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Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3831-3838
Endothelial Cells Undergoing Apoptosis Become Proadhesive for
Nonactivated Platelets
By
Thomas Bombeli,
Barbara R. Schwartz, and
John M. Harlan
From the Division of Hematology, University of
Washington, Seattle, WA; and the Division of Hematology, the Department
of Medicine, University Hospital of Zurich, Zurich, Switzerland.
 |
ABSTRACT |
Under normal conditions, platelets do not adhere to endothelium.
However, when platelets or endothelial cells are stimulated by thrombin
or cytokines, respectively, platelets bind avidly to endothelium.
Because there is accumulating evidence that endothelial cells may
become apoptotic under certain proinflammatory or prothrombotic conditions, we investigated whether endothelial cells undergoing apoptosis may become proadhesive for nonactivated platelets. Human umbilical vein endothelial cells (HUVEC) were induced to undergo apoptosis by staurosporine, a nonspecific protein kinase inhibitor, or
by culture in suspension with serum-deprivation. After treatment of
HUVEC or platelets with different receptor antagonists, nonactivated, washed human platelets were allowed to adhere to HUVEC for 20 minutes.
To exclude matrix involvement, platelet binding was measured in
suspension by using flow cytometry. Independent of the method of
apoptosis induction, there was a marked increase in platelet binding to
apoptotic HUVEC. Although HUVEC exhibited maximal adhesiveness for
platelets after 2 to 4 hours, complete DNA fragmentation of HUVEC
occurred only several hours later. Adhesion assays after blockade of
different platelet receptors showed only involvement of
 1-integrins. Platelet binding to apoptotic HUVEC was
inhibited by more than 70% when platelets were treated with blocking
anti-1 antibodies. Treatment of apoptotic HUVEC with
blocking antibodies to different potential platelet receptors,
including known ligands for 1-integrins, did not affect
platelet binding. As assessed by determination of -thromboglobulin
and platelet factor 4 in the supernatants, platelets bound to apoptotic
HUVEC became slightly activated. However, significant expression of
platelet P-selectin (CD62P) was not found. These data provide further
evidence that endothelial cells undergoing apoptosis may contribute to
thrombotic events.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
UNPERTURBED endothelial cells provide
potent anticoagulant properties, preventing the adhesion of platelets
as well as the initiation of coagulation. However, when exposed to
proinflammatory stimuli the endothelium is rapidly transformed into a
proadhesive and procoagulant surface, promoting formation of
thrombi.1,2 Several lines of evidence indicate that, under
similar conditions, endothelial cells may also undergo apoptosis.
Vascular cell death has been observed primarily in the environment of
hypertension, inflammation, and arteriosclerosis.3,4 Thus,
it is suggestive that apoptotic endothelial cells may contribute to the
pathophysiology of thrombosis. In a recent in vitro study, we have
found that endothelial cells undergoing apoptosis became highly
procoagulant because of the loss of anticoagulant membrane components
and the exposure of negatively charged phospholipids, which accelerated activation of coagulation factors.5 In addition, two other reports showed that endothelial cells markedly increased cell surface
tissue factor activity on induction of apoptosis.6,7 Vascular smooth muscle cells have similarly been found to promote thrombin generation during the process of cell death.8
Hence, it is conceivable that, in vivo, an area of vascular cells
undergoing apoptosis may contribute to the initiation of thrombosis.
For years it has been shown that activated platelets readily adhere to
normal endothelium.9 We have recently elucidated the
receptors responsible for this binding mechanism and showed that
platelet adhesion is mediated by a bridging mechanism involving platelet GPIIbIIIa (CD41a/CD61), different adhesive proteins, and three
different endothelial cell-counter receptors.10
Perturbation of endothelial cells has similarly been found to alter
their reactivity with platelets. In vivo studies have shown that
nonactivated platelets roll on stimulated endothelium, thereby
interacting with endothelial P-selectin and
E-selectin.11,12 Consequently, stimulated, but structurally
intact, endothelium has been suggested to contribute significantly to
the formation of thrombosis.13
The purpose of this study was to determine whether endothelial cells
undergoing apoptosis would become proadhesive for nonactivated platelets and whether platelets would become activated through contact
with apoptotic endothelial cells. With a flow cytometry-based binding
assay, we show that nonactivated platelets bind to apoptotic human
umbilical vein endothelial cells (HUVEC) to the same extent as
thrombin-activated platelets bind to normal HUVEC. [The mechanism by
which HUVEC apoptosis was induced was not critical, since the extent of
nonactivated platelet adhesion was similar using either staurosporine-treated or suspended and starved HUVEC.] Binding was
inhibited only by treating platelets with
anti-1-integrin antibodies, indicating that
platelet 1 receptors were primarily responsible for adhesion. The 1-receptor ligands on
apoptotic HUVEC remain undefined, but collagen, fibronectin, and
RGD-dependent ligands are not involved. Platelets bound to apoptotic
HUVEC became slightly activated as demonstrated by the release of thromboglobulin and platelet factor 4, but P-selectin was not expressed
significantly. These data provide further evidence that endothelial
cells undergoing apoptosis may acquire prothrombotic properties.
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MATERIALS AND METHODS |
Cell culture.
HUVEC were obtained by collagenase treatment of umbilical cord veins as
previously described.14 Cells were cultured on
gelatin-coated dishes and propagated in RPMI 1640 medium supplemented
with 20% bovine calf serum, 90 µg/mL heparin (Sigma Chemical Co, St
Louis, MO), and 50 µg/mL endothelial cell growth factor prepared from bovine hypothalamus.15 For flow cytometry assays, HUVEC
derived from passage two or three were allowed to grow to confluence in 12-well dishes. Before the adhesion assay, HUVEC were washed twice with
RPMI medium and incubated with receptor antagonists in phenol red-free
RPMI medium for 30 minutes at 37°C. Where indicated, HUVEC were
stimulated with 0.5 U/mL human thrombin (Sigma) for 15 minutes at
37°C.
Apoptosis induction.
Apoptosis of HUVEC was induced either by 200 nmol/L staurosporine
(Sigma) in growth medium or by culture in suspension in the absence of
heparin, serum, and growth factors. To prevent cells from adherence,
tissue culture plates were coated with a 2% agarose gel. Both methods
induce apoptosis in about 50% of the cells within 6 to 8 hours.5
Platelet preparation.
Blood was obtained by venipuncture from healthy adult volunteers
according to a protocol approved by the Human Subjects Division of the
University of Washington, Seattle. The volunteers did not take any
drugs for the previous 10 days. Isolation of platelets was performed as
described by Baenziger and Majerus.16 In brief, blood was
drawn into polypropylene syringes containing one-tenth volume
of 0.11 mol/L sodium citrate and centrifuged at 1,000g for
4 minutes to obtain platelet-rich plasma. Platelets were
sedimented by centrifugation at 2,000g for 10 minutes and
washed twice with 10 mL of HEPES buffer (10 mmol/L HEPES, 0.5 mmol/L
MgCl2, 130 mmol/L NaCl, 4 mmol/L KCl, 1 mmol/L
CaCl2, 5 mmol/L glucose, pH 7.4). To pellet erythrocytes
selectively, platelets were resuspended in the same buffer and
centrifuged twice at 120g for 3 minutes. Subsequently, the
content of erythrocytes was less than 1% as calculated with a
hemocytometer. Platelets were stained with 2.5 µmol/L
calcein-acetoxymethyl ester (Molecular Probes, Eugene, OR) in the dark
for 15 minutes. To avoid platelet activation, all
centrifugations were done at room temperature (RT) and in the presence
of 1 µmol/L prostaglandin E1 (Alprostadil, Prostin VR Pediatric;
Upjohn, Kalamazoo, MI). After a washing step, platelets were adjusted
to a final concentration of 2 × 109/mL and incubated with
receptor antagonists for 30 minutes at RT. Where indicated, platelets
were activated with 0.5 U/mL human thrombin for 10 minutes. Thrombin
was then inactivated with 2 U/mL hirudin (Sigma) for 10 minutes.
Antibodies and other receptor antagonists.
To block platelet or HUVEC receptors, the following monoclonal
antibodies (MoAbs) were used (the corresponding references refer to
studies in which the MoAbs were found to have blocking functions):
P4C10 (1 integrin, CD29, IgG1) (Life
Technologies Inc, Gaithersburg, MD)17; 2A11
(1 integrin, CD29) produced in our laboratory (B.R.
Schwartz, unpublished observations); P1E6 ( 2, CD49b,
IgG1) (Chemicon International, Inc, Temecula,
CA)18; P1D6 (5, CD49e, ascites fluid) (Life
Technologies Inc)19; GoH3 (6, CD49f,
IgG2A) (Endogen Inc, Woburn, MA)20; LM609
(v3 integrin, CD51/CD61,
IgG1) (Chemicon)21; P2 (GPIIbIIIa,
CD41a/CD61, IgG1) (Immunotech Inc, Westbrook,
ME)22; SZ2 and 2E4 (GPIb, CD42b, IgG1)
(Immunotech and Dr G.J. Roth, Veterans Affairs Medical Center, Seattle,
WA, respectively)23; FA6.152 (GPIV, CD36,
IgG1) (Immunotech); R6.5 and 2D5 (ICAM-1, CD54,
IgG1) provided by Dr R. Rothlein, Department of
Immunologic Diseases, Boehringer Ingelheim Pharmaceuticals Inc,
Ridgefield, CT and Dr D.C. Altieri, The Boyer Center for Molecular
Medicine, Yale University School of Medicine, New Haven, CT,
respectively24,25; WAPS12.2 and G1 (P-selectin, CD62P,
IgG1) (Endogen Inc and Biosource Int, Camarillo, CA,
respectively26,27; 1.2B6 and H18-7 (E-selectin, CD62E)
(Endogen Inc and Becton Dickinson, San Jose, CA,
respectively)28,29; E1-6 and 1G11 (VCAM-1; CD106, IgG1) (Becton Dickinson and Immunotech,
respectively).29,30 JC70A and M89D3 (PECAM-1, CD31,
IgG1) (Accurate Chemicals Co, Westbury, NY, and Dr M.R.
Zocchi, Laboratory of Adoptive Immunotherapy, Milan, Italy,
respectively); and anti-human fibronectin MoAb type 2 from Calbiochem
(La Jolla, CA).31 All of these MoAbs were used at a final
concentration of 30 µg/mL. The anti-human collagen IV MoAb COL-94
(IgG1; Sigma) and the anti-human von Willebrand factor
(vWF) rabbit polyclonal antibody (IgG fraction; Sigma) were used at a
final dilution of 1:250.10 A nonspecific isotype control
was performed with a mouse IgG1k MoAb (Sigma).
Gly-Arg-Gly-Asp-Ser (GRGDS) and Gly-Arg-Gly-Glu-Ser (GRGES) peptides
(Peninsula Laboratories, Belmont, CA) and recombinant annexin V
(provided by Dr J.F. Tait, Department of Laboratory Medicine,
University of Washington, Seattle, WA) were used at a final
concentration of 50 µg/mL and 5 µmol/L, respectively.
Adherence assay.
HUVEC (either adherent or suspended) were washed twice with RPMI medium
and incubated with 600 µL of HEPES buffer (same buffer as described
above, but with 2 mmol/L CaCl2) and 40 µL of the suspension of stained platelets for 20 minutes at 37°C. The final platelet concentration was 1.25 × 108/mL. When suspended
HUVEC were used, the adhesion assay was performed in a 1.5-mL Eppendorf
tube placed on a shaking table to avoid sedimentation. Adherent HUVEC
were harvested mechanically by vigorous pipetting. After washing, HUVEC
were fixed immediately with 80% ethanol on ice for 30 minutes. To
differentiate HUVEC from residual unbound platelets, HUVEC were stained
with the DNA binding dye, propidium iodide. The cells were resuspended
in 200 µL phosphate-buffered saline pH 7.4 and 0.1% Triton-X 100 (Sigma) containing 5 µg/mL propidium iodide (Sigma) and 50 µg/mL
ribonuclease A (R-6513; Sigma). Subsequently, specimens were analyzed
by a FACScan (Becton Dickinson, Mountain View, CA). At least 10,000 cells that stained positive for propidium iodide (FL-2-H) were
evaluated. Platelet adhesion to HUVEC was expressed by the
median fluorescence (FL-1-H) of the entire HUVEC population. Based on
the distribution of the DNA content (FL-2-H), HUVEC were routinely
tested for apoptosis (designated A0 region below
G0/G1 peak) and homotypic aggregate formation
(region above G2/M peak). On average, less than 5% of the
cells exhibited more than 4C DNA, indicating that they were clumped.
Statistical significance was determined by using Student's t-test.
Determination of P-selectin expression.
Suspended HUVEC, platelets, or platelet-HUVEC aggregates were incubated
with the anti-P-selectin MoAb WAPS12.2 (IgG1; Endogen Inc)
at a final concentration of 10 µg/mL for 30 minutes at RT. After
washing twice, the secondary fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Caltag Lab, San Francisco, CA)
was incubated for 30 minutes (final dilution of 1:200). At least 10,000 cells were then analyzed by a FACScan (Becton Dickinson). Cells treated
with FITC-conjugated isotype-specific IgG were used as negative controls.
Determination of release of platelet factor 4 and
-thromboglobulin from platelets.
After incubation of platelets with staurosporine-treated and washed
HUVEC in HEPES-buffer (see above) for 20 minutes at 37°C, the
supernatants were carefully removed and centrifuged at 2,000g for 5 minutes to remove residual unbound platelets. Before
centrifugation, a stop solution was added to a final concentration of 5 mmol/L EDTA (Sigma), 1 µmol/L prostaglandin E1 (see above), and 3%
paraformaldehyde (Sigma) to prevent centrifuge-induced secretion from
the residual unbound platelets. -thromboglobulin and platelet factor
4 were then determined by using enzyme-linked immunosorbent assay
(ELISA) kits, according to the manufacturer's instructions
(Asserachrom BTG and Asserachrom PF4, Diagnostica Stago,
Asnieres-Sur-Seine, France).
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RESULTS |
Apoptotic HUVEC bind platelets.
Because platelets adhere primarily to matrix proteins, determination of
platelet adhesion to adherent HUVEC may produce spurious results if the
monolayer is not completely confluent. This may be a particular problem
with apoptotic HUVEC, because endothelial cell death is accompanied by
retraction and also detachment. To avoid this problem, HUVEC were
mechanically detached after the binding assay and platelet binding was
determined in suspension with flow cytometry. As we have previously
shown, this method allows us to completely exclude involvement of
matrix proteins.10 Unstimulated live HUVEC did not bind
nonactivated platelets in a significant manner (Fig
1A). However, HUVEC treated
with staurosporine, a nonspecific protein kinase inhibitor, for 8 hours
(Fig 1B) or kept in suspension and serum deprived also for 8 hours (Fig
1C) bound nonactivated platelets to the same degree as normal HUVEC incubated with thrombin-activated platelets (Fig 1D). Within this period of time, both methods induce cell death in at least 50% of the
cells as demonstrated by the occurrence of apoptosis-associated DNA
degradation.5 Platelet adhesion to apoptotic HUVEC was not
further increased when thrombin-activated platelets were used (data not
shown). On stimulation with 0.5 U/mL human thrombin for 15 minutes,
HUVEC also exhibited an increased fluorescence which, however, was not
as high as with apoptotic HUVEC (Fig 1E). This proadhesive effect of
thrombin could be blocked by treating HUVEC with 2 U/mL
hirudin for 15 minutes before adding the platelets (data not
shown). Higher thrombin concentrations (up to 2 U/mL) did not increase
further platelet adhesion (data not shown). These data indicate that
HUVEC undergoing apoptosis may become more proadhesive for platelets
than on direct stimulation with a prothrombotic stimulus.
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| Fig 1.
Platelet binding to HUVEC. Nonactivated or
thrombin-activated, washed, and calcein-loaded platelets were allowed
to adhere to normal, apoptotic or thrombin-stimulated HUVEC for 20 minutes. Apoptosis was induced either by staurosporine or by suspending
cells with serum deprivation. Platelet binding was then determined by
flow cytometry to exclude involvement of subendothelial matrix
proteins. Binding is expressed as the median fluorescence (FL-1-H) of
the entire HUVEC population. Apoptotic HUVEC bound nonactivated
platelets as effective as normal HUVEC bound thrombin-activated
platelets. A representative experiment of three performed is shown.
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Time course of platelet binding.
To differentiate HUVEC from residual unbound platelets in flow
cytometry, HUVEC were stained with the DNA-binding dye propidium iodide
after the adhesion assay. This procedure allowed us to also determine
the percentage of HUVEC with a hypodiploid DNA content that is
characteristic of apoptosis. Thus, it was possible to correlate the
time course of the increase in platelet adhesion with the occurrence of
apoptotic DNA fragmentation. As shown in Fig
2, the onset of platelet adhesion did not
correlate with DNA fragmentation, but preceded it by several hours. The
maximal increase in platelet adhesion was observed after 2 to 4 hours
by using either staurosporine or suspension/starvation for apoptosis
induction, whereas complete DNA fragmentation was observed only after
16 or more hours. These results indicate that during the course of HUVEC apoptosis important functionalchanges occur several hours before
structural alterations of the nucleus are completed.
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| Fig 2.
Time-course of platelet binding to apoptotic HUVEC.
Washed and calcein-loaded nonactivated platelets were allowed to adhere
to HUVEC undergoing apoptosis induced either by staurosporine or by
keeping cells in suspension with serum-deprivation. After the adhesion
assay, HUVEC were permeabilized and stained for DNA with propidium
iodide. HUVEC were then assayed by flow cytometry to determine platelet
binding and, simultaneously, apoptosis-induced DNA fragmentation.
Platelet binding is expressed as the median fluorescence (FL-1-H) of
the entire HUVEC population. DNA fragmentation is expressed as
percentage of cells with hypodiploid DNA. A representative experiment
of three performed is shown.
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Platelet receptors.
To determine the platelet receptors involved in the binding of
nonactivated platelets to apoptotic HUVEC, platelets were incubated with different blocking MoAbs for 30 minutes before the adhesion assay.
Apoptosis of HUVEC was induced by treating the cells with staurosporine
for 6 hours. Inhibition of platelet adhesion was observed only when
platelets were treated with the anti-1-integrin antibodies P4C10 or 2A11 (Fig 3), both of
which inhibited platelet binding by more than 70%. To clarify further
which type of platelet 1-integrin
(21, 51, or
61) would be involved in the binding, platelets were treated with blocking moAbs to either the
2-, 5-, or 6-subunit.
However, platelet binding was not reduced significantly when one of the
-subunits was blocked. Significant inhibition of platelet binding
(about 20%) was observed only when all three anti--MoAbs were used
in combination. RGD peptides also were without effect at concentrations
that inhibit GPIIaIIIb-dependent adhesion of thrombin-activated
platelets to HUVEC.10 Blockade of other important receptors
for platelet-subendothelium interactions, including GPIb, GPIV, and
v3-integrin, as well as
activation-dependent receptors, including GPIIbIIIa and P-selectin, did
not inhibit platelet binding to staurosporine-treated HUVEC.
Furthermore, incubation of platelets with annexin V to block negatively
charged phospholipids did not affect platelet adherence.
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| Fig 3.
Platelet adhesion to apoptotic HUVEC after blockade of
platelet receptors. Nonactivated platelets treated with different
receptor antagonists were allowed to adhere to a HUVEC monolayer
treated with staurosporine for 6 hours. Platelet binding was determined
by flow cytometry as described above. The following concentrations were
used: 5 µmol/L annexin V, 50 µg/mL RGE and RGD peptides, 30 µg/mL
MoAb, and a dilution of 1:250 for polyclonal antibodies. Results are
expressed as the mean ± SD of the median fluorescence (FL-1-H) of at
least three experiments. *P < .01 by Student's
t-test, compared with adhesion of nonactivated platelets
treated with the isotype-specific control MoAb.
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HUVEC receptors.
To determine a counter receptor on apoptotic HUVEC, cells were treated
with a variety of blocking MoAbs to different adhesion receptors known
to be important for the binding of leukocytes or activated platelets.
However, none of the tested MoAbs was found to inhibit adherence
significantly. Blockade of both constitutively expressed receptors
(v3-integrin, 1-integrin,
GPIb, ICAM-1, and PECAM-1) and inducible receptors (VCAM-1,
E-selectin, and P-selectin) did not affect platelet binding (Fig
4). In accordance with this finding, we
found that HUVEC undergoing apoptosis induced by staurosporine did not
show increased expression of ICAM-1 or induction of expression of
E-selectin or VCAM-1 (B.R. Schwartz, unpublished
observations). Based on the fact that platelet
1-integrins mediated adhesion to apoptotic
HUVEC, it was conceivable that HUVEC would expose particular
platelet-binding matrix proteins such as collagen or fibronectin that
promoted binding. However, treatment of apoptotic HUVEC with blocking
antibodies to collagen IV or fibronectin did not reduce adhesion.
Because a previous report has shown that nonactivated platelet adhesion
to virally infected endothelial cells was mediated by endothelial
exposure of von Willebrand factor (vWF),32 platelet
adhesion was also tested after treatment of apoptotic HUVEC with a
blocking polyclonal anti-vWF antibody. However, platelet binding was
not altered, consistent with the fact that vWF is not a known ligand
for 1-integrins.
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| Fig 4.
Platelet adhesion to apoptotic HUVEC after blockade of
HUVEC receptors. Nonactivated platelets were allowed to adhere to HUVEC
treated with staurosporine for 6 hours. Before the adhesion assay,
HUVEC were treated with different receptor antagonists. Platelet
binding was determined by flow cytometry as described above. The
following concentrations were used: 5 µmol/L annexin V, 50 µg/mL
RGE and RGD peptides, 30 µg/mL MoAb, and a dilution of 1:250 for
polyclonal antibodies. Results are expressed as the mean ± SD of the
median fluorescence (FL-1-H) of at least two experiments.
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HUVEC-bound platelets become slightly activated.
The question of whether platelets would become activated through the
contact with apoptotic HUVEC was also addressed. As shown in Fig
5, the supernatants of apoptotic HUVEC with
bound platelets contained a significant amount of -thromboglobulin
and platelet factor 4 as compared with the supernatants of normal or
apoptotic HUVEC alone, nonactivated platelets alone, or normal HUVEC
incubated with nonactivated platelets. However, the concentration of
-thromboglobulin and platelet factor 4 was almost three times higher
when HUVEC were incubated with thrombin-activated platelets.
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| Fig 5.
Release of activation markers of HUVEC-bound platelets.
To determine whether platelets were activated through the contact with
apoptotic HUVEC, the supernatants were assayed for the presence of
platelet-specific activation markers. After incubation of the platelets
with HUVEC for 20 minutes, the supernatants were tested for
-thromboglobulin and platelet factor 4 using an ELISA assay.
Apoptosis of HUVEC was induced by treatment with staurosporine for 6 hours. Results are expressed as the mean ± SD of at least three
experiments. *P < .01 by Student's t-test, compared
with the release of activation markers by nonactivated platelets
incubated with normal HUVEC.
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Likewise, normal or apoptotic HUVEC alone, nonactivated platelets
alone, or normal HUVEC incubated with nonactivated platelets did not
express P-selectin, as determined by flow cytometry (Fig 6A-C). Complexes between nonactivated
platelets and apoptotic HUVEC as well as residual unbound platelets
derived from the supernatant of apoptotic HUVEC were not found to
express P-selectin in any significant manner (Fig 6B and C). Only when
preactivated with thrombin, platelets or platelet-HUVEC complexes
exhibited increased expression of P-selectin (Fig 6B and C). These data
indicate that platelets do indeed become slightly activated on binding
to apoptotic HUVEC. This is evidenced by release of a small quantity of
activation markers, although expression of P-selectin is not
significant.
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| Fig 6.
Expression of P-selectin of platelets and HUVEC. To
determine the degree of platelet activation through the contact with
apoptotic HUVEC, platelets were tested for the expression of
P-selectin. After incubation of the platelets with HUVEC for
20 minutes, the cells (whether adherent or unbound) were stained
separately with anti-P-selectin MoAb (WAPS 12.2) followed
by an FITC-conjugated anti-mouse MoAb. Cells were then assayed by flow
cytometry to determine P-selectin expression. As controls, nonactivated
and activated platelets and HUVEC were also measured separately. A
representative experiment of three performed is shown.
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| |
DISCUSSION |
Whereas endothelium normally controls platelet reactivity, recent in
vitro and in vivo studies have showed that, under certain conditions,
endothelial cells may develop properties that render them adhesive for
platelets. Once activated, platelets are well known to rapidly adhere
to both microvascular and macrovascular endothelial cells, recruit
additional platelets, and eventually form increasing larger
aggregates.9,10,33 Recently, we further characterized the
adhesion of activated platelets to endothelial cells, showing that it
is mediated by adhesive proteins involving platelet GPIIbIIIa and
different endothelial cell counter receptors.10 Furthermore, platelet-endothelial interactions may also occur when the
endothelium, but not the platelets, are activated. Two recent in vivo
studies have showed that nonactivated platelets roll on the endothelium
when it is activated with proinflammatory stimuli.11,12
Stimulation of the endothelium by laser light has similarly been shown
to induce adhesion of nonactivated platelets.13 This report
shows that during the process of apoptotic cell death, endothelial
cells also become proadhesive for nonactivated platelets.
Independent of the method of apoptosis induction, both adherent HUVEC
and HUVEC kept in suspension dramatically increased adhesion of
nonactivated platelets. Interestingly, the amount of binding was
similar to that of normal HUVEC incubated with thrombin-activated
platelets. Because we have previously shown that activated platelets
bound to normal HUVEC in the entire circumference,10 it is
conceivable that apoptotic HUVEC may be covered with platelets to a
similar extent. This hypothesis is supported by the fact that
incubation of apoptotic HUVEC with thrombin-activated platelets did not
further increase the fluorescence.
As shown in Fig 2, the acquisition of an adhesive phenotype during the
process of apoptosis preceded DNA fragmentation by several hours. This
is not a surprising finding, as it is well known that the translocation
of negatively-charged phospholipids to the external leaflet of the cell
membrane is an early event in apoptosis.5,34 Furthermore,
we have previously shown that apoptosis-induced changes of the HUVEC
surface membrane occurred well before detectable alterations of the
nucleus.5 Hence, the rapid binding of platelets to
apoptotic HUVEC may reflect early structural modifications of the cell
membrane such as the expression of new, presynthesized molecules or the
loss of constitutively expressed, platelet-repellent components. In
particular, treatment of valvular endothelial cells with heparinase or
neuraminidase has been found to render the cells proadhesive for
nonactivated platelets, suggesting that the loss of membrane sialyl
residues and heparan sulfate may induce a prothrombotic phenotype in
endothelial cells.35
So far, several receptors have been shown to be involved in the
interaction of live and apoptotic cells. Macrophages and other phagocytic cells recognize and bind apoptotic cells primarily via
v3-integrin or the CD36 receptor. Binding
is mediated by thrombospondin, which is secreted by the macrophages,
forming a bridge to a so far unidentified counter
receptor.36 Binding of apoptotic cells by phagocytes has
also been found to involve CD14 and the fibronectin receptor
51-integrin, which is thought to be
regulated by
v3-integrin.37,38 In our
studies, blockade of platelet v3-integrin
or the thrombospondin receptor CD36 (GPIV) did not inhibit binding to
apoptotic HUVEC. As well, platelet adhesion was not reduced by
treatment with an antithrombospondin MoAb. However, when platelets were
treated with anti-1 MoAbs, binding to apoptotic HUVEC
was inhibited by more than 70%. The fact that only the combination of
all three anti--MoAbs but not any one anti--MoAb alone revealed a
blocking effect suggests that all three -subunits might recognize a
ligand(s) on apoptotic HUVEC. It is important to note that the
anti-1-MoAbs blocked platelet binding more effectively
than the combination of the three anti--MoAbs. Importantly, all
three anti--MoAbs (P1E6, P1D6, and GoH3) have been found to block
platelet adhesion to matrix proteins.18-20 However, because
treatment of HUVEC with blocking MoAbs to fibronectin or collagen IV or
addition of RGD peptides did not reduce platelet binding, it is not
likely that matrix ligands were involved. Based on the lack of evidence
in the literature, we believe that another -subunit is
involved.39 Furthermore, studies in our laboratory have
shown that adhesion of leukocytes to endothelial cells undergoing
apoptosis is similarly mediated by multiple 1-integrins
(B.R. Schwartz, A. Karsan, T. Bombeli, J.M. Harlan,
submitted). Possibly, platelet binding to the apoptotic
ligand(s) on HUVEC uses regions of the -subunits different from
those used for known ligands.
Unfortunately, our attempts failed to determine the ligand(s) on
apoptotic HUVEC to which nonactivated platelets bind. Blockade of the
HUVEC receptors ICAM-1, v3-integrin, and
GPIb, which we have previously found to mediate binding of
thrombin-activated platelets,10 did not reduce platelet
binding. In addition, we did not find any evidence for the involvement
of endothelial vWF or PECAM-1, which have been suggested to be
responsible for the binding of nonactivated platelets to endothelial
cells stimulated with cytokines or laser light.32,40 Recent
in vivo studies have shown that nonactivated platelets roll on
postischemic or inflamed endothelium.11,12,41 These
interactions were found to be dependent on endothelial
P-selectin and E-selectin, whereas the counter receptors on
platelets have, so far, not been characterized. In our studies,
treatment of apoptotic HUVEC with blocking anti-P-selectin or
E-selectin MoAbs did not reduce nonactivated platelet binding. When
compared with leukocyte-endothelial interactions, these findings are
not surprising because rolling is known to involve different receptors
than firm adhesion.42 Therefore, we expected other endothelial adhesion receptors to be involved, possibly VCAM-1 (binds
1-integrins) and ICAM-1. Both receptors have recently been suggested to be responsible for the hyperadhesiveness of apoptotic
HUVEC for monocytic cells.43 However, blockade of these
receptors did not affect platelet adhesion to apoptotic HUVEC.
Moreover, we did not find any evidence that VCAM-1 and ICAM-1 became
significantly upregulated on induction of apoptosis (B.R. Schwartz,
unpublished observations). It has further been demonstrated that binding and phagocytosis of apoptotic cells can be
mediated by phosphatidylserine, which is rapidly upregulated on
induction of apoptosis.44,45 However, treatment of
apoptotic HUVEC with saturating concentrations of the
phosphatidylserine-binding protein annexin V did not reduce platelet
adhesion. These findings indicate that nonactivated platelet
adhesion to HUVEC undergo-ing apoptosis involves a, so far,
unidentified endothelial cell ligand(s) recognized by multiple
1-integrins.
When bound to subendothelial matrix proteins, platelets become readily
activated as characterized by the shape change, the release reaction,
and the activation of adhesion receptors. Thus, it seemed important to
evaluate whether binding of platelets to apoptotic endothelial cells
would similarly induce activation of the platelets. Based on the
increase of -thromboglobulin and platelet factor 4 in the
supernatant of apoptotic HUVEC with bound platelets, it is evident that
platelets become activated. However, apoptotic HUVEC seem to be a
relatively weak agonist, because the bound platelets did not
significantly express P-selectin. This slight activation may be
explained by the observation that only one type of platelet receptors
(1-integrins) is involved and that the
ligand(s) on HUVEC is probably not a matrix protein. Platelet binding
to matrix proteins and subsequent activation involves several
receptors.46 Hence, apoptotic HUVEC appear to bind
nonactivated platelets as effectively as the subendothelium. However,
after binding of a single layer, the recruitment of additional platelets into the evolving thrombus would likely be less effective, as
compared with subendothelial matrix proteins. Based on our previous
findings that apoptotic endothelial cells also become highly
procoagulant,5 it is tempting to speculate that endothelium undergoing apoptosis may support thrombotic events.
| |
ACKNOWLEDGMENT |
The authors gratefully acknowledge Dr R. Rothlein (Department of
Immunologic Diseases, Boehringer Ingelheim Pharmaceuticals Inc,
Ridgefield, CT), Dr D.C. Altieri (The Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT), Dr M.R.
Zocchi (Laboratory of Adoptive Immunotherapy, Milan, Italy), and Dr
G.J. Roth (Veterans Affairs Medical Center, Seattle, WA) for providing
antibodies. We also thank Dr J.F. Tait (Department of Laboratory
Medicine, University of Washington, Seattle, WA) for the generous gift
of recombinant annexin V and Tom Eunson for preparing cell cultures.
| |
FOOTNOTES |
Submitted June 18, 1998; accepted January 27, 1999.
Supported by the United States Public Health Service Grant Nos. HL
18645 and HL 03174. T.B. was supported by the Swiss Foundation for
Medical and Biological Grants of Switzerland.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Thomas Bombeli, MD, Division of Hematology,
Department of Medicine, Raemistrasse 100, University Hospital of
Zurich, 8091 Zurich, Switzerland; e-mail: haembomb{at}usz.unizh.ch.
| |
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ABSTRACT
INTRODUCTION