *, p < 0.05.
Effect of LaCl3, and miconazole on TG-evoked Ca2+ entry
View larger version (21K):
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Fig. 5.
The significance of actin cytoskeleton
integrity on Ca2+ entry and externalization of PS.
A, histograms showing the decrease in Fluo-3 fluorescence in
HEL cells stimulated by A23187 (2 µM) in the presence
versus absence of cytochalasin D (CytD, 10 µM). Cells were incubated for 30 min with CytD or not
prior activation by Ca2+ ionophore. B, cells
were preincubated with CytD in the presence of EGTA in the external
medium, with subsequent reintroduction of CaCl2 where
indicated, in the continuous presence of TG. Results are expressed as
the mean of fluorescence ± S.E. of five to seven experiments.
C, prothrombinase activity measured in HEL cells and
corresponding supernatants. Cells were preincubated with CytD (10 µM) for 30 min at 37 °C and were either nonstimulated
or stimulated by 2 µM A23187 for 10 min at 37 °C, in
the presence of 1 mM external CaCl2. Data are
representative of seven independent experiments (each with duplicate
samples). *, p < 0.05; **, p < 0.01.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported in part by grants from the Institut National de la Santé et de la Recherche Médicale and the Université Louis Pasteur.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by a doctoral fellowship from the Ministère de la Recherche et de la Technologie, France.
Supported by a fellowship from the Fondation pour la Recherche
Médicale, France.
** To whom correspondence should be addressed: Institut d'Hématologie et d'Immunologie, Faculté de Médecine, 4 rue Kirschleger, 67085 Strasbourg, France. Tel.: 33-3-88-24-33-39; Fax: 33-3-88-25-58-83; E-mail: secr600@pharma.u-strasbg.fr.
Published, JBC Papers in Press, November 13, 2000, DOI 10.1074/jbc.M007924200
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ABBREVIATIONS |
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The abbreviations used are:
[Ca2+]i, cytoplasmic free Ca2+
concentration;
SOCE, store-operated Ca2+ entry;
PS, phosphatidylserine;
SKF-96365, 1-[-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole
hydrochloride;
Fluo-3, fluo-3/acetoxymethyl ester;
TG, thapsigargin;
CytD, cytochalasin D;
annexin VFITC, annexin V conjugated
with fluorescein isothiocyanate;
ABC, ATP-binding cassette;
HEL cells, human erythroleukemia cells.
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