© 1995 American Heart Association, Inc.
Articles |
Procoagulant Human Monocytes Mediate Tissue Factor/Factor VIIa–Dependent Platelet-Thrombus Formation When Exposed to Flowing Nonanticoagulated Human Blood
From Nycomed Bioreg AS and the Department of Clinical Chemistry, Ulleval University Hospital (P.K., A.-B.W), Oslo, Norway.
Correspondence to R. Marius Barstad, MD, Nycomed Bioreg AS, Gaustadalléen 21, N-0371 Oslo, Norway.
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Abstract |
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Abstract Tissue factor (TF) on monocyte and macrophage surfaces is a nonproteolytic cofactor for factor VIIa (FVIIa)–induced coagulation. Monocyte-derived macrophages in atherosclerotic plaques express TF, which, after plaque disruption or rupture, may complex with FVII/VIIa from the bloodstream, resulting in activation of extrinsic coagulation. We studied the effect of TF expression on human monocytes on arterial thrombus formation in a model system of thrombogenesis. Thawed, cryopreserved human monocytes adherent to plastic coverslips were stimulated with lipopolysaccharide (0.5 µg/mL) to express TF and subsequently exposed to flowing nonanticoagulated human blood in a parallel-plate perfusion chamber. The wall shear rate at the cell surface was 650 seconds-1, corresponding to that of average-sized coronary arteries. The stimulated monocytes elicited pronounced fibrin deposition and platelet-thrombus formation. The platelet-thrombus volume was as large as that triggered by human type III collagen fibrils under similar experimental conditions. In contrast, the monocytes elicited much more fibrin deposition than the collagen surface. However, inclusion of an anti-TF monoclonal antibody that blocks the complexation of FVII/FVIIa with TF virtually abolished the fibrin deposition (P<.03) and reduced platelet-thrombus formation by more than 70% (P<.04). Thus, arterial thrombus formation induced by stimulated monocytes was almost completely blocked by the anti-TF antibody, suggesting that inhibition of TF/FVIIa complex formation on monocytes and macrophages at sites of plaque rupture or after percutaneous transluminal coronary angioplasty procedures may reduce intravascular thrombotic complications.
Key Words: thrombosis model • procoagulant monocytes • tissue factor • fibrin deposition • platelet-thrombus formation
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Introduction |
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Tissue factor (TF) is a cell-surface transmembrane protein with an extracellular domain that complexes with coagulation factor VIIa (FVIIa) and functions as a cofactor for the catalytic activity of the FVIIa moiety. Such catalytic complexes form at vascular lesions, resulting in proteolytic activation of factors IX and X, subsequent thrombin generation, and eventually fibrin formation and deposition.1 2 3 Platelets are activated concomitantly by thrombin through the platelet-thrombin receptor,4 which enhances platelet recruitment at the lesion.
TF is apparently present in subendothelium,5 6 and disruption of the endothelial layer of blood vessels in experimental animal models activates coagulation and triggers thrombus formation. Inclusion of monoclonal anti-TF antibodies (MAbs) efficiently blocks this thrombus formation,7 8 indicating a prominent role for TF/FVIIa–dependent coagulation in thrombus formation, at least in these models. Furthermore, TF expressed by stimulated endothelium9 and TF present in the endothelial extracellular matrix10 11 activate coagulation and induce thrombus formation at venous9 and arterial10 11 blood flow conditions because anti-TF MAbs efficiently block both coagulation and thrombus formation on these surfaces.9 10 11
Macrophages dominate the lipid core of atherosclerotic plaques,12 and these cells have been shown to express TF.13 14 These observations led us to study the effect of TF expression on immobilized peripheral human blood monocytes on arterial thrombus formation in flowing native human blood. Thawed, cryopreserved human peripheral blood monocytes15 (±lipopolysaccharide stimulation) adherent to plastic coverslips were placed in a parallel-plate perfusion chamber16 17 and exposed to native human blood for 5 minutes at an arterial wall shear rate of 650 seconds-1. The procoagulant monocytes elicited pronounced fibrin deposition and platelet-thrombus formation that was dependent on the expression of TF.
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Methods |
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Human Monocytes
Peripheral blood mononuclear cells from healthy subjects were isolated by Lymphoprep (Nycomed Pharma), frozen in aliquots of 6x106 cells/mL at -135°C, and thawed according to the method described by Osnes et al.15 This cryopreservation does not affect the potential for TF synthesis. Mononuclear cells were suspended in RPMI-1640 (Whittaker) with 5% fetal calf serum (FCS) (GIBCO, Life Technologies Ltd), and the number of cells was adjusted to 2.4x106/mL, giving approximately 30% monocytes, as determined with nonspecific esterase staining (Bürker-chamber) and flow cytometry with CD14. Two milliliters of the cell suspension (4.8x106 mononuclear cells) were seeded onto 4-cm2 plastic coverslips (Thermanox, Miles Laboratories) placed in multiwell (six-well) plates (NUNC AS). The incubation medium was RPMI-1640 (Whittaker) supplemented with 5% FCS (GIBCO)±0.5 µg/mL lipopolysaccharide from E. Coli OB 55 (Sigma Chemical Co). Incubation was at 37°C and 5% CO2 for 2 or 4 hours. Subsequently, the coverslips were washed three times in phosphate-buffered saline (PBS, 3x2 mL) (Whittaker) to remove nonadherent cells. The adherent cells on the coverslips consisted of more than 90% monocytes, as judged by nonspecific esterase staining.
The coverslips with adherent lipopolysaccharide-stimulated monocytes were incubated for 30 minutes in 2.5 mL fresh RPMI-1640 with or without 25 µg/mL anti-TF MAb (MAb AD 4509, American Diagnostica). This anti-TF MAb blocks the complexation of FVII/FVIIa to TF. The adherent cells were washed three times in RPMI-1640 and immediately used in blood perfusion experiments (see below) or in a one-stage coagulation assay (Thrombotrack 4, Nycomed Pharma) (see below).
The number of adherent monocytes on the coverslips after 2 or 4 hours of incubation and after a 5-minute perfusion was determined. Each coverslip was photographed at a magnification of x63 with a light microscope (Zeiss Axiophot 405M) in combination with a video camera (DXC-750D, Sony Corp) and color video printer (UP-5000p, Sony) at the upstream edge before and after perfusion experiments with RPMI-1640. The number of adherent monocytes was subsequently counted from the printed images. The monocyte count on the coverslips was similarly determined after 5-minute perfusions with native blood (larger viscous forces) over nonstimulated monocytes and lipopolysaccharide-stimulated and anti-TF MAb–incubated monocytes (2 hours of incubation). The relatively modest thrombus formation under these experimental conditions did not interfere with identification of the Thermanox-adherent monocytes and the cell counting. However, the number of adherent cells after 4 hours of incubation and blood perfusion could not be determined because of thrombus formation that obscured the view of the Thermanox-adherent monocytes.
Cell viability after thawing, seeding on coverslips, lipopolysaccharide stimulation, and incubation with the anti-TF MAb was determined by the trypan blue exclusion test.
Testing for Endotoxin
The growth medium of RPMI-1640 and FCS is tested for endotoxin
contamination by the manufacturers and has been routinely tested for
endotoxin at the Department of Clinical Chemistry at Ullevål
University Hospital, Oslo. The levels of endotoxin have been below the
detection limit of 30 pg/mL.18 Thermanox coverslips,
provided sterile from the manufacturer, were kept in 96% ethanol to
avoid bacterial contamination. Each coverslip was handled under sterile
conditions in a laminar air-flow hood. No detectable endotoxin (<30
pg/mL) was measured in growth medium incubated for 4 hours in
multiwells in the presence or absence of coverslips.18
However, we cannot exclude the possibility of trace amounts of
endotoxin present.
Monocyte Procoagulant Activity
The procoagulant activity of adherent nonstimulated and adherent
lipopolysaccharide-stimulated monocytes with and without the inclusion
of an anti-TF MAb was determined by a one-stage clotting assay. The
cells were scraped off the Thermanox coverslips with a rubber policeman
(NUNC AS) and resuspended in 400 µL PBS (Whittaker). Subsequently, 50
µL cell suspension (four parallels) was preincubated with 50 µL of
25 mmol/L CaCl2 for 2 minutes (37°C). Clotting time (in
seconds) was measured automatically after addition of 50 µL of a pool
of normal human plasma (37°C) by a Thrombotrack 4 coagulometer
(Nycomed Pharma). Six dilutions of a standard TF preparation from
rabbit brain (Nycomed Pharma) was used to make a standard curve.
The protein content of the resuspended cells (200 µL) was determined by a Micro BCA Protein Assay Reagent Kit according to the manufacturer (Pierce Chemical Co). The monocyte procoagulant activity was expressed in arbitrary units of TF activity per microgram of protein because the anti-TF MAb blocked virtually all procoagulant activity. A control MAb (Dako M734, mouse anti-human transferrin receptor MAb, Dacopatts) of the same subclass (IgG1) as the anti-TF MAb was included in the coagulation studies.
Blood Donors
The blood donors were healthy nonsmoking individuals, who denied
any drug intake for at least 14 days before the blood perfusion
experiments. Hematologic parameters (hemoglobin, hematocrit, and
leukocyte and platelet counts) were within the normal ranges (Auto
Counter AC 920, Swelab Instrument).
Perfusion Experiments
Ex vivo perfusion experiments19 were performed in
the parallel-plate perfusion model of thrombogenesis, as described in
detail previously.16 17 20 A Thermanox plastic coverslip
with adherent monocytes was positioned in the perfusion chamber and
subsequently exposed to flowing nonanticoagulated human blood for 5
minutes. An antecubital vein was punctured with a 19-gauge butterfly
infusion set (Abbot Lab), and the blood was drawn (10 mL/min) directly
from the vein through the perfusion chamber by an occlusive roller pump
(Gilson Minipuls 3, Villiers Le Bell) placed distally to the perfusion
chamber. The wall shear rate at the cell surface was 650
seconds-1. During the perfusion experiments, blood was
sampled downstream from the adherent monocytes to assess the plasma
levels of fibrinopeptide A (FPA) and ß-thromboglobulin
(ß-TG) after 4 and 4.5 minutes, respectively, of perfusion time (see
below). A rubber sleeve wrapped around Silastic tubing (Dow Corning
Corp) immediately distal to the perfusion chamber allowed repeated
punctures of the tubing without leakage. Each perfusion was terminated
with a 20-second perfusion (10 mL/min) with RPMI-1640 at 37°C,
followed by a 40-second perfusion (10 mL/min) with freshly prepared
fixation solution consisting of 2.5% glutaraldehyde per 0.1 mol/L
cacodylate in deionized water (pH 7.4) at 23°C. The specimens were
stored in 7% sucrose per 0.1 mol/L cacodylate buffer at 4°C and
finally embedded in Epon.21
Morphometry
Semithin Epon sections (1 µm) were prepared perpendicular to
the direction of the blood flow 1 mm from the upstream edge of the
coverslip.22 The sections were stained with toluidine blue
and basic fuchsin. Thrombus formation was quantified as percent surface
coverage with fibrin by light microscopy at x1000 magnification
(Standard 25, Zeiss),19 and platelet-thrombus volume
(micrometers cubed per micrometers squared) was derived from thrombus
area (micrometers squared per micrometer) assessed by computer-assisted
morphometry (Kontron Vidas, Eching) at x2500 magnification, as
previously described.23
FPA and ß-TG Plasma Levels
The plasma levels of FPA and ß-TG were determined in blood
samples (0.9 mL) drawn immediately distal to the perfusion chamber. The
blood samples were drawn directly into 2-mL syringes (Becton Dickinson)
prefilled with 0.1 mL anticoagulant consisting of 1000 IU heparin (LEO,
Baldrup) and 1000 KIU Trasylol (Bayer Leverkusen, Germany) per 1 mL
saline for FPA and an anticoagulant according to Ludlam and
Cash24 for ß-TG. Further processing of the blood samples
was essentially according to the manufacturers of the respective
radioimmunoassay kits: FPA from Imco and ß-TG from Amersham, UK.
Statistical Analysis
Statistical analysis was performed with two-tailed
Student's t test and Mann-Whitney U test. A
value of P<.05 was considered significant.
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Results |
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Monocyte-Thermanox Adhesion and Cell Viability
Monocytes adhered singly and randomly to the plastic coverslips (Thermanox). Cell aggregate formation on the coverslip was not encountered. The cell density after 2 hours of incubation was on average 500 cells/mm2 (Table 1
). When adherent
monocytes were perfused with RPMI-1640 for 5 minutes, approximately 50
cells/mm2 were washed off, corresponding to 10% cell loss
(n=4, P<.01). Perfusion with native human blood, which has
a higher viscous force, increased the cell loss to about 75
cells/mm2, corresponding to 15% cell loss (n=8,
P<.05). Neither stimulation with lipopolysaccharide nor
inclusion of the anti-TF MAb influenced cell adhesion. However, the
number of adherent monocytes increased by 67% to approximately 840
cells/mm2 (n=8, P<.01) when the incubation
period was prolonged from 2 to 4 hours. The trypan blue exclusion test
revealed less than 1% cell death after the incubation periods and
before the perfusion experiments.
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Clotting Assays: TF/FVIIa–Induced Coagulation
Nonstimulated monocytes adherent to plastic coverslips for 2 hours
showed very little procoagulant activity, whereas a threefold increase
in activity was observed after 4 hours of adhesion
(P<.0001) (Fig 1). The anti-TF MAb reduces this
activity by more than 90% (n=4, P<.005). In contrast, the
lipopolysaccharide-stimulated adherent monocytes elicited considerable
procoagulant activity after incubation for 2 hours and almost twice as
much after 4 hours (P<.0001). When adherent cells
stimulated with lipopolysaccharide for 2 or 4 hours were incubated with
the anti-TF MAb, the coagulant activity was almost abolished
(P<.0001). Inclusion of the anti–transferrin receptor MAb
did not significantly affect the cellular procoagulant activity.
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Perfusion Studies
Perfusion studies with Thermanox-adherent monocytes using
nonanticoagulated human blood were performed for 5 minutes at a wall
shear rate of 650 seconds-1. Thrombus formation was
morphometrically assessed as percent surface coverage with fibrin
(fibrin deposition) and as platelet-thrombus volume.
Fibrin Deposition
Fig 2A gives data on monocyte, collagen, and
Thermanox-induced fibrin deposition. Lipopolysaccharide-stimulated
cells triggered substantial fibrin deposition, which was most
pronounced on and around cells stimulated for 4 hours
(P<.03). Fibrin deposition was almost completely abolished
when the cells were incubated with the anti-TF MAb (P<.03).
Nonstimulated cells elicited virtually no detectable fibrin deposition.
When blood was perfused over uncoated Thermanox coverslips or over
coverslips coated with type III collagen fibrils, virtually no
detectable fibrin deposition was observed.
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Platelet-Thrombus Volume
Fig 2B
summarizes the data on platelet-thrombus volume elicited by
monocytes, collagen fibrils, and Thermanox. Blood perfusions of
2-hour-adherent and nonstimulated monocytes resulted in very little
platelet-thrombus formation, whereas 4-hour-adherent and nonstimulated
monocytes triggered pronounced platelet-thrombus formation. Monocytes
stimulated with lipopolysaccharide for 2 hours triggered pronounced
formation of platelet thrombi compared with the nonstimulated cells
(P<.03). There was no difference in platelet-thrombus
formation on cells stimulated with lipopolysaccharide for 2 versus 4
hours or between monocytes that were not stimulated and those
stimulated for 4 hours.
However, inclusion of the anti-TF MAb inhibited platelet-thrombus formation on cells stimulated 2 and 4 hours with lipopolysaccharide by 86% (P<.05) and 71% (P<.01), respectively.
Type III collagen fibrils triggered platelet-thrombus formation as efficiently as the monocytes. However, no platelet thrombi formed on uncoated Thermanox coverslips.
FPA
Plasma levels of FPA were measured distal to the perfusion chamber
(Table 2). There was no significant difference in the
average plasma levels of FPA after perfusions of cells stimulated for 2
hours with lipopolysaccharide or nonstimulated cells. Nor did blocking
of the TF/FVIIa complex formation with the anti-TF MAb produce any
significant change. The average plasma levels of FPA increased with
time of monocyte adherence to the Thermanox and with time of
lipopolysaccharide stimulation, although not significantly. However,
prolonged time of adherence with and without stimulation resulted in
about a twofold increase in adherent cells, which presumably affected
the FPA levels.
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Inclusion of the anti-TF MAb reduced the average FPA levels but not significantly.
ß-TG
Plasma levels of ß-TG were measured distal to the perfusion
chamber (Table 2). The levels were increased (P<.01) after
4 hours of polysaccharide stimulation of monocytes compared with the
nonstimulated cells. The increased ß-TG levels paralleled the
increase in platelet-thrombus volume (Fig 2B). In contrast, no increase
was observed after perfusion of cells stimulated for 2 hours, despite
the fact that the measured thrombus volume was increased significantly
over that of nonstimulated cells (Fig 2B).
Inclusion of the anti-TF MAb significantly decreased the plasma ß-TG levels for cells stimulated for 4 hours with lipopolysaccharide (P<.04) but not for the cells stimulated for 2 hours.
Morphology
Monocytes adhered singly and randomly to the Thermanox coverslips.
However, confluence at 2 or 4 hours of incubation was not achieved with
the number of cells seeded, thus leaving some Thermanox material
exposed to blood flow. The platelet thrombi always formed in
association with adherent monocytes and deposited fibrin, giving the en
face appearance of more clustering of the thrombotic deposits than on
collagen fibrils (Fig 3). Inspection of the sections
revealed fibrin strands sprouting out from monocytes and frequently
covering parts of the uncoated Thermanox coverslip. Platelet thrombi
formed on fibrin strands in close proximity to adherent monocytes (Fig 4A) or apparently directly on the monocyte surface (Fig 4B).
Occasionally, fibrin strands formed on lipopolysaccharide-stimulated
monocytes incubated with the anti-TF MAb, but in general fibrin
deposition and platelet-thrombus formation were suppressed (Fig 4C).
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Discussion |
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The normal vessel wall possesses TF that complexes with FVII/FVIIa from plasma and acts as a cofactor for activation of the extrinsic coagulation.5 6 TF also is found in atherosclerotic plaques25 and generally in association with monocyte-derived macrophages.13 14 The location of TF in healthy and sick vascular compartments separated from the blood flow by the endothelial layer only and the apparent role of FVII/FVIIa in thrombogenesis26 have spurred much interest in TF/FVIIa–induced coagulation and thrombosis.
The role of TF associated with lipopolysaccharide- or cytokine-stimulated endothelium or corresponding extracellular endothelial matrices,9 10 normal arterial intima, or deeper compartments of the vessel wall7 8 has been investigated in various studies of thrombogenesis. Blocking TF/FVIIa activity by anti-TF MAbs interrupts this thrombus formation efficiently.7 8 9 10 11 However, peripheral blood monocytes also express TF when stimulated, and together with TF of monocyte-derived macrophages in arterial plaques, these cells may provide the primary source of TF in several pathological conditions,27 28 such as disseminated intravascular coagulation, plaque rupture, and plaque disruption after percutaneous transluminal coronary angioplasty (PTCA) procedures. Therefore, we found it of interest to investigate the role of TF expression of monocytes15 in arterial thrombus formation. For that purpose, human monocytes were immobilized on plastic coverslips and exposed to flowing nonanticoagulated human blood in a parallel-plate perfusion chamber device16 17 20 at an arterial wall shear rate of 650 seconds-1.
We observed that lipopolysaccharide-stimulated monocytes elicited pronounced fibrin deposition and platelet-thrombus formation. However, inclusion of an anti-TF MAb, which blocks the complexation of TF with FVII/FVIIa, almost abolished fibrin deposition and efficiently reduced platelet-thrombus formation. The procoagulant state of these cells was also confirmed by the one-stage clotting assay and by the inclusion of the same anti-TF MAb, which efficiently blocked the procoagulant activity. An indifferent antibody of the same subclass had no significant effect, indicating that the anti-TF MAb specifically affected the monocyte TF procoagulant activity only. Thus, the monocyte procoagulant activity and its significant impact on arterial thrombus formation were predominantly TF/FVIIa dependent.
However, nonstimulated monocytes adherent for 4 hours to plastic coverslips expressed low levels of TF activity. Induction of TF by adherence has been confirmed on the cellular mRNA level (unpublished observation). Because endotoxin was not detected in either media or on coverslips, these findings indicate that the adherence process itself may initiate TF expression. Also, this observation is in accordance with findings reported by others,29 30 showing that adherence of monocytes and macrophages to artificial surfaces triggers cellular procoagulant activity in the absence of exogenous agonists. It is interesting to note, however, that these non–lipopolysaccharide-stimulated adherent monocytes, which expressed low levels of TF and elicited platelet-thrombus formation under arterial blood flow conditions, may trigger similar events when adhering to graft material in vivo and thus play a role in artificial graft failure.
Despite the pronounced effect of the anti-TF MAb on monocyte-mediated coagulation and thrombus formation, platelets apparently adhered directly to the monocytes, which subsequently resulted in platelet-thrombus formation. These platelet thrombi formed on the cell surface in areas that on morphological inspection revealed no signs of fibrin deposition. However, we cannot exclude the possibility that the cells bound and activated factor X,28 31 resulting in minute undetectable fibrin deposition that served as a nidus for platelet-thrombus formation, or that the 1-µm-thick sections revealed platelet thrombi growing outside the fibrin mesh and thus gave the impression of direct platelet-monocyte adhesion. Nevertheless, from our experiments, it appeared that TF is the key initiator of monocyte-mediated coagulation and arterial thrombus formation, at least during the early stage of thrombus formation.
The material of the Thermanox coverslips is virtually nonthrombogenic.17 Exposure of this material to flowing nonanticoagulated human blood results in minimal activation of platelets and coagulation. Fibrin deposits are not detected, and platelets are generally making contact with the Thermanox surface by a small portion of their plasma membrane. Platelet aggregates are never observed.17 Thus, it is obvious that fibrin deposition and platelet-thrombus formation elicited by the Thermanox-adherent monocytes were due entirely to the cells. The larger thrombus formation triggered by 4-hour-adherent and/or stimulated cells relative to the 2-hour-adherent cells was apparently related to higher amounts of expressed TF and to the higher density of adherent cells. The size of these platelet thrombi was in the range of that previously observed on human type III collagen fibrils.17 32 Thus, procoagulant monocytes trigger platelet-thrombus formation as efficiently as fibrillar collagen.
Plasma activation markers of coagulation (FPA) and platelets (ß-TG) collected distally to the perfusion chamber after 4 hours of lipopolysaccharide stimulation showed a nearly sixfold and fourfold increase in the average FPA and ß-TG levels, respectively. Inclusion of the anti-TF MAb reduced these levels to those measured with nonstimulated cells. Thus, the levels of the activation markers measured complemented the morphological data obtained with adherent cells stimulated with lipopolysaccharide for 4 hours. However, the situation was different for the 2-hour-adherent cells. Lipopolysaccharide stimulation increased procoagulant activity and fibrin deposition without affecting the plasma FPA levels. Furthermore, the platelet-thrombus formation was enhanced more than 10-fold, but this was not reflected in the modest nonsignificant rise of the plasma ß-TG. The reason remains unknown. However, it should be emphasized that the morphometric approach is an end point measurement, while measurements of the plasma activation markers represent observations taken at one defined point of time during thrombus formation. Thus, dissimilarities between such measured parameters may occur.
Our data suggest that thrombotic complications following situations such as plaque disruption after PTCA, rupture of arterial plaques, or artificial graft failure can be reduced by blocking monocyte procoagulant activity. However, it might prove necessary to include a platelet antagonist as well because other thrombogenic components such as collagens or even the monocyte itself33 may trigger platelet-dependent thrombus formation.
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Acknowledgments |
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We acknowledge Merete Thune Wiiger, Biotechnology Centre of Oslo, University of Oslo, for sharing her experience with measurements of cell procoagulant activity.
Received June 1, 1994; accepted October 20, 1994.
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