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Prepublished online as a Blood First Edition Paper on April 17, 2002; DOI 10.1182/blood-2001-11-0140.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Holland Laboratory, American Red Cross,
Rockville, MD and Nara Medical University, Kashihara, Nara,
Japan.
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 low-density 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 oxLDL-treated 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
formation. 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) Human atherosclerosis involves 2 distinct
pathologic processes 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 Membrane-bound
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 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
Antibodies
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 serum-free 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 µg/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 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 µM, 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 µM for all cell types; desipramine, 1 µM for SMCs and 10 µM for macrophages and HAECs; Z-VAD-FMK, 100 µM for SMCs and HAECs and 10 µM 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 µM). The kinetics of thrombin formation were measured as described above.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/µg, 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-VAD-FMK 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 µg/mL trypsin (Invitrogen), 50 µg/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 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 µg/mL, Vector Laboratories). The fVIII-stained sections
were subsequently stained for SMCs or macrophages using mouse
anti-
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 HAECsto
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 thrombin-activated 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 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.
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 with an increase in PS content in both synthetic and physiologic membranes,20,50 the observed elevated fVIII binding to oxLDL-treated 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.
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 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 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
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
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 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 oxLDL-treated 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 oxLDL-treated 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.
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 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 oxLDL-treated cells, is also one of indicators of apoptosis.32,62 Several 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 macrophage-derived 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.
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.
Submitted November 30, 2001; accepted February 7, 2002.
Prepublished online as Blood First Edition Paper, April 17, 2002; DOI 10.1182/blood-2001-11-0140.
Supported by National Institutes of Health-National Heart, Lung and Blood Institute RO1 grant HL66101 awarded to E.L.S.
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.
Reprints: Evgueni L. Saenko, Department of Biochemistry, Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855; e-mail: saenko{at}usa.redcross.org.
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Top
Abstract
Introduction
) or in the presence of 100 µg/ml oxLDL (
) or
native LDL (
) 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.
