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J Biol Chem, Vol. 275, Issue 8, 5987-5996, February 25, 2000
From the Apoptosis is a physiological program for the
deletion of cells in which caspases govern events leading to safe
clearance by phagocytes. However, a growing weight of evidence now
suggests that not all forms of programmed cell death are
caspase-dependent. We now report a complete and
constitutive but caspase-independent program for the specific
phagocytic clearance of intact effete platelets, anucleated blood cells
of critical importance in health and disease. Platelets aged in
vitro not only exhibited increased expression of proapoptotic Bak
and Bax but also evidenced constitutive diminution of function such as
decreased aggregation to ADP, which was accelerated by culture in the
absence of plasma. This abrogation of cell function in plasma-deprived
platelets was associated with morphological and biochemical features
similar to those of granulocyte apoptosis, that is, cytoplasmic
condensation, plasma membrane changes including exposure of
phosphatidylserine and the granule protein P-selectin, and recognition
by phagocyte scavenger receptors. However, and in contrast with
constitutive death of other inflammatory blood cells by apoptosis,
these events were not affected by caspase inhibitors, nor was there
evidence of caspase-3 activation either by hydrolysis of analog peptide
substrates or Western blot analysis, serving to emphasize that neither
programmed cell death nor clearance by phagocytes need involve caspases.
Apoptosis has attracted intense scrutiny as a self-contained and
physiological program for deletion of unwanted cells (1, 2). Caspase
activation has emerged as a key effector mechanism responsible for many
of the classical phenomena of apoptosis (3-5) including the first
biochemical marker of this type of cell death, endonuclease activation
(6, 7). Furthermore, caspases have been persuasively implicated in
directing plasma membrane changes that lead to the key physiological
outcome of apoptosis, the nonphlogistic recognition and uptake of the
intact dying cell by phagocytes (8).
However, the study of caspase-mediated cell death by apoptosis has been
dominated by experiments that have frequently relied on artificially
induced death in transformed cells and that have rarely paid attention
to their recognition and clearance by phagocytes. Nevertheless, these
criticisms have been addressed in studies of primary blood cells
freshly isolated from healthy human donors (9). For example, neutrophil
granulocytes purified by methods designed to minimize artifactual
activation (10) undergo constitutive apoptosis that clearly directs
specific recognition by phagocytes (11, 12). Furthermore, not only is
there evidence that "machinery" caspases such as caspase-3 are
activated as neutrophils aged in culture undergo constitutive apoptosis
(13-15), but it is also possible to inhibit both apoptosis and
recognition by phagocytes using broad spectrum caspase inhibitors such
as zVAD-fmk1 (16, 17).
Indeed, in this system, zVAD-fmk inhibits even the very earliest plasma
membrane changes that lead to binding of aged neutrophils by phagocytes
(17).
The platelet is another blood cell with a short in vivo
half-life (18). Platelets are critical for normal hemostasis, but disorders of platelet number and function are common, giving rise to a
range of bleeding or thrombotic disorders, including stroke and
myocardial infarction. Given their importance, it is remarkable that
there has been very little study of the constitutive death program that
likely accounts for platelet deletion in vivo. An important
preliminary study (19) suggested that platelets may undergo an
apoptotic program because increases in the expression of proapoptotic
members of the Bcl-2 family of death-regulating proteins were observed
following treatment with ionomycin, a calcium ionophore that induces
apoptosis in a range of cell types, most notably lymphocytic cells (20,
21). Interestingly, this study showed that ionomycin-induced increases
in Bax and Bak, as well as the cell surface expression of PS, were
unaffected by zVAD-fmk and were hence caspase-independent.
To investigate whether platelets exhibited a constitutive death
program, freshly isolated platelets were cultured for up to 24 h
at 37 °C. In the presence of plasma we observed an increase in
levels of proapoptotic Bax and Bak, which, in keeping with an earlier
report (19), was consistent with the suggestion that platelets might
engage a death program. In support of this, we also observed a
constitutive loss of aggregation and spreading functions. By depriving
platelets of plasma, a putative source of survival factors, not only
was loss of function accelerated, but there was also revelation of a
cell death program characterized by cytoplasmic condensation, retention
of plasma membrane integrity, display at the cell surface of
phosphatidylserine and P-selectin, and specific recognition by
phagocyte scavenger receptors. However, there was no evidence of
caspase-3 activation, emphasizing that constitutive cell death in
platelets represents a complete but caspase-independent program leading
to phagocyte clearance of intact effete cells.
Materials--
All chemicals were of analytical reagent grade
and purchased from Sigma unless stated otherwise. Percoll was obtained
from Amersham Pharmacia Biotech; sodium citrate solution was from
Pharma Hameln GmbH (Hanover, Germany); HBSS without Ca2+
and Mg2+, pH 6.4, RPMI 1640, Iscove's modified Dulbecco's
medium, and supplements (penicillin, streptomycin, glutamine, and fetal
bovine serum) were from Life Technologies, Inc.; FITC and/or
phycoerythrin-conjugated mAbs to CD61 (clone BL-E6) and CD62P (clone
CRC81) were from Caltag Laboratories (TCS Biologicals Ltd., Botolph
Clayton, UK); anti-CD42a (clone GRP-P) was from Serotec Ltd
(Kidlington, UK); unconjugated anti-CD62P clone CRC81 and G1-4 were
from Caltag Laboratories and Ancell Corporation (Bayport, MN),
respectively; FITC-conjugated annexin-V was from BioWhittaker UK Ltd
(Wokingham, UK); pan interleukin-1 Platelet Isolation and Culture--
Freshly drawn venous blood
was obtained from aspirin-free healthy donors, and PRP was prepared
following citration and centrifugation at 300 × g for
20 min. PPP was prepared from PRP by centrifugation at 1200 × g. Washed platelets were prepared by diluting PRP with 5 volumes of HBSS, pH 6.4, containing EDTA (4 mM) into a
round-bottom capped polystyrene centrifuge tube before centrifugation
at 280 × g for 20 min. Platelets were resuspended and
washed in HBSS, pH 6.4, before finally resuspending in either PPP,
Iscove's Dulbecco's modified Eagle's medium, or HBSS, pH 6.4. Platelets were maintained at 37 °C in a closed (capped) tube at
3 × 108/ml.
Following isolation, for those experiments involving phagocytic
recognition of aged platelets, platelets were first incubated in HBSS
at 5 × 109/ml for 10 min with 1.8 µM
CM-Orange. Unincorporated dye was removed with two washes of HBSS, pH
6.4, before resuspending at 5 × 108/ml in HBSS, pH
6.4. Platelet preparations were routinely checked by microscopy for the
presence of aggregates, which if found resulted in the experiment being discarded.
Platelet Aggregation Experiments--
Platelet aggregation
studies were performed in a PAP4 aggregometer (Bio-Data Corporation) in
which 0.5-ml aliquots of platelets, resuspended in PPP, were incubated
for 2 min while stirring at 37 °C before the direct addition of
agonist. Prior to aggregation experiments, washed platelets were
harvested by centrifugation at 280 × g for 20 min and
resuspended in autologous PPP. Agonists used were ADP (10 µM), thrombin (10 units/ml), platelet-activating factor
(10 µM), and U46619 (10 µM). In some
instances, platelets were preincubated for 1 min with the
anti-aggregating reagent MK852 (10 µM) prior to the
addition of agonist.
Platelet Adhesion and Cell Spreading on Collagen--
Glass
microscope slides were coated with either collagens I or IV (100 mg/ml
solutions in PBS, pH 7.4). After 30 min the slides were washed
exhaustively with PBS before applying a volume of suspended platelets
in cultured media. Following a 20-min incubation, the slides were
flicked free of media and nonadherent cells and snap-frozen with
methanol and acetone (1:1). The slides were then air-dried before
staining with phalloidin-FITC in PBS (2 µg/ml). Slides were examined
by epifluorescent microscopy (Nikon Diaphot 300).
Sample Preparation for Transmission Electron
Microscopy--
Cell suspensions were prepared for electron microscopy
by first washing the platelets with PBS and fixing with 2.5% (v/v) glutaraldehyde in PBS for at least 1 h at 4 °C. Cells were then pelleted at 280 × g before resuspending in a sodium
cacodylate buffer, pH 7.4, containing 2.5% glutaraldehyde and leaving
overnight. Samples were recentrifuged at 280 × g, the
supernatant was removed, and the cells were overlaid with a minimal
volume of PRP-derived serum, which in turn was overlaid with the
cacodylate buffer for at least 1 h at room temperature. The pellet
was then transferred to fresh fixative and treated as normal resected
tissue for TEM.
Monocyte Isolation and Culture--
Human monocytes were
isolated from freshly drawn venous blood following citration, dextran
sedimentation, and plasma-Percoll density gradient centrifugation as
described previously (10, 11). Human monocyte-derived macrophages
(M Phagocytic Recognition of Aged Platelets--
Platelets labeled
with CM-Orange and aged in culture were washed free of conditioned
media and resuspended in HBSS before addition to a prewashed monolayer
of adherent phagocyte cell lines cultured in 24-well plates. Typically
5 × 107 platelets were incubated with 1 × 105 phagocytes at 37 °C for 10 min for platelets aged in
the absence of plasma and 30 min for platelets aged in citrated plasma.
Following the incubation period, the phagocyte monolayer was washed
free of noninteracting platelets, and any adherent platelets were
removed by treatment with trypsin at 37 °C for 5 min followed by 5 mM EDTA at 4 °C, to recover the human M Immunolabeling and Fluorescence-activated Cell Sorter Analysis of
Platelets--
Immunofluorescent labeling of intact platelets for
surface expression of CD42a, CD61, or CD62P was typically performed by resuspending 5 µl of cultured platelets with 40 µl of the
appropriate FITC-conjugated mAb (diluted 1:1000 with 10% new born calf
serum in PBS) for 10 min before adding 400 µl of
fluorescence-activated cell sorter sheath fluid and sampling by flow
cytometry using a single laser FACScan (Becton-Dickinson, Mountain
View, CA).
Intracellular immunolabeling of the Bcl family of proteins was
performed following fixation and permeabilization of the platelets with
PermeaFix (1 ml/5 × 108 platelets for 30 min on ice).
Excess fixative was then removed with two washes of PBS before
resuspending with 10% new born calf serum in PBS at 5 × 108/ml. 5 × 106 platelets were then
incubated overnight with neat antibodies to Bak (1 µl), Bax (1 µl),
Bcl-2 (1 µl), Bcl-x (1 µl), and Mcl-1 (1 µl) or their appropriate
negative controls. All primary antibodies were detected with
FITC-conjugated F(ab')2 fragments of sheep anti-rabbit
polyclonal antibodies or goat anti-mouse polyclonal antibodies from Sigma.
Phosphatidylserine exposure by intact platelets was determined by
resuspending 5 µl of cultured platelets in 400 µl of HBSS containing 10 mM Ca2+ and 10 µg/ml of
FITC-conjugated annexin-V. All labeling steps were maintained at
4 °C.
SDS-Polyacrylamide Gel Electrophoresis and Protein
Blotting--
For detection of caspase-3, platelets were lysed by
resuspending in SDS Laemmli sample buffer and immediately boiled for 5 min. For cytochrome c detection, washed platelets were
resuspended in ice-cold 10 mM Hepes buffer (containing 1 mM EDTA, 1 mM 1,10-phenanthroline, 1 mM phenylmethylsulphonylfluoride, 1 mM
benzamidine, 10 µM pepstatin, 10 µM
leupeptin, 10 µM antipain, pH 8.0) and lysed following
two rounds of freeze-thaw. The lysed platelets were then centrifuged at
13,000 × g to yield a cytosolic supernatant (S13), and
the pellet was resuspended in lysis buffer before centrifuging again and dissolving the pellet in 1% TX-100 and recentrifuging at
13,000 × g to yield soluble protein from intact
mitochondria (T13). The S13 and T13 fractions were then precleared with
a control IgG and protein G-agarose before immunoprecipitating
cytochrome c with clone 6H2.B4 and boiling in SDS sample
buffer (Laemmli). Protein was analyzed on the basis of equal numbers of
extracted cells rather than on the amount of protein loaded.
Immunodetection of transblotted protein to polyvinylidene difluoride
membranes was performed as described previously (13).
Spectrophotometric Assay for Lactate Dehydrogenase--
Aged
washed platelets were incubated overnight and pelleted by
centrifugation at 280 × g. The supernatant was
removed, and the pellet was resuspended to the same volume in HBSS and
sonicated. Stock solutions of NADH (0.2 mM) and sodium
pyruvate (1.6 mM) were freshly prepared in a Tris (81.3 mM)/NaCl (203.3 mM) buffer, pH 7.2. The assay
was initiated by the addition of platelet supernatant or sonicate (40 µl) to a quartz silica cuvette maintained at 37 °C and containing
420 µl of NADH and 80 µl of pyruvate. LDH activity was measured as
the time-dependent and pyruvate-dependent
decrease in the absorbance of NADH at 339 nm. LDH activity was
expressed as µmoles of NADH consumed per min per ml of platelet
supernatant or sonicate.
Cultured Platelets Exhibit Increased Levels of Proapoptotic Bak and
Bax--
An important determinant of whether a cell will undergo
apoptosis is its intracellular balance between anti-apoptotic members of the Bcl-2 protein family such as Bcl-2 and Mcl-1 and proapoptotic members such as Bax and Bak (23, 24). The possibility that platelets
might be able to undergo an apoptosis-like death has been raised by
Vanags et al. (19) who reported that ionomycin, which
triggers apoptosis in many cell types (20, 21, 25), caused an increase
in the expression of proapoptotic Bax and Bak, but not Bcl-2. To extend
the findings of Vanags et al. we decided to study a
different model system in which apoptosis-like death might occur.
Granulocytes "aged" in culture undergo constitutive death that is
accelerated in the absence of survival factors (26-28) and is
correlated both with increased levels of Bax (29, 30), but not Bak
(31), and decreased levels of Mcl-1 (32). We therefore tested the
hypothesis that prolonged culture of platelets might also lead to an
increase in the expression of proapoptotic Bcl-2 family members and a
susceptibility to apoptosis-like death.
By immunofluorescence flow cytometry we confirmed that freshly isolated
platelets expressed Bax, Bak, and Mcl-1, but not Bcl-2 (Fig.
1). The ability of each antibody to
recognize its antigen was verified both by flow cytometry and Western
blot analysis with appropriate control cell
lines.2 Interestingly, the
levels of immunodetectable Bak and Bax increased by 3.4- and 2.4-fold,
respectively, as platelets were aged for 18 h in citrated plasma
(Fig. 1, B and C). No significant changes in
Mcl-1 were apparent. In keeping with studies of ionomycin stimulation of platelets (19), these data suggest that platelets cultured for
18 h can adopt a more proapoptotic balance and further suggest that aging in culture, as originally reported for neutrophils (11), was
likely to be a useful model for platelet cell death. Furthermore, a
tendency to apoptosis-like death during culture was reinforced by the
observations that after 18 h of culture, aged platelets exhibited
diminished mitochondrial membrane potential as measured by JC-1 and
release of cytochrome c into the cytoplasm (data not
presented).
Because plasma is a potential source of exogenous survival factors, we
went on to seek evidence that plasma deprivation, which resulted in
increased levels of Bax and Bak comparable with those observed for
platelets aged in the presence of plasma (data not shown), might
accelerate and therefore reveal a constitutive death program overlooked
in previous short term culture experiments on platelets.
Aged Platelets Exhibit Impaired Function--
Platelets possess
many of the functional responses exhibited by other inflammatory blood
cells and a key feature of apoptosis in leukocytes that are cultured
overnight in the presence of serum is loss of the ability to respond to
external stimuli and mount pro-inflammatory responses (33).
Interestingly, abrogation of cell function is characteristic of
platelets that have been stored at 37 °C in the presence of plasma,
especially the inability to respond to weak agonists such as ADP. With
the use of an aggregometer, which measures the light transmittance of a
stirred platelet suspension, we were able to confirm that platelets
lost the ability to aggregate but not to undergo a shape change in
response to ADP when cultured overnight in the presence of citrated
plasma (Fig. 2).
Treatment of freshly isolated (viable) platelets with 10 µM ADP resulted in an immediate shape change, observed as
a slight decrease in light transmittance (upward deflection in Fig.
2A), followed by an irreversible decrease in light
transmittance that was indicative of a full aggregation response (Fig.
2A). By reducing the concentration of ADP to 3 µM and in accordance with the biphasic nature of platelet
aggregation (34), we observed a reversal of the initial wave of
aggregation because of the absence of endogenous agonists such as ADP
being secreted by the weakly activated platelets (Fig. 2A).
In the presence of 1 µM ADP there was no evidence of aggregation, although shape change, which is the most sensitive response of platelets, persisted. Aggregation, but not shape change, could also be blocked by preincubating freshly isolated platelets with
the anti-GPIIb reagent MK852 (data not presented). In comparison, platelets aged over a 24-h period in plasma lost the ability to aggregate (t1/2 = 12 ± 2 h) but not to
undergo a shape change in response to ADP (Fig. 2B). The
inability of aged platelets to respond was also observed with the
agonist platelet-activating factor and the endoperoxide analog U46619 (data not presented).
In keeping with the hypothesis that plasma might contain survival
factors that normally retard constitutive platelet death, we found that
the loss of an aggregation response to ADP was markedly accelerated
when platelets were washed and cultured in the absence of plasma
(t1/2 = 1.5 ± 0.5 h). This rapid loss in
ADP-induced aggregation by washed platelets was prevented and returned
to rates comparable with those of unwashed platelets maintained in
plasma by reconstituting the washed platelets with PPP
(t1/2 = 12 ± 2 h) (Fig. 2C).
This indicated that the loss in platelet response was not dependent on
the washing procedure per se but rather on the absence of
permissive factors within plasma. Culturing washed platelets in the
presence of the megakaryocyte differentiation factor thrombopoietin and
known survival factors in other cell systems such as platelet-derived growth factor, granulocyte-macrophage colony-stimulating factor, and
insulin-like growth factor-1, had no effect. As a control for oncotic
effects we tested bovine serum albumin at 4 mg/ml and found no
protective effect and could eliminate glucose as a confounding factor
given that its concentration in HBSS (1 mg/ml) is equivalent to plasma
concentrations (0.7-1.1 mg/ml). Initial investigations to characterize
the putative soluble plasma survival factor(s) by dialysis have
revealed the activity to be of 50 kDa or greater in molecular size and
stable to long term storage at 4 °C and
Constitutive loss of platelet function on incubation at 37 °C was
further confirmed by assessing the ability of aged platelets to adhere
and spread on collagen coated surfaces with the formation of
lammelipodia and filopodia. Microscopic examination of
phalloidin-FITC-stained preparations revealed that freshly isolated
platelets readily adhered to both collagen I- and IV-coated glass
slides, whereas aged platelets, whether cultured in the presence or
absence of plasma protein, did not (data not presented).
Aged Platelets Maintain Plasma Membrane
Integrity--
Down-regulation of cell function is a feature of
programmed cell death in other blood cells (33, 35, 36) but might also have reflected necrosis. Assessing necrosis was not straightforward in
platelets because their small size precluded the use of vital dyes such
as Trypan Blue because admission of dye could not be confidently
assessed by light microscopy. Similarly, the absence of a nucleus
precluded the use of DNA staining vital dyes such as Hoechst and
propidium iodide. Nevertheless, flow cytometry revealed that the
forward and side scatter properties of fresh and aged platelets,
whether cultured in HBSS or PPP, were virtually superimposable (data
not presented). This contrasted with the deliberate impairment of
plasma membrane integrity by hypotonic lysis, thermal treatment, or
mild acid treatment that invariably resulted in the appearance of
cellular debris and the loss of platelets as assessed by forward and
side scatter (data not shown).
Further evidence against necrosis being a confounding factor in the
constitutive loss of platelet function was the use of phalloidin-FITC
as an actin-binding "vital dye" where greater than 99% of both
fresh and aged platelets excluded the dye when assessed by flow
cytometry (Fig. 3A). As a
positive control to reveal the potential level of intracellular
staining, aged platelets were permeabilized with the fixative PermeaFix
prior to staining with phalloidin-FITC. Fluorescence microscopy
confirmed that phalloidin-FITC had stained intracellular F-actin of
permeabilized platelets (data not presented). These data were a strong
indication that aged platelets were capable of maintaining plasma
integrity under the conditions employed in this study.
As further confirmation of platelet integrity, we assessed the level of
LDH activity, an abundant intracellular enzyme of platelets, in the
supernatants of cultured platelets (Fig. 3B). Importantly,
less than 4% of total LDH activity was found in the supernatant of
platelets aged for 18 h in HBSS in the absence of serum. To
confirm that soluble LDH maintained under comparable conditions was
stable, we cultured whole cell lysates from fresh platelets either on
its own or with erythrocytes. These experiments revealed that soluble
LDH was stable in culture over a 24-h period (Fig. 3B). The
75% loss in LDH activity in the cytosol of intact aged platelets
appeared likely to reflect intracellular catabolism of retained LDH or
its inactivation by, for example, transglutaminases. Unfortunately,
given the high levels of LDH in plasma, we were unable to reliably
assess the release of LDH from platelets aged in plasma. Taken together
with the phalloidin data, these results provide strong evidence against
the possibility that aged platelets may have undergone necrosis but
rather may have undergone a form of programmed cell death.
Transmission Electron Microscopy of Aged Platelets Is Suggestive of
a Cell Death Program--
To further eliminate the possibility of
necrosis we prepared samples for TEM. Freshly isolated platelets,
whether washed or not, exhibited characteristic discoid-like features
of nonactivated platelets (Fig. 4). This
was confirmed by the absence of filopodia and cell surface protrusions
or a centrally clumped body of organelles surrounded by a
circumferential band of a constricting microtubular network. A
cross-section through the platelets also revealed the typical
distribution of dense bodies,
However, platelets aged in culture under conditions leading to loss of
function exhibited dramatic morphological changes. Control platelets
maintained in citrated plasma for 24 h exhibited few changes from
the freshly isolated state, except that the majority of cells assumed a
spherical rather than a discoid shape. However, when aged for 12 h
in HBSS in the absence of serum, when loss of function was complete,
there was remarkable condensation of cytoplasm and granules with
submembrane vacuolization reminiscent of that reported during apoptosis
in megakaryocytes (37). Interestingly, cultured platelets did not
exhibit plasma membrane blebbing that is a common feature of many cell
types undergoing apoptosis, but lack of surface blebbing is a notable
feature of granulocyte apoptosis (11). Furthermore, in keeping with
evidence of granule fusion with the plasma membrane as aging neutrophil
granulocytes progress to an intact late apoptotic state prior to
secondary necrosis (38), platelets aged for 24 h exhibited fusion
of granules with the plasma membrane. In view of these morphological
changes suggestive of an apoptosis-like program of platelet death with
similarities to that observed in granulocytes, we went on to seek
comparable plasma membrane changes.
Aged Platelets Exhibit Cell Surface Changes of
Apoptosis--
Although exposure of phosphatidylserine (PS) in the
outer membrane of platelets was originally demonstrated to be a marker of platelet activation with procoagulant properties (39), PS exposure
is also recognized as a reliable marker of cells undergoing caspase-dependent cell death (40-42). In granulocytes, PS
exposure and caspase activation are tightly linked to nuclear changes
typical of apoptosis (14, 16, 17). We therefore sought to determine by
flow cytometry the level of PS exposure using FITC-conjugated annexin-V, a high affinity probe for PS (43). Flow cytometric analysis
of control platelets aged for 24 h in citrated plasma and labeled
with annexin-V-FITC revealed a bimodal distribution with typically no
more than 8% found positive (Fig.
5A). Although a background
level of 0.2-0.5% of fresh platelets bound annexin-V, any increases
were not apparent until at least 8 h of culture where annexin-V
binding increased steadily to 8% by 24 h with minimal increases
seen thereafter. In contrast, washed platelets aged in the absence of
plasma, which again contained few annexin-V binding cells in the first
6 h of culture, rapidly switched after 8 h to be >80%
positive by 18 h (Fig. 5A). To address the confounding possibility that PS exposure was simply a result of the cell having insufficient energy to maintain the "flippase" activity, washed platelets were aged in the presence of varying concentrations of
glucose (1 mg/ml to 10 mg/ml). Following assessment of the level of PS
exposure by Annexin-V binding and flow cytometry, no significant
differences were seen (data not presented). The inability of aged
platelets to maintain an asymmetric distribution for PS was strong
evidence of an apoptosis-like constitutive cell death program because
activated platelets expressing PS possess a translocase that
re-establishes an asymmetric distribution, unless platelets have
undergone secondary events of aggregation (44).
Furthermore, we sought to confirm morphological evidence of granule
fusion with the plasma membrane during constitutive platelet death seen
by TEM (Fig. 4) because this is an important cell surface feature of
later stages of constitutive apoptosis in neutrophils (38). To assess
for this possibility we probed for the intracellular Constitutive Platelet Death Is Caspase-independent--
Activation
of caspases is reported to be upstream of plasma membrane changes
associated with apoptosis including PS exposure (8, 16, 17, 40, 42).
Although Western blot analysis confirmed that caspase-3 was present as
a 32-kDa species in platelets that was reduced in aged preparations
(Fig. 6), we were unable to identify
caspase-mediated cleavage to an active fragment (17 kDa) as was
observed with apoptotic Jurkat T cells. Interestingly, aged platelets
did reveal the presence of proteolyzed species of molecular weights
just less than the p32 parent band, which a very recent report (46)
indicates is due to calpain-mediated processing to nonactivated
species. In keeping with these and other results (19, 46), and in
contrast to apoptotic Jurkat T cells, we were also unable to detect
caspase activity using fluorogenic-AFC or chromogenic-pNA substrates
for either caspase-1 or caspase-3 (data not presented).
In further agreement with these data we also found that a number of
protease inhibitors, including the caspase inhibitors Asp-Glu-Val-Asp-fluoromethylketone and zVAD-fmk, had no effect on the
rate of PS exposure (Table I).
Additionally, we also found that the inhibitors had no effect on the
refractiveness of aged platelets to ADP-induced aggregation (data not
presented) whether cultured either in the presence or absence of
plasma. Similarly, phagocyte recognition of washed platelets aged in
the presence of caspase inhibitors was not different from control,
confirming the caspase-independent nature of phagocyte clearance of
aged platelets (Table I). Combined, these results suggest that caspases do not have a major role in the constitutive cell death of platelets and complement recent studies on apoptosis-like events associated with
platelet activation (19, 46).
Aged Platelets Are Ingested by Phagocytes via Scavenger
Receptors--
In vivo, the most important feature of the
apoptotic cell death program is that intact effete cells are recognized
and rapidly ingested by professional and semi-professional phagocytes.
In keeping with this, we found that platelets cultured for 18 h in the absence of plasma were readily ingested by 6-day-old human M
To quantitate platelet ingestion by a range of phagocytes, we developed
a flow cytometric method (Fig. 8) that
was dependent on incubating the phagocytes with platelets that had been
prelabeled with an orange fluorescing reagent (CM-Orange) (Fig.
8A). Phagocytes were then sorted by flow cytometry where
they were readily resolved from platelets by forward and side scatter
(Fig. 8B) with any shift in orange fluorescence (FL2)
attributed to interacting platelets (Fig. 8C). To
discriminate between adherence and ingestion we also labeled the
phagocytes prior to flow cytometry with an FITC-conjugated anti-CD61
mAb. CD61, also known as glycoprotein IIIa, is a specific cell surface
marker for platelets (Fig. 8A) in which its expression was
not found to alter as platelets were aged in culture whether in the
presence or absence of plasma. Interestingly, we observed that
anti-CD61 mAb routinely failed to label our phagocyte populations, suggesting that platelets were ingested, in keeping with TEM (Fig. 7)
and that any adherent platelets were removed prior to flow cytometry.
Assessment of cytospin preparations by confocal microscopy also
confirmed that platelets were contained within phagocytes (data not
presented). In contrast and as a control comparison, we confirmed that
freshly isolated platelets adhere to the surface of freshly isolated
monocytes (47) (Fig. 8D).
Although fresh platelets on microscopic examination were observed to
adhere to all phagocytic cell lines tested, only aged platelets were
found to be ingested following flow cytometric analysis (Table
II). We also observed that the degree of
phagocytosis was always greater for platelets aged in the absence of
plasma survival factors than those aged in citrated plasma. Typically, after a 30-min interaction, greater than 90% of human M
The degree to which cell lines ingested aged platelets, whether
cultured in the presence or absence of plasma, was found to be
unaffected by various well characterized inhibitors of recognition (Table II) (48). These included the integrin inhibitor RGDS, the PS
receptor-competitor phospho-L-serine, the cationic sugars glucosamine and galactosamine, the anti-CD36 mAb SM
Because our studies had also shown that aged platelets expressed
P-selectin (Fig. 5B), which mediates adhesion of activated platelets to monocytes (50), and given that fucoidan is known to bind
the lectin domain of P-selectin, we explored the role of P-selectin in
platelet recognition. By using a function-blocking mAb (clone G1) to
P-selectin, which recognizes the lectin domain, we found that the
recognition of aged platelets by human M Platelets play a crucial role in hemostasis and thrombosis, with
the consequence that they are of central importance in common disorders
such as myocardial infarction and stroke. However, little is known of
candidate mechanisms for safe clearance of these anucleated blood
elements. Prompted by earlier work on constitutive apoptosis in other
key blood cells, granulocytes, we sought evidence for a constitutive
death program available to platelets. In keeping with earlier work (19)
we confirmed that platelets expressed members of the Bcl-2 family of
cell death-regulating proteins and observed that there was an apparent
proapoptotic shift in platelets aged for 18 h in citrated plasma.
We also observed that aged platelets lost the ability to aggregate in
response to weak agonists such as ADP and failed to adhere and spread
on collagen coated surfaces.
Given that serum is a rich source of survival factors for granulocytes,
with which platelets have much in common, we reasoned that the
physiological milieu of platelets, plasma, was likely to also contain
factors capable of retarding their programmed death. We therefore
sought evidence of accelerated programmed cell death when platelets
were cultured in the absence of plasma, finding that they not only
exhibited an accelerated loss of function but also displayed many
features in common with programmed death of granulocytes. Such changes
included morphological evidence of cytoplasmic condensation and cell
surface expression of the "eat me" signal phosphatidylserine (52)
and granule components such as P-selectin. Importantly, however, there
was strong evidence that such aged platelets retained plasma membrane
integrity because there was no detectable release of the cytoplasmic
marker enzyme lactate dehydrogenase, nor did the aged cells admit
actin-binding phalloidin-FITC. Furthermore, aged platelets were
recognized and ingested by all professional and semi-professional
phagocytes tested by a mechanism in which phagocyte scavenger receptors predominated.
An important conclusion to be drawn from these studies is that
constitutive death in plasma-deprived platelets represents a
caspase-independent program for phagocyte clearance of effete cells.
Not only did broad spectrum caspase inhibitors fail to affect all
phenomena associated with platelet death, including recognition by
phagocytes, but there was no evidence that caspase pseudo-substrates
were cleaved. Given earlier studies in various models of apoptotic cell
death, in which the display of cell surface "eat me" signals such
as phosphatidylserine exposure appears to be mediated by caspases (8,
40, 42), we believe that demonstration of a complete
caspase-independent program for cell death and clearance is an
important addition to examples of caspase-independent cell death where
recognition of such cells by phagocytes has not been previously studied
(53-56).
With regard to platelet cell death, these studies also complement
recent reports suggesting that ionomycin stimulation of platelets, a
model of activation-induced cell death, can recapitulate many of the
features found in apoptosis (19, 46). Although there is some evidence
that caspases may participate in platelet activation (57), Wolf
et al. (46) suggest that the effects of ionomycin on
platelets are independent of caspases because calpains disable
activation by partial cleavage of their pro-domains. Similar mechanisms
could be at play in constitutive platelet cell death given its
independence from caspases despite the presence of cytochrome
c in supernatants from cell lysates, which can activate caspases (46), and the remarkably similar evidence of partial caspase-3
cleavage on Western blot. This possibility is now under active
investigation, especially as further studies are clearly required to
characterize the caspase-independent mechanisms by which aged platelets
express PS because this was not affected by the calpain inhibitors
acetyl-leucyl-leucylnorleucinal and calpeptin.
It will also be important to define in more detail the molecular
mechanisms by which phagocyte class A scavenger receptors mediate
recognition and ingestion of aged platelets, as clearly indicated by
the inhibitor studies presented in this report. Although the role of
the scavenger receptor has already been implicated in the clearance of
apoptotic thymocytes by mouse macrophages (58) the ligands displayed by
dying cells, which lead to their recognition by scavenger receptors,
are currently unknown. Furthermore, although PS is widely recognized as
an "eat me" signal (52), it is not properly understood why various
phagocyte types will apparently ignore PS in favor of other yet to be
characterized signals for ingestion (59).
Because constitutive death in plasma-deprived platelets was
caspase-independent and could not be assessed for typical nuclear changes because these cells have no nucleus, we feel it is not appropriate to label this form of cell death as "apoptosis."
However, we believe that the data indicate that platelets can undergo a form of programmed cell death that can be regulated by exogenous influences, in particular plasma-derived survival factors. In keeping
with this, we observed that the return of plasma-deprived platelets to
plasma slowed the phenomena of cell death, most notably returning the
rate of loss of aggregation and levels of PS exposure to that observed
for platelets cultured in plasma. Ongoing work is directed at the
biochemical characterization of the survival activity present in plasma
because a range of candidate cytokine survival factors could not
substitute for plasma. Nevertheless it should be emphasized that plasma
deprivation appeared merely to accelerate a constitutive death program
that was already active in platelets at 37 °C and evident after
18-24 h of culture in plasma. However, study of constitutive death in
plasma-replete platelets will be difficult because our preliminary work
demonstrated progressive loss of platelets from populations cultured
for >24 h, presumably reflecting relatively rapid secondary necrosis
of that proportion of cultured platelets undergoing programmed death each day.
In conclusion, we have provided in vitro evidence that human
platelets can undergo a constitutive program of cell death that was
caspase-independent and that resulted in the specific recognition of
effete cells by phagocytes employing the scavenger receptor as a
recognition mechanism. Although our findings have potentially important
implications for understanding platelet kinetics and the related
pathogenesis of thrombotic and bleeding disorders, no firm conclusions
on in vivo relevance can be drawn from the current data.
Nevertheless, these data raise the exciting prospect that platelet
lifespan and clearance is amenable to exogenous regulation for
therapeutic purposes.
We kindly thank Prof. Stan Heptinstall
(Division of Cardiovascular Medicine, University Hospital, Nottingham)
for many helpful discussions and the staff of the Rayne Laboratory
(Medical School, Edinburgh) for platelet-rich plasma.
*
This work was supported in part by Wellcome Trust Program
Grant 047273 (to J. S.) and a National Kidney Research Foundation studentship (to M. C. H. C.).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.
§
These authors contributed equally to this work.
¶
To whom correspondence should be addressed: Centre for
Inflammation Research, Medical School, University of Edinburgh, Teviot Place, Edinburgh EH8 9AG, UK. Tel.: 44-131-6511577; Fax:
44-131-6511607; E-mail: Simon.Brown@ed.ac.uk.
2
L. Magowan, unpublished observations.
The abbreviations used are:
zVAD-fmk, carbobenzoxy-Val-Ala-Asp-fluoromethylketone;
CM-Orange, ((4-chloromethyl)benzoyl)aminotetra-methylrhodamine;
FITC, fluorescien
isothiocyanate;
HBSS, Hanks' balanced salt solution;
LDH, lactate
dehydrogenase;
mAb, monoclonal antibody;
M
Constitutive Death of Platelets Leading to Scavenger
Receptor-mediated Phagocytosis
A CASPASE-INDEPENDENT CELL CLEARANCE PROGRAM*
§¶,§,
,
Centre for Inflammation Research, Department
of Clinical & Surgical Sciences (Internal Medicine), Royal
Infirmary, Edinburgh, EH3 9YW and the Divisions of Renal & Inflammatory Disease and ** Cardiovascular Medicine, Department of
Medicine, University Hospital,
Nottingham NG7 2UH, United Kingdom
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-converting enzyme (Ab-1, catalog
number PC84), caspase-1-pNA substrate (400025), -AFC substrate
(688225), caspase-3-pNA substrate (235400), -AMC substrate (235425),
acetyl-leucyl-leucylnorleucinal, calpeptin, zVAD-fmk, and
Asp-Glu-Val-Asp-fluoromethylketone were from Calbiochem Novachem
(Nottingham, UK); anti-human caspase-3 polyclonal antibody (catalog
number 65906E) and anti-cytochrome c mAbs (clones 6H2.B4 and
7H8.2C12) were from PharMingen (Becton Dickinson, Cowley, UK);
PermeaFix was from Orthodiagnostics; and JC-1 (catalog number T3168)
and CM-Orange (catalog number C2927) were from Molecular Probes
(Leiden, The Netherlands).
) were obtained by the standard technique of culturing adherent
monocytes for 5-7 days in Iscove's Dulbecco's modified Eagle's
medium plus 10% PRP-derived serum (11, 22).
, and
trypsin/EDTA treatment at 37 °C for 15 min for all other cell lines
tested, before flow cytometric and epifluorescent microscopic analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (29K):
[in a new window]
Fig. 1.
The proapoptotic balance of Bcl homologs is
accentuated in aged platelets. A, typical
immunofluorecence flow cytometry histograms of fixed permeabilized
(PermeaFix) cells that demonstrate that freshly isolated platelets
express the proapoptotic Bcl homologs Bak and Bax but not the
anti-apoptotic homolog Bcl-2. The negative controls for mouse (Bcl-2)
and rabbit (Bak, Bax, and Mcl-1) antibodies were superimposable and no
different from autofluorescent controls, confirming an absence of
nonspecific binding. B, overlays of representative
histograms showing the shift in fluorescence for Bak and Bax expression
between freshly isolated and aged platelets. Autofluorescence and
control irrelevant staining for fresh and aged platelets were
essentially indistinguishable. C, the mean peak fluorescence
for various members of the Bcl family is presented as the arithmetic
mean ± 95% confidence interval for n = 4 separate experiments (three different donors). Each experiment was
performed in duplicate on samples permeabilized with PermeaFix with
fresh citrated platelets (open columns) or platelets
cultured in the presence of citrated plasma for 18 h (solid
columns).
View larger version (32K):
[in a new window]
Fig. 2.
Agonist-induced aggregation is down-regulated
in aged platelets. Photometric measurement of stirred suspensions
of freshly isolated platelets (A) or platelets aged in
citrated plasma for 24 h (B) were monitored for changes
in light transmittance over a 10-min period following activation with
ADP to a final concentration of 10 µM (i), 3 µM (ii), and 1 µM
(iii). The upward deflection (decreased transmittance) in
the traces immediately following the addition of ADP is indicative of
shape change, whereas increases in transmittance are indicative of
reversible (ii) or full aggregation (i). The
chart recorder moved from left to right with a
1-min interval represented by the horizontal bar in
B. C, freshly isolated platelets were maintained
for 4 h at 37 °C in either citrated plasma (i) or,
after being washed free of plasma, in HBSS (ii) containing
either thrombopoietin (5 ng/ml) (iv), platelet-derived
growth factor (10 ng/ml) (v), insulin-like growth factor-1
(100 ng/ml) (vi), or bovine serum albumin (4 mg/ml)
(vii). Alternatively, washed platelets were returned to PPP
(iii). ADP was then added to a final concentration of 10 µM, and the percentage of change in light transmittance
was recorded for 10 min. Error bars represent the 95%
confidence interval for five separate experiments.
20 °C.
View larger version (30K):
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Fig. 3.
Aged platelets maintain plasma membrane
integrity. A, flow cytofluorimetric analysis for
platelet membrane integrity of fresh and aged platelets using
actin-binding phalloidin-FITC as a vital dye. Suspensions of platelets
aged in the presence (Aged PRP) or absence of plasma
(Aged Washed) were washed free of conditioned media and
incubated for 10 min on ice with phalloidin-FITC at 2 µg/ml in PBS
before flow analysis. The FL1 channel (FITC) on a Becton-Dickinson
FACScan was set to the autofluorescence of unlabeled freshly isolated
washed platelets. As a positive control for phalloidin-FITC staining,
aged platelets cultured in the absence of serum were permeabilized with
PermeaFix (see "Experimental Procedures"). B, freshly
isolated platelets were resuspended in HBSS and cultured in the absence
of plasma for either 1 or 18 h before separating the cells from
the conditioned supernatant and resuspending the cells in an equivalent
volume of fresh HBSS and sonicating. Clarified supernatants of fresh
(i) and aged (ii) washed platelets or their cell
lysates (iii and iv, respectively) were monitored
for LDH activity. Cell lysates from fresh platelets were also incubated
for 18 h in HBSS either on their own (v) or in the
presence of erythrocytes (vi). The level of LDH in PPP is
shown for comparison (vii). Error bars represent
the 95% confidence interval of four separate experiments each done in
duplicate.
-granules, mitochondria, and glycogen
particles.
View larger version (81K):
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Fig. 4.
Aged platelets exhibit cytoplasmic
condensation. Transmission electron micrographs of platelet
preparations are shown: washed platelets fixed immediately following
isolation; aged platelets maintained in citrated plasma for 24 h;
and washed platelets maintained in HBSS for 12 and 24 h,
respectively. Note evidence of granule fusion with the plasma membrane
(arrows). The white scale bar in each panel
represents 500 nm.
View larger version (36K):
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Fig. 5.
Aged platelets express phosphatidylserine and
P-selectin at their surface. Fresh washed platelets, platelets
aged in HBSS in the absence of serum protein (aged washed), and
platelets aged in citrated plasma (aged PRP) were assessed by flow
cytometry for phosphatidylserine exposure using annexin-V-FITC
(A) and granule cell fusion with the plasma membrane by
monitoring for cell surface P-selectin expression using a
FITC-conjugated anti-P selectin mAb CRC81 (B). The FL1-H
channel (FITC) was set to the autofluorescence of unlabeled platelets
and confirmed that nonspecific binding of control antibodies was
minimal.
and dense
granule marker P-selectin (45) (Fig. 5B). Reassuringly we
found that platelets maintained in citrated plasma did not express any
cell surface P-selectin in the first 8 h of culture, evidence
against platelet activation. Indeed, after aging for 24 h in the
presence of plasma we found that only a small proportion (~10%) of
platelets expressed P-selectin. However, washed platelets cultured in
HBSS in the absence of serum rapidly mobilized intracellular stores of
P-selectin following 6 h of in vitro culture so that all cells were positive by 7-10 h.
View larger version (28K):
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Fig. 6.
Caspase-3 is not cleaved as platelets age in
culture. Cytosolic extracts taken from fresh (iii) and
aged platelets (iv) cultured in the absence of serum were
probed for caspase-3 by Western blot with a polyclonal antibody that
recognized both the 32-kDa (p32) precursor and the17-kDa
subunit of the activated enzyme (note lack of the latter). As positive
controls, cell lysates were prepared from untreated Jurkat T-cells
(ii) that exhibited low levels (10%) of constitutive
apoptosis as judged by Giemsa stained cytospins, and those induced to
undergo apoptosis (i) following a 5-h treatment with
staurosporine (2 µM) (approximately 65% apoptosis).
Positions for the 30-, 23-, and 16.5-kDa mass markers were determined
with the use of Rainbow Markers (Sigma).
Effect of protease inhibitors on PS exposure and recognition of aged
washed platelets
following a 30-min phagocytosis assay as evidenced by TEM (Fig. 7).
View larger version (169K):
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Fig. 7.
Human monocyte-derived macrophages readily
ingest aged platelets. A representative TEM showing a cytoplasmic
region just beneath the plasma membrane of a human macrophage that
contains distinct evidence of having ingested two (solid
arrow) and possibly three (open arrow) platelets. The
white scale bar represents 500 nm.
View larger version (50K):
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Fig. 8.
Ingestion of aged platelets is readily
quantitated with the development of a flow cytometric method.
A, human platelets prelabeled with CM-Orange (i)
and aged in HBSS in the absence of plasma were confirmed to express the
platelet specific marker CD61 with an FITC-conjugated mAb
(ii) whose expression remained unaltered relative to freshly
isolated CM-Orange unlabeled platelets (iii). B,
CM-Orange labeled platelets (i), incubated with human
macrophages (ii), or with Bowes melanoma cells (iii) for
times indicated elsewhere were readily resolved from the phagocytes
according to their forward and side scatter properties in flow
cytometry. C, CM-Orange-labeled platelets that associated
with the phagocyte population are seen as a distinct subpopulation that
is shifted in orange fluorescence (y axis). These platelets
were predominantly ingested given that they failed to dual label with
the FITC-conjugated mAb to CD61. Similar results were found with mAbs
to other platelet markers such as CD42a. D, in contrast,
freshly isolated monocytes readily bind but do not ingest freshly
isolated CM-Orange-labeled platelets.
and Bowes melanoma cells ingested platelets aged in the absence of plasma compared with only 30 ± 12% and 20 ± 6%, respectively,
when aged in the presence of citrated plasma. Moreover, time course
experiments repeatedly showed that in a fixed time phagocytosis assay
the maximal level of ingestion by human M and Bowes melanoma cells was achieved with platelets aged for 12 h in the absence of plasma or 24 h for those cultured in citrated plasma.
Platelet ingestion is mediated by the scavenger receptor
, 0-5%
inhibition; +, 5-20% inhibition; +, 30-50% inhibition; +++,
75-90% inhibition. A typical interaction assay resulted in 40 ± 8% of M ingesting platelets aged in the absence of plasma after a
10-min assay and 38 ± 6% after 40 min for platelets aged in the
presence of plasma, in contrast to BOWES where the level of ingestion
was 30 ± 12% and 20 ± 6%, respectively.
, and the anti-CD14 mAb 61D3 (Table II). Conclusive evidence against a role for
CD36 in phagocytosis was confirmed with the use of Bowes melanoma cells
stably transfected with CD36, which exhibited no increase in
phagocytosis when compared with control Bowes melanoma cell lines (49).
However, greater than 75% inhibition was observed with fucoidan, a
recognized inhibitor of the scavenger receptor pathway, in contrast to
the lack of inhibition by dextran at the same concentration, which
served as control. Polyinositol, another inhibitor of the scavenger
receptor pathway, but not its standard control polycytidine also
inhibited recognition, although not as effectively as fucoidan (Table
II). Nevertheless, further confirmation of a major role for the
scavenger receptor was obtained with the anti-murine scavenger receptor
mAb 2F8, which inhibited mouse peritoneal macrophage uptake of aged platelets.
and Bowes melanoma cells
was only weakly affected, and no synergy with polyinositol was observed
(Table II). This suggests that although P-selectin had a minor role in
the phagocytosis of aged platelets it was not primarily responsible for
mediating phagocytic recognition and clearance, a conclusion in
agreement with others (51).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
, monocyte-derived
macrophage;
PBS, phosphate-buffered saline;
PPP, platelet poor plasma;
PRP, platelet-rich plasma;
TEM, transmission electron microscopy;
PS, phosphatidylserine.
REFERENCES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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