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Blood, Vol. 96 No. 1 (July 1), 2000:
pp. 170-175
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Transfer of tissue factor from leukocytes to platelets is
mediated by CD15 and tissue factor
Ursula Rauch,
Diana Bonderman,
Bernd Bohrmann,
Juan J. Badimon,
Jacques Himber,
Markus A. Riederer, and
Yale Nemerson
From the Division of Thrombosis Research, Department of Medicine,
and The Zena and Michael A. Wiener Cardiovascular Institute, Mount
Sinai School of Medicine, New York, NY 10029; and F. Hoffmann La Roche
Ltd, Pharma Division, Preclinical Research, Basel, Switzerland.
 |
Abstract |
We describe thrombogenic tissue factor (TF) on leukocyte-derived
microparticles and their incorporation into spontaneous human thrombi.
Polymorphonuclear leukocytes and monocytes transfer TF+
particles to platelets, thereby making them capable of triggering and
propagating thrombosis. This phenomenon calls into question the
original dogma that vessel wall injury and exposure of TF within the
vasculature to blood is sufficient for the occurrence of arterial
thrombosis. The transfer of TF+ leukocyte-derived
particles is dependent on the interaction of CD15 and TF with
platelets. Both the inhibition of TF transfer to platelets by
antagonizing the interaction CD15 with P-selectin and the direct
interaction of TF itself suggest a novel therapeutic approach to
prevent thrombosis.
(Blood. 2000;96:170-175)
© 2000 by The American Society of Hematology.
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Introduction |
Vessel wall injury and exposure of tissue factor (TF)
within the vascular wall to blood are considered necessary for the
initiation of arterial thrombosis.1-3 This paradigm has
been broadened by the observation that TF is also present in
circulating blood.4-6 We have previously demonstrated the
presence of TF in thrombi and have shown that ex vivo thrombus and
fibrin formations were reduced by the addition of a TF inhibitor to
blood. These results indicate that circulating TF is potentially
active,4 which implies the existence of a thrombogenic pool
of circulating TF. In our study, TF in thrombi appeared mostly on
membranous structures that were proximate to platelets. In fact,
polymorphonuclear (PMN) leukocytes from both circulating blood and
rabbit liver tested positive for TF by staining.4,7 In
addition, leukocytes coincubated with platelets generate more
procoagulant activity than either cell type alone.8,9 Taken
together, these data suggest that leukocytes are a potential source of
TF microparticles that adhere to platelets within a thrombus.
Leukocytes and platelets are known to interact via CD15 (a leukocyte
membrane-bound carbohydrate known as sialyl
Lewisx) with CD62P (P-selectin), which is an  granule-derived activation-dependent adhesion molecule found on
platelets.10-13
This study examines whether (1) a cell line that was derived from
monocytes (THP-1 cells) can transfer TF+ fragments to
platelets during thrombus formation and (2) the above-mentioned
adhesion molecules are involved in the formation of platelet-TF
hybrids. Furthermore, inhibitory monoclonal antibodies (mAbs) to the
identified membrane proteins were used to inhibit the transfer of
preexisting TF to platelets. Our data suggest that monocytes and
possibly PMN leukocytes are the source of circulating TF which is
transferred to platelets, thereby making F platelets capable of triggering and propagating thrombosis. This transfer process
is mediated by the interaction of CD15 with platelets and by TF itself.
The inhibition of TF transfer to platelets suggests a novel therapeutic
approach to prevent thrombosis.
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Materials and methods |
Reagents
We used the following in this study: human recombinant factor VIIa
(FVIIa) (gift from Novo-Nordisk, Gentofte, Denmark); Spectrozyme Xa
(American Diagnostica, Greenwich, CT); monomeric bovine type I collagen
(Vitrogen; Collagen Corp, Palo Alto, CA); Tyrode's solution, sterile
bovine serum albumin (BSA), citrate-dextrose solution (ACD), tumor
necrosis factor (TNF-), and prostaglandin E1 (Sigma
Chemical Co, St Louis, MO); Roswell Park Memorial
Institute medium (RPMI 1640) with L-glutamine, Hanks' balanced salt
solution (HBSS), phosphate-buffered saline (PBS), and a
penicillin-streptomycin combination (Gibco Life Technologies,
Braunschwedg, Germany); THP-1 cells (American
Type Culture Collection, Bethesda, MD); and DC Protein
Assay (Bio-Rad Laboratories, Hercules, CA). Purified factor X was prepared from human plasma.14 Immunopurified
rabbit antihuman TF polyclonal antibody (pAb-TF) and
digoxygenin-labeled FVIIa (Dig-VIIa) were prepared as
reported.15 Anti-TF mAb (hTF1) has been
described.16
Preparation of platelets and THP-1 cells
Platelet-rich plasma was prepared from citrated blood of healthy
donors (who had given informed consent) by centrifugation (Sorvall RT
6000B Refrigerated Centrifuge; DuPont, Wilmington, Delaware) at 250g for 10 minutes. The plasma was mixed
with modified Tyrode's solution I (Tyrode's solution, ACD 1:10
vol/vol, 0.35% BSA [pH 6.5], and 20 µmol/L prostaglandin E1).
Isolated platelets were washed 2 times in modified Tyrode's solution
II (Tyrode's solution, 0.35% BSA [pH 6.5], and 20µmol/L
prostaglandin E1) followed by a final washing in modified Tyrode's
solution III (Tyrode's solution, 0.35% BSA, 2 mmol/L magnesium, and
100 U/mL penicillin/streptomycin). The platelet preparations were used
for TF transfer experiments after 30 minutes at rest. THP-1 cells from
a human monocytic cell line were cultured in RPMI 1640 supplemented
with 10% fetal bovine serum (FBS), L-glutamine, and 2%
penicillin/streptomycin. Before induction of TF, THP-1 cells were
thoroughly washed in HBSS and then incubated with 1 µg/mL TNF-
(cell concentration, 20 000 to 30 000 cells per µL) overnight.
Perfusion experiments on collagen- and platelet-coated glass
slides
Microscope slides (Superfrost/Plus, Fisher Scientific,
Springfield, NJ) were incubated with 10 µg/mL collagen overnight at 4 °C followed by washing 3 times in PBS and blocking in 1%
BSA/PBS for 30 minutes. Perfusion systems for the slides
were previously reported.17 Two approaches for the
assessment of TF transfer to platelets were established: (1)
Collagen-coated glass slides were mounted in a parallel plate flow
chamber.17 The induced THP-1 cells were mixed with
platelets and perfused at a shear rate of 500s 1 over
collagen-coated slides. (2) A platelet carpet was first deposited on
the collagen-coated slide by incubating isolated platelets in the
presence of 1 mmol/L magnesium chloride and 2.5 mmol/L calcium chloride
for 2 hours at 37°C. The platelets deposited on collagen were then
perfused with TF-expressing THP-1 cells. All perfusions were performed
for 10 minutes at room temperature.
Immunocytochemistry
Immediately after perfusion, the slides were removed from the
chamber, gently rinsed in PBS, placed in freshly prepared 4% phosphate-buffered paraformaldehyde (pH 7.4), and fixed for 1 hour at
room temperature. The slides were washed in PBS, blocked with 1.2%
peroxide in methanol and goat serum, and incubated with 1 µg/mL
anti-TF pAb (pAb-TF, anti-sTF). Bound primary antibody was detected
using a biotin streptavidin-amplified detection system (BioGenex
Laboratories, San Ramon, CA) developed with 3,3-diaminobenzidine tetra-hydrochloride. After counter staining with hematoxylin, the
slides were examined microscopically. Controls for immunostaining consisted of a nonspecific antibody as the primary antibody.
Immunoelectron microscopy
Preembedding with double-immunogold labeling.
Human blood was perfused over collagen-coated slides to obtain thrombi
as described.4 The thrombi were then fixed in 4% paraformaldehyde. Nonspecific binding was reduced by washing the thrombi in 1.5% BSA and 0.5% ovalbumin in PBS (pH 7.4)
twice for 5 minutes each washing. Incubation with 10 µg/mL rabbit
anti-hTF pAb was performed in 0.1% BSA/PBS for 1 hour. Simultaneously, we used 10 µg/mL anti-CD15 antibody (mouse monoclonal
antineutrophil/monocyte carbohydrate epitope, immunoglobulin M
[IgM])(Dako, Hialeah, FL). The samples were then washed
3 times in 0.1% BSA/PBS for 5 minutes each washing. The samples were
incubated with secondary goat antirabbit IgG coupled to 5-nm colloidal
gold particles (Biocell Laboratories, Rancho Dominguez,
CA) or goat antimouse IgM coupled to 10-nm colloidal gold particles.
The samples were then diluted 1:40 in 0.1% BSA/PBS and 0.05% Tween 20 (pH 7.4) for 1 hour.
Alternatively, primary and secondary antibodies were applied in a
single step and lead to identical results. After 3 washes in PBS for 5 minutes each, the samples were fixed in 2% glutaraldehyde in 0.1 mol/L
sodium cacodylate buffer (pH 7.4) for 1 hour, briefly washed in 0.1 mol/L Na cacodylate buffer, and postfixed in 2% osmium tetroxide. The
samples were then dehydrated in a series of aqueous ethanol solutions,
exposed to propylene oxide, and embedded in Epon 812 according to Luft.18 Ultrathin sections were
cut on a Reichert Ultracut S and stained with 5% aqueous uranyl acetate followed by lead citrate.19 Electron
micrographs were acquired with a JEOL 1210 at 100 kV. The
controls, which were treated with mouse preimmune serum or nonspecific
monoclonal IgG, revealed no significant stain, and a
negligible background of fewer than 10 gold grains was
noted in cells greater than 10 µm2.
On-section immunogold labeling with ultrathin
cryosections.
Human venous thrombi were obtained from a 38-year-old patient. Samples
were fixed overnight with 4% formaldehyde in 0.1 mol/L PBS (pH 7.4).
After storage in PBS for 6 days, samples were dissected into small
fragments not more than 1 µL and postfixed in 3% formaldehyde with
0.1% glutaraldehyde in PBS for 2 hours. Sample preparation was done as
described by Tokuyasu20 with the following modifications: neutralization of free aldehydes was achieved by rinsing in PBS and
incubating in 50 mmol/L ammonium chloride in PBS for 1 hour. The
samples were immersed overnight in cryoprotection buffer consisting of
1.7 mol/L sucrose and 15% polyvinyl-pyrrolidone (10 000 MW) in 10 mmol/L PBS (pH 7.4) at 4°C and frozen in liquid nitrogen.
Ultrathin (9-nm) cryosections were prepared at  100°C with a
Reichert Ultracut S + FCS using a diamond knife
(Diatome) in the presence of an antistatic line (Diatome). Cryosections
were collected in 2.3 mol/L aqueous sucrose onto formvar/carbon-coated 200-mesh nickel grids. The sections were thawed over PBS prior to
immunolabeling, which was performed at room temperature. The sections
were floated on 0.05 mol/L glycine in PBS for 15 minutes to inactivate
free aldehyde groups and then on 2.5% BSA/PBS and 2.5% ovalbumin for
15 minutes to block unspecific binding sites. Sections were incubated
with 10 µg/mL hTF1 in 2% BSA/PBS for 1 hour. After 6 washes in
BSA/PBS, the sections were incubated with secondary goat antimouse IgG
(Amersham, Arlington Heights, IL), conjugated to 10-nm gold at 1:20
dilution in 2% BSA with 0.1% Tween 20 in PBS for 1 hour, and washed
in BSA/PBS. For a control, we used sections treated with normal mouse
serum, which resulted in a negligible background of not more than
10 gold grains in cells larger than 10 µm.2
The sections were postfixed in 2% glutaraldehyde in PBS for 5 minutes,
washed, and stained with 0.4% uranyl acetate in 1.8% methyl cellulose
(25 cP) for 5 minutes.
THP-1 cell-derived cell fragments and measurement of TF
activity
Conditioned medium from TNF--induced THP-1 cells was used as a
source of microparticles. THP-1 cells were removed by centrifugation at
2000g for 10 minutes, and the THP-1 cell pellet was washed in HBSS and
pelleted again. Cell particles and fragments present in the supernatant
were concentrated by centrifugation at 16 000g for 15 minutes. The
absence of THP-1 cells in the particle suspension was confirmed by
light microscopy. The protein concentration of the particles was
determined according to the manufacturer's instructions using a
microplate protocol (DC Protein Assay, Bio-Rad Laboratories). Absorbance was read at 650 nm, and sample protein concentrations were
calculated from a standard curve using BSA. We assayed 40 µL
concentrated particles for TF activity using HEPES
(4-[2-Hydroxyethyl]-1-piperazineethanesulfonic acid) buffer with 1 nmol/L FVIIa, 150 nmol/L factor X, and 5 mmol/L calcium
chloride. At intervals, samples were transferred to a microtiter plate
in which each well contained 100 µL EDTA (ethylenediamine tetraacetic
acid) buffer (50 mmol/L Bicine [pH 8.5], 20 mmol/L EDTA, and 1 mg/mL
BSA), which terminates production of factor Xa. Chromogenic substrate
Spectrozyme Xa (final concentration 0.5 mmol/L) was added to each well,
and the increase in OD was monitored at 405 nm for 10 minutes by using
a kinetic enzyme-linked immunoabsorbent assay (ELISA) plate reader at
35°C (Tmax, Molecular Devices, Sunnyvale, CA).
Activity was quantified by reference to purified human rTF of known concentration.
Flow cytometry
Particles from TNF--induced THP-1 cells were freshly prepared
and resuspended in PBS for flow cytometric analysis of surface membrane
proteins. Particles were blocked in normal goat serum and incubated
with an mAb to 20 µg/mL CD15 (clone AHN1.1, Ancell), 10 µg/mL TF (hTF-1), and 10 µg/mL CD18 (clone IB4, Ancell, Bayport, MN), respectively, for 2 hours at room temperature. Mouse IgG1 served
as a control. After 2 washings in 1% BSA/PBS, samples were incubated
with a secondary rabbit antimouse antibody conjugated to phycoerythrin
(PE) (Molecular Probes, Eugene, OR) for 1 hour at room
temperature. Flow cytometry was performed immediately thereafter, and
10 000 events were acquired for each measurement.
To minimize the amount of antibodies needed to inhibit the TF
transfer, the parallel flow chamber was replaced by a cone and plate
viscometer that was suitable for small sample volumes
(250 µL instead of 4 mL).21 Isolated platelets were
activated on collagen-coated glass plates within plastic wells;
TF-containing particles were added, and the mixture was subjected to
500s1 wall shear rates in the viscometer21
for 30 minutes. To examine whether blocking antibodies to the
identified adhesion proteins on the microparticle surface inhibit the
attachment of TF to platelets, TF-containing microparticles were
preincubated with either a blocking antibody to 200 µg/mL CD15
(anti-LewisX, clone AHN1.1, Ancell) or to 200 µg/mL TF (hTF-1) for 15 minutes at room temperature before adding the
microparticles to the platelets. After exposing the platelet-particle
mixture to shear stress, platelet-particle hybrids were fixed in 1%
paraformaldehyde for 15 minutes, washed in PBS, incubated with 0.02%
Triton X-100 in PBS for 5 minutes at 37°C, and washed twice with PBS.
Platelet-particle hybrids were further blocked with 1% BSA/PSA and
normal goat serum, then stained with 1 µg/mL rabbit anti-TF pAb for 2 hours at room temperature or overnight at 4°C. After rinsing twice
with PBS, samples were incubated with a secondary goat antirabbit
antibody conjugated to PE. To double stain for TF and a specific
platelet antigen on platelet-particle hybrids, the sample was
simultaneously incubated with 1 µg/mL mouse anti-platelet glycoprotein IIb/IIIa complex (anti-GPIIb/IIIa) mAb (anti-CD41a, Ancell) directly conjugated to fluorescein isothiocyanate (FITC). Rabbit IgG, followed by the secondary antibody described above, and
mouse IgG-FITC were used as a negative staining control. The incubation
was performed at 4°C for 2 hours. Flow cytometry was performed
immediately thereafter.
Attachment of the TF-containing particles to platelets was inhibited by
hTF-1 (see "Results"). To ensure that the mAb did not inhibit
detection of TF by the pAb, preliminary experiments binding pAb with
and without the addition of hTF-1 were performed. The presence of hTF-1
had no significant effect on the binding.
Confocal laser scanning microscopy
Platelet-TF hybrids were examined using a Leica TCS-SP
(ultraviolet [UV]) confocal laser scanning microscope (Leica,
Heidelberg, Germany) equipped with a 4-channel spectrophotometer scan
head and 4 lasers (argon-UV [Ar-UV], Ar, krypton, and helium-neon
[HeNe]). For these studies, the platelet-TF hybrids were illuminated
simultaneously with the  488- and 568-nm laser lines. The pinhole
size was adjusted such that resultant "optical sections" were
approximately 0.25- to 0.3-µm thick. To ensure that the signal did
not "spill over" from one channel into another, the
acousto-optical tunable filter was adjusted, and the spectrophotometer
windows in each channel were set so that signals from one channel were
not detected in the other channel.
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Results |
TF+ microparticles are transferred from
leukocytes to platelets
To further visualize the structure of TF on platelets,
immunoelectronmicroscopy was performed on thrombi generated ex vivo by
perfusing human blood directly from a donor's arm over collagen-coated slides. TF+ pseudopodia extending from PMN leukocytes were
found to contact platelets within the thrombus (Figure
1). Adhesion molecules present on the
leukocyte surface, which may possibly be involved in the adhesion to
platelets during thrombus formation, were also identified. A
colocalization of CD15 and TF on pseudopodia and small membrane particles was demonstrated by double immunolabeling (Figure 1). To
confirm the presence of TF+ particles within thrombi, a
human venous thrombus obtained from a patient was examined by
immunoelectronmicroscopy. TF+ particles were present within
the thrombus and also intermingled with fibrin strands (Figure
2).
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| Fig 1.
Collagen-coated glass slides were perfused with
native human blood, and the deposited thrombi were double
immunostained for TF and CD15.
(A) Electronmicroscopy showing platelets and PMN leukocytes within a
thrombus. Note the PMN leukocyte-derived membrane attached to the
platelet surface depicted in the boxed field. (B) Closeup examination
of the boxed field: the structure double stains for CD15 (10-nm gold
grains) and TF (5-nm gold grains). (C) TF on small particle-like
structures in proximity to PMN leukocytes.
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| Fig 2.
Ultrathin cryosections through a human venous thrombus.
TF within the thrombus was detected by on-section labeling using mAb
anti-TF (hTF1) followed by goat antimouse IgG conjugated to 10-nm
colloidal gold. (A) Electronmicroscopy shows TF+ cell
fragments adjacent to the fibrin strands. (B) Closeup of the region in
the boxed field: a clearly discernible bilayered cell membrane and
organelle substructure are indicative of the cellular origin of the
TF+ structures.
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THP-1 cells as a model to study the transfer of
TF+ cell fragments to platelets
We then established a perfusion model to study whether
leukocytic cells can transfer TF+ particles to platelets.
Platelets isolated from human blood were deposited on collagen-coated
slides that were then mounted in a parallel plate flow chamber. THP-1
cells were treated with TNF- to induce the production of TF
microparticles22,23; the suspension was then perfused at
500s1 over the previously formed platelet carpet. In
this model system, platelets, which usually do not contain TF (Figure
3A), became TF+ after being
exposed to TF+ THP-1 cells and THP-1 cell-derived cell
fragments (Figure 3B).
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| Fig 3.
Results of immunocytochemistry performed on
platelets.
(A) The platelets were isolated from circulating blood and
deposited on collagen-coated slides. Immunocytochemistry was performed
to detect TF. When stained, the platelets did not test positive for TF.
Hemotoxylin was used for counter staining. (B) THP-1 cells induced to
produce TF and also TF+ microparticles by treatment with
TNF- were perfused over a platelet carpet on a collagen-coated glass
slide that was mounted in a parallel plate flow chamber.14
The induced THP-1 cells were perfused over the platelets at
500s1. Note that platelets turned a brown color,
indicating TF+, after exposure to TF containing THP-1 cells
(original magnification × 200).
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Confocal microscopy demonstrated the colocalization of TF+
cell fragments and platelets. After coincubating activated platelets with THP-1-derived TF particles overnight, platelet-particle hybrids were then stained for TF and CD41. TF fragments on platelets were visualized by anti-TF pAbs that were detected with a
secondary antirabbit antibody conjugated to PE (Figure
4A), whereas the platelets were recognized
by FITC-conjugated anti-GPIIb (CD41) antibodies (Figure 4B). Confocal
microscopy revealed a high degree of colocalization between TF and
platelets (Figure 4C). Identical results were obtained using an anti-TF
mAb (not shown).
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| Fig 4.
Platelets activated on collagen were incubated overnight
with THP-1 cell-derived TF particles.
The platelet-particle hybrids were then double stained: TF
particles on platelets were visualized by anti-TF pAb followed by
staining with a secondary goat antirabbit antibody conjugated to PE.
The platelets were simultaneously stained by an FITC-conjugated mAb
directed against GPIIb. (A) Confocal microscopy reveals TF particles
stained red. (B) GPIIb on platelets were stained green. (C) An overlay
of panels A and B shows the colocalization of TF and platelets.
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The TF cell fragments derived from stimulated THP-1 cells were further
characterized by qualitative and quantitative flow cytometric analysis.
Conditioned medium from TNF--induced THP-1 cells was used as a
source of particles. CD15 and TF were abundantly present on the
microparticle surface (detectable in approximately 60%
and 45% of the particles, respectively; Figure
5A and 5B), whereas CD18 was only
detectable in approximately 14% of the particles (Figure 5C). In
addition, measurement of the Xa generation by a chromogenic assay
showed that THP-1 cell-derived fragments produced 370 pµ Xa per min
in suspension, which reflects the high procoagulant activity of
leukocyte-derived TF particles.
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| Fig 5.
Characterization of THP-1 cell-derived microparticles by
flow cytometry.
Using mAbs, we detected CD15, TF, and CD18 and acquired 10 000 events
for each measurement. Plots show the PE staining on particles
(immunofluorescence 2 [LFL-2]). Staining showed that
(A) CD15+ was present in 60% of the particles tested, (B)
TF was present in 45%, and (C) CD18+ was present in
14%.
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The role of CD15 and TF on the transfer of TF+
cell fragments to platelets
At the platelet-PMN leukocyte contact site (Figure 1), an
intense cluster of TF and CD15 labeling was evident. This suggests a
possible involvement of these molecules in the transfer of
TF+ particles from leukocytes to platelets. For these
experiments we employed a microcone and microplate viscometer using
small sample volumes of 250 µL. Anti-CD15 and anti-hTF (hTF-1) mAbs reduced the formation of platelet-TF hybrids (Figure
6).
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| Fig 6.
Inhibiting the transfer of TF cell fragments to
platelets.
Platelets were activated on collagen, and (A)
platelet-particle hybrids were double stained. TF was visualized by
rabbit pAbs against hTF followed by goat antirabbit antibodies
conjugated to PE, whereas platelets were stained with an
FITC-conjugated mAb directed against GPIIb. Plots in panels B, C, and D
show the GPIIb+ particles (platelets). (B) TF was found on
14% of platelets. (C) A blocking anti-CD15 antibody inhibited the
attachment of TF-containing particles to platelets. (D) The anti-TF mAb
(hTF-1) also reduced TF adherence to platelets.
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Discussion |
The presence of thrombogenic TF activity in circulating blood has
recently been demonstrated by us,4 but the source of platelet-associated TF and the mechanism of its transfer have not yet
been identified. In this study we present data indicating that
monocytes and possibly PMN leukoctyes were involved in the transfer of
TF+ particles to platelets. Studies on THP-1 cells revealed
that the adherence of TF+ particles to platelets was
mediated by CD15. The role of P-selectin in thrombogenesis was
previously shown in a baboon model in which antibodies to P-selectin
inhibited fibrin deposition in a polyester fiber
arteriovenous shunt,24 although the formation of
TF-platelet hybrids was not considered. Moreover, it has been shown
that P-selectin knockout mice show a hemostatic
defect.25 The addition of anti-CD15 mAbs or
oligosaccharides containing sialyl Lewisx inhibits CD15
binding to P-selectin on platelets. This action has been shown to
prevent both platelet-leukocyte adherence10,26,27 and the
recurrence of arterial thrombosis in animal
models.27 The data we present in this paper provide an
explanation for these observations, namely that the CD15 and P-selectin
interactions mediate the formation of highly procoagulant platelet
aggregates containing TF particles from leukocytic cells.
Platelets became TF+ after exposure to TF+
particles (Figure 3). Our findings substantiate previous ex vivo
observations suggesting that leukocytes are one possible source of
TF+ structures in thrombi.4 Monocytes have been
documented to express TF. Although TF+ neutrophils have
repeatedly been observed by us and others, we do not know whether these
cells synthesize TF. Further studies are needed to evaluate whether PMN
leukocytes acquire TF from other cells and then deposit it onto
platelet thrombi or whether the leukocytes themselves are able to
express TF.
Notably, it has been suggested that circulating microparticles are a
thrombogenic species.28,29 Previously it was shown that TF
modulates the migration of TF-containing monocytes through an
endothelial cell monolayer. Antibodies to TF and TF fragments were
inhibitory, thus implicating TF as an adhesive molecule and suggesting the presence of a TF receptor on endothelial
cells.30 A role for TF in cell adhesion and migration
mediated by interaction with intracellular actin-binding protein 280 was also recently reported.31 Ultrastructural data on the
location of TF expressed by various cell lines demonstrated that TF was
at the cell surface and appeared in a spotty pattern at the
base and apex of budding processes and in close proximity to
actin-rich regions.32 Immobilized ligands for TF
accelerated the adhesion and spreading of TF-expressing cells,
which indicates that cellular TF may be involved in cell adhesion.32 Taken together, these observations implicate
TF-leukocyte interactions in TF-dependent coagulation and
thrombotic events and support the concept of TF as an adhesive molecule.
Suspensions of TF+ particles exhibited considerable TF
activity, which points to the potential thrombogenicity of
leukocyte-derived TF-bearing particles. It should be emphasized that
under normal conditions, most cell surface TF is encrypted, which means
that the TF binds to FVIIa and anti-TF, but it is not capable of
initiating coagulation. Encrypted TF allows circulating TF+
leukocytes to be present in the circulation without generalized coagulation ensuing.33-36 The transfer of TF+
microparticles to platelets during thrombogenesis clearly favors the
propagation of thrombosis. The activation of coagulation factors IX and
X occurs very close to platelet surfaces, thereby favoring the
formation of factors IXa:VIIIa and Xa:Va complexes on their highly
procoagulant surfaces. In our experiments, platelets seem to acquire TF
after thrombus formation has been initiated and therefore perpetuate
the process of thrombus growth. Whether platelet-associated TF may be
able to participate in the initiation of thrombus formation remains to
be clarified.
Our findings demonstrate that TF+ microparticles
are present on platelets within thrombus-forming platelet-TF hybrids.
Thus, the data presented here show monocytes and possibly PMN
leukocytes as sources of TF+ membrane particles that are
transferred to platelets, thereby enabling them to trigger and
propagate thrombosis. The TF transfer process is mediated by the
interaction of CD15 and TF with platelets, and the inhibition of TF
transfer may be a novel therapeutic approach to prevent thrombosis.
| |
Acknowledgments |
We thank Professor J. T. Fallon, Department of
Pathology, Mount Sinai School of Medicine (MSSM), New York, NY, for his
excellent assistance, and Veronia Gulle for her extensive technical
support. We also thank David Varon, Sheba Medical Center, Tel-Hashomer, Israel, for the use of his viscometer, and Scott Henderson,
MSSM-CLSM core facility, for help with confocal laser
scanning microscopy.
| |
Footnotes |
Submitted September 27, 1999; accepted February 23, 2000.
Supported in part by grants HL 29019 and HL 54469 from the
National Heart Lung and Blood Institute, National Institutes of Health
(NIH), Bethesda, MD; shared instrumentation grant 1 S10 RR0 9145-01 from NIH; and instrumentation grant DBI-9724504 from the
National Science Foundation (Arlington, VA) Major Research.
Reprints: Yale Nemerson, Division of Thrombosis Research,
Department of Medicine, Box 1269, 1 Gustave L. Levy Pl, New York, NY
10029; e-mail: yale.nemerson{at}mssm.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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(20):
e109 - e109.
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R. R. Dalton, J. S. Krauss, D. G. Falls III, and G. K. Fuller
Granulocytic Fragments in Sepsis
Ann. Clin. Lab. Sci.,
October 1, 2001;
31(4):
365 - 368.
[Abstract]
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Z. Mallat and A. Tedgui
Current Perspective on the Role of Apoptosis in Atherothrombotic Disease
Circ. Res.,
May 25, 2001;
88(10):
998 - 1003.
[Abstract]
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U. Rauch, J. I. Osende, V. Fuster, J. J. Badimon, Z. Fayad, and J. H. Chesebro
Thrombus Formation on Atherosclerotic Plaques: Pathogenesis and Clinical Consequences
Ann Intern Med,
February 6, 2001;
134(3):
224 - 238.
[Abstract]
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P. Andre, D. Hartwell, I. Hrachovinova, S. Saffaripour, and D. D. Wagner
Pro-coagulant state resulting from high levels of soluble P-selectin in blood
PNAS,
December 5, 2000;
97(25):
13835 - 13840.
[Abstract]
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Top
Abstract
Introduction