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Environment
Original article
Components of ambient air pollution affect thrombin generation in healthy humans: the RAPTES project
  1. Maciej Strak1,2,
  2. Gerard Hoek2,
  3. Maaike Steenhof2,
  4. Evren Kilinc3,
  5. Krystal J Godri4,5,
  6. Ilse Gosens1,
  7. Ian S Mudway4,
  8. René van Oerle3,
  9. Henri M H Spronk3,
  10. Flemming R Cassee1,
  11. Frank J Kelly4,
  12. Roy M Harrison5,6,
  13. Bert Brunekreef2,7,
  14. Erik Lebret1,2,
  15. Nicole A H Janssen1
  1. 1Centre for Environmental Health (MGO), National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands
  2. 2Division of Environmental Epidemiology and Division of Toxicology, Institute for Risk Assessment Sciences (IRAS), Utrecht University, Utrecht, The Netherlands
  3. 3Laboratory for Clinical Thrombosis and Haemostasis, Department of Internal Medicine, Cardiovascular Research Institute Maastricht, Maastricht University Medical Center, Maastricht, The Netherlands
  4. 4Environmental Research Group, MRC-HPA Centre for Environmental Health, King's College London, London, UK
  5. 5Division of Environmental Health and Risk Management, School of Geography, Earth & Environmental Sciences, University of Birmingham, Edgbaston, Birmingham, UK
  6. 6Department of Environmental Sciences, Center of Excellence in Environmental Studies, King Abdulaziz University, Jeddah, Saudi Arabia
  7. 7Julius Centre for Health Sciences and Primary Care, University Medical Centre Utrecht, Utrecht, The Netherlands
  1. Correspondence to Dr Nicole A H Janssen, Centre for Environmental Health (MGO), National Institute for Public Health and the Environment (RIVM), P.O. Box 1, Bilthoven 3720BA, The Netherlands; nicole.janssen{at}rivm.nl

Abstract

Objectives Increases in ambient particulate matter (PM) have been associated with an elevated risk of stroke, myocardial ischaemia and coronary heart disease, with activation of blood coagulation likely playing an important role. PM-mediated activation of two major activation pathways of coagulation provides a potential mechanism for the observed association between PM and cardiovascular disease. However, it remains unclear which specific characteristics and components of air pollution are responsible.

Methods In order to investigate those characteristics and components, we semiexperimentally exposed healthy adult volunteers at five different locations with increased contrasts and reduced correlations among PM characteristics. Volunteers were exposed for 5 h, exercising intermittently, 3–7 times at different sites from March to October 2009. On site, we measured PM mass and number concentration, its oxidative potential (OP), content of elemental/organic carbon, trace metals, sulphate, nitrate and gaseous pollutants (ozone, nitrogen oxides). Before and 2 and 18 h after exposure we sampled blood from the participants and measured thrombin generation using the calibrated automated thrombogram.

Results We found that thrombin generation increases in the intrinsic (FXII-mediated) blood coagulation pathway in relation to ambient air pollution exposure. The associations with NO2, nitrate and sulphate were consistent and robust, insensitive to adjustment for other pollutants. The associations with tissue factor-mediated thrombogenicity were not very consistent.

Conclusions Ex vivo thrombin generation was associated with exposure to NO2, nitrate and sulphate, but not PM mass, PM OP or other measured air pollutants.

  • experimental exposure
  • thrombin generation
  • PM

https://doi.org/10.1136/oemed-2012-100992

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    What this paper adds

    • Ambient PM has been associated with an elevated risk of stroke, myocardial ischaemia or coronary heart disease.

    • ·  Air pollution is a complex mixture and its specific characteristics and components responsible for health effects remain unclear.

    • Air pollution can activate FXII-dependent blood coagulation pathway ex vivo.

    • Using a semiexperimental volunteer exposure design we identified independent contributions of particularly NO2, nitrate and sulphate.

    Introduction

    Epidemiological studies have shown associations of exposure to ambient air pollution with adverse cardiovascular effects1 ,2 and prothrombotic changes are considered to play an important role.1 ,3 ,4 Next to well-established links of particulate matter (PM) air pollution with arrhythmia, stroke and myocardial infarction, recent studies also demonstrated the association between PM exposure and increased risk of deep vein thrombosis.5 ,6 One of the possible mechanisms is PM-driven activation of the blood coagulation cascade leading to a hypercoagulable state:3 ,7 Baccarelli et al8 showed associations between exposure to air pollution (PM10, CO, NO2) and shorter prothrombin time. Rudez et al9 found that increased thrombin generation was associated with increased levels of CO, NO and NO2, which can represent the associations with those gases itself, but since those can be considered markers for traffic-related air pollution—also with other air pollutants with which these gases are highly correlated. Bonzini et al10 reported that exposure to PM was associated with decreased prothrombin time and increased thrombin generation. The latter was also associated with PM10 exposure at residence address in a study by Emmerechts et al.11

    Currently, two scenarios involving the blood coagulation are considered; in the first one, PM causes an inflammatory response in the lungs, resulting in IL-6 release, which in turn increases cellular expression of tissue factor (TF) that further activates factor (F) VII (extrinsic pathway), eventually leading to conversion of prothrombin into thrombin.2 ,3 ,12 ,13 Both epidemiological11 and toxicological studies14 ,15 showed that increase in TF was associated with elevated PM concentrations. In the second scenario, particles translocate across the lung–blood barrier and initiate the intrinsic pathway of blood coagulation through activation of factor (F) XII (and possibly FXI), leading to thrombin formation.3 ,12 ,16 A recent toxicological study demonstrated plausibility of this pathway through ultrafine particles-mediated activation of the FXII into FXIIa in mice.16 Thus, PM-mediated activation of the two major coagulation pathways could provide a potential mechanism for the observed associations between PM and cardiovascular disease.

    However, air pollution is a complex, heterogeneous mixture of PM and gaseous compounds, and its specific characteristics and components responsible for activation of blood coagulation remain unclear. The aim of the ‘Risk of Airborne Particles: a Toxicological-Epidemiological Hybrid Study’ (RAPTES project) was to assess the independent contribution of individual PM characteristics to various health outcomes. Using a short-term, well-defined exposure of healthy volunteers to ambient PM at real-world locations with previously established substantial differences in PM characteristics,17 we investigated the effects of air pollution components on PM toxicity in vitro,18 as well as on the acute respiratory19 and cardiovascular20 changes in human volunteers. The present study focuses on the effects of PM and gaseous pollutants on both extrinsic (TF-mediated) and intrinsic (FXII- and FXI-mediated) pathways of blood coagulation in healthy human subjects. We hypothesised that PM would increase the magnitude of ex vivo thrombin generation and shorten the time necessary to invoke it.

    Materials and methods

    Study design

    We used a semiexperimental design in which 31 healthy human volunteers were exposed to ambient air pollution at five locations with differences in air pollution characteristics. A detailed characterisation of air pollution was performed on-site during the exposure of volunteers. Pre-exposure and postexposure blood samples of volunteers were taken to assess thrombin generation. Online supplementary figure S1 in the online supplementary material presents a timeline of a typical sampling day. The five selected locations were: an underground train station, a continuous traffic and a stop-and-go traffic location, a farm and an urban background site. All were located in The Netherlands and we chose them based on different source characteristics, so that contrast in exposure could be increased and correlations between PM characteristics could be reduced.17 The locations were not further than 70 km away from the collection point located at the Utrecht University campus, where pre-exposure and a part of postexposure health measurements took place. To minimise exposure during transport of participants between the collection point and the sampling locations, we equipped a minibus with a custom-made high-efficiency cabin air filter. To estimate the traffic-related air pollution during transport, we measured the particle number concentration (PNC) in the minibus during each commute using a portable, real-time condensation particle counter (CPC 3007; TSI, St. Paul, Minnesota, USA). The mean PNC concentration during transport was 12 904 particles/cm3, with a SD of 3903 particles/cm3, which is low considering that transport occurred during rush hours at highways. It is also low compared with the on-site exposures which moreover lasted longer and involved exercise increasing the inhaled dose.

    Data were collected on 30 sampling days between March and October 2009. On each sampling day, a maximum of eight participants were present. Participants were exposed multiple times at five sampling locations, and each made at least three visits one of which was to the underground site. This was motivated by the results of a preceding screening phase, in which the underground location appeared to have substantially higher concentrations of almost every measured PM characteristic compared with the outdoor sites.17 To avoid potential carry-over effects from previous exposure sessions, there were at least 14 days between two consecutive exposures of an individual volunteer. Exposure of participants lasted 5 h and always started between 09:00 and 09:30. During each hour of the exposure, participants cycled for 20 min on a stationary bicycle to increase the inhaled dose of ambient air pollution. The dose was kept similar among the participants by instructing them to cycle at a heart rate corresponding to a minute ventilation rate of 20 l/min/body surface area (m2). This ventilation rate was determined before the start of the study for each participant using a previously described method.21 Blood samples were taken at the collection point at three different times: during the morning check-up before exposure (t=0 h), 2 h after the end of 5-h long exposure (t=9) and the following morning (t=25).

    Study population

    We recruited 31 healthy, young participants among students of Utrecht University. All of them lived on the campus so that the background air pollution concentrations were similar and exposure to traffic-related air pollution when travelling to the collection point was minimised.

    The potential participants filled in an online screening questionnaire. We did not include participants who were smoking or living in a household with a smoker, had a history of cardiovascular disease, diabetes mellitus, lifetime diagnosis of asthma or chronic obstructive pulmonary disease, or were pregnant. These exclusion criteria were also chosen based on other (respiratory, cardiovascular) health effects investigated in the RAPTES project. Each participant qualified for the study attended a check-up by a physician to obtain medical clearance for participation.

    The study was approved by the ethics committee at University Medical Center Utrecht. Written informed consent was provided by all participants.

    Air pollution measurements

    We described the air pollution measurements in detail previously.17 Summarising, on each sampling day at the location, while the participants were being exposed, we measured mass concentrations of PM10 and PM2.5. Those samples were later used to determine their absorbance using a smoke stain reflectometer. The mass concentration of the coarse PM fraction (PM2.5−10) was calculated as the differences between PM10 and PM2.5. Using an limulus amebocyte lysate (LAL) assay we measured endotoxin content of PM10 samples. We also measured PNC, a proxy of ultrafine particles, using a condensation particle counter. Additionally, PM2.5−10 and PM2.5 samples were collected using a high volume sampler. We measured in those samples concentrations of elemental carbon and organic carbon (OC), water-soluble and ‘total’ acid-extracted fractions of trace metals: iron (Fe), copper (Cu), nickel (Ni), vanadium (V) and inorganic components of PM (nitrate, sulphate). Oxidative potential (OP) of PM samples was determined in vitro as an extent of ascorbate (OPAA) and reduced glutathione (OPGSH) depletion.22 The sum of both metrics is presented as OPTOTAL. We included the measurements of OP as oxidative stress was suggested as a mechanism driving negative PM health effects23 and OP is a promising metric potentially integrating the effects of different PM characteristics.24 OP and metals levels were analysed and presented as aggregated across individual PM size fractions. We additionally measured the concentrations of gaseous pollutants: O3 and nitrogen oxides (NO2, NOX).

    Blood collection

    Blood was drawn at t=0, t=9 and t=25 by venipuncture in the antecubital vein using the Vacutainer system (Becton Dickinson, Plymouth, UK) containing sodium citrate (final concentration, 3.2%). Platelet-poor plasma was obtained by two centrifugation steps: first at 2000 g for 15 min and subsequently at 11 000 g for 5 min. Plasma aliquots were stored at −80°C until further analysis, which took place after the end of the sampling campaign.

    Thrombin generation analysis

    Thrombin generation was measured in the citrated platelet-poor plasma collected from the participants (ie, ex vivo) by means of the Calibrated Automated Thrombogram method (Thrombinoscope BV, Maastricht, The Netherlands), as described previously.25 In brief, for TF-mediated thrombin generation, 20 μl trigger reagent (6 pM TF, 24 μM phospholipids at 20 : 20 : 60 mol% PS:PE:PC) was added to 80 μl plasma sample. After 10 min incubation at 37°C, 20 μl FluCa (fluorogenic substrate, calcium chloride) was added to start recording the thrombin generation. FXII-mediated thrombin generation was assessed through addition of the trigger reagent containing only phospholipids (24 µM). To avoid influences of endogenous TF, active site inhibited FVIIa (ASIS) was added at a final concentration of 30 nM. A combination of ASIS (30 nM) and corn trypsin inhibitor (40 µg/ml; specific inhibitor of FXIIa) were added to plasma to assess the contribution of FXIa in thrombin generation. Thrombin generation curves were calculated with Thrombinoscope software (Thrombinoscope BV, Maastricht, The Netherlands) and three parameters were derived from the curves: endogenous thrombin potential (ETP; area under the curve), peak height (PH) and lag time (LT). LT is defined as the time to reach 1/6 of the PH, and ETP and PH reflect the potential of plasma to generate thrombin and their elevated state has been suggested to indicate hypercoagulability.9

    Statistical analysis

    We analysed the associations between air pollution concentrations and the difference in thrombin generation between postexposure (t=9, t=25) and pre-exposure (t=0). As independent variables, we used the 5-h average concentrations of air pollutants measured during the exposure of participants at the locations. To account for the influence of repeated observations per subject we used mixed linear regression models (with compound symmetry of the residuals). Differences between postexposure and pre-exposure in LT in the TF-dependent pathway were log-transformed as those were skewed. First, we specified single-pollutant models. Next, to disentangle the individual effects of different pollutants, we specified two-pollutant models with all possible combinations of measured pollutants. In the results and discussion sections we largely focus on the two-pollutant models. We did not interpret the models in which a Spearman's R between two pollutants was larger than 0.7 as we considered those to be highly correlated. Since we measured a large number of air pollutants over multiple time points, thus defining a large number of models, we focus rather on the consistency of significant associations than single isolated significant associations. As we measured a substantial difference in some exposure parameters between outdoor locations and the underground location we analysed the data separately for all locations and the outdoor locations. We adjusted for potential confounders, such as temperature and relative humidity measured at the location during sampling, the season in which the sampling day occurred (before or after the start date of the calendar summer), and the use of oral contraceptives, as those are known to increase hypercoagulability.26 We did not include in the analysis the observations from the participants who used oral contraceptives but did not take them in the 24 h preceding the baseline measurement on the sampling day. This involved 17 observations in the intrinsic pathway and 23 observations in the extrinsic pathway, broadly equally distributed over the five sites. We excluded these observations as they had a substantial impact on the measured thrombin generation parameters (if use stopped, in the intrinsic pathway ETP at t=0 was 12% higher, whereas at t=9 and t=25 it was 38% higher).

    We did not investigate the associations of ETP, PH and LT with air pollutants in the FXI-dependent thrombin generation since, depending on the time point, in 66%–72% of all those samples there was no thrombin generation.

    The impact of influential values on the regression results was assessed by comparing effect estimates with and without the 1% of observations with the highest Cook's Distance value.

    All data analyses were carried out using SAS 9.2 (SAS Institute, Cary, North Carolina, USA).

    Results

    We obtained a total of 170 observations (table 1). Each participant was exposed at least three and at most seven times.

    View this table:
    Table 1

    Population characteristics (N=170 observations)

    Air pollution measurements

    At the underground train station we measured high concentrations of PM mass and several PM components, especially concentrations of Fe, Cu and OPTOTAL (see online supplementary table S1); the highest PNC was measured at the continuous traffic site and the highest levels of endotoxin at the farm. The highest NO2 concentrations were measured at the stop-and-go traffic site.

    Online supplementary table S2 presents correlations between air pollution concentrations. For a more extensive discussion, see.17 ,19 Correlations between pollutants varied widely, and as described in the Materials and methods section, we interpret the results of the two-pollutant analyses in the light of these correlations.

    FXII-mediated thrombin generation (intrinsic pathway)

    After adjusting for other pollutants in the two-pollutant models, we saw consistent, significant, positive associations of NO2, nitrate and sulphate with ETP and PH 2 h after exposure (table 2, see online supplementary tables S3 and S7). The estimates of these associations remained consistent in the outdoor-only dataset, although not all of them remained significant (table 3, see online supplementary tables S4 and S8). Effect estimates for nitrate and sulphate were reduced after mutual adjustment. We observed no associations with LT at this time point (see online supplementary tables S11–S12). In the morning following the exposure, there were positive associations of NO2 and PNC with LT (tables 4 and 5, see online supplementary tables S13 and S14). Associations of NO2 and PNC became weaker after adjusting for each other. There were no consistent associations of air pollution with next morning ETP and PH, except for the negative associations with coarse-fraction OC (see online supplementary tables S5, S6, S9 and S10). Since ETP and PH were highly correlated (Spearman's r>0.85), we present all the tables with the associations for PH in the online supplementary material.

    View this table:
    Table 2

    Associations between exposure to air pollution and changes (t=9–t=0) in endogenous thrombin potential in FXII-mediated thrombin generation pathway (all sites)

    View this table:
    Table 3

    Associations between exposure to air pollution and changes (t=9–t=0) in endogenous thrombin potential in FXII-mediated thrombin generation pathway (outdoor sites)

    View this table:
    Table 4

    Associations between exposure to air pollution and changes (t=25–t=0) in lag time in FXII-mediated thrombin generation pathway (all sites)

    View this table:
    Table 5

    Associations between exposure to air pollution and changes (t=25–t=0) in lag time in FXII-mediated thrombin generation pathway (outdoor sites)

    TF-mediated thrombin generation (extrinsic pathway)

    In the two-pollutant models, when investigating the components that showed consistent and significant associations in single-pollutant models, there were virtually no positive associations with any measured indicators of thrombin generation, neither 2 h after exposure nor the following morning (see online supplementary tables S15–S24), except for the association between LT and water-soluble V at the latter time point (see online supplementary tables S25 and S26). We observed a number of negative associations, particularly with metals, but these associations were not observed at the same time points across the thrombin generation parameters. Additionally, those associations were either present in the full- or the outdoor-only datasets, but not across both. There was a consistent negative association between fine-fraction OC and LT in the morning following the exposure (see online supplementary tables S25 and S26).

    Discussion

    In this study, we observed that 5-h long exposure to NO2, nitrate and sulphate resulted in FXII-mediated thrombin generation. The associations with TF-mediated thrombogenicity were not very consistent.

    According to our knowledge, this is the first report studying so extensively the role of components of ambient air pollution in mediating the thrombin generation in healthy human subjects. Our study design, with multiple components measured multiple times at locations with different ambient air pollution characteristics, allowed us to use two pollutant-models to investigate independent effects of individual components of the air pollution mixture. The associations we found with NO2 and NOX were overall robust and did not change after adjustment for a suite of other pollutants, including many PM characteristics not usually available in these types of studies. Associations remained consistent when the underground observations with high PM concentrations were removed from the analysis. There is still an ongoing debate whether the effects of NO2 seen in the epidemiological studies are due to its direct effects, or rather the PM characteristics correlated with it.27 Our results suggest that for some aspects of thrombin generation, associations with nitrogen oxides represent a direct effect or one mediated by co-pollutants not included in this study. In a time-series study in Rotterdam, The Netherlands, Rudez and colleagues9 found a strong, significant association between exposure to NO2 and NO 24–48 h before and increased ETP and PH (2%–8% increase above baseline) and shortened LT (2%–3% decrease below baseline) in healthy adults. However, those associations were found in the TF-mediated blood coagulation pathway, while in our study the associations between thrombin generation and nitrogen oxides were only present in the FXII-mediated pathway (without TF).

    In the current study we also found consistent associations of the FXII pathway with nitrate and sulphate, which is in line with our previous investigation of the acute vascular inflammatory and coagulative parameters in the RAPTES project.20 In human and animal experiments, exposure to nitrates and sulphates at higher than ambient concentrations does not have demonstrable effects.28 ,29 Many epidemiological studies have reported associations of nitrate and sulphate with mortality and hospital admissions.30–33 In these studies, sulphates and nitrates may have served as a proxy for other causally related air pollutants. However, a recent Statement by the American Heart Association did not exclude the possibility that particle sulphate could play a direct role in cardiovascular events.1 In our study of ambient mixtures, we could not disentangle direct effects from other unmeasured components.

    Similarly to our study, Bonzini and colleagues10 observed no short-term associations between thrombin generation and PM1–10 or PM1. Rudez and colleagues9 also observed no association with PM10, which they suggested was possibly related to PM mass metric being a poor measure of biological activity of PM. In our study we included measurements of PM10 OP, but found no consistent associations with it either. It is possible, as we noted previously,19 ,20 that this is a limitation of the OP determination method applied in our study. This method examines the intrinsic PM potential to drive oxidation reactions in an acellular model, which reflects the redox-active transition metals and quinones content of PM. As PM upon interaction with airway cells could possibly elicit oxidative stress through other pathways, the method used would only account for a fraction of PM biological activity in vivo.

    We could not explain the fairly consistent negative associations with OC in the morning following the exposure: fine-fraction OC was associated with LT in the TF pathway, whereas coarse-fraction OC was associated with ETP and PH in the FXII-dependent pathway. The latter associations almost disappeared after adjusting for coarse PM in the outdoor-only dataset (too highly correlated to investigate in the full dataset). In the TF-dependent pathway, there were some not very consistent negative associations with few metal PM components, like water-soluble Cu.

    In the current study, we focused on the consistency of associations in the three investigated thrombin generation parameters (ETP, PH, LT) with the investigated air pollutants, rather than single (significant) associations. This way, our conclusions are less affected by the limitations of multiple comparisons and possible chance findings, but represent associations seen across the dataset. Our results may support future investigations of blood coagulation mechanism as a result of ambient air pollution exposure, and the independent components of the air pollution mixture responsible.

    In conclusion, ex vivo thrombin generation in the FXII-dependent blood coagulation pathway was associated with exposure to NO2, nitrate and sulphate, but not PM mass, PM OP or other measured air pollutants.

    Acknowledgments

    We are grateful to all study participants; John Boere, Paul Fokkens, Daan Leseman, Lise van den Burg, Veerle Huijgen, Maartje Kleintjes, Marja Meijerink and Jet Musters, for their excellent support in data collection; Eef van Otterloo, for his help with participant's recruitment; and Miriam Groothoff, for medical supervision.

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    Supplementary materials

    • Supplementary Data

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    Footnotes

    • Contributors MStr participated in the design of the study, carried out the fieldwork, performed statistical analysis and drafted the manuscript. GH participated in the design of the study, participated in the coordination of the study, assisted in statistical analyses and helped to draft the manuscript. MSte participated in the design of the study and carried out the fieldwork. EK performed the thrombin generation measurements and helped to draft the manuscript. KJG performed chemical and oxidative potential analyses in the study. IG and FRC participated in the design and coordination of the study. ISM participated in the design of the study, and assisted in the oxidative potential analyses in the study. RvO assisted in the thrombin generation measurements. HMHS supervised the thrombin generation measurements. FJK participated in the design of the study, and supervised the oxidative potential analyses in the study. RMH participated in the design of the study and supervised the chemical analyses in the study. BB and EL participated in the design of the study, participated in the coordination of the study and helped to draft the manuscript. NAHJ was the project leader of RAPTES, designed the study, assisted in statistical analyses and helped to draft the manuscript. All authors read and approved the manuscript.

    • Funding The RAPTES project was funded by the RIVM Strategic Research Program (S630002).

    • Competing interests None.

    • Patient consent Obtained.

    • Ethics approval University Medical Center Utrecht.

    • Provenance and peer review Not commissioned; externally peer reviewed.