Tobacco Smoke Induces the Generation of Procoagulant
Microvesicles From Human Monocytes/Macrophages
Mingzhen Li, Demin Yu, Kevin Jon Williams, Ming-Lin Liu
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Objective—To investigate whether exposure of human monocytes/macrophages to tobacco smoke induces their release of
membrane microvesicles (MVs) that carry tissue factor (TF) released from cells, whether smoke-induced MVs are
procoagulant, and what cellular processes might be responsible for their production.
Methods and Results—We found that exposure of human THP-1 monocytes and primary human monocyte– derived
macrophages to 3.75% tobacco smoke extract (TSE) significantly increased their total and TF-positive MV
generation. More importantly, MVs released from TSE-treated human monocytes/macrophages exhibited 3 to 4
times the procoagulant activity of control MVs, as assessed by TF-dependent generation of factor Xa. Exposure
to TSE increased TF mRNA and protein expression and cell surface TF display by both THP-1 monocytes and
primary human monocyte– derived macrophages. In addition, TSE exposure caused activation of C-Jun-N-terminal
kinase (JNK), p38, extracellular signal regulated kinase (ERK) mitogen-activated protein kinases (MAPK), and
apoptosis (a major mechanism for MV generation). Treatment of THP-1 cells with inhibitors of ERK, MAP kinase
kinase (MEK), Ras, or caspase 3, but not p38 or JNK, significantly blunted TSE-induced apoptosis and MV
generation. Surprisingly, neither ERK nor caspase 3 inhibition altered the induction of cell surface TF display by
TSE, indicating an effect solely on MV release. Inhibition of ERK or caspase 3 essentially abolished TSE-induced
generation of procoagulant MVs from THP-1 monocytes.
Conclusion—Tobacco smoke exposure of human monocytes/macrophages induces cell surface TF display, apoptosis, and
ERK- and caspase 3– dependent generation of biologically active procoagulant MVs. These processes may be novel
contributors to the pathological hypercoagulability of active and secondhand smokers. (Arterioscler Thromb Vasc Biol.
2010;30:1818-1824.)
Key Words: apoptosis 䡲 coagulation 䡲 smoking 䡲 microvesicles 䡲 monocytes
A
s of 2002, an estimated 1.3 billion people in the world
actively smoked cigarettes, and even more individuals
are exposed to secondhand tobacco smoke.1,2 Tobacco smoke
substantially increases the risk of atherothrombotic disease in
coronary, cerebral, and peripheral arteries1,3– 8; and of venous
thrombosis.9 –11 Public smoking bans are associated with
rapid and significant reductions in atherothrombotic cardiovascular events,3,12 and this beneficial effect promptly disappeared in 1 municipality when it lifted its ban.13 Moreover,
much of the decline in cardiovascular events in America and
Europe during the past 2 to 3 decades has been attributed to
management of the major conventional cardiovascular risk
factors, including reductions in cigarette smoking.12,14,15
Despite its wide importance, the underlying pathological
mechanisms for cardiovascular harm from tobacco smoke
remain poorly understood.3 Herein, we focused on microvesicles (MVs), also called microparticles, which are
released from the plasma membrane during cell activation or
apoptosis and have played an important role in thrombus
formation.16 –20 MVs transport tissue factor (TF), a transmembrane molecule that initiates coagulation in vivo.16 –20 Several
studies have shown that cigarette smoking increases TF
expression on peripheral monocytes,21 by cultured mouse
alveolar macrophages,22 and in atherosclerotic lesions.23
Smokers have higher plasma concentrations of TF than do
nonsmokers, and smoking just 2 cigarettes in a row increases
their TF levels even further.24 Nevertheless, we are aware of
no prior reports characterizing cellular mechanisms for increased plasma TF in smokers. In the current study, we
sought to determine whether exposure of human monocytes/
macrophages to tobacco smoke induces the release of MVs,
whether these smoke-induced MVs are procoagulant, and
what cellular processes might be responsible for their
production.
Received on: October 3, 2009; final version accepted on: June 7, 2010.
From the Section of Endocrinology, Diabetes and Metabolism, Department of Medicine (M.L., K.J.W., and M.-L.L.), Temple University School of
Medicine, Philadelphia, Pa; and The Metabolic Disease Hospital, Tianjin Medical University (D.Y.), Tianjin, China.
Correspondence to Ming-Lin Liu, MD, PhD, Section of Endocrinology, Diabetes and Metabolism, Temple University School of Medicine, 3500 N
Broad St, Room 480A, Philadelphia, PA 19140. E-mail Minglin.Liu@Temple.edu
© 2010 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
1818
DOI: 10.1161/ATVBAHA.110.209577
Li et al
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Figure 1. TSE increases the generation
of total, TF-positive, and procoagulant
MVs from human monocytes and primary human monocyte– derived macrophages. A through D, Exposure of
human THP-1 monocytes (A and C) or
primary hMDMs (B and D) to TSE for 20
hours significantly increased total MV
generation. A, Dose-dependent
responses are shown. B, Responses to
0% (control) or 3.75% TSE are shown. C
and D, Exposure to 3.75% TSE induces
the generation of MVs, as measured by
ELISA. E and F, Concentrations of
TF-positive MVs released from control
and TSE-treated THP-1 cells (E) or
hMDMs (F). G and H, The procoagulant
activities of MVs isolated from culture
supernatants of THP-1 monocytes (G)
and hMDMs (H) after 20 hours of exposure to control medium or medium supplemented with 3.75% TSE, as indicated. Data are given as the mean⫾SEM
(n⫽4 to 6). In A, P⬍0.001 by ANOVA;
significance levels for individual pairwise
comparisons by the Student-NewmanKeuls (SNK) test are displayed. NS indicates not significant. In B through H, the
displayed probability values were computed using the t test.
400
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Methods
Results
The human THP-1 monocytic cell line (ATCC, Manassas, Va) was
maintained in RPMI medium 1640 with 10% FBS. Primary human
monocyte– derived macrophages (hMDMs) were prepared from
fresh buffy coats by selecting monocytes by adherence followed by
differentiation into macrophages.20 Tobacco smoke extract (TSE)
was prepared as previously described.25,26 At the beginning of each
experiment, THP-1 monocytes or hMDMs were transferred to
serum-free RPMI medium 1640 plus BSA supplemented with
different concentrations of TSE, ranging from 0% (control) to 3.75%
(vol/vol), and then incubated at 37°C for 2 to 20 hours (0 hours
denotes harvest immediately before adding TSE). In time-course
studies of kinase activation, cells were placed into serum-free
medium simultaneously; TSE was added at different times; and all
cells were harvested simultaneously. In experiments using kinase or
caspase 3 inhibitors, the compounds were added to cells 1 hour
before the addition of TSE and remained until the end of the
experiment, at concentrations as follows: SP600125, 10 ␮mol/L;
SB202190, 10 ␮mol/L; U0126, 10 ␮mol/L; PD98059, 20 ␮mol/L;
farnesyl transferase inhibitor (FTI), 20 ␮mol/L; and caspase 3
inhibitor Z-DEVD-FMK, 100 ␮mol/L. Flow cytometric characterization of MVs and cells was performed according to previously
published protocols.20 Additional experimental details are provided in the supplemental materials (available online at
http://atvb.ahajournals.org).
We found that exposure of human THP-1 monocytes to TSE
significantly increased total MV release, in a dose- (Figure
1A) and a time-dependent manner (supplemental Figure I).
Smoke-induced MV release was confirmed by 2 independent
assay methods (Figure 1A and C). Likewise, TSE significantly stimulated total MV generation from primary hMDMs
(Figure 1B and D). In addition, the numbers of TF-positive
MVs released from TSE-treated human THP-1 cells (Figure
1E) and hMDMs (Figure 1F) were significantly higher than
those from control cells incubated without TSE. More importantly, we found that TSE treatment of THP-1 cells (Figure
1G) or hMDMs (Figure 1H) for 20 hours tripled the procoagulant activity of their MVs.
Because MVs are released from the plasma membrane, we
examined whether tobacco smoke affects expression and cell
surface display of TF on human THP-1 monocytes and
hMDMs. We found that exposure to 3.75% TSE for 2 hours
increased TF mRNA levels in both THP-1 cells (Figure 2A)
and primary hMDMs (Figure 2B), measured by real-time
quantitative PCR. Exposure of THP-1 cells and hMDMs to
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Figure 2. Exposure of human monocytes/macrophages to TSE induces TF
mRNA, protein, and cell surface display.
A and B, Levels of TF mRNA, measured
by quantitative RT-PCR in THP-1 cells
(A) and hMDMs (B) after a 2-hour exposure to control medium or 3.75% TSE.
C and D, Quantifications and representative immunoblots of total TF content of
THP-1 cells (C) and hMDMs (D) after a
6-hour exposure to control medium or
3.75% TSE, as indicated. E and F, Flow
cytometric histograms of cell surface TF
display by THP-1 cells (E) and hMDMs
(F) after a 6-hour incubation in control
media (dashed lines) or 3.75% TSE
(heavy black lines). Cells that were
treated with 3.75% TSE for 6 hours but
stained with an isotype control antibody
are shown as thin black lines (E and F).
The experiments in each of the panels
were repeated at least 3 times, and the
column graphs display mean⫾SEM
(A, C, and D) or medians in box-andwhisker plots (B). In A, C, and D, the displayed probability values were computed
using the t test. The Mann-Whitney ranksum test was used for B. FITC indicates
fluorescein isothiocyanate.
TSE for 6 hours substantially increased their content of TF
protein (Figure 2C and D) and stimulated cell surface TF
display (Figure 2E and F). In a time course, display of TF on
the surface of THP-1 cells peaked at 6 hours after the addition
of TSE and was still elevated at 20 hours, compared with
baseline (supplemental Figure II). The decline in cell-surface
TF from 6 to 20 hours may reflect, in part, the release of
cellular TF into the medium on MVs.
Tobacco smoke has been reported to activate a number of
intracellular signaling pathways,27–33 but their roles in smokeinduced MV generation are unknown. Consistent with prior
literature in other cell types,27,34,35 we found that exposure of
THP-1 monocytes to TSE increased the phosphorylation of 3
major mitogen-activated protein (MAP) kinases (ie, c-Jun-Nterminal kinase [JNK], p38, and extracellular signal regulated
kinase [ERK]) (Figure 3). The time courses differed considerably. Phosphorylated JNK was increased at 0.5 hours
but returned to nearly its baseline level after 1 to 6 hours
of TSE exposure (Figure 3A). In contrast, phosphorylated
p38 and phosphorylated ERK were partially induced at 0.5
hours and peaked at 2 to 4 hours; the increase lasted
throughout the entire 6-hour period of TSE exposure
(Figure 3B and C). Then, we sought to determine which, if
any, of these activated MAP kinases might be involved in
TSE-induced generation of biologically active MVs. Inhibitors of ERK or its upstream molecules (ie, MEK and
Ras) significantly decreased total MV generation from
TSE-exposed THP-1 cells, in some cases to levels statistically indistinguishable from control, as measured by flow
cytometry (supplemental Figure III) or ELISA (Figure
Figure 3. Exposure of THP-1 monocytes to TSE activates MAP kinases. Displayed are quantifications and representative immunoblots
of phosphorylated and total JNK (A), p38 (B), and ERK (C) after the indicated periods of exposure of human THP-1 cells to 3.75% TSE.
All cells were harvested simultaneously; TSE was added at the indicated times before harvest. Quantifications in the column graphs
indicate the ratios of band intensities of phosphorylated to total forms, averaged from 3 independent experiments. In each panel,
P⬍0.001 by ANOVA; *P⬍0.05 and **P⬍0.01 vs values at 0 hours by the SNK test.
Li et al
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Figure 4. Inhibition of ERK blocks TSEinduced generation of total, TF-positive,
and procoagulant MVs. A, Treatment of
THP-1 monocytes with the ERK inhibitor
U0126 (ERKi) substantially inhibited the
ability of 3.75% TSE to induce the generation of total MVs, as measured by
ELISA. B through D, The ERK inhibitor
blocked TSE-induced generation of
TF-positive MVs, assessed by flow
cytometry (B), and essentially abolished
the TSE-induced increase in procoagulant activity (PCA) of purified MVs (C and
D). The PCA results in C are from 3 to 5
independent experiments. D, Representative kinetic curves of TF-dependent
factor Xa generation by isolated MVs in
vitro, indicating that our measurements
were taken without saturation of the
assay. P⬍0.01 by ANOVA for A through
C, and individual pairwise comparisons
by SNK are displayed.
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F-positive Mic
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MV-ELIS
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Tobacco Smoke–Induced Microvesicle Release
10
20
4A). However, inhibitors of JNK or p38 did not affect
TSE-induced MV generation, and none of the previously
mentioned inhibitors significantly affected MV generation
in the absence of TSE (Figure 4A [ERK inhibitor] and data
not shown [remainder of the inhibitors]). Surprisingly,
treatment of THP-1 cells with the ERK inhibitor did not
significantly affect TSE-induced display of TF on the cell
surface (supplemental Figure IV); however, ERK inhibition decreased TSE-induced generation of TF-positive
MVs to a level indistinguishable from control, implying an
effect solely on MV release (Figure 4B). Most important,
ERK inhibition essentially blocked the ability of TSE to
stimulate the release of procoagulant MVs from THP-1
monocytes (Figure 4C and D). Thus, TSE activates the
ERK MAP kinase pathway, and this activation is crucial
for the generation of procoagulant MVs.
Next, we examined the role of programmed cell death in
the production of procoagulant MVs after TSE exposure.
Consistent with prior literature36 –39 in other cell types, TSE
exposure of THP-1 monocytes caused cell surface exposure
of phosphatidylserine (supplemental Figure VA and B) and
dose-dependent DNA fragmentation detected by TUNEL
staining (Figure 5A and B). Likewise, exposure of human
primary hMDMs to 3.75% TSE also substantially increased
apoptosis, as determined by TUNEL staining (supplemental
Figure VC and D). The inhibition of ERK, MEK, or Ras,
but not JNK or p38, partially decreased TSE-induced
apoptosis, as assessed by TUNEL staining (Figure 5C and
D); none of these inhibitors affected TUNEL staining of
THP-1 cells in the absence of TSE (data not shown).
Finally, the caspase 3 inhibitor, Z-DEVD-FMK, substantially inhibited TSE-induced apoptosis (Figure 6A and B)
and decreased TSE-induced generation of total MVs,
measured by flow cytometry (Figure 6C) and MV-ELISA
30
40
Time ((min))
50
60
(Figure 6D). Z-DEVD-FMK, like the ERK inhibitor, did
not affect TSE-induced TF display on the cell surface (data
not shown) but decreased TSE-induced procoagulant activity of the purified MVs released from THP-1 cells
essentially to baseline (Figure 6E).
Discussion
Our results demonstrate that exposure of human monocytes
and primary human macrophages to tobacco smoke provokes
the generation of highly procoagulant membrane MVs, in a
process requiring ERK activation and apoptosis. These cellular mechanisms may contribute to the link, in clinical and
population studies, between tobacco smoke exposure (both
active and secondhand) and a high risk of thrombotic
events.3,21,23,24,40 – 42 Our results may also help explain why
short-term cigarette smoking increases plasma TF concentrations in current smokers and why active smokers have higher
levels of circulating TF than nonsmokers.24 Consistent with
this model, exposure of healthy nonsmokers to secondhand
smoke was recently reported to increase circulating concentrations of MVs, although their potential prothrombotic effects were not evaluated.43 Additional studies36 –39 reported
that exposure of experimental animals to tobacco smoke
induces apoptosis of several different cell types, which we
infer should increase their production of MVs. Thus, our
findings are likely to be relevant in vivo.
Regarding intracellular signaling pathways, we found that
TSE exposure caused transient activation of JNK and sustained activation of p38 and ERK (Figure 3), but only ERK
appeared to be involved in TSE-induced apoptosis and MV
generation. ERK MAP kinases are known for their involvement in cell survival, but recent evidence suggests that ERK
activation can also contribute to cell death under certain
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Figure 5. Tobacco smoke exposure
induces apoptosis and MV generation in
part through the Ras/MEK/ERK pathway. A,
representative histograms from flow cytometry of TUNEL staining of THP-1 monocytes
after exposure to control medium (0% TSE,
filled gray curve) or medium supplemented
with 1.25% (dotted line), 2.5% (thin black
line), or 3.75% (heavy black line) TSE for 20
hours. FITC indicates fluorescein isothiocyanate. B, Quantification of TUNEL-positive
THP-1 cells by flow cytometry after exposure to the indicated concentrations of TSE.
C, Representative histograms of TUNEL
staining of THP-1 monocytes after exposure
to control medium (0% TSE, filled gray
curve) or medium supplemented with 3.75%
TSE without (thin black line) or with (heavy
black line) indicated inhibitors for 20 hours;
the x-axis scale is logarithmic. The horizontal
lines indicate intensities considered to be
TUNEL positive for quantification in D. B and
D, Quantifications of TUNEL-positive cells
from 3 to 4 independent experiments.
P⬍0.001 by ANOVA for B and D. Individual
pairwise comparisons by SNK are displayed.
conditions.44,45 For example, the Ras/MEK/ERK pathway
was reported to play a key role in apoptosis induced by UV-A
irradiation46 or a calcium ionophore.44 Based on our results,
TSE induces TF expression and cell surface display (Figure
2) through a process independent of ERK or caspase 3. In
other circumstances, TF expression is either regulated47,48 or
not regulated49 through ERK activation. Then, our TSEexposed cells underwent apoptosis and extensive apoptotic
Figure 6. Apoptosis is required for TSE-induced MV generation by human THP-1 monocytes. A, Representative histograms of TUNEL
staining of THP-1 cells after exposure to 0% TSE (control, filled gray curve) or 3.75% TSE without (heavy black line) or with (dashed
line) caspase-3 inhibition by Z-DEVD-FMK (caspase3i). As an additional control, the thin black line shows TUNEL-stained cells after
treatment with caspase3i without TSE. The horizontal line (M1) indicates intensities considered to be TUNEL positive for quantification
in B. FITC indicates fluorescein isothiocyanate. B, Quantifications of TUNEL-positive THP-1 cells after exposure to the indicated
agents. C and D, Total MV generation from THP-1 cells, measured by flow cytometry (C) or ELISA (D), after exposure to control
medium (Control), 3.75% TSE, and/or the Caspase3i, as indicated. E, The procoagulant activity of isolated MVs released by THP-1
cells after exposure to control medium (Control), 3.75% TSE, and/or the Caspase3i, as indicated. In B through E, results from 3 to 6
independent experiments are shown. P⬍0.01 by ANOVA for each of those panels; and the individual pairwise comparisons were performed by SNK. In all panels, TSE exposure lasted 20 hours and treatment with Caspase3i began 1 hour before the addition of TSE
and remained until the end of the study.
Li et al
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vesiculation, which depends on ERK and caspase 3 and leads
to the release of biologically active TF on highly procoagulant MVs (Figures 1, 4, and 6). In addition, the presence of
phosphatidylserine on the MV surface is a key identifying
characteristic during flow cytometry and ELISA analyses,
and this unique surface phospholipid promotes the catalytic
efficiency of complexes in the blood coagulation cascade,19,20,50 especially for apoptotic MVs.20,51–53
The relative importance of TSE-induced TF released on
MVs or remaining on cells may depend on the context. For
example, biologically active TF transported by monocytederived MVs can become targeted into developing thrombi
via the interaction of P-selectin glycoprotein ligand-1
(PSGL1) on the MV surface, with P-selectin displayed by
newly activated platelets.17 In contrast, the direct recruitment
of intact monocytes or macrophages into early experimental
clots appears to be minimal.54,55 In a pathophysiologic setting,
atherosclerotic plaques contain MV-associated TF56 and cellassociated TF,23,57 both of which we presume would increase
in smokers23,24 and thereby contribute to thrombus formation
and acute vessel occlusion on plaque rupture.
Overall, our findings indicate that exposure of human
monocytes/macrophages to tobacco smoke induces cell surface TF display, apoptosis, and ERK-, and caspase 3– dependent generation of biologically active procoagulant MVs.
These processes may be novel contributors to pathological
hypercoagulability caused by active and secondhand exposure to tobacco smoke.
Acknowledgments
We thank Elias G. Argyris, PhD, for generously providing the
primary hMDMs. This study was presented at the 2008 American
Heart Association Scientific Sessions (Circulation. 2008;118[suppl
2]:S478).
Sources of Funding
This study was supported by grant HL73898 from the National
Institutes of Health (Dr Williams); an American Heart Association–
Great Rivers Affiliate Beginning Grant-In-Aid (Dr Liu); and a
Temple University Department of Medicine Career Development
Award (Dr Liu).
Disclosures
None.
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Tobacco Smoke Induces the Generation of Procoagulant Microvesicles From Human
Monocytes/Macrophages
Mingzhen Li, Demin Yu, Kevin Jon Williams and Ming-Lin Liu
Arterioscler Thromb Vasc Biol. 2010;30:1818-1824; originally published online June 17, 2010;
doi: 10.1161/ATVBAHA.110.209577
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SUPPLEMENTARY MATERIALS
For
Tobacco Smoke Induces the Generation of Procoagulant Microvesicles from Human
Monocyte/Macrophages
Mingzhen Li, Demin Yu, Kevin Jon Williams, Ming-Lin Liu
From the Section of Endocrinology, Diabetes and Metabolism, Department of Medicine, Temple
University School of Medicine, Philadelphia, PA, USA (M.L., K.J.W., M.-L. L.); The Metabolic
Disease Hospital, Tianjin Medical University, Tianjin, China (D.Y).
Correspondence to:
Ming-Lin Liu, Section of Endocrinology, Diabetes & Metabolic Diseases, Temple University School
of Medicine, 3500 N. Broad Street, room 480A, Philadelphia, PA 19140, Telephone: 215-707-3387,
FAX: 215-707-3484, eMAIL: Minglin.Liu@Temple.edu
1
SUPPLEMENTAL METHODS
Reagents and antibodies
PhosphoPlus antibody kits against three major mitogen-activated protein kinases (MAPK), namely,
Jun N-terminal kinase (JNK, catalog #9250), p38 MAPK (cat#9210), and extracellular signalregulated kinase (ERK, cat#9100), were purchased from Cell Signaling Technology (Beverly, MA,
USA). Monoclonal antibodies (mAb) against human TF, with or without FITC labeling, were from
American Diagnostica (Stamford, CT). Phycoerythrin (PE)-labeled annexin-V was from BD
Pharmingen (San Jose, CA). Inhibitors of JNK (SP600125, Cat#5567), p38 (SB202190, Cat#7067),
ERK (U0126, Cat#U120), MAP kinase/ERK kinase (MEK, PD98059), Ras (farnesyl transferase
inhibitor, FTI) were obtained from Sigma Chemical Company (St. Louis, MO). The caspase 3
inhibitor, Z-DEVD-FMK, was from R&D Systems (Minneapolis, MN).
Preparation of tobacco smoke extract
Research-grade cigarettes were obtained from the Reference Cigarette Program at the University of
Kentucky. Tobacco smoke extract (TSE, 100%) was prepared by using a device to bubble
mainstream smoke from five cigarettes through 10 ml of serum-free RPMI medium containing 0.2%
BSA (RPMI/BSA), at one cigarette per 7-8min, simulating the burning rate of typical smoking.1-4
The TSE was adjusted to a pH of 7.4 and then sterilized by passage through a filter with a 0.22-µm
size cut-off. Our initial studies indicated that a single freeze-thaw cycle did not alter the biologic
effects of TSE on cultured cells, and so TSE was aliquoted and stored at -80°C. To ensure
consistency among preparations, each batch of TSE was standardized according to its absorbance at
320nm5, 6 by comparison with aliquots of a standard preparation that was made at the beginning of
the study.
Cell culture
The human THP-1 monocytic cell line (ATCC, Manassas, VA) was maintained in suspension culture
in RPMI-1640 supplemented with 10% FBS and 1% penicillin/streptomycin, as recommended by the
ATCC. Primary human monocyte-derived macrophages (hMDMs) were prepared from fresh Buffy
coats by selecting monocytes by adherence followed by differentiation into macrophages as
described.7-9 At the beginning of each experiment, THP-1 monocytic cells or hMDMs were
transferred to serum-free RPMI/BSA supplemented with different concentrations of TSE, ranging
from 0% (control) to 3.75% (v:v), and then incubated at 37ºC for 2-20 h (0h denotes harvest
immediately before adding TSE). In time-course studies of kinase activation, cells were placed into
serum-free medium simultaneously and then harvested simultaneously; TSE was added at different
times before harvest. In experiments using kinase or caspase 3 inhibitors, the compounds were added
to cells 1h before the addition of TSE and remained until the end of the study, at concentrations of
100 µM Z-DEVD-FMK, 10 µM SP600125, 10 µM SB202190, 10 µM U0126, 20 µM PD98059, and
20 µM FTI.
Flow cytometric characterization of microvesicles and cells, Total MV quantification by ELISA
Quantifications of MV generation, TF display by MVs and cells, and markers of early and late
apoptosis were performed according to our published protocols.9, 10 In brief, at the end of each
incubation, THP-1 cells, which grow in suspension, were fixed in their conditioned medium by the
addition of paraformaldehyde to a final concentration of 1%. Conditioned medium from hMDMs was
also fixed and then supplemented with latex beads as a reference for MV counts. The number of MVs
2
in each sample was quantified by flow cytometry (FACSCalibur, Becton Dickinson), using gating
criteria based on particle size, as detected by forward scatter, and surface exposure of PS, as detected
by staining with PE-labeled annexin-V (BD Pharmingen).9, 10 For THP-1 cell suspensions, results are
reported as MVs per 1000 cells. Because hMDMs are adherent, we added 25000 15-µm latex beads
(Molecular Probes) to each 100 µL of their conditioned medium as a reference, and our results are
reported as absolute numbers of MVs per ml of conditioned culture medium. The portion of these PSpositive MVs that were also TF-positive was quantified by simultaneous staining with a FITClabeled anti-human TF mAb (Cat# 4508CJ, American Diagnostica)9.
As a second, independent method, the Zymuphen MP-Activity ELISA kit (Anira, Mason, OH) was
used to quantify total PS-positive MVs in culture supernatants that we prepared by low-speed
centrifugation to remove cells. The MV ELISA assessment was performed according to the
manufacture’s instruction by using 20-fold dilutions of THP-1-conditioned media and 5-fold
dilutions of hMDM-conditioned media. Results are expressed as PS equivalents (nM PS eq).
Cell-surface display of TF was analyzed by flow cytometry using the same FITC-labeled anti-TF
mAb.9 Cell-surface exposure of PS, an early marker of apoptosis, was detected by staining of cells
with PE-labeled annexin-V following by flow cytometric analysis. Late-stage apoptosis was assessed
by terminal deoxynucleotidyl transferase FITC-dUTP nick end labeling of the cells (TUNEL; APODIRECT kit, BD Pharmingen), also quantified by flow cytometry.
Assessments of tissue factor mRNA and protein expression and tissue factor procoagulant activity
Total RNA from 2 x 106 cells was isolated with 1 ml Trizol® reagent (GiBCo BRL, Carlsbad, CA)
according to the manufacturer’s instructions. Tissue factor mRNA expression was assessed by realtime quantitative reverse-transcriptase PCR (qPCR). Primers and probes for qPCR were synthesized
by the Gene Expression Facility at the University of North Carolina (Dr. Hyung Suk Kim, Director;
Chapel Hill, NC), using the following sequences:
Human tissue factor mRNA:
5’- gat aaa gga gaa aac tac tgt ttc ag -3’ (sense primer)
5’- tac cgg gct gtc tgt act ct -3’ (antisense primer)
F-5’- tca agc agt gat tcc ctc ccg aac ag -3’-Q (probe, where F and Q denote the positions of the
fluorophore and quencher)
Human ß-actin mRNA:
5’- ggt cat cac cat tgg caa tg -3’ (sense primer)
5’- tag ttt cgt gga tgc cac ag -3’ (antisense primer)
F-5’- cag cct tcc ttc ctg ggc atg ga -3’-Q (probe)
Tissue factor procoagulant activity (PCA) was measured with fresh (unfixed, unfrozen) samples
using a chromogenic assay for the activation of clotting factor X (active factor Xa; Actichrome TF
activity kit, American Diagnostica). In brief, cell-culture supernatants were obtained by low-speed
centrifugation of conditioned medium from THP-1 cells or hMDMs after treatment without or with
TSE. MVs from 4ml of each supernatant were purified by high-speed centrifugation (100,000 x g at
4ºC for 1h), then washed, re-isolated, and re-suspended in 0.2ml of 50 mM Tris-HCl and 100 mM
NaCl. The PCA of these purified MVs was analyzed according to the manufacturer’s instructions,
3
except that to avoid saturation of the assay, we measured the absorbance at 405 nm of the reaction
solutions in a 96-well plate every minute for 60 min with a BioTek Powerwave XS
spectrophotometer. The linear portion of the curve was used to calculate the PCA.
Immunoblots
Cells were extracted into a lysis buffer containing 20 mM Tris/HCl, Ph 7.4, 150 mM NaCl, 1mM
EGTA, 1% Triton X-100, 1% sodium deoxycholate, 1 mM EDTA, 2.5mM sodium pyrophosphate,
1mM Na3VO4, and a commercial protease inhibitor cocktail tablet (Roche Diagnostics GmbH,
Germany) for 30 min on ice. Samples containing the same amount of total protein were
electrophoresed through a 10% SDS-polyacrylamide gel, followed by transfer to a nitrocellulose
membrane (Bio-Rad). The membranes were then blocked with skim milk (5%, w:v) and probed with
primary antibodies against human JNK, p38, ERK, and their phosphorylated (active) forms, or
against TF or ß-actin. Thereafter, detection was accomplished with horseradish peroxidaseconjugated secondary antibodies, to generate a chemiluminescent product (AmershamTM ECLTM
Western blot analysis system, GE Healthcare, USA). Signals were quantified using Image J
densitometry software and normalized in each independent experiment to values from control (0%
TSE) cells.
Statistical analyses
Normally distributed data are shown as means ± SEM (n = 4-6). Comparisons amongst three or more
groups were performed using one-way analysis of variance (ANOVA), followed by the StudentNewman-Keuls (SNK) test, with p<0.05 considered significant. Comparisons between two groups
used the Student’s unpaired, two-tailed t-test. Comparisons between two groups of non-normally
distributed data used the Mann-Whitney rank-sum test.
4
Total Microvesicles (per 1000 cells)
SUPPLEMENTAL FIGURES
8000
3.75% TSE
Control
6000
4000
*
*
‡
*
2000
0
0
2
4
6
8 10 12 14 16 18 20
Time (h)
Supplemental Figure I. Exposure of human THP-1 monocytes to TSE increases total MV
generation in a time-dependent manner. THP-1 cells were treated for 0 to 20h without (control) or
with 3.75% TSE, as indicated, and then samples were analyzed by flow cytometry. *p < 0.05, ‡p <
0.01 compared by Student’s t-test to control values.
Supplemental Figure II. The figure shows flow cytometric histograms of cell-surface TF display by
THP-1 cells after 0h (black curve), 6h (red curve) or 20h (orange curve) of incubation with 3.75%
TSE. THP-1 cells treated with 3.75% TSE for 6h but stained with an isotype control antibody are
shown by the blue curve.
5
Total Microvesicles (per 1000 cells)
NS
P<0.01
P<0.01
P<0.05
P<0.05
10000
8000
6000
4000
2000
0
ol
ntr
Co
E
SE
SE
SE
TS
i+T
i+T
i+T
s
K
K
a
R
ER
ME
Supplemental Figure III. Treatment of THP-1 monocytes with inhibitors of ERK (U0126, indicated
as ERKi) or the upstream molecules MEK (PD98059, indicated as MEKi) or Ras (FTI, indicated as
Rasi) essentially blocked the ability of TSE (3.75%) to induce the generation of total MVs, as
measured by flow cytometry. P< 0.001 by ANOVA; individual pairwise comparisons were
performed by SNK.
Supplemental Figure IV. No effect of ERK inhibition on TSE-induced display of TF on the surface
of THP-1 monocytes. The black curve shows a representative histogram from flow cytometry of TF
display on the surface of control THP-1 cells incubated for 6h without TSE. The blue curve shows
the surface TF staining of THP-1 cells after 6h treatment with ERK inhbitor U0126. The red curve
shows surface TF staining of THP-1 cells after a 6h treatment with 3.75% TSE. The green curve
shows surface TF staining of THP-1 cells treated with both the ERK inhbitor U0126 and 3.75% TSE.
6
TUNEL Positive Cells (%)
D
P < 0 .0 0 1
25
20
15
10
5
0
C o n tro l
TSE
Supplemental Figure V. TSE treatment induces cell-surface PS exposure and nuclear TUNEL
staining. Panel A (dose-response) shows representative histograms from flow cytometry of Annexin
V staining after 20-h incubations of THP-1 cells with 0 (filled grey), 1.25% (dash line), 2.5% (light
line), and 3.75% (heavy line) TSE. Panel B (time course) shows histograms of Annexin V staining
after 0h (filled grey), 6h (dash line), or 20h (heavy line) incubations of THP-1 cells with 3.75% TSE.
Panel C shows representative histograms of TUNEL staining of hMDMs after 20-h exposure to 0%
TSE (filled grey curve) or 3.75% TSE (black line). The horizontal line (M1) indicates intensities
considered to be TUNEL-positive for quantification in Panel D. Panel D indicates quantification of
TUNEL-positive hMDMs after exposure to 0% (Control) or 3.75% TSE (TSE). The P-value was
computed using Student’s t-test.
7
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