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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Intrinsic pathway of blood coagulation contributes to thrombogenicity
of atherosclerotic plaque
Natalya M. Ananyeva, Diana V. Kouiavskaia, Midori Shima, and Evgueni L. Saenko
Thrombosis is the major mechanism underlying acute complications of atherosclerosis. Although thrombogenicity of
atherosclerotic plaques has been ascribed to activation of the extrinsic pathway of blood coagulation, in the present
study we investigated contribution of the
intrinsic factor VIII (fVIII)–dependent pathway. We found that in vitro exposure of
human macrophages and smooth muscle
cells (SMCs) to atherogenic oxidized lowdensity lipoprotein (oxLDL) enhances
their ability to support activity of 2 major
complexes of the intrinsic pathway, Xase
and prothrombinase, leading to a 20- and
10-fold increase in thrombin formation,
respectively. In contrast, human aortic
endothelial cells were less responsive to
oxLDL. The increase in the intrinsic procoagulant activity was related to formation
of additional fVIII binding sites due to
enhanced translocation of phosphatidylserine to the outer surface of oxLDLtreated cells and a 5-fold higher affinity of
interaction between components of the
Xase complex, activated factors VIII and
IX. Processes occurring at early apoptotic stages, including changes in the cell
membrane induced by free radicals, may
be related to activation of the intrinsic
pathway as suggested by effects of inhibitors of early apoptosis on thrombin forma-
tion. Immunohistochemical studies on human atherectomy specimens revealed the
presence of fVIII in the vicinity of macrophages and SMCs in atheromatous regions with massive deposits of oxLDL,
supporting the possible involvement of
the intrinsic pathway in thrombus formation in vivo. Our data predict that the
intrinsic pathway significantly enhances
thrombogenicity of atherosclerotic lesions after removal of the endothelial
layer and exposure of SMCs and macrophages to blood flow. (Blood. 2002;99:
4475-4485)
© 2002 by The American Society of Hematology
Introduction
Human atherosclerosis involves 2 distinct pathologic processes—
conventional atherogenesis at early stages, and atherothrombosis at advanced stages, which is responsible for acute manifestations of the disease.1,2 Atherogenesis is initiated by oxidation of
low-density lipoproteins (LDL) and recruitment of monocytes to
the intima of the vessel wall leading to accumulation of oxidized
LDL (oxLDL) in macrophages and smooth muscle cells (SMCs)
and their transformation into lipid-laden foam cells.2 This
process is accompanied by extensive cell proliferation and
elaboration of extracellular matrix components accounting for
atheroma progression.3 However, severe clinical complications
of atherosclerosis, including myocardial infarction, ischemic
stroke, and sudden cardiac death, are caused by atherothrombosis manifested in thrombus formation primarily on ruptured
advanced atherosclerotic plaques, which finally leads to occlusion of the vessel lumen.4,5
A major role in determining thrombogenicity of human
atherosclerotic lesions has been ascribed to the extrinsic, tissue
factor (TF)–dependent pathway of blood coagulation.6,7 TF is
expressed at high levels in macrophages and SMCs within
human atherosclerotic plaques and its activity correlates with
plaque progression and thrombin generation.8-10 Membranebound complex of TF with activated factor VII (fVIIa) proteolytically activates factors IX (fIX) and X (fX). In its turn,
activated fX (fXa) participates in conversion of a zymogen
prothrombin into thrombin, the key enzyme of the coagulation cascade.11
Although the TF-dependent pathway is unequivocally responsible for initial generation of fXa, the intrinsic pathway catalyses
fX activation approximately 50-fold more efficiently, thus dramatically amplifying coagulation events triggered by the TF-dependent
pathway.11 In the intrinsic pathway, fX activation is provided by a
membrane-bound Xase complex formed by activated factors VIII
(fVIIIa) and IX (fIXa). The fVIII molecule consists of 3 homologous A
domains, 2 homologous C domains, and the unique B domain, which
are arranged in the order A1-A2-B-A3-C1-C2. Thrombin or fXa
activates fVIII by intramolecular cleavages producing heterotrimeric fVIIIa (A1/A2/A3-C1-C2). Although the role of the intrinsic
pathway in determining thrombogenicity of atherosclerotic plaque
has not been studied, its possible contribution is suggested by a
number of clinical studies, which demonstrated correlation between elevated levels of fVIII and fIX and the risk of coronary
heart disease,12-14 myocardial infarction,15 and ischemic stroke.16
On the other hand, several clinical surveys revealed a significantly
reduced risk of myocardial infarction and 5-fold lower mortality
from ischemic heart disease in fVIII-deficient patients with hemophilia A, suggesting that the lowering of fVIII level reduces
development of thrombotic complications in atherosclerosis.17,18
The functioning of the intrinsic Xase complex requires its
assembly on the phospholipid surface, the major role of which is to
From the Holland Laboratory, American Red Cross, Rockville, MD and Nara
Medical University, Kashihara, Nara, Japan.
Reprints: Evgueni L. Saenko, Department of Biochemistry, Holland
Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD
20855; e-mail: [email protected].
Submitted November 30, 2001; accepted February 7, 2002. Prepublished
online as Blood First Edition Paper, April 17, 2002; DOI 10.1182/blood-200111-0140.
Supported by National Institutes of Health-National Heart, Lung and Blood
Institute RO1 grant HL66101 awarded to E.L.S.
BLOOD, 15 JUNE 2002 䡠 VOLUME 99, NUMBER 12
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.
© 2002 by The American Society of Hematology
4475
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BLOOD, 15 JUNE 2002 䡠 VOLUME 99, NUMBER 12
ANANYEVA et al
direct interaction between the components of the Xase complex
from 3- to 2-dimensional space. This results in a dramatic acceleration of
fXa generation due to decrease in the Michaelis constant (Km) for
fX19 and increase in the catalytic constant of the reaction (kcat).20
Although the phospholipid surface is classically provided by
membranes of activated platelets,21 3 major cell constituents of
atherosclerotic lesion, that is, macrophages,22,23 SMCs,23 and
endothelial cells (ECs)23,24 also support Xase assembly in vitro, yet
far less effectively than activated platelets. Within atherosclerotic
lesions, all these cell types are exposed to oxLDL, which triggers
transformation of macrophages and SMCs to lipid-laden foam
cells.2 At later stages of atherogenesis, oxLDL induces apoptosis,
as evidenced by an increased occurrence of apoptotic macrophages
and SMCs in human atherosclerotic lesions,25,26 especially in
ruptured plaques.27 Induction of apoptosis by oxLDL was also
demonstrated in vitro for SMCs,28,29 ECs,30,31 and macrophages.32,33 Because the complexes of both extrinsic and intrinsic
pathways of coagulation are highly dependent on the presence of
phosphatidylserine (PS) in cell membranes,20 translocation of this
anionic phospholipid from the inner to the outer leaflet of the
membrane during apoptosis is likely to be an important factor
defining the procoagulant activity of the cells in atherosclerotic
lesions. Apoptotic macrophages and SMCs were shown to have an
increased procoagulant activity in the TF-dependent pathway,34 and
induction of apoptosis in ECs by staurosporin was reported to
increase the intrinsic Xase activity.35 However, the link between
oxLDL-induced apoptosis and the activity of the intrinsic pathway
in the environment of atherosclerotic lesion remains to be elucidated.
In the present study we investigated whether in vitro exposure
of major cell constituents of the vessel wall to oxLDL alters their
ability to support the activity of the intrinsic Xase and prothrombinase complexes. We compared relative susceptibility of SMCs,
macrophages, and ECs to oxLDL and demonstrated that exposure
of macrophages and SMCs to atherogenic levels of oxLDL resulted
in a significant increase in the rates of fXa and thrombin formation.
Materials and methods
Materials
Human plasma–derived fVIII was purified from therapeutic fVIII concentrates (AHF concentrate, American Red Cross, Rockville, MD) as described.36 Human coagulation factors V, IXa, X, and Xa, human prothrombin, and thrombin were purchased from Enzyme Research Laboratories
(South Bend, IN). Native LDL and fully oxidized LDL were from Intracel
(Rockville, MD). A general caspase inhibitor (Z-VAD-FMK) was purchased from R & D Systems (Minneapolis, MN). An antioxidant butylated
hydroxytoluene (BHT) and an inhibitor of acidic sphingomyelinase,
desipramine, were from Sigma (St Louis, MO).
Antibodies
Mouse monoclonal antibody (mAb) ESH8 recognizing residues 2248-2285
of the C2 domain of fVIII37 was purchased from American Diagnostica
(Greenwich, CT). The mAb NMC-VIII/10 recognizing residues 1675-1684
within the acidic region of the light chain of fVIII was produced as
described.38 Biotinylation of mAbs ESH8 and NMC-VIII/10 was performed using EZ-Link Sulfo-NHS-LC-Biotinylation kit (Pierce, Rockford,
IL). Mouse mAb OXL41.1 specifically recognizing oxLDL and not
cross-reacting with native LDL was purchased from Neomarkers (Fremont,
CA). Mouse mAb to vascular ␣-actin 1A4 conjugated with a red fluorophore Cy3 was from Sigma. Mouse mAb to macrophage-specific marker
CD68 was purchased from Dako (Carpinteria, CA) and coupled with a red
fluorophore Alexa Fluor 594 (Molecular Probes, Eugene, OR).
Cell culture
Primary cultures of human aortic SMCs and human aortic endothelial cells
(HAECs) were purchased from Biowhittaker (Walkersville, MD) and used
at passages 4 through 10. SMCs were propagated in SmGM-2 BulletKit
Medium supplemented with 10% fetal bovine serum (FBS), human basic
fibroblast growth factor (FGF), human epidermal growth factor (EGF), and
insulin (Biowhittaker) at 37°C in 6% CO2. HAECs were propagated in
modified endothelial cell basal medium-2 supplemented with 2% FBS,
human basic FGF, human vascular endothelial growth factor (VEGF),
human EGF, insulinlike growth factor 1 (IGF-1), heparin, ascorbic acid, and
hydrocortisone (Biowhittaker) as above. SMCs and HAECs were used in
experiments at 80% to 90% confluence. Human monocytes were isolated
from mononuclear leukocyte preparations obtained by apheresis procedure
performed by Research Blood Staff at the Holland Laboratory, American
Red Cross under approved institutional review board protocol. The
population of monocytes was enriched to 97% by positive selection on
CD14 beads (Miltenyi, Auburn, CA). Differentiation of monocytes into
macrophages was promoted by addition of macrophage colony-stimulating
factor (Sigma) in RPMI supplemented with 10% human AB serum
(Biowhittaker).
Prior to experiments, SMCs were transferred to low serum (0.5% FBS)
SmGM-2 BulletKit Medium, HAECs to defined human endothelial serumfree growth medium supplemented with human basic FGF (20 ng/mL) and
human EGF (10 ng/mL; Invitrogen, Carlsbad, CA) and macrophages to
defined human macrophage serum-free growth medium (Invitrogen).
Incubation of the cells with lipoproteins was performed under serum-free
(for HAECs and macrophages) or low serum (for SMCs) conditions.
Measurement of the intrinsic pathway activities
Factor Xa generation assay. Cells were incubated without LDLs or with
100 ␮g/mL of either oxLDL or native LDL for increasing time intervals,
and the ability of the cell surface to support conversion of fX to fXa was
measured in a chromogenic assay performed at 37°C as described.39 The
reaction was performed in 20 mM Hepes buffer, pH 7.4, containing 0.15 M
NaCl, 5 mM CaCl2, and 0.5% bovine serum albumin (HBS). FVIII and fIXa
were added to final concentrations of 1 nM and 2 nM, respectively. After
activation of fVIII by thrombin (0.5 U/mL) for 30 seconds, the reaction was
initiated by addition of fX (170 nM). The aliquots were taken at defined
time intervals, the reaction was stopped with 0.05 M EDTA, followed by
determination of generated fXa from the rate of conversion of a chromogenic substrate S-2765 (Chromogenix, Milan, Italy). The increase in
absorbance was read at 405 nm using a Labsystems multiscan microplate
reader (Labsystems, Franklin, MA) in a kinetic mode and converted into
fXa concentration using a purified fXa standard. Maximal rates of fX
activation were calculated from individual kinetic curves by linear regression of fXa concentration over time using a SigmaPlot 1.02 (Jandel
Scientific, Chicago, IL) computer program. In a control experiment,
thrombin activation of fVIII was stopped by 1.5-molar excess of hirudin
prior to addition to oxLDL-treated cells. In an additional control experiment, the Xase reaction was performed in wells incubated with oxLDL in
the absence of cells.
Determination of parameters of interaction of the Xase components.
Following incubation of the cells in the absence or presence of oxLDL, fXa
generation assays were performed as described above at a constant fVIII
concentration (1 nM) and increasing concentrations of either fIXa or fX. In
fIXa titration experiment, fX concentration was 170 nM, and fIXa
concentrations increased from 0.05 to 5 nM; in fX titration experiment,
fIXa concentration was 1 nM and fX concentrations ranged from 1 to 200
nM. Initial rates of fX activation for each condition were determined as
described above and plotted versus concentration of fX or fIXa. The
apparent affinity of fVIIIa/fIXa interaction (Kapp) was determined according to a standard equilibrium binding model described in the corresponding
figure legend. Km and the maximal rate (Vmax) of fX conversion by
fVIIIa/fXa complex were determined by fitting the initial rates of fX
generation versus fX concentration using a Michaelis-Menton model.
Thrombin generation assay. Activation of prothrombin to thrombin on
the surface of control and oxLDL- or native LDL-treated cells was
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BLOOD, 15 JUNE 2002 䡠 VOLUME 99, NUMBER 12
measured as described.40 The components of the Xase and prothrombinase
complexes, fVIII, fIXa, and fV, were added in HBS to final concentrations
of 1 nM, 2 nM, and 20 nM, respectively. The reaction was initiated by
simultaneous addition of fX and prothrombin to final concentrations of 170
nM and 1.4 ␮M, respectively. FV was activated by fXa generated by the
Xase complex. Thrombin formation was measured by the rate of conversion
of a chromogenic substrate S-2238 specific for thrombin (Chromogenix)
registered as an increase in absorbance at 405 nm and converted to thrombin
concentration using purified thrombin standard. The maximal rates of
thrombin generation were determined as in the fXa generation assay. In
additional experiments, the cells were incubated with various inhibitors of
apoptosis for 1 hour prior to addition of oxLDL. The inhibitors were used at
the following concentrations: BHT, 50 ␮M for all cell types; desipramine, 1
␮M for SMCs and 10 ␮M for macrophages and HAECs; Z-VAD-FMK, 100
␮M for SMCs and HAECs and 10 ␮M for macrophages.
To examine the effect of oxLDL on the activity of the prothrombinase
complex, thrombin formation on oxLDL-treated cells was determined in the
presence of fV (20 nM), fXa (8 nM), and prothrombin (1.4 ␮M). The
kinetics of thrombin formation were measured as described above.
THROMBOGENICITY OF ATHEROSCLEROTIC PLAQUE
4477
OXL41.1 and ESH8, respectively. The antibodies were visualized using
horseradish peroxidase–based Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA), and sections were counterstained with hematoxylin.
In double-staining experiments, oxLDL was detected as above, followed by
staining for fVIII using ESH8 and alkaline phosphatase–based Vectastain
ABC-AP kit (Vector Laboratories). The double-stained sections were
counterstained with nuclear fast red.
In double-label immunofluorescence staining, tissue autofluorescence was quenched using sodium borohydride as described.46 Staining
for fVIII was performed by incubating atherectomy sections with
biotinylated mAb ESH8 or mAb NMC-VIII/10, followed by visualization with fluorescein-conjugated avidin DCS (5 ␮g/mL, Vector Laboratories). The fVIII-stained sections were subsequently stained for SMCs
or macrophages using mouse anti-␣-smooth muscle actin mAb 1A4
conjugated with Cy3 fluorophore or antihuman CD68 mAb conjugated
with Alexa Fluor 594. Lipofuscinlike autofluorescence of doublelabeled sections was eliminated using 1% Sudan black B in 70%
ethanol.47 Air-dried sections were mounted in ProLong Antifade medium (Molecular Probes) and microscopy was performed as above using
Cell-mediated fVIII binding assay
Factor VIII was labeled with Na125I (100 mCi/mL [3700 MBq], Amersham
Pharmacia Biotech, Uppsala, Sweden) using lactoperoxidase beads (Worthington Biochemical, Lakewood, NJ) as described.41 FV and annexin V
(MBL International, Watertown, MA) were labeled with Na125I by IODOGEN method as described.42,43 The specific radioactivities of 125I-fVIII,
125I-fV, and 125I-annexin V were 8.7 ⫻ 106, 2.9 ⫻ 106, and 2.6 ⫻ 106
cpm/␮g, respectively.
125I-FVIII binding assay was performed as generally described.44 After
treatment of the cells with native LDL or oxLDL for increasing time
intervals, the cells were incubated with 125I-fVIII (0.5 nM) in 10 mM Hepes,
pH 7.4 containing 137 mM NaCl, 4 mM KCl, 11 mM glucose, 5 mM CaCl2,
and 2% BSA for 2 hours at 10°C. In some experiments, the cells were
incubated with oxLDL in the presence of BHT, desipramine, or Z-VADFMK as above. After washing the cells with the binding buffer without
125I-fVIII, surface-bound 125I-fVIII was determined as radioactivity released after treatment of the cells with 50 ␮g/mL trypsin (Invitrogen), 50
␮g/mL proteinase K (Boehringer Mannheim, Mannheim, Germany), and 5
mM EDTA. The 125I-fVIII binding level was corrected for nonspecific
125I-fVIII binding measured in the presence of 500-fold excess of unlabeled
fVIII. The binding experiments with 125I-fV and 125I-annexin V were
performed analogously.
Detection of PS by annexin V binding
Cells were grown in Lab-Tek II chamber slides (Nalge Nunc International,
Naperville, IL) in the absence or presence of oxLDL for 12 hours.
Translocation of PS from the inner to the outer leaflet of the cell membrane
was tested with annexin V fused with enhanced green fluorescent protein
(annexin V-EGFP) using ApoAlert annexin V-EGFP apoptosis kit (Clontech, Palo Alto, CA). The binding of annexin V-EGFP was performed at
room temperature for 15 minutes on unfixed cells to avoid cell membrane
perforation and possible annexin V penetration into the cells. In a positive
control the cells were incubated with apoptosis-inducing agents, 20 nM
staurosporin (Clontech) or with 20 ng/mL human recombinant tumor
necrosis factor-␣ (R & D Systems). Annexin V⫹ cells were detected in an
Eclipse E800 microscope (Nikon, Melville, NY) equipped with a set of
fluorescent filter blocks and a digital SPOT RT camera (Diagnostic
Instruments, Sterling Heights, MI).
Immunohistochemistry
Coronary atherectomy specimens were obtained from patients (mean age,
64 ⫾ 11 years) who underwent directional coronary atherectomy at the
Washington Hospital Center (Washington, DC). The specimens were
processed as described.45 Almost all specimens had regions of normal
media, which served as built-in controls for quiescent SMCs. The atherectomy sections were stained for oxLDL or fVIII using mouse mAbs
Figure 1. Effect of oxLDL on the procoagulant activity of human aortic SMCs,
macrophages, and aortic ECs. SMCs (A), macrophages (B), or HAECs (C) in the
amount of 2 ⫻ 105/well were incubated in the absence of lipoproteins (E) or in the
presence of 100 ␮g/ml oxLDL (F) or native LDL (Œ) for 12 hours at 37°C. Following
incubation, the conversion of fX (170 nM) into fXa catalyzed by fIXa (2 nM) and
thrombin-activated fVIII (1 nM) was measured in a chromogenic assay as described
in “Materials and methods.” In the control experiment (〫), the Xase reaction was
performed in wells incubated with oxLDL in the absence of cells. In the additional
control experiment (⫹), the Xase reaction was performed using fVIII activated by
thrombin, which was subsequently inactivated by excess of hirudin. Each data point
represents the mean value ⫾ SD of triplicates.
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ANANYEVA et al
Table 1. Maximal rates of generation of fXa and thrombin in the intrinsic pathway by oxLDL-treated cells
Rate of fXa generation, V (nM/min)
Rate of thrombin generation, Vthr (nM/min)
Cell type
Control, V0
oxLDL-treated, V
V/ V0 ratio
Control, Vthr0
Macrophages
0.218 ⫾ 0.05
1.768 ⫾ 0.022
8.1 ⫾ 1.8
16.6 ⫾ 0.43
SMCs
0.176 ⫾ 0.016
1.06 ⫾ 0.056
6.0 ⫾ 0.63
18.3 ⫾ 0.6
HAECs
0.33 ⫾ 0.012
0.51 ⫾ 0.035
1.55 ⫾ 0.12
33.5 ⫾ 0.82
OxLDL-treated,
Vthr
326 ⫾ 11.2
Vthr/Vthr0 ratio
19.6 ⫾ 0.84
187.5 ⫾ 4.7
10.2 ⫾ 0.42
74 ⫾ 3.1
2.2 ⫾ 0.11
After incubation of the cells with oxLDL for 12 hours, generation of fXa and thrombin was determined as described in “Materials and methods.” Maximal rates were
determined from individual kinetic curves shown in Figures 1 and 7 by linear regression of fXa or thrombin concentration over time using a SigmaPlot 1.02 computer program.
selective fluorescent filter blocks. Simultaneous visualization of fVIII
and ␣-actin or CD68 was performed by merging single-dye images using
SPOT Advance Program Mode (Diagnostic Instruments).
fXa generation was not due to the presence of necrotic cells. Thus,
of 3 cell types comprising atherosclerotic lesion, exposure of SMCs
and macrophages to oxLDL led to a significant acceleration of
fVIII-dependent generation of fXa, whereas endothelial cells were
far less responsive to oxLDL.
Results
Oxidized LDL increases activity of intrinsic Xase complex
assembled on human SMCs and macrophages
We first compared the effect of oxLDL on the ability of major cell
types forming the arterial wall—SMCs, macrophages, and
HAECs—to support fX activation by the intrinsic coagulation
pathway. In the absence of lipoproteins, all cell types had a low
ability to support fXa generation in the presence of thrombinactivated fVIII and fIXa (Figure 1). Incubation of SMCs and
macrophages with oxLDL for increasing time intervals up to 24
hours led to a gradual increase in fXa formation (data not shown).
The maximal effect was achieved at 12 hours of incubation
resulting in a 6- and 8.1-fold increase in the maximal rates of fXa
generation for oxLDL-treated SMCs and macrophages, respectively (Figure 1A,B and Table 1). Notably, incubation of HAECs
with oxLDL resulted only in a 1.6-fold increase in the rate of fXa
generation (Figure 1C and Table 1). In contrast to oxLDL, native
LDL had a slight effect on the procoagulant properties of the tested
cell types. The control experiment confirmed that acceleration of
Xase was mediated by the effect of oxLDL on the cells, because no
generation of fXa was detected in the wells incubated with oxLDL
in the absence of cells (Figure 1). In another control experiment,
fVIII was activated prior to addition to the cells, and thrombin was
subsequently inactivated by excess of hirudin. Because kinetics of
fXa generation on oxLDL-treated cells were not affected by
hirudin, the possible effect of thrombin on fXa formation by
oxLDL-treated cells was excluded. OxLDL-treated cells stained
negative with trypan blue, indicating that the registered increase in
FVIII binding to oxLDL-treated cells is increased
Because the binding of fVIII to the phospholipid membrane is
required for assembly of the Xase complex, the increased procoagulant activity may be related to the increased binding of fVIII to
oxLDL-treated cells. We, therefore, measured 125I-fVIII binding to
the cells treated with oxLDL for increasing time intervals. Incubation of SMCs and macrophages with oxLDL for 12 hours led to a
maximal 4-fold and 5-fold increase in 125I-fVIII binding, respectively, compared to untreated cells (Figure 2A,B), whereas for
HAECs this increase was only 1.5-fold (Figure 2C). In contrast,
incubation of the cells with native LDL did not result in appreciable
increase in fVIII binding (Figure 2).
Because oxLDL itself can support assembly of the intrinsic
Xase complex,48 we verified that accelerated fXa generation and
formation of additional fVIII-binding sites on macrophages and
SMCs were due to changes in cell membrane caused by internalization of oxLDL. This was confirmed by lack of appreciable increase
in the Xase activity and 125I-fVIII binding to the cells incubated
with oxLDL at 10°C, when internalization was suppressed (data
not shown). Thus, the increased ability of oxLDL-treated cells to
support activity of the intrinsic Xase complex (Figure 1) correlated
with the level of fVIII bound to their surface.
oxLDL induces translocation of PS in the cell membrane
Binding of fVIII to phospholipid membranes is mediated by PS,49
which is ultimately required for the formation of fVIII binding
sites.50 Because the number of fVIII binding sites sharply increases
Figure 2. Effect of oxLDL treatment on 125I-fVIII binding to the cell surface. Human SMCs (A), macrophages (B), or HAECs (C) in the amount of 2 ⫻ 105/well were
incubated in the absence of lipoproteins (open bars) or in the presence of 100 ␮g/mL native LDL (hatched bars) or 100 ␮g/mL oxLDL (black bars) for the time intervals indicated.
Subsequently, 125I-fVIII was added to a final concentration of 0.5 nM and its binding was determined as described in “Materials and methods.” Each data point represents the
mean value ⫾ SD of triplicate determinations.
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BLOOD, 15 JUNE 2002 䡠 VOLUME 99, NUMBER 12
with an increase in PS content in both synthetic and physiologic
membranes,20,50 the observed elevated fVIII binding to oxLDLtreated cells may be related to translocation of PS from the inner to
the outer leaflet of the cell membrane. We compared exposure of PS
on the surface of oxLDL-treated and untreated cells using annexin
V–EGFP as a specific probe for PS.51 Microscopic analysis
revealed that incubation of SMCs and macrophages with oxLDL
for 12 hours led to a pronounced increase in fluorescence intensity,
reflecting intensive translocation of PS to the outer membrane
leaflet (Figure 3A-D). For HAECs, surface-bound annexin V–EGFP
was detected in a few untreated cells but the difference in
fluorescence intensity of control and oxLDL-treated cells was less
pronounced (Figure 3E,F). Thus, oxLDL-induced translocation of
PS in macrophages and SMCs may account for the increased
125I-fVIII binding to these cells.
THROMBOGENICITY OF ATHEROSCLEROTIC PLAQUE
4479
FVIIIa interacts with fIXa with a higher affinity on
oxLDL-treated cells
Because the PS content in membrane strongly affects the affinity of
fVIIIa for fIXa and the ability of the assembled Xase complex to
catalyze activation of fX into fXa,20 we examined the effect of
oxLDL treatment of SMCs on the parameters characterizing
fVIIIa/fIXa interaction and the activity of the resulting Xase
complex. The dependence of fXa formation on fIXa concentration
at constant concentration of fVIIIa and fX was adequately fitted to
a standard equilibrium binding model (Figure 4A). The value of
Kapp, characterizing the apparent affinity of fVIIIa for fIXa in the
functional Xase assay, was 0.41 ⫾ 0.17 nM for untreated and
0.08 ⫾ 0.022 nM for oxLDL-treated SMC. Thus, treatment of the
cells with oxLDL not only increases the concentration of cell
Figure 3. Effect of oxLDL on translocation of PS to the outer leaflet of cell membranes. Human SMCs (A,B), macrophages (C,D), and HAECs (E,F) were grown in
Lab-Tek II chamber slides and incubated in the absence (A,C,E) or presence (B,D,F) of 100 ␮g/mL oxLDL for 12 hours. Exposure of PS on cell membranes was tested by
immunofluorescence using annexin V coupled with EGFP as described in “Materials and methods.” Microscopy was performed using an Eclipse E800 microscope (Nikon)
equipped with fluorescent filter blocks at magnification ⫻ 600.
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4480
ANANYEVA et al
Figure 4. Effect of oxLDL treatment of SMC on assembly and activity of the
Xase complex. (A) Determination of the apparent affinity of fVIIIa/fIXa interaction.
SMCs in the amount of 2 ⫻ 105/well were incubated in the absence (E) or presence
(F) of 100 ␮g/mL oxLDL for 12 hours. The initial rates of fXa generation were
determined in the chromogenic assay performed at constant concentrations of fVIII
(1 nM) and fX (140 nM) and indicated concentrations of fIXa. Each data point
represents the mean value ⫾ SD of triplicates. The experimental data were fitted to
an equation describing the equilibrium binding V ⫽ Vsat[fIXa]/Kapp ⫹ [fIXa], where V is
the initial rate of fX activation, [fIXa] is the concentration of fIXa, Vsat is the rate of fX
activation at the saturating [fIXa], and Kapp is the apparent affinity of fVIIIa for fIXa.
The data were fitted to this equation using Marquart algorithm and SigmaPlot 1.02
computer program. (B) Determination of the kinetic parameters of fX activation. FVIII
(1 nM), fIXa (1 nM), and indicated concentrations of fX were added to SMCs
incubated in the absence (E) or presence (F) of oxLDL, which was followed by
determination of the initial rates of fXa generation as in panel A. Each data point
represents the mean value ⫾ SD of triplicates. The curves show the best fit of the
data to the Michaelis equation V ⫽ Vmax[fX]/Km ⫹ [fX], where V is the initial rate of fX
activation, [fX] is the concentration of fX, Vmax is the rate of fX activation at its
saturating concentration, and Km is the Michaelis constant. The data were fitted to the
above equation as in panel A.
surface-bound fVIII but also leads to a 5-fold increase in the
affinity of fVIIIa/fIXa interaction, which provides a higher concentration of functional fVIIIa/fIXa complex on the surface of
oxLDL-treated cells.
The parameters characterizing activity of the Xase complex
assembled on untreated and oxLDL-treated cells were determined
by varying fX concentration at constant concentrations of fVIIIa
and fIXa. The kinetics of fX activation on both treated and
untreated cells (Figure 4B) were adequately described by the
Michaelis equation. The obtained value of Vmax ⫽ 4.9 ⫾ 0.4 nM/
min for oxLDL-treated cells was 4.7 times higher than that for
control cells, Vmax ⫽ 0.98 ⫾ 0.108 nM/min, consistent with a
higher concentration of membrane-bound fVIIIa/fIXa complex. In
contrast, the difference between Km values for treated and control
SMCs was insignificant (22.3 ⫾ 4.7 nM and 19.42 ⫾ 4.9 nM,
respectively), suggesting that oxLDL does not affect the affinity of
fX for the fVIIIa/fIXa complex.
FVIII is associated with macrophage- and SMC-derived foam
cells in atherosclerotic lesions
The elevated fVIII binding to oxLDL-treated macrophages and
SMCs in vitro suggests that in human atherosclerotic lesions fVIII
may be associated with lipid-laden macrophage- or SMC-derived
foam cells. We first examined the patterns of distribution of fVIII
and oxLDL in human atherosclerotic lesion by staining 19 human
atherectomy specimens for oxLDL or fVIII. Figure 5 shows a
representative atherectomy specimen containing regions of both
normal tissue and atheroma. Normal tissue containing nonpathologic quiescent SMCs showed no staining for oxLDL (Figure 5A),
whereas in the atheromatous region both intracellular and extracellular deposits of oxLDL were detected (Figure 5B), consistent with
reported massive accumulation of oxLDL in advanced lesions.52
We next performed staining of serial sections of the same specimen
BLOOD, 15 JUNE 2002 䡠 VOLUME 99, NUMBER 12
for fVIII. We attempted to detect the A1/A3-C1-C2 dimer of
fVIIIa, which remains membrane-associated on fVIIIa inactivation caused by dissociation of the A2 subunit.53 We used mAb
ESH8, which binds to the C2 domain of fVIII and does not
prevent its binding to the phospholipid membrane.37 Notably,
intensive staining for fVIII was observed only in the atheromatous regions (Figure 5D), but not in the normal tissue (Figure
5C). Double staining for oxLDL and fVIII revealed large areas
staining black in atheromatous regions (Figure 5F), which
resulted from the superimposing of positive staining for oxLDL
(brown) and fVIII (blue). In contrast, regions of normal tissue
were negative for both markers (Figure 5E). These results
indicate that fVIII is present in human atherosclerotic lesions
and its accumulation correlates with deposition of oxLDL.
We next performed double-label immunofluorescence staining of 6 atherectomy specimens, where massive deposits of
oxLDL were detected, for fVIII using mAb ESH8 and for
␣-actin or CD68, which are specific markers of SMCs and
macrophages, respectively. A representative specimen is shown
in Figure 6. Regions of normal media containing spindle-shaped
␣-actin–positive SMCs (red fluorescence, Figure 6A) did not
show fVIII positivity (Figure 6B and merged image in panel C).
In oxLDL-rich regions of advanced lesions we selected areas
enriched in SMCs (Figure 6D) and macrophages (Figure 6G),
and fVIII presence in both regions was evidenced by green
fluorescence (Figure 6E and 6H, respectively). Corresponding
merged images (Figure 6F,I) revealed that fVIII was present in
the vicinity of SMCs and macrophages. In the control experiment, we performed the staining for fVIII using mAb NMCVIII/10 with the epitope within the acidic region of fVIII light
chain, which is removed on fVIII activation. Lack of appreciable fVIII-related fluorescence in the serial section of the
same specimen (data not shown) confirmed the specificity of
ESH8 staining for fVIIIa. Thus, immunohistochemical studies
of human atherosclerotic tissues revealed accumulation of
fVIII in the vicinity of macrophages and SMCs within oxLDLrich regions.
oxLDL dramatically increases thrombin formation on
macrophages and SMCs
To assess the magnitude of the overall procoagulant effect of
oxLDL, we determined the maximal rates of thrombin formation
on the cells incubated with oxLDL for 12 hours, when maximal
stimulation of the Xase activity was observed. Thrombin formation
was measured in the presence of components of both Xase (fVIII,
fIXa, and fX) and prothrombinase (fV and prothrombin) complexes. Incubation with oxLDL led to a 10-fold and 20-fold
increase in the maximal rates of thrombin formation on the surface
of SMC and macrophages, respectively (Figure 7A,B and Table 1).
The effect was much less pronounced for HAECs, where the
increase was only 2.2-fold (Figure 7C). Native LDL moderately
increased the rates of thrombin generation on all cell types, which
may be due to cell-mediated oxidation of LDL.54,55
The contribution of prothrombinase in the overall increase of
thrombin formation was assessed by performing the assay in the
presence of components of the prothrombinase complex only.
The concentrations of fV and prothrombin were as in Figure 7
and fXa concentration was 8 nM, which constitutes approximately 50% of maximal fXa concentration formed in the Xase
assay (Figure 1). The maximal rates of thrombin formation on
SMCs and macrophages were increased by oxLDL by 2.2-fold
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BLOOD, 15 JUNE 2002 䡠 VOLUME 99, NUMBER 12
THROMBOGENICITY OF ATHEROSCLEROTIC PLAQUE
4481
Figure 5. Patterns of localization of fVIII and oxLDL in atherosclerotic lesions. Human coronary atherectomy sections containing normal media (A,C,E) and atheromatous
regions (B,D,F) were stained for oxLDL (A,B) or fVIII (C,D) using mAbs OXL41.1 and ESH8, respectively, and double stained for both components (E,F). The single and double
staining were performed as described in “Materials and methods.” Positive staining for oxLDL and fVIII in panels B and D is brown; cell nuclei in panels A through D are
counterstained with hematoxylin. In double-stained images, panels E and F, accumulations of fVIII (blue) and oxLDL (brown) frequently overlap as evidenced by presence of
areas stained black. Cell nuclei in panels E and F were counterstained with nuclear fast red. Microscopy was performed using an Eclipse E800 microscope at
magnification ⫻ 600.
and 3.1-fold, respectively (Figure 8). This indicates that acceleration of thrombin formation on oxLDL-treated cells (Figure 7)
results mainly from the increase of the intrinsic Xase activity
and less from the increase of the prothrombinase activity.
Comparison of 125I-fV binding to control and oxLDL-treated
SMCs and macrophages revealed a moderate 1.6-fold and
2.1-fold increase in the binding (data not shown). Thus, oxLDL
dramatically increases the ability of macrophages and SMCs to
support thrombin formation in the intrinsic pathway and this
effect is mainly due to activation of the intrinsic Xase.
Relationship between procoagulant activity of oxLDL-treated
cells and apoptosis
Because oxLDL may induce apoptosis in SMCs,28,29 macrophages,32,33 and ECs,30,31 we tested whether the observed increase
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4482
BLOOD, 15 JUNE 2002 䡠 VOLUME 99, NUMBER 12
ANANYEVA et al
Figure 6. Detection of fVIII, macrophages, and SMCs in atherosclerotic lesions. Sections of a human coronary atherectomy specimen containing normal media (A-C) and
atheromatous regions (D-I) were double stained for fVIII (B,E,H, green fluorescence) and ␣-actin (A,D, red fluorescence) or CD68 (G, red fluorescence) as specific markers for
SMCs and macrophages, respectively. Staining procedure is described in “Materials and methods. The single-stained images were obtained in an Eclipse E800 microscope at
magnification ⫻ 1000 using selective fluorescent filter blocks. The merged images (C,F,I) were obtained by superimposing single-stained images using SPOT Advance
Program Mode. Arrows in panels F and I point to fVIII staining (green fluorescence) on the periphery of selected SMCs and macrophages, respectively (red fluorescence). The
staining patterns similar to those presented in this figure were found in all specimens analyzed.
in the procoagulant activity of oxLDL-treated cells is related to
apoptosis. TUNEL assay did not reveal DNA fragmentation in
macrophages and ECs and only 3% to 4% SMCs showed positive
nuclear staining (data not shown). This indicates that fragmentation
of chromatin, typical for advanced apoptotic stages, is not required
for development of the maximal procoagulant activity of oxLDLtreated cells. We next tested the effect of inhibitors of early stages
of apoptosis, including a scavenger of free radicals BHT, an
inhibitor of acidic sphingomyelinase (the major enzyme involved
in ceramide generation) desipramine, and a general caspase inhibitor Z-VAD-FMK, on thrombin generation. BHT and desipramine
had comparable inhibitory effects on thrombin formation on
oxLDL-treated macrophages and SMCs (Figure 9A), suggesting
that free radical–mediated processes and ceramide generation are
related to an increase in their procoagulant activity. Notably, a
general caspase inhibitor was more effective on SMCs compared to
macrophages, indicating that the maximal increase in the procoagulant activity of SMC may correspond to a more advanced stage of
apoptosis. Neither of the inhibitors affected thrombin generation on
oxLDL-treated HAECs.
Because the increase in the procoagulant activity of oxLDLtreated cells correlated with the increase in fVIII binding, we tested
whether inhibitors of early apoptosis affect this binding. Noteworthy, BHT, desipramine, and a general caspase inhibitor attenuated
fVIII binding in a pattern similar to their effects on thrombin
formation as shown in panels B and A, respectively, of Figure 9.
Binding of 125I-annexin V to oxLDL-treated SMCs was inhibited
by 35%, 22% and 30% by BHT, desipramine, and Z-VAD-FMK,
respectively (data not shown). The above experiments suggest that
the processes associated with early apoptotic stages (free radical
formation, ceramide generation, and activation of caspases) may be
related to development of the maximal procoagulant activity of
oxLDL-treated cells in the intrinsic pathway.
Discussion
Although increased thrombogenicity of advanced atherosclerotic
plaques has been linked to activation of the extrinsic, TF-dependent
pathway of blood coagulation, the present study suggests that the
intrinsic pathway significantly contributes to thrombus formation.
We found that exposure of macrophages and SMCs to oxLDL
significantly enhanced their ability to support activity of 2 major
complexes of the intrinsic pathway of blood coagulation, the Xase
and prothrombinase complexes. This resulted in a 20- and 10-fold
increase in thrombin formation for macrophages and SMCs,
respectively, mainly due to activation of the intrinsic Xase complex. The magnitude of the effect of oxLDL on the procoagulant
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BLOOD, 15 JUNE 2002 䡠 VOLUME 99, NUMBER 12
THROMBOGENICITY OF ATHEROSCLEROTIC PLAQUE
4483
Figure 8. Effect of oxLDL on the prothrombinase activity. Human SMCs (A) or
macrophages (B) were incubated in the absence (E) or in the presence (F) of oxLDL
as described in Figure 7. Following incubation, the conversion of prothrombin (1.4
␮M) into thrombin in the presence of fXa (8 nM) and fV (20 nM) was measured in a
chromogenic assay as described in “Materials and methods.” In the control experiment (〫), the reaction was performed in wells incubated with oxLDL in the absence
of the cells. Each data point represents the mean value ⫾ SD of triplicates.
Figure 7. Effect of oxLDL on thrombin generation via the intrinsic pathway.
Human SMCs (A), macrophages (B), or HAECs (C) in the amount of 2 ⫻ 105/well
were incubated in the absence of lipoproteins (E) or in the presence of 100 ␮g/mL
oxLDL (F) or native LDL (Œ) for 12 hours at 37°C. Following incubation, the
conversion of prothrombin (1.4 ␮M) into thrombin in the presence of fVIII (1 nM), fIXa
(2 nM), fV (20 nM), and fX (170 nM) was measured in a chromogenic assay as
described in “Materials and methods.” In the control experiment (〫), the reaction was
performed in wells incubated with oxLDL in the absence of cells. Each data point
represents the mean value ⫾ SD of triplicates.
activity of macrophages and SMCs in the intrinsic pathway
determined in our study is comparable to the reported increase in
the activity of the extrinsic coagulation pathway, which constituted
30-fold for cultured human macrophages,56 24-fold for macrophages isolated from atherosclerotic plaques,57 and 5- to 6-fold for
cultured SMCs.58 The modest oxLDL-induced increase in the
intrinsic procoagulant activity of HAECs determined by us is
similar to that previously determined for human umbilical vein
endothelial cells,23 however, less pronounced than the reported
4-fold increase in the extrinsic procoagulant activity.59 Although
oxLDL induces TF expression in macrophages60 and ECs61 and
up-regulates its expression in SMCs,58 the effects observed in our
study were solely due to oxLDL-induced activation of the intrinsic
pathway, because fVIIa, the counterpart of TF, was not present in
the assays. We demonstrated that the increase in the intrinsic
procoagulant activity is related to formation of additional fVIII
binding sites due to intensive translocation of PS to the outer
surface of oxLDL-treated cells. This was evidenced by a pronounced increase in the binding of 125I-fVIII and annexin V, a
specific probe for PS, to the surface of oxLDL-treated cells.
Furthermore, we found that interaction between Xase components,
fVIII and fIXa, on the surface of oxLDL-treated SMC occurs with a
5-fold higher affinity. Both phenomena are consistent with the
reported dependence of the number of fVIII binding sites50 and
fVIIIa/fIXa affinity20 on the PS content in phospholipid membranes. Together, the increased fVIII binding and a higher affinity
of fVIIIa for fIXa provide a higher concentration of functional
Xase complex on the surface of oxLDL-treated cells. The detected
modest 2-fold increase in fV binding to oxLDL-treated cells may
be related to a lower sensitivity of fV binding to the PS content of
membrane.50
Consistent with high levels of fVIII bound to the surface of
oxLDL-treated macrophages and SMCs in vitro, immunohistochemical studies on human coronary atherectomy specimens
revealed the presence of fVIII in the vicinity of macrophages and
SMCs in oxLDL-enriched atheromatous regions but not in the
normal tissue. These data are in favor of possible involvement of
the intrinsic coagulation pathway in thrombus formation at the site
of atherosclerotic lesion.
Redistribution of PS, which is likely responsible for the
observed increase in the intrinsic procoagulant activity of oxLDLtreated cells, is also one of indicators of apoptosis.32,62 Several
Figure 9. Effects of inhibitors of early stage of apoptosis on thrombin
generation and fVIII binding. Human macrophages, SMCs, or HAECs in the
amount of 2 ⫻ 105/well were incubated with 100 ␮g/mL oxLDL in the absence or
presence of BHT (open bars), desipramine (gray bars), or a general caspase inhibitor
Z-VAD-FMK (hatched bars) for 12 hours at 37°C as described in “Materials and
methods.” (A) Thrombin generation in the presence of inhibitors. Following incubation, the conversion of prothrombin into thrombin in the intrinsic pathway was
measured as described in Figure 7. The maximal rate of thrombin formation on
oxLDL-treated cells in the absence of inhibitors was arbitrary defined as 100%
(control). The maximal rate of thrombin formation on oxLDL-treated cells in the
presence of inhibitors is expressed as percent of control. (B) fVIII binding in the
presence of inhibitors. Binding of 125I-fVIII to oxLDL-treated cells in the absence (solid
bars) or presence (open bars), of BHT desipramine (gray bars), or a general caspase
inhibitor Z-VAD-FMK (hatched bars) was performed as described in Figure 2. Each
bar in panels A and B represents the mean ⫾ SD of 3 determinations.
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4484
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ANANYEVA et al
reports state that apoptotic cells become procoagulant.35,63,64 Because oxLDL can induce apoptosis in macrophages,32,33 SMCs,26,28
and ECs,30,31 we explored a link between the increased procoagulant activity of oxLDL-treated cells and apoptosis. We found that
processes occurring at early apoptotic stages, including free
radical–induced changes in the cell membrane and generation of
ceramide, are related to activation of the intrinsic pathway. This
was demonstrated by the ability of BHT (a scavenger of free
radicals), desipramine (an inhibitor of acid sphyngomyelinase
responsible for ceramide generation), and a general caspase
inhibitor to decrease the rates of thrombin generation and fVIII
binding to oxLDL-treated macrophages and SMCs. We did not
detect, however, DNA fragmentation in oxLDL-treated cells,
typical for the terminal apoptotic stage. We cannot exclude,
therefore, that activation of the intrinsic pathway on macrophages
and SMCs may be related to nonapoptotic translocation of PS
induced by components of the oxLDL particle. This possibility is
suggested by a recent report that oxidative derivatives of phosphatidylethanolamine, the active component of oxLDL, are responsible
for augmentation of the platelet prothrombinase activity by inducing exposure of PS on their surface.65
Our results suggest that the intrinsic coagulation pathway, in
addition to the TF-dependent pathway, accounts for the dramatic
increase in thrombogenicity of atherosclerotic lesion in the course
of its progression. The observed modest 2-fold increase in thrombin formation by oxLDL-treated HAECs in the intrinsic pathway
implies that thrombogenicity of lesions with the preserved endothelial layer is mainly determined by oxLDL-induced TF expression in
endothelial cells leading to a 4- to 7-fold increase in the extrinsic
procoagulant activity.59 This moderate increase in the overall
procoagulant activity of dysfunctional endothelium is consistent
with its role as the protective barrier preventing plaque components
from direct contact with blood. In light of our findings, a dramatic
increase in plaque thrombogenicity with the loss of the endothelial
layer integrity is determined by more than an order of magnitude
increase in the procoagulant activity of SMCs and macrophages
both in the intrinsic and extrinsic pathways. At the final stage,
plaque rupture results in massive exposure to blood flow of plaque
material depleted in SMCs and enriched in lipid-laden macrophagederived foam cells. The highest oxLDL-induced increase in the
procoagulant activity of this cell type in both the extrinsic pathway
(24- to 30-fold56,57) and intrinsic pathway (20-fold in our experiments) may explain the burst of thrombogenicity of vulnerable
atheromatous plaques, which is an acknowledged cause of sudden
cardiac death.
In conclusion, we demonstrated that in vitro treatment of human
macrophages and SMC with oxLDL dramatically increases their
ability to support thrombin formation via the intrinsic pathway,
which is likely to be an additional mechanism determining
thrombogenicity of the atherosclerotic plaque.
Acknowledgments
We are indebted to Dr Christian Haudenschild (Department of
Pathology, Holland Laboratory, American Red Cross, Rockville,
MD) for providing human atherectomy specimens. We also express
our gratitude to Dr Edward Tuddenham (MRC Clinical Sciences
Center, Imperial College School of Medicine, London, United
Kingdom), Drs Andrey Sarafanov, Larisa Cervenakova, Alexey
Khrenov and Alexey Belkin (Holland Laboratory) for their critical
review of the manuscript and helpful discussions.
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2002 99: 4475-4485
doi:10.1182/blood-2001-11-0140 originally published online
April 17, 2002
Intrinsic pathway of blood coagulation contributes to thrombogenicity of
atherosclerotic plaque
Natalya M. Ananyeva, Diana V. Kouiavskaia, Midori Shima and Evgueni L. Saenko
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