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Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2725-2734
Circulating Activated Platelets Assist THP-1 Monocytoid/Endothelial
Cell Interaction Under Shear Stress
By
Gregor Theilmeier,
Tim Lenaerts,
Claude Remacle,
Désiré Collen,
Jos Vermylen, and
Marc F. Hoylaerts
From the Center for Molecular and Vascular Biology, Katholieke
Universiteit Leuven, Leuven, Belgium; and the Laboratoire de Biologie
Cellulaire, Universite Catholique de Louvain, Louvain-la-Neuve,
Belgium.
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ABSTRACT |
Circulating complexes of leukocytes and activated platelets are
markers for atherosclerosis, but their interaction with the arterial
endothelial lining has not been studied. Therefore, the effect of
activated platelets on rolling and adhesion of labeled human THP-1
monocytoid cells to human umbilical vein endothelial cell (HUVEC)
monolayers was studied by epifluorescence microscopy in a parallel
plate flow chamber. In the absence of activated platelets, THP-1
rolling on resting HUVEC was negligible at shear rates greater than 300 s 1. Activation of HUVEC with 100 nmol/L phorbol
myristate acetate (PMA) increased THP-1 cell adhesion at
shear rates less than 400 s1. Therefore, a shear rate of
400 s1 was identified as a threshold for THP-1 adhesion.
THP-1 rolling on activated HUVEC was reduced by 64% after L-selectin
inhibition but was not affected by P-selectin inhibition. The addition
of 1 to 50 thrombin receptor-activating peptide
(TRAP)-activated platelets per THP-1 cell enhanced
interactions between THP-1 cells and HUVEC, resulting in a steep
bell-shaped dose-response curve, with a peak of 10 ± 3 rolling
cells/50 seconds at 3 platelets per THP-1 cell (P < .01 v control) with a concomitant 2- to 3-fold increase of firmly
adhering cells (P < .01 v control). In reconstituted blood, low numbers of activated platelets had the same effect on THP-1
rolling and adhesion. P-selectin inhibition reduced platelet/THP-1 cell
interaction in suspension and deposition of the complexes on the
endothelial monolayer. Inhibition of both P- and L-selectin reduced
THP-1/HUVEC interactions to 14% (P < .01, n = 4).
Sialidase digestion and removal of terminal sialic acid residues from
HUVEC or THP-1 cells but not from platelets abolished the platelet
mediated augmentation of THP-1 cell adhesion. Thus, THP-1 rolling on
HUVEC is shear-dependent and largely mediated by L-selectin. P-selectin expressed on activated platelets increases monocytoid cell adhesion to
endothelial cells at shear rates found in coronary arteries through
interactions with both endothelial and monocytoid cells and may
facilitate macrophage accumulation in the vessel wall.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PLATELETS HAVE BEEN implicated in the
early development and the progression of atherosclerosis. After plaque
rupture, platelets adhere to the prothrombotic material of the plaque
interior and set off the cascade of thrombus formation and vessel
occlusion.1-3 A contribution of platelets to endothelial
injury and leukocyte extravasation into the subendothelial space during
the early stages of atherogenesis before any morphological changes of
the vessel wall can be observed has been postulated, but the mechanism
of the phenomenon remains to be defined.4,5
Activated platelets mediate leukocyte/endothelial cell interactions,
such as lymphocyte rolling on high endothelial venules of
lymphnodes.6 Platelets roll on P-selectin expressed by
endothelial cells in mesenteric venules in vivo.7 Platelets
adherent to subendothelial matrix components support rolling, adhesion,
and transmigration of leukocytes.8,9 Furthermore, platelets
deposited on subendothelial matrix exposed between shear stress
strained human umbilical vein endothelial cells (HUVEC) have been
reported to mediate leukocyte delivery to the endothelial
cells.10
Circulating activated platelets and leukocytes have been observed in
animal models and in patient populations at risk for atherosclerosis.11-14 Increased levels of low-density
lipoprotein (LDL) cholesterol induce platelet activation, as assessed
by expression of P-selectin.15 Expression of adhesion
molecules on endothelial cells is increased in conditions predisposing
to atherosclerosis.16,17 Circulating complexes of activated
platelets and leukocytes have been observed in unstable angina
pectoris18 and after mechanical revascularization of
stenosed coronary arteries,19,20 suggesting that they may
be markers of vessel wall disease and thrombogenic atherosclerotic lesions.
Injection of chemically oxidized LDL into hamsters induced complex
formation between platelets and granulocytes and increased leukocyte
rolling on microvascular endothelium exposed in a skinfold chamber.21 Thus, circulating activated platelets may
deliver leukocytes to the vessel wall under low shear conditions. The functional consequences of circulating leukocyte/platelet complexes, their adhesive properties, and the adhesion molecules involved in their
delivery to the arterial vessel wall have not yet been elucidated.
The present study evaluates whether circulating activated platelets
form complexes with monocytes and thereby enable the adhesion of
monocytes to endothelial cells under shear conditions present in the
arterial circulation. A videomicroscopic in vitro system was used,
employing cosuperfusion over resting or activated HUVEC of activated
human platelets and the human monocytoid cell line THP-1.
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MATERIALS AND METHODS |
Reagents and materials.
Cell culture reagents, medium 199, RPMI 1640, Hank's balanced salt
solution (HBSS), phosphate-buffered saline (PBS), trypsin/EDTA, fetal
bovine serum and penicillin/streptomycin (10,000/10,000 U/mL) were
purchased from GIBCO, Lifetechnologies Inc (Paisley, UK). Human serum
was from Biowhittaker (Walkersville, MD). Paraformaldehyde and
microscopic glass coverslips 24 × 50 mm from VEL (Haasrode, Belgium) were sterilized in 70% propanol and washed in PBS. Tissue culture dishes were from Becton Dickinson Labware (Maylan Cedex, France) and uncoated Petri dishes were from Sterilin (Suffordshire, UK). For flow chamber experiments, rinsing of HUVEC monolayers and
preparation of the cell suspensions, 1% bovine serum albumin (Boehringer Mannheim, Mannheim, Germany), 200 µg/mL purified human fibrinogen, 2 mmol/L CaCl2, and 2 mmol/L MgCl2
(Sigma, St Louis, MO) were added to HBSS. This buffer is referred to as
binding buffer throughout the text. Tissue culture grade calf skin
collagen I was purchased from Boehringer Mannheim. Neuraminidase
(sialidase at 1 U/100 µL) was from Boehringer Mannheim or Sigma. For
the preparation of pH 6.5 ACD, Na3-citrate (75 mmol/L) and
dextrose (100 mmol/L) were purchased from VEL. Citric acid (38 mmol/L) was obtained from Sigma. Phorbol myristate acetate
(PMA) was dissolved in dimethyl sulfoxide
(DMSO), stored at  20°C, and added to media 1:1,000 for a final concentration of 100 nmol/L to activate HUVEC. 2',
7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM; Molecular Probes Europe, Leiden, The Netherlands) was
dissolved in DMSO, aliquoted, and stored at 20°C until used. The 14 amino acid form (S-F-L-L-R-N-P-N-D-K-Y-E-P-F) of thrombin receptor-activating peptide (TRAP) was either purchased from Sigma or
custom-synthesized by Eurogentech (Seraing, Belgium).
Monoclonal antibodies.
The L-selectin blocking22 monoclonal antibody DREG56 (mouse
IgG1; Endogen, Woburn, MA) was used at 2.5 µg/mL. The monoclonal anti-P-selectin antibody CLB-Thromb/623 (mouse IgG1;
Immunotech, Marseille, France) was used at concentrations ranging from
0 to 2.5 µg/mL. For fluorescence-activated cell sorting
(FACS) analysis of P-selectin expression, the same
antibody was purchased as fluorescein isothiocyanate (FITC) conjugate.
The anti-CD34 control antibody, Birma-K324 (mouse IgG1;
Dako, Glostrup, Denmark) was used at concentrations corresponding to
those of the blocking antibodies. The anti-GP IIb/IIIa antibody
MA-16N7C2 was raised and characterized in our laboratory as described
elsewhere.25 The GPIb blocking antibody G19H10F6 was raised
against the soluble GPIb domain glycocalicin by immunization of mice
according to standard techniques.26,27 This antibody
specifically binds to GPIb in enzyme-linked immunosorbent assay
(ELISA) and blocks ristocetin- and botrocetin-induced
platelet aggregation. von Willebrand factor-dependent platelet
deposition on coated collagen at 1,300 s1 was
inhibited by this antibody in flow chamber experiments with an
IC50 of 5 µg/mL. The RGD-mimetic G4120, used at 1 µg/mL, was a generous gift from Genentech (San Francisco, CA).
Culture of HUVEC and THP-1.
HUVEC were obtained by collagenase treatment28 and expanded
in Medium 199 supplemented with 10% fetal bovine serum, 10% human
serum, 100 U penicillin, and 100 U streptomycin on gelatin-coated dishes and frozen in liquid nitrogen after 2 passages. HUVEC were grown
to confluency in tissue culture flasks coated with calf skin collagen
I, trypsinized, and homogeneously seeded at high density on
collagen-coated glass coverslips placed in noncoated Petri dishes.
HUVEC were used at passage 3 to 5 for perfusion experiments. Cells were
allowed to adhere for 2 hours before media was added and cells were
grown to confluency on the coverslip. Whenever indicated, HUVEC were
activated 4 hours before the experiment by the addition of 100 nmol/L
PMA to the media. Coverslips were then carefully rinsed with binding
buffer and mounted in the flow chamber. Confluency of each coverslip
was confirmed microscopically before and after perfusion.
THP-1 cells were maintained in suspension in RPMI 1640 supplemented
with 10% fetal calf serum, 100 U penicillin, and 100 U streptomycin.
Cells were maintained at densities between 0.2 and 1.0 × 106/mL. Before perfusions, cells were washed and
resuspended in HBSS. Trypan blue exclusion showed a viability of
greater than 98%. BCECF-AM (1 µmol/L) was added to 1 × 106 THP-1/mL in HBSS for 45 minutes at 37°C. After
labeling, cells were washed and resuspended in HBSS at 10 × 106/mL and kept in the dark at room temperature until use.
For flow chamber experiments, THP-1 cells were added to binding buffer at 2 × 105/mL and allowed to equilibrate for 20 minutes at 37°C. Final concentrations of Ca2+ and
Mg2+ were 2 mmol/L. In a further set of experiments, THP-1
cells were used at 2 × 105/mL, 4 × 105/mL, and 6 × 105/mL in the presence or
absence of 3:1 activated platelets to examine whether the
platelet/THP-1 ratio or the number of platelets was the determining
factor of augmented THP-1 cell adhesion.
Platelet isolation.
Blood was drawn from healthy volunteers by puncture of an antecubital
vein with an 18G needle and freely drained on 0.1 vol citrate (108 mmol/L). Whole blood was centrifuged at 150g for 10 minutes to
obtain platelet-rich plasma (PRP), which was diluted in
1:1 vol ACD and centrifuged at 600g for 10 minutes. The
resulting pellet was resuspended in HBSS and 0.3 vol ACD was added
before the final wash at 600g for 10 minutes. Platelets were
counted and resuspended in HBSS after removing residual ACD at
300,000/µL and stored at room temperature until use within 3 hours.
An aliquot of platelets was diluted to 50,000/µL in binding buffer
and activated by the addition of 100 µmol/L TRAP for 10 minutes and
then immediatelely added to the THP-1 suspension for perfusion experiments.
FACS analysis of P-selectin expression.
Washed platelets (2.5 × 105/µL) from 3 different
donors were activated with 100 µmol/L TRAP for 10 minutes in the
presence of 1 µg/mL G4120. Samples of resting and activated platelets
were fixed at 4°C for 2 hours with 1% paraformaldehyde in
Tyrode's buffer. Platelets were washed and resuspended in Tyrode's
buffer and labeled for 20 minutes with the FITC-conjugated P-selectin antibody Thromb/6. Expression of P-selectin on the platelet membrane was analyzed using the FACS Calibur (Becton Dickinson) equipped with an
argon ion laser at wavelength 488 nm.
Flow chamber perfusion studies.
HUVEC on glass coverslips were washed free of media with binding buffer
and mounted on the bottom of a parallel plate flow chamber with a
chamber height of 0.2 mm.29 Defined shear rates between 100 and 700 s1 were generated with a precision pump in
withdrawal mode (Harvard Instruments, South Natick, MA). The flow
chamber was placed on the stage of an inverted epifluorescence
microscope (Diaphot; Nikon, Melville, NY) equipped with a 20× and
a 4× long working distance lens, allowing direct, real time,
videomicroscopic observation of interactions between fluorophor labeled
platelets and/or monocytes with the endothelial monolayer. Labeling of
monocytes and exposure to the fluorescent light did not alter numbers
of rolling or adhering cells (data not shown). Blocking antibodies and
antagonists were added 20 minutes before the perfusion at the indicated
final concentrations to the THP-1 suspension before activated platelets
were added. Single passage perfusions with platelets, THP-1 cells, or
both were performed for 5 minutes. During this period, 5 video
recordings spanning in total 50 seconds were obtained at defined time
intervals from 5 different high-power fields of 0.06 mm2
each with a Cohu CCD video camera (COHU Inc, San Diego, CA). Images
were read directly into the memory of an attached computer (Apple
Computers Inc, Palo Alto, CA) equipped with a Scion LG3 frame grabber
(Scion Corp, Frederick, ML) and stored on the hard drive for later
off-line analysis of the numbers of rolling cells. The movies were
recorded at a set rate of 20 frames per second. After 5 minutes, the
coverslips were rinsed with binding buffer for 5 minutes at a shear
rate of 400 s1 and 15 high-power fields were recorded.
Platelet labeling and superfusion.
For direct visualization of platelet/endothelial cell interactions,
washed platelets were incubated with 1 µmol/L BCECF-AM for 20 minutes
at 300,000/µL in HBSS, activated with 100 µmol/L TRAP for 10 minutes, and then diluted to 10,000/µL in binding buffer. For
superfusions with reconstituted blood, platelets were resuspended at
10,000/µL in a mixture of packed red blood cells (purchased from the
Red Cross Blood Bank, Leuven, Belgium) and binding buffer to obtain a
hematocrit of 40%. In these experiments, no monocytes were present. In
separate sets of experiments, activated platelets were superfused at
600/µL in binding buffer for 5 minutes, followed by 5 minutes of
rinsing. Then, 2 × 105 THP-1 cells/mL were superfused
and adhesion was quantitated.
Perfusion studies with reconstituted blood and fibrinogen-coated
albumin microcapsules.
In these experiments, THP-1 cells were resuspended at 2 × 105/mL with or without 3:1 platelets in reconstituted
blood. Packed red blood cells were washed at 150g with binding
buffer to remove residual platelets before the hematocrit was adjusted
to 40%. Alternatively, THP-1 cells were resuspended in binding buffer containing 10,000 fibrinogen-coated albumin microcapsules per microliter before activated platelets were added. These microcapsules have been demonstrated to closely resemble platelets in size
distribution and recruitment to forming thrombi.30 To
exclude the possibility that rheological changes in the presence of
corpuscular particles in the perfusate caused the inhibitory effect on
THP-1 cell adhesion at higher platelet THP-1 ratios, we chose
this approach instead of nonactivated platelets to avoid interference
by mildly activated platelets generated during the platelet isolation,
because very low concentrations of activated platelets in relation to
THP-1 cells in our studies already affected THP-1 adhesion.
Deposition of platelets.
Human blood was drawn on 3.82% citrate and superfused for 5 minutes at
400 s1 over coverslips coated with 1 mg/mL calf skin
collagen I. This protocol yielded homogeneous deposition of platelet
microaggregates on the coverslip. The coverslips were rinsed until no
red blood cells were observed microscopically and were then immediately used for perfusion experiments with 1 × 105 THP-1
cell/mL suspensions.
Contribution of shear to formation of platelet/THP-1 complexes.
To elucidate whether complex formation between platelets and THP-1
cells would occur in suspension and whether exposure to shear forces
during the superfusion in the flow chamber would enhance this complex
formation, platelets were labeled with BCECF-AM, activated with 100 µmol/L TRAP for 10 minutes, and added in a ratio of 3:1 to 2 × 105 nonlabeled THP-1 cells/mL binding buffer. The number of
THP-1 cells bearing labeled platelets and the overall number of THP-1 in the cell suspension was assessed microscopically in mixed white and
fluorescent light in a Fuchs-Rosenthal counting chamber
(Electron Microscopy Sciences, Fort Washington, PA) before and after
passage of the flow chamber. The fraction of platelet decorated THP-1 cells deposited on HUVEC after 5 minutes of superfusion and 5 minutes
of buffer rinse was also counted. For each of these experiments, at
least 100 THP-1 cells on the monolayer were counted in mixed white and
fluorescent light and the fraction of platelet bearing THP-1 cells was
assessed. Similar experiments were performed in the presence of the
P-selectin blocking antibody Thromb/6 (2.5 µg/mL).
Sialidase digestion.
HUVEC were overlayed with sialidase at 0.1 U/mL and kept in an
incubator for 30 minutes. For platelet incubations, the enzyme was added to the platelets after activation to reach a final
concentration of 0.1 U/mL and then diluted greater than 100-fold in
THP-1 cell suspension after 30 minutes. THP-1 cells (10 × 106/mL) were incubated with 0.4 U/mL sialidase for 60 minutes and then diluted to 2 × 105/mL in binding
buffer for perfusion experiments. Cell suspensions were kept in a
37°C waterbath during the incubation period and the experiment.
Data analysis and statistical methods.
The recorded movies were analyzed off-line with NIH Image 1.61 (developed at the Regional Services Branch of the National Institute of
Mental Health, Bethesda, MD). The movies were reduced to
every fourth frame for analysis after detecting brightly labeled THP-1
cells. Movies were black-white inverted to facilitate further image
processing. These frames were overlayed, with the resulting image
depicting rolling cells as series of dark blobs. Rolling cells were
defined as cells moving more than their own diameter during the movie,
before arresting on the endothelium or leaving the endothelial cells to
return back to the speed of the flowing cells. The number of cells in
the 5 movies of 1 experiment spanning the full duration of the
superfusion was defined as rolling THP-1 cells per 50 seconds. Adhering
cells were recorded on 15 high-power fields of 0.06 mm2
each after 5 minutes of buffer rinse and were saved as a stack of
single frames on the hard drive of the attached computer. Because after
this time rolling cells were no longer observed, cells that were still
present on the endothelium were defined as adhering THP-1 cells/0.9
mm2. Data were processed in InStat 2.03, GraphPad Software
Inc (San Diego, CA). For comparison between groups, a
Kruskal-Wallis nonparametric ANOVA test, followed by a Dunn's multiple
comparison test, was performed. To assess differences in platelet
decoration of THP-1 cells, a  2 test was performed on all
cells that were counted under the different conditions, and the
individual fractions of platelet bearing THP-1 cells were then compared
by Fisher's exact test. A P value less than .05 was considered
significant. Data are reported as the mean ± standard error of the
mean (SEM).
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RESULTS |
Shear dependence of THP-1 adhesion to HUVEC.
Resting HUVEC did not support rolling and adhesion of THP-1 cells at
shear rates greater than 400 s1. Activation of HUVEC
with PMA led to a significant increase of rolling and adhesion at shear
rates of 300 and 200 s1
(Table 1). Because 400 s1 appeared to be a threshold shear rate for
monocyte endothelial cell interaction in our flow chamber system, the
following studies were performed at this shear rate and after
activation of HUVEC monolayers with PMA to examine if activated
platelets would enhance THP-1/endothelial cell interactions.
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Table 1.
Activation of HUVEC With 100 µmol/L PMA for 4 Hours
Increases THP-1 Rolling and Adhesion Only at Shear Rates Less Than
400 s1 (n = 3/6)
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P-selectin expression on activated platelets.
TRAP activation of washed platelets induced a significant shift
of mean fluorescence values (9.2 ± 0.8 v 39 ± 5.8, n = 3, P < .05), indicating pronounced expression of P-selectin
on the platelet membrane.
Activated platelets augment THP-1 rolling and adhesion.
The addition of 1 to 50 TRAP-activated platelets per THP-1 cell to the
superfusions at 400 s1 on PMA-activated HUVEC showed
a steep, bell-shaped dose-response curve at very low platelet/leukocyte
ratios, with a peak at 3 platelets/THP-1 cell and an inhibitory effect
compared with the control experiments starting at a ratio of 10 activated platelets/THP-1. At 50 platelets per leukocyte, rolling and
adhesion were virtually abolished (Fig 1).
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| Fig 1.
Activated platelets augment THP-1 rolling and adhesion.
Addition of 1 to 50 TRAP-activated platelets per THP-1 cell showed a
steep bell-shaped dose-response curve of monocyte rolling (A) and
adhesion (B) on PMA-activated HUVEC, with a narrow peak at 3 platelets
per monocyte. Inhibition of monocyte margination was observed at ratios
= 10 platelets per monocyte (n = 4, *P < .01 v no
platelet control).
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The addition of 3 activated platelets per THP-1 cell caused a marked
shift of the adhesive interactions towards higher shear resistance. At
300 s1, the addition of platelets to the suspension
still increased margination of THP-1 cells from 9.3 ± 5.3 to
37 ± 3.7 rolling monocytes/50 seconds at 300 s1 (P < .01, n = 6). These increases in
dynamic interactions resulted in a corresponding increase of firmly
adhering THP-1 cells. The threshold shear rate (400 s1) yielded 184 ± 55 versus 65 ± 13 adhering
THP-1 cells per 0.9 mm2 (P < .01, n = 6;
Fig 2). THP-1 cells (2 × 105) were superfused in reconstituted blood to titrate a
similar threshold shear rate as for the experiments in buffer (data not shown). At a shear force of 5 dyn, a similar augmentation of THP-1 rolling and adhesion was observed: 1.4 ± 0.8 THP-1 cells
rolled in the absence of platelets. This rolling was augmented to 6.2 ± 1.3 rolling THP-1/50 seconds, when 3:1 TRAP-activated platelets were added to the suspension (n = 5, P < .01). Adhesion was
likewise increased from 14.8 ± 7.5 to 50.4 ± 10 adhering THP-1
cells per 0.9 mm2 (n = 5, P < .01).
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| Fig 2.
Addition of 3:1 platelets to the THP-1 suspension causes
a left shift in the shear response. THP-1 cell rolling and adhesion to
PMA-activated HUVEC ( ) was only detectable at shear rates less than
400 s1. The addition of 3 TRAP-activated platelets per
THP-1 cell ( ) supported dynamic and firm interactions up to 600 s1. Rolling (A) and adhesion (B) of THP-1 cells
similarly increased with the addition of activated platelets to the
suspension (n = 6, for each condition, *P < .01 v PMA-activated HUVEC in the absence of platelets).
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When the THP-1 cell count in binding buffer was increased stepwise from
2 × 105 to 4 × 105 and 6 × 105 /mL, while the ratio of activated platelets was kept at
3:1, there was an increase in basal rolling and adhesion as a function of THP-1 cell number, even in the absence of platelets. However, the
significant augmentation of THP-1 cell rolling and adhesion to HUVEC by
activated platelets was conserved (Fig 3).
To rule out that rheological changes were responsible for the
inhibitory effect of higher numbers of platelets per THP-1 cell,
fibrinogen-coated albumin microparticles were included in the
superfusion to mimick the presence of excess resting platelets. The
augmentation of THP-1 adhesion by activated platelets was conserved,
even when 10,000 microcapsules per microliter (a ~20-fold excess in
comparison to the ratio 3:1) were present during the superfusion (1.8 ± 0.8 v 11 ± 3.3 rolling THP-1/50 seconds and 63 ± 6.5 v 191 ± 64 adhering THP-1/0.9 mm2, n = 4, P < .05, in the absence or presence of activated platelets).
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| Fig 3.
When THP-1 cell numbers were stepwise increased from
2 × 105 to 4 × 105 and 6 × 105, basal rolling and adhesion increased progressively
(). The addition of 3:1 activated platelets ( ) further augmented
THP-1 cell rolling (A) and adhesion (B) irrespective of absolute THP-1
cell number (n = 4, * P < .01).
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THP-1 rolling on deposited platelets.
When THP-1 cells were superfused at 400 s1 over
immobilized platelets, significant rolling occurred. Upon addition of
increasing numbers of activated platelets to the superfusate, a
progressive inhibition of THP-1 cell rolling was observed, with an
IC50 of 940 platelets/µL, corresponding to approximately
10 platelets per THP-1 cell. The addition of nonactivated platelets
also caused inhibition of THP-1 rolling, with an IC50 of
22,300 platelets/µL. THP-1 cell rolling on deposited platelets was
also dose-dependently inhibited by the P-selectin blocking antibody
Thromb/6, whereas control antibodies against CD34 or platelet GPIb did
not affect THP-1 rolling (Fig 4). Because
2.5 µg/mL Thromb/6 virtually abolished leukocyte rolling on
deposited, activated platelets, this concentration of blocking antibody
was used for all further experiments. Therefore, the inhibition of
THP-1 rolling over coated platelets by high resting platelet numbers
seems to be due to mild activation and P-selectin expression on a
subset of platelets as a consequence of the isolation procedure.
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| Fig 4.
THP-1 cells rolled on deposited platelets in a
P-selectin-dependent fashion (anti-CD62P). There was no interaction in
the absence of platelets on the coverslip (collagen). Anti-CD34 and
anti-GPIb antibodies did not interfere with rolling (n = 4, *P < .05 v platelets).
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Interaction of activated platelets with HUVEC.
Whether the platelet/THP-1 cell interactions that augmented
shear-dependent monocyte margination occurred in suspension or with
deposited platelets on the HUVEC surface was studied as follows. Platelet adhesion on HUVEC was directly determined by epifluorescence microscopy. Ten thousand TRAP-activated platelets per microliter, perfused at 400 s1, did not interact with
PMA-activated HUVEC when suspended in binding buffer: 3.7 ± 0.7 platelets rolled within 50 seconds and 34 ± 7.4 platelets arrested on 0.15 mm2 of PMA-activated HUVEC.
After suspension of activated platelets in reconstituted blood,
37 ± 1.9 translocating platelets per 50 seconds and 1,103 ± 113 adhering platelets/0.15 mm2
(Fig 5, n = 4; P < .001) were
counted. In binding buffer in which activated platelets supported THP-1
cell delivery to HUVEC, ie, at 600 platelets/µL and 200 THP-1
cells/µL, direct interactions of individual platelets with HUVEC were
virtually absent (data not shown).
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| Fig 5.
Rolling and adhesion to activated endothelial cells of
10,000 platelets/µL were only observed in the presence of packed red
blood cells (PRBC) but not in binding buffer (n = 4, **P < .001 v PRBC).
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To further support the conclusion that platelet deposition and
subsequent THP-1 adhesion was not the mechanism of platelet assistance
to monocyte adhesion, platelet perfusion and THP-1 perfusion were
separated. A prior superfusion of activated platelets at 600/µL did
not yield an increase in subsequent monocyte adhesion (72 ± 2.9 v 76 ± 8.8 THP-1 cells/0.9 mm2, n = 4, P = not significant), indicating that the increased THP-1 adhesion in the presence of platelets was not due to adhesion of THP-1
to previously deposited platelets.
Activated platelets and THP-1 cells form complexes under shear
conditions.
Most of the monocytoid cells had 1 or 2 platelets attached. THP-1 cells
decorated with more than 3 platelets were very rare. Complexes of
several THP-1 cells bridged by platelets were only rarely observed and
not entered in the analysis. After passage of the flow chamber, the
THP-1 number in the effluent was 123,000 ± 1,600/mL in the absence
of platelets and 92,000 ± 16,000 (n = 4) when 3:1 activated
platelets were present. Thus, 38% and 54% of the THP-1 cells were
trapped on the HUVEC monolayer. When labeled platelets were activated
and coperfused with nonlabeled THP-1 cells, 160 of 635 (n = 5 experiments) counted THP-1 cells entering the flow chamber were
decorated with platelets. The number of THP-1 cells that were decorated
with platelets in the effluent was significantly higher (318 of 764, n = 5, P < .001). Moreover, THP-1 cells recruited to the
monolayer also were preferentially decorated with platelets. Upon
addition of the P-selectin blocking antibody, the percentage of
platelet-decorated THP-1 cells in the pre-perfusion suspension was
reduced as was the recruitment of these complexes to the HUVEC and the
increase of THP-1 cell decoration in the effluent
(Table 2 and
Fig 6).
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| Fig 6.
Visualization of platelet/THP-1 complexes. Fluorescently
labeled activated platelets were added to the suspension of nonlabeled
THP-1 cells in a ratio of 3:1. Complex formation between platelets and
THP-1 cells was assessed by combined light and fluorescence microscopy.
The panels in row (A) depict micrographs of THP-1/platelet complexes
adhering to the endothelial monolayer (40×). Row (B) shows typical
examples of sparsely decorated THP-1 cells in the suspension after
passage through the flow chamber (40×).
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L-selectin and P-selectin mediate rolling of leukocyte/platelet
complexes.
The rolling of labeled THP-1 cells perfused over PMA-activated HUVEC in
the absence of activated platelets was inhibited by 64% after the
addition of 2.5 µg/mL of the L-selectin blocking antibody to the
superfused THP-1 cells. In the presence of 3 activated platelets
(600/µL) per THP-1 cell (200/µL), the inhibition of the increased
rolling by this antibody was 60%. The anti-P-selectin antibody did
not block rolling in the absence of platelets, but blocked 40% of the
rolling after platelets were added. Both antibodies together blocked
86% of the rolling observed in the presence of platelets, reducing the
number of rolling monocytes to control levels after L-selectin blockade
(P < .01, n = 4). Control antibody against CD34, matched for
isotype, did not block rolling or adhesion in the presence or absence
of platelets. The anti-GP IIb/IIIa antibody inhibited rolling
(Fig 7).
View larger version (19K):
[in this window]
[in a new window]
| Fig 7.
Rolling of THP-1 cells on PMA-activated HUVEC was to a
large extent mediated by L-selectin. P-selectin inhibition did not
yield a blocking effect. After the addition of activated platelets
(ratio 3:1), L- and P-selectin blocking resulted in an inhibition of
rolling and a concomitant reduction of adhering monocytes. GPIIb/IIIa
blockade resulted in reduced rolling, whereas GPIb inhibition reduced
adhesion of complexes without affecting rolling of THP-1 cells in the
presence of platelets (n = 4 for each condition, *P < .05).
|
|
Role of GPIb and GPIIb/IIIa for firm adhesion of
platelet/THP-1-complexes.
Blockade of the GPIb pathway caused a trend (not significant) towards
reduced rolling of THP-1 cells in the presence of platelets. Firm
adhesion was significantly reduced by 40% (P < .05, n = 4). On the other hand, blocking of GPIIb/IIIa did not reduce monocyte adhesion in the presence of platelets (Fig 7).
Sialidase treatment of HUVEC and THP-1 abolishes rolling of
platelet/THP-1 complexes.
Treatment of platelets with 0.1 U/mL sialidase for 30 minutes did not
result in an inhibition of platelet-assisted monocyte adhesion. When
HUVEC or THP-1 cells were incubated with sialidase, the numbers of
rolling and adhering THP-1 cells in the presence of 3:1 activated
platelets at 400 s1 were reduced to the level found
in the absence of activated platelets (Fig
8).
View larger version (18K):
[in this window]
[in a new window]
| Fig 8.
HUVEC and THP-1 treatment with sialidase abolished the
platelet-mediated (ratio 3:1) augmentation of rolling and subsequent
adhesion of THP-1 cells at 400 s1. Sialidase treatment
of platelets had no effect. Combined sialidase digestion of HUVEC and
platelets did not further reduce THP-1/endothelial cell interaction in
the presence of activated platelets (n = 4, *P < .05).
|
|
| |
DISCUSSION |
The primary finding of the present study is that activated platelets
augment adhesion of monocytoid cells to cultured activated HUVEC in a
flow chamber system under moderate shear forces. Adhesion of THP-1
cells alone was prevented by shear forces resembling those found in
arterial vessels that are affected by atherosclerotic lesion
development in vivo. However, the addition of very small numbers of
activated platelets enabled interactions between THP-1 and endothelial
cells at higher shear rates as a consequence of decoration of the
monocytoid cells with platelets in suspension. Platelet-assisted THP-1
delivery to confluent endothelial layers was observed at shear rates of
up to 600 s1. Thus, activated platelets can assist
monocytoid cell adhesion to endothelial cells at shear forces that
preclude direct monocyte adhesion.
A steep bell-shaped dose-response curve was observed, demonstrating
platelet-supported THP-1 adhesion in a narrow range of platelet/THP-1
ratios. Inhibition of THP-1 adhesion in the presence of excess
platelets suggested that the interaction between platelets and THP-1
cells in suspension was mediated at least partially by the same
adhesion molecule as the interaction of the complex with the
endothelial cell. The significant augmentation of THP-1 adhesion with
increasing numbers of THP-1 cells but at a constant ratio of 3:1
activated platelets indicated that the platelet/THP-1 ratio was the
critical parameter, not the absolute platelet number, suggesting that
the observed platelet-mediated augmentation of THP-1 adhesion could
occur across the whole physiological range of monocyte counts in vivo,
provided that circulating activated platelets are present. Counts of
activated platelets can be as high as 10% after acute coronary
syndromes and interventional revascularization or
thrombolysis.14 Lower fractions of activated platelets have
been reported in the earlier stages of the disease process.15,18 The presence of truly resting platelets does not interfere with the observed effect of low numbers of activated platelets, because in the experiments with albumin microcapsules, a
similar increase of rolling and adhesion was observed when 3 TRAP-activated platelets were added in the presence or absence of these
artificial platelets. This rules out that the inhibition of THP-1
rolling and adhesion in the presence of greater than 10 activated
platelets was caused by changes in the numbers of cells in the
perfusate or by changes in viscosity.
Kuijper et al10 have reported neutrophil adhesion to
platelets that had been previously deposited on extracellular matrix between HUVEC strained by shear stress. In this system, leukocyte adhesion occurred preferentially to platelets instead of endothelial cells.10 However, this study did not include platelets in
the superfusate.
To further characterize the interaction between the 3 cell types, the
THP-1/endothelial cell, platelet/endothelial cell, and THP-1/platelet
interactions were separated. Platelet adhesion to activated HUVEC
required a hemorheological cell distribution as in blood, in which the
presence of red blood cells drives platelets into the periphery of the
perfused lumen, thus increasing the density of platelets in the
immediate vicinity of the endothelial lining.31
The classical finding that platelet/matrix interaction requires red blood cells was extended to the adhesion of activated platelets to activated endothelium by adding labeled platelets to
reconstituted blood, which immediately resulted in ample translocation and adhesion of individual activated platelets to activated endothelial cells. When THP-1 cells were resuspended in reconstituted blood, the
addition of 3:1 activated platelets yielded similar increases in
rolling and adhesion as in the buffer system. However, under these
conditions, a contribution of independently deposited platelets to
THP-1 adhesion cannot be ruled out. Therefore, all other experiments were performed in buffer, in which the rheological parameters did not
allow platelets to adhere by themselves.
Monocytes are known to roll on and adhere to predeposited
platelets.9 Antibody studies showed that THP-1 adhesion to
deposited platelets was primarily mediated by P-selectin, as has been
reported by several groups.9,21,32 However, the presence of
activated platelets in the suspension inhibited monocyte adhesion,
providing additional evidence for an interaction between THP-1 cells
and platelets in suspension and suggesting P-selectin to be one of the
candidate molecules involved in complex formation between circulating
platelets and monocytes.
The rate of complex formation in the THP-1/platelet solution before
perfusion through the flow chamber was rather low at approximately 25%, whereas passage through the chamber significantly increased decoration of THP-1 cells with platelets, confirming the impact of
shear on the association of activated platelets with leukocytes as
reported before.33 The P-selectin blocking antibody
decreased the percentage of platelet-decorated THP-1 cells in the cell
suspension before passage through the chamber and prevented the
shear-induced increase in THP-1 cell decoration after passage through
the flow chamber. Importantly, P-selectin inhibition virtually
abolished the preference for the recruitment of decorated THP-1 cells
to the HUVEC monolayer. This finding is in agreement with and confirms previous findings,32,34,35 suggesting that engagement of
selectins with their ligands requires threshold shear forces. However,
it cannot be excluded that monocytes binding platelets through
P-selectin may become activated and express adhesion molecules that may
mediate adhesion at higher shear rates.36 P-selectin
engagement in flow mediated delivery of THP-1 cells by activated
platelets to the endothelium, as evidenced by reduced deposition of
these complexes after P-selectin inhibition. P-selectin contributes to
the decoration of monocytes with platelets as well as to their delivery
to endothelial layers.
Rolling of THP-1 cells on HUVEC in the absence of platelets was, as
expected, largely mediated by L-selectin,37 with no effect
of P-selectin blockade. However, in the presence of 3 activated platelets per THP-1 cell, P-selectin accounted for 40% of the rolling
of the complexes. Because the total number of rolling cells increased
in the presence of platelets, 60% inhibition of rolling by
anti-L-selectin antibodies indicated that the decoration of THP-1
cells with platelets also favored direct THP-1 cell interactions with
the endothelium. Combination of both antibodies nearly abolished rolling and adhesion, indicating that the margination of THP-1 cells by
platelets allowed the engagement of L-selectin with its ligands. These
findings also demonstrate that the flow chamber model used for these
studies very closely resembles leukocyte endothelial cell interactions
in vivo, because the disruption of the initial selectin mediated
tethering and rolling also abolished firm adhesion of leukocytes, as
postulated in the multistep model of leukocyte adhesion.38
Because P-selectin is expressed on activated platelets as well as on
endothelial cells and the ligand PSGL-1 is present on leukocytes and
endothelium, THP-1, platelets, or HUVEC were exposed to neuraminidase
to eliminate the respective selectin ligands. Treatment of platelets
with the enzyme did not inhibit the augmented THP-1/HUVEC interactions,
whereas digestion of sialyl sugars on THP-1 or HUVEC reduced THP-1
adhesion in the presence of platelets to the level found in the absence
of activated platelets. These results clearly show that platelet
P-selectin in a first step engages sialyl sugars on THP-1 cells to form
complexes. In a second step, P-selectin on the platelet travelling with
the THP-1 engages sialyl sugars present on the endothelial cell.
Removal of either receptors on THP-1 or endothelial cells completely
disrupts this triangular interaction.
The inhibitory effect of the GPIb blocking antibody on firm THP-1 cell
adhesion in the presence of platelets indicates a role for von
Willebrand factor anchored on endothelial cells in mediating monocyte/platelet adhesion to endothelial cells. Another putative ligand for platelet GPIb is intercellular adhesion molecule-1 (ICAM-1), expressed after activation of endothelial
cells.39 Because GPIb inhibition did not affect THP-1
interactions with deposited platelets, the reduced adhesion in the
presence of the blocking antibody suggests an involvement of platelets
in firm THP-1 cell adhesion to HUVEC via this pathway. GPIIb/IIIa,
which has been suggested to be the only platelet membrane receptor
mediating platelet/HUVEC interactions in a static adhesion
assay,39 does not seem to play a role in the phenomenon
under investigation here. As for the increased contribution of
L-selectin to rolling in the presence of platelets, it seems likely
that monocyte adhesion molecules37 and their ligands
mediate monocyte/endothelial cell interactions after platelet
decoration has led to a P-selectin-mediated margination of the complex.
In conclusion, our data support the hypothesis that activated platelets
assist monocyte adhesion to endothelial cells at shear rates that do
not allow monocytes by themselves to adhere to activated endothelium.
The interaction of platelets with monocytes occurs in the circulation;
it is mediated by platelet P-selectin and dependent on ambient shear
forces. The margination of platelet decorated monocytes occurs through
P-selectin, presented by the platelets, but also augments direct
monocyte/endothelial cell interaction. These findings suggest that
circulating activated platelets, as found in hypercholesterolemia in
humans, play a role in margination, adhesion, and extravasation of
monocytes at sites where shear forces would preclude
monocyte/endothelial cell interaction.
| |
ACKNOWLEDGMENT |
Fibrinogen-coated albumin microcapsules were a generous gift from Dr
Marcel Levi (Academisch Medisch Centrum, Amsterdam, The Netherlands).
The authors are grateful to Erik Spaepen for excellent technical assistance.
| |
FOOTNOTES |
Submitted December 14, 1998; accepted June 14, 1999.
Supported by the Interuniversitaire Attractiepolen, (P4/34). J.V. is
holder of the Dr. J. Choay Chair in Hemostasis Research. G.T. is on
temporary leave from the Department for Anesthesiology and Surgical
Intensive Care Medicine, Faculty of Medicine, University of
Münster Germany. G.T. received a postdoctoral fellowship from the
IMF, University of Münster, Germany.
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 Marc F. Hoylaerts, PhD, Center for
Molecular and Vascular Biology, Katholieke Universiteit Leuven, Campus
Gasthuisberg, O&N, Herestraat 49, B-3000 Leuven, Belgium; e-mail:
marc.hoylaerts{at}med.kuleuven.ac.be.
| |
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A. Nemmar, B. Nemery, P. H. M. Hoet, N. Van Rooijen, and M. F. Hoylaerts
Silica Particles Enhance Peripheral Thrombosis: Key Role of Lung Macrophage-Neutrophil Cross-Talk
Am. J. Respir. Crit. Care Med.,
April 15, 2005;
171(8):
872 - 879.
[Abstract]
[Full Text]
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C. C.M. Appeldoorn, A. Bonnefoy, B. C.H. Lutters, K. Daenens, T. J.C. van Berkel, M. F. Hoylaerts, and E. A.L. Biessen
Gallic Acid Antagonizes P-Selectin-Mediated Platelet-Leukocyte Interactions: Implications for the French Paradox
Circulation,
January 4, 2005;
111(1):
106 - 112.
[Abstract]
[Full Text]
[PDF]
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J. N. Ghansah and J. T. Murphy
Complications of Major Aortic and Lower Extremity Vascular Surgery
Seminars in Cardiothoracic and Vascular Anesthesia,
December 1, 2004;
8(4):
335 - 361.
[Abstract]
[PDF]
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M. M. Burdick and K. Konstantopoulos
Platelet-induced enhancement of LS174T colon carcinoma and THP-1 monocytoid cell adhesion to vascular endothelium under flow
Am J Physiol Cell Physiol,
August 1, 2004;
287(2):
C539 - C547.
[Abstract]
[Full Text]
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H. Shiratsuchi and M. D. Basson
Extracellular pressure stimulates macrophage phagocytosis by inhibiting a pathway involving FAK and ERK
Am J Physiol Cell Physiol,
June 1, 2004;
286(6):
C1358 - C1366.
[Abstract]
[Full Text]
[PDF]
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R. J. Ludwig, B. Boehme, M. Podda, R. Henschler, E. Jager, C. Tandi, W.-H. Boehncke, T. M. Zollner, R. Kaufmann, and J. Gille
Endothelial P-Selectin as a Target of Heparin Action in Experimental Melanoma Lung Metastasis
Cancer Res.,
April 15, 2004;
64(8):
2743 - 2750.
[Abstract]
[Full Text]
[PDF]
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K. Nishijima, J. Kiryu, A. Tsujikawa, K. Miyamoto, M. Honjo, H. Tanihara, A. Nonaka, K. Yamashiro, H. Katsuta, S. Miyahara, et al.
Platelets Adhering to the Vascular Wall Mediate Postischemic Leukocyte-Endothelial Cell Interactions in Retinal Microcirculation
Invest. Ophthalmol. Vis. Sci.,
March 1, 2004;
45(3):
977 - 984.
[Abstract]
[Full Text]
[PDF]
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P. da Costa Martins, N. van den Berk, L. H. Ulfman, L. Koenderman, P.L. Hordijk, and J.J. Zwaginga
Platelet-Monocyte Complexes Support Monocyte Adhesion to Endothelium by Enhancing Secondary Tethering and Cluster Formation
Arterioscler Thromb Vasc Biol,
January 1, 2004;
24(1):
193 - 199.
[Abstract]
[Full Text]
[PDF]
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H. Koyama, T. Maeno, S. Fukumoto, T. Shoji, T. Yamane, H. Yokoyama, M. Emoto, T. Shoji, H. Tahara, M. Inaba, et al.
Platelet P-Selectin Expression Is Associated With Atherosclerotic Wall Thickness in Carotid Artery in Humans
Circulation,
August 5, 2003;
108(5):
524 - 529.
[Abstract]
[Full Text]
[PDF]
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L. H. Ulfman, D. P. H. Joosten, C. W. van Aalst, J.-W. J. Lammers, E. A. van de Graaf, L. Koenderman, and J. J. Zwaginga
Platelets Promote Eosinophil Adhesion of Patients with Asthma to Endothelium under Flow Conditions
Am. J. Respir. Cell Mol. Biol.,
April 1, 2003;
28(4):
512 - 519.
[Abstract]
[Full Text]
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P. C. Burger and D. D. Wagner
Platelet P-selectin facilitates atherosclerotic lesion development
Blood,
April 1, 2003;
101(7):
2661 - 2666.
[Abstract]
[Full Text]
[PDF]
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C. C. M. Appeldoorn, T. J. M. Molenaar, A. Bonnefoy, S. H. van Leeuwen, P. A. H. Vandervoort, M. F. Hoylaerts, T. J. C. van Berkel, and E. A. L. Biessen
Rational Optimization of a Short Human P-selectin-binding Peptide Leads to Nanomolar Affinity Antagonists
J. Biol. Chem.,
March 14, 2003;
278(12):
10201 - 10207.
[Abstract]
[Full Text]
[PDF]
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A. Bonnefoy, H. Yamamoto, C. Thys, M. Kito, J. Vermylen, and M. F. Hoylaerts
Shielding the front-strand beta 3 of the von Willebrand factor A1 domain inhibits its binding to platelet glycoprotein Ibalpha
Blood,
February 15, 2003;
101(4):
1375 - 1383.
[Abstract]
[Full Text]
[PDF]
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T. J. M. Molenaar, C. C. M. Appeldoorn, S. A. M. de Haas, I. N. Michon, A. Bonnefoy, M. F. Hoylaerts, H. Pannekoek, T. J. C. van Berkel, J. Kuiper, and E. A. L. Biessen
Specific inhibition of P-selectin-mediated cell adhesion by phage display-derived peptide antagonists
Blood,
November 15, 2002;
100(10):
3570 - 3577.
[Abstract]
[Full Text]
[PDF]
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E. M. Conway, M. Van de Wouwer, S. Pollefeyt, K. Jurk, H. Van Aken, A. De Vriese, J. I. Weitz, H. Weiler, P. W. Hellings, P. Schaeffer, et al.
The Lectin-like Domain of Thrombomodulin Confers Protection from Neutrophil-mediated Tissue Damage by Suppressing Adhesion Molecule Expression via Nuclear Factor {kappa}B and Mitogen-activated Protein Kinase Pathways
J. Exp. Med.,
September 2, 2002;
196(5):
565 - 577.
[Abstract]
[Full Text]
[PDF]
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M. J. Quinn, E. F. Plow, and E. J. Topol
Platelet Glycoprotein IIb/IIIa Inhibitors: Recognition of a Two-Edged Sword?
Circulation,
July 16, 2002;
106(3):
379 - 385.
[Full Text]
[PDF]
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K. Hahnenkamp, G. Theilmeier, H. K. Van Aken, and C. W. Hoenemann
The Effects of Local Anesthetics on Perioperative Coagulation, Inflammation, and Microcirculation
Anesth. Analg.,
June 1, 2002;
94(6):
1441 - 1447.
[Full Text]
[PDF]
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G. Theilmeier, C. Michiels, E. Spaepen, I. Vreys, D. Collen, J. Vermylen, and M. F. Hoylaerts
Endothelial von Willebrand factor recruits platelets to atherosclerosis-prone sites in response to hypercholesterolemia
Blood,
May 29, 2002;
99(12):
4486 - 4493.
[Abstract]
[Full Text]
[PDF]
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A. Tokumura, Y. Kanaya, M. Kitahara, M. Miyake, Y. Yoshioka, and K. Fukuzawa
Increased formation of lysophosphatidic acids by lysophospholipase D in serum of hypercholesterolemic rabbits
J. Lipid Res.,
February 1, 2002;
43(2):
307 - 315.
[Abstract]
[Full Text]
[PDF]
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P. Holvoet, K. Peeters, S. Lund-Katz, A. Mertens, P. Verhamme, R. Quarck, D. Stengel, M. Lox, E. Deridder, H. Bernar, et al.
Arg123-Tyr166 Domain of Human ApoA-I Is Critical for HDL-Mediated Inhibition of Macrophage Homing and Early Atherosclerosis in Mice
Arterioscler Thromb Vasc Biol,
December 1, 2001;
21(12):
1977 - 1983.
[Abstract]
[Full Text]
[PDF]
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J. Vermylen, M. Hoylaerts, J. Arnout, D. P. Chew, D. L. Bhatt, E. J. Topol, and S. Sapp
Increased Mortality With Long-Term Platelet Glycoprotein IIb/IIIa Antagonists: An Explanation? Response
Circulation,
November 13, 2001;
104
(20):
e109 - e109.
[Full Text]
[PDF]
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N. Methia, P. Andre, C. V. Denis, M. Economopoulos, and D. D. Wagner
Localized reduction of atherosclerosis in von Willebrand factor-deficient mice
Blood,
September 1, 2001;
98(5):
1424 - 1428.
[Abstract]
[Full Text]
[PDF]
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K. Freson, K. Devriendt, G. Matthijs, A. Van Hoof, R. De Vos, C. Thys, K. Minner, M. F. Hoylaerts, J. Vermylen, and C. Van Geet
Platelet characteristics in patients with X-linked macrothrombocytopenia because of a novel GATA1 mutation
Blood,
July 1, 2001;
98(1):
85 - 92.
[Abstract]
[Full Text]
[PDF]
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T. Kawasaki, M. Dewerchin, H. R. Lijnen, I. Vreys, J. Vermylen, and M. F. Hoylaerts
Mouse Carotid Artery Ligation Induces Platelet-Leukocyte-Dependent Luminal Fibrin, Required for Neointima Development
Circ. Res.,
February 2, 2001;
88(2):
159 - 166.
[Abstract]
[Full Text]
[PDF]
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T. Dickfeld, E. Lengyel, A. E May, S. Massberg, K. Brand, S. Page, C. Thielen, K. Langenbrink, and M. Gawaz
Transient interaction of activated platelets with endothelial cells induces expression of monocyte-chemoattractant protein-1 via a p38 mitogen-activated protein kinase mediated pathway: Implications for atherogenesis
Cardiovasc Res,
January 1, 2001;
49(1):
189 - 199.
[Abstract]
[Full Text]
[PDF]
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G. THEILMEIER, B. DE GEEST, P. P. VAN VELDHOVEN, D. STENGEL, C. MICHIELS, M. LOX, M. LANDELOOS, M. J. CHAPMAN, E. NINIO, D. COLLEN, et al.
HDL-associated PAF-AH reduces endothelial adhesiveness in apoE-/- mice
FASEB J,
October 1, 2000;
14(13):
2032 - 2039.
[Abstract]
[Full Text]
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T. Katayama, Y. Ikeda, M. Handa, T. Tamatani, S. Sakamoto, M. Ito, Y. Ishimura, and M. Suematsu
Immunoneutralization of Glycoprotein Ib{alpha} Attenuates Endotoxin-Induced Interactions of Platelets and Leukocytes With Rat Venular Endothelium In Vivo
Circ. Res.,
May 26, 2000;
86(10):
1031 - 1037.
[Abstract]
[Full Text]
[PDF]
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O. Shpilberg, I. Rabi, K. Schiller, R. Walden, D. Harats, K.S. Tyrrell, B. Coller, and U. Seligsohn
Patients With Glanzmann Thrombasthenia Lacking Platelet Glycoprotein {alpha}IIb{beta}3 (GPIIb/IIIa) and {alpha}v{beta}3 Receptors Are Not Protected From Atherosclerosis
Circulation,
March 5, 2002;
105(9):
1044 - 1048.
[Abstract]
[Full Text]
[PDF]
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J. Sarma, C. A. Laan, S. Alam, A. Jha, K. A.A. Fox, and I. Dransfield
Increased Platelet Binding to Circulating Monocytes in Acute Coronary Syndromes
Circulation,
May 7, 2002;
105(18):
2166 - 2171.
[Abstract]
[Full Text]
[PDF]
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TOP
ABSTRACT
INTRODUCTION