Mingzhen Li, Demin Yu, Kevin Jon Williams and Ming-Lin LiuMonocytes/Macrophages

Tobacco Smoke Induces the Generation of Procoagulant Microvesicles From Human

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Tobacco Smoke Induces the Generation of ProcoagulantMicrovesicles From Human Monocytes/Macrophages

Mingzhen Li, Demin Yu, Kevin Jon Williams, Ming-Lin Liu

Objective—To investigate whether exposure of human monocytes/macrophages to tobacco smoke induces their release ofmembrane microvesicles (MVs) that carry tissue factor (TF) released from cells, whether smoke-induced MVs areprocoagulant, 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– derivedmacrophages to 3.75% tobacco smoke extract (TSE) significantly increased their total and TF-positive MVgeneration. More importantly, MVs released from TSE-treated human monocytes/macrophages exhibited 3 to 4times the procoagulant activity of control MVs, as assessed by TF-dependent generation of factor Xa. Exposureto TSE increased TF mRNA and protein expression and cell surface TF display by both THP-1 monocytes andprimary human monocyte– derived macrophages. In addition, TSE exposure caused activation of C-Jun-N-terminalkinase (JNK), p38, extracellular signal regulated kinase (ERK) mitogen-activated protein kinases (MAPK), andapoptosis (a major mechanism for MV generation). Treatment of THP-1 cells with inhibitors of ERK, MAP kinasekinase (MEK), Ras, or caspase 3, but not p38 or JNK, significantly blunted TSE-induced apoptosis and MVgeneration. Surprisingly, neither ERK nor caspase 3 inhibition altered the induction of cell surface TF display byTSE, indicating an effect solely on MV release. Inhibition of ERK or caspase 3 essentially abolished TSE-inducedgeneration of procoagulant MVs from THP-1 monocytes.

Conclusion—Tobacco smoke exposure of human monocytes/macrophages induces cell surface TF display, apoptosis, andERK- and caspase 3–dependent generation of biologically active procoagulant MVs. These processes may be novelcontributors to the pathological hypercoagulability of active and secondhand smokers. (Arterioscler Thromb Vasc Biol.2010;30:1818-1824.)

Key Words: apoptosis � coagulation � smoking � microvesicles � monocytes

As of 2002, an estimated 1.3 billion people in the worldactively smoked cigarettes, and even more individuals

are exposed to secondhand tobacco smoke.1,2 Tobacco smokesubstantially increases the risk of atherothrombotic disease incoronary, cerebral, and peripheral arteries1,3–8; and of venousthrombosis.9–11 Public smoking bans are associated withrapid and significant reductions in atherothrombotic cardio-vascular events,3,12 and this beneficial effect promptly disap-peared in 1 municipality when it lifted its ban.13 Moreover,much of the decline in cardiovascular events in America andEurope during the past 2 to 3 decades has been attributed tomanagement of the major conventional cardiovascular riskfactors, including reductions in cigarette smoking.12,14,15

Despite its wide importance, the underlying pathologicalmechanisms for cardiovascular harm from tobacco smokeremain poorly understood.3 Herein, we focused on mi-crovesicles (MVs), also called microparticles, which are

released from the plasma membrane during cell activation orapoptosis and have played an important role in thrombusformation.16–20 MVs transport tissue factor (TF), a transmem-brane molecule that initiates coagulation in vivo.16–20 Severalstudies have shown that cigarette smoking increases TFexpression on peripheral monocytes,21 by cultured mousealveolar macrophages,22 and in atherosclerotic lesions.23

Smokers have higher plasma concentrations of TF than dononsmokers, and smoking just 2 cigarettes in a row increasestheir TF levels even further.24 Nevertheless, we are aware ofno prior reports characterizing cellular mechanisms for in-creased plasma TF in smokers. In the current study, wesought to determine whether exposure of human monocytes/macrophages to tobacco smoke induces the release of MVs,whether these smoke-induced MVs are procoagulant, andwhat cellular processes might be responsible for theirproduction.

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 DOI: 10.1161/ATVBAHA.110.209577

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MethodsThe human THP-1 monocytic cell line (ATCC, Manassas, Va) wasmaintained in RPMI medium 1640 with 10% FBS. Primary humanmonocyte–derived macrophages (hMDMs) were prepared fromfresh buffy coats by selecting monocytes by adherence followed bydifferentiation into macrophages.20 Tobacco smoke extract (TSE)was prepared as previously described.25,26 At the beginning of eachexperiment, THP-1 monocytes or hMDMs were transferred toserum-free RPMI medium 1640 plus BSA supplemented withdifferent concentrations of TSE, ranging from 0% (control) to 3.75%(vol/vol), and then incubated at 37°C for 2 to 20 hours (0 hoursdenotes harvest immediately before adding TSE). In time-coursestudies of kinase activation, cells were placed into serum-freemedium simultaneously; TSE was added at different times; and allcells were harvested simultaneously. In experiments using kinase orcaspase 3 inhibitors, the compounds were added to cells 1 hourbefore the addition of TSE and remained until the end of theexperiment, 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 3inhibitor Z-DEVD-FMK, 100 �mol/L. Flow cytometric character-ization of MVs and cells was performed according to previouslypublished protocols.20 Additional experimental details are pro-vided in the supplemental materials (available online athttp://atvb.ahajournals.org).

ResultsWe found that exposure of human THP-1 monocytes to TSEsignificantly increased total MV release, in a dose- (Figure1A) and a time-dependent manner (supplemental Figure I).Smoke-induced MV release was confirmed by 2 independentassay methods (Figure 1A and C). Likewise, TSE signifi-cantly stimulated total MV generation from primary hMDMs(Figure 1B and D). In addition, the numbers of TF-positiveMVs released from TSE-treated human THP-1 cells (Figure1E) and hMDMs (Figure 1F) were significantly higher thanthose from control cells incubated without TSE. More impor-tantly, we found that TSE treatment of THP-1 cells (Figure1G) or hMDMs (Figure 1H) for 20 hours tripled the proco-agulant activity of their MVs.

Because MVs are released from the plasma membrane, weexamined whether tobacco smoke affects expression and cellsurface display of TF on human THP-1 monocytes andhMDMs. We found that exposure to 3.75% TSE for 2 hoursincreased TF mRNA levels in both THP-1 cells (Figure 2A)and primary hMDMs (Figure 2B), measured by real-timequantitative PCR. Exposure of THP-1 cells and hMDMs to

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Figure 1. TSE increases the generationof total, TF-positive, and procoagulantMVs from human monocytes and pri-mary human monocyte–derived macro-phages. A through D, Exposure ofhuman THP-1 monocytes (A and C) orprimary hMDMs (B and D) to TSE for 20hours significantly increased total MVgeneration. A, Dose-dependentresponses are shown. B, Responses to0% (control) or 3.75% TSE are shown. Cand D, Exposure to 3.75% TSE inducesthe generation of MVs, as measured byELISA. E and F, Concentrations ofTF-positive MVs released from controland TSE-treated THP-1 cells (E) orhMDMs (F). G and H, The procoagulantactivities of MVs isolated from culturesupernatants of THP-1 monocytes (G)and hMDMs (H) after 20 hours of expo-sure to control medium or medium sup-plemented with 3.75% TSE, as indi-cated. Data are given as the mean�SEM(n�4 to 6). In A, P�0.001 by ANOVA;significance levels for individual pairwisecomparisons by the Student-Newman-Keuls (SNK) test are displayed. NS indi-cates not significant. In B through H, thedisplayed probability values were com-puted using the t test.

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TSE for 6 hours substantially increased their content of TFprotein (Figure 2C and D) and stimulated cell surface TFdisplay (Figure 2E and F). In a time course, display of TF onthe surface of THP-1 cells peaked at 6 hours after the additionof TSE and was still elevated at 20 hours, compared withbaseline (supplemental Figure II). The decline in cell-surfaceTF from 6 to 20 hours may reflect, in part, the release ofcellular TF into the medium on MVs.

Tobacco smoke has been reported to activate a number ofintracellular signaling pathways,27–33 but their roles in smoke-induced MV generation are unknown. Consistent with priorliterature in other cell types,27,34,35 we found that exposure ofTHP-1 monocytes to TSE increased the phosphorylation of 3major mitogen-activated protein (MAP) kinases (ie, c-Jun-N-terminal kinase [JNK], p38, and extracellular signal regulated

kinase [ERK]) (Figure 3). The time courses differed con-siderably. Phosphorylated JNK was increased at 0.5 hoursbut returned to nearly its baseline level after 1 to 6 hoursof TSE exposure (Figure 3A). In contrast, phosphorylatedp38 and phosphorylated ERK were partially induced at 0.5hours and peaked at 2 to 4 hours; the increase lastedthroughout the entire 6-hour period of TSE exposure(Figure 3B and C). Then, we sought to determine which, ifany, of these activated MAP kinases might be involved inTSE-induced generation of biologically active MVs. In-hibitors of ERK or its upstream molecules (ie, MEK andRas) significantly decreased total MV generation fromTSE-exposed THP-1 cells, in some cases to levels statis-tically indistinguishable from control, as measured by flowcytometry (supplemental Figure III) or ELISA (Figure

Figure 2. Exposure of human mono-cytes/macrophages to TSE induces TFmRNA, protein, and cell surface display.A and B, Levels of TF mRNA, measuredby quantitative RT-PCR in THP-1 cells(A) and hMDMs (B) after a 2-hour expo-sure to control medium or 3.75% TSE.C and D, Quantifications and representa-tive immunoblots of total TF content ofTHP-1 cells (C) and hMDMs (D) after a6-hour exposure to control medium or3.75% TSE, as indicated. E and F, Flowcytometric histograms of cell surface TFdisplay by THP-1 cells (E) and hMDMs(F) after a 6-hour incubation in controlmedia (dashed lines) or 3.75% TSE(heavy black lines). Cells that weretreated with 3.75% TSE for 6 hours butstained with an isotype control antibodyare shown as thin black lines (E and F).The experiments in each of the panelswere repeated at least 3 times, and thecolumn graphs display mean�SEM(A, C, and D) or medians in box-and-whisker plots (B). In A, C, and D, the dis-played probability values were computedusing the t test. The Mann-Whitney rank-sum test was used for B. FITC indicatesfluorescein isothiocyanate.

Figure 3. Exposure of THP-1 monocytes to TSE activates MAP kinases. Displayed are quantifications and representative immunoblotsof 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 graphsindicate 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.

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4A). However, inhibitors of JNK or p38 did not affectTSE-induced MV generation, and none of the previouslymentioned inhibitors significantly affected MV generationin the absence of TSE (Figure 4A [ERK inhibitor] and datanot shown [remainder of the inhibitors]). Surprisingly,treatment of THP-1 cells with the ERK inhibitor did notsignificantly affect TSE-induced display of TF on the cellsurface (supplemental Figure IV); however, ERK inhibi-tion decreased TSE-induced generation of TF-positiveMVs to a level indistinguishable from control, implying aneffect solely on MV release (Figure 4B). Most important,ERK inhibition essentially blocked the ability of TSE tostimulate the release of procoagulant MVs from THP-1monocytes (Figure 4C and D). Thus, TSE activates theERK MAP kinase pathway, and this activation is crucialfor the generation of procoagulant MVs.

Next, we examined the role of programmed cell death inthe production of procoagulant MVs after TSE exposure.Consistent with prior literature36–39 in other cell types, TSEexposure of THP-1 monocytes caused cell surface exposureof phosphatidylserine (supplemental Figure VA and B) anddose-dependent DNA fragmentation detected by TUNELstaining (Figure 5A and B). Likewise, exposure of humanprimary hMDMs to 3.75% TSE also substantially increasedapoptosis, as determined by TUNEL staining (supplementalFigure VC and D). The inhibition of ERK, MEK, or Ras,but not JNK or p38, partially decreased TSE-inducedapoptosis, as assessed by TUNEL staining (Figure 5C andD); none of these inhibitors affected TUNEL staining ofTHP-1 cells in the absence of TSE (data not shown).Finally, the caspase 3 inhibitor, Z-DEVD-FMK, substan-tially 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

(Figure 6D). Z-DEVD-FMK, like the ERK inhibitor, didnot affect TSE-induced TF display on the cell surface (datanot shown) but decreased TSE-induced procoagulant ac-tivity of the purified MVs released from THP-1 cellsessentially to baseline (Figure 6E).

DiscussionOur results demonstrate that exposure of human monocytesand primary human macrophages to tobacco smoke provokesthe generation of highly procoagulant membrane MVs, in aprocess requiring ERK activation and apoptosis. These cel-lular mechanisms may contribute to the link, in clinical andpopulation studies, between tobacco smoke exposure (bothactive and secondhand) and a high risk of thromboticevents.3,21,23,24,40–42 Our results may also help explain whyshort-term cigarette smoking increases plasma TF concentra-tions in current smokers and why active smokers have higherlevels of circulating TF than nonsmokers.24 Consistent withthis model, exposure of healthy nonsmokers to secondhandsmoke was recently reported to increase circulating concen-trations of MVs, although their potential prothrombotic ef-fects were not evaluated.43 Additional studies36–39 reportedthat exposure of experimental animals to tobacco smokeinduces apoptosis of several different cell types, which weinfer should increase their production of MVs. Thus, ourfindings are likely to be relevant in vivo.

Regarding intracellular signaling pathways, we found thatTSE exposure caused transient activation of JNK and sus-tained activation of p38 and ERK (Figure 3), but only ERKappeared to be involved in TSE-induced apoptosis and MVgeneration. ERK MAP kinases are known for their involve-ment in cell survival, but recent evidence suggests that ERKactivation can also contribute to cell death under certain

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Figure 4. Inhibition of ERK blocks TSE-induced generation of total, TF-positive,and procoagulant MVs. A, Treatment ofTHP-1 monocytes with the ERK inhibitorU0126 (ERKi) substantially inhibited theability of 3.75% TSE to induce the gen-eration of total MVs, as measured byELISA. B through D, The ERK inhibitorblocked TSE-induced generation ofTF-positive MVs, assessed by flowcytometry (B), and essentially abolishedthe TSE-induced increase in procoagu-lant activity (PCA) of purified MVs (C andD). The PCA results in C are from 3 to 5independent experiments. D, Represen-tative kinetic curves of TF-dependentfactor Xa generation by isolated MVs invitro, indicating that our measurementswere taken without saturation of theassay. P�0.01 by ANOVA for A throughC, and individual pairwise comparisonsby SNK are displayed.

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conditions.44,45 For example, the Ras/MEK/ERK pathwaywas reported to play a key role in apoptosis induced by UV-Airradiation46 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. Inother circumstances, TF expression is either regulated47,48 ornot regulated49 through ERK activation. Then, our TSE-exposed cells underwent apoptosis and extensive apoptotic

Figure 5. Tobacco smoke exposureinduces apoptosis and MV generation inpart through the Ras/MEK/ERK pathway. A,representative histograms from flow cytom-etry of TUNEL staining of THP-1 monocytesafter exposure to control medium (0% TSE,filled gray curve) or medium supplementedwith 1.25% (dotted line), 2.5% (thin blackline), or 3.75% (heavy black line) TSE for 20hours. FITC indicates fluorescein isothiocya-nate. B, Quantification of TUNEL-positiveTHP-1 cells by flow cytometry after expo-sure to the indicated concentrations of TSE.C, Representative histograms of TUNELstaining of THP-1 monocytes after exposureto control medium (0% TSE, filled graycurve) or medium supplemented with 3.75%TSE without (thin black line) or with (heavyblack line) indicated inhibitors for 20 hours;the x-axis scale is logarithmic. The horizontallines indicate intensities considered to beTUNEL positive for quantification in D. B andD, Quantifications of TUNEL-positive cellsfrom 3 to 4 independent experiments.P�0.001 by ANOVA for B and D. Individualpairwise comparisons by SNK are displayed.

Figure 6. Apoptosis is required for TSE-induced MV generation by human THP-1 monocytes. A, Representative histograms of TUNELstaining of THP-1 cells after exposure to 0% TSE (control, filled gray curve) or 3.75% TSE without (heavy black line) or with (dashedline) caspase-3 inhibition by Z-DEVD-FMK (caspase3i). As an additional control, the thin black line shows TUNEL-stained cells aftertreatment with caspase3i without TSE. The horizontal line (M1) indicates intensities considered to be TUNEL positive for quantificationin B. FITC indicates fluorescein isothiocyanate. B, Quantifications of TUNEL-positive THP-1 cells after exposure to the indicatedagents. C and D, Total MV generation from THP-1 cells, measured by flow cytometry (C) or ELISA (D), after exposure to controlmedium (Control), 3.75% TSE, and/or the Caspase3i, as indicated. E, The procoagulant activity of isolated MVs released by THP-1cells after exposure to control medium (Control), 3.75% TSE, and/or the Caspase3i, as indicated. In B through E, results from 3 to 6independent experiments are shown. P�0.01 by ANOVA for each of those panels; and the individual pairwise comparisons were per-formed by SNK. In all panels, TSE exposure lasted 20 hours and treatment with Caspase3i began 1 hour before the addition of TSEand remained until the end of the study.

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vesiculation, which depends on ERK and caspase 3 and leadsto the release of biologically active TF on highly procoagu-lant MVs (Figures 1, 4, and 6). In addition, the presence ofphosphatidylserine on the MV surface is a key identifyingcharacteristic during flow cytometry and ELISA analyses,and this unique surface phospholipid promotes the catalyticefficiency of complexes in the blood coagulation cas-cade,19,20,50 especially for apoptotic MVs.20,51–53

The relative importance of TSE-induced TF released onMVs or remaining on cells may depend on the context. Forexample, biologically active TF transported by monocyte-derived MVs can become targeted into developing thrombivia the interaction of P-selectin glycoprotein ligand-1(PSGL1) on the MV surface, with P-selectin displayed bynewly activated platelets.17 In contrast, the direct recruitmentof intact monocytes or macrophages into early experimentalclots appears to be minimal.54,55 In a pathophysiologic setting,atherosclerotic plaques contain MV-associated TF56 and cell-associated TF,23,57 both of which we presume would increasein smokers23,24 and thereby contribute to thrombus formationand acute vessel occlusion on plaque rupture.

Overall, our findings indicate that exposure of humanmonocytes/macrophages to tobacco smoke induces cell sur-face TF display, apoptosis, and ERK-, and caspase 3–depen-dent generation of biologically active procoagulant MVs.These processes may be novel contributors to pathologicalhypercoagulability caused by active and secondhand expo-sure to tobacco smoke.

AcknowledgmentsWe thank Elias G. Argyris, PhD, for generously providing theprimary hMDMs. This study was presented at the 2008 AmericanHeart Association Scientific Sessions (Circulation. 2008;118[suppl2]:S478).

Sources of FundingThis study was supported by grant HL73898 from the NationalInstitutes of Health (Dr Williams); an American Heart Association–Great Rivers Affiliate Beginning Grant-In-Aid (Dr Liu); and aTemple University Department of Medicine Career DevelopmentAward (Dr Liu).

DisclosuresNone.

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1

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

2

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 signal-regulated 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

3

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 PS-positive MVs that were also TF-positive was quantified by simultaneous staining with a FITC-labeled 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; APO-DIRECT 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 real-time 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,

4

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 peroxidase-conjugated 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 Student-Newman-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.

5

SUPPLEMENTAL FIGURES 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.

0 2 4 6 8 10 12 14 16 18 200

2000

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*

‡

**

3.75% TSE Control

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6

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ERKi+TSE

MEKi+TSE

Rasi+TSE0

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4000

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P<0.05P<0.05

P<0.01

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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.

7

C o n t r o l T S E05

1 01 52 02 5

DP < 0 .0 0 1

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(%)

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.

8

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serum elastase-inhibitory capacity by fresh cigarette smoke and its prevention by antioxidants. Am Rev Respir Dis. 1978;118:617-621.

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