Airborne Fine Particles Induce Hematological Effects through

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Ecotoxicology and Human Environmental Health

Airborne Fine Particles Induce Hematological Effects through Regulating the Crosstalk of the Kallikrein-Kinin, Complement and Coagulation Systems Xiaoting Jin, Qianchi Ma, Zhendong Sun, Xuezhi Yang, Qunfang Zhou, Guangbo Qu, Qian Liu, Chunyang Liao, Zhuoyu Li, and Guibin Jiang Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 11 Feb 2019 Downloaded from http://pubs.acs.org on February 11, 2019

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Environmental Science & Technology

1

Airborne Fine Particles Induce Hematological Effects through

2

Regulating the Crosstalk of the Kallikrein-Kinin, Complement and

3

Coagulation Systems

4 5

Xiaoting Jin,†, ‡ Qianchi Ma,†, § Zhendong Sun,†, § Xuezhi Yang,†, § Qunfang Zhou,*, †, §, ‡+

6

Guangbo Qu,†, § Qian Liu,†, § Chunyang Liao,† Zhuoyu Li,‡ and Guibin Jiang†, §

7 8

†

9

Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, P. R. China

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for

10

‡

Institutes of Biomedical Sciences, Shanxi University, Taiyuan 030006, P. R. China

11

§

College of Resources and Environment, University of Chinese Academy of Sciences, Beijing

12

100049, P. R. China

13

‡+

Institute of Environment and Health, Jianghan University, Wuhan 430056, P. R. China

14 15 16 17 18 19 20 21 22

*Correspondence to:

23

Dr. Qunfang Zhou, State Key Laboratory of Environmental Chemistry and Ecotoxicology,

24

Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085,

25

China.

1

ACS Paragon Plus Environment


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ABSTRACT

27

Particulate air pollution caused by human activities has drawn global attention due to

28

its potential health risks. Considering the inevitable contact of inhaled airborne fine

29

particulate matter (PM) with the plasma, the hematological effects of PM are worthy

30

of being studied. In this study, the potential effect of PM on hematological

31

homeostasis through triggering the crosstalk of the kallikrein-kinin system (KKS),

32

complement, and coagulation systems in plasma was investigated. The ex vivo, in

33

vitro and in vivo KKS activation assays confirmed that PM samples could efficiently

34

cause the cascade activation of key zymogens in the KKS, wherein, the particles

35

coupled with lipopolysaccharide (LPS) attachment provided substantial contribution.

36

The binding of Hageman factor XII (FXII) with PM samples and its subsequent

37

auto-activation initiated this process. The crucial elements in the complement cascade,

38

including complement 3 (C3) and complement 5 (C5), and coagulation system

39

(prothrombin) were also found to be actively induced by PM exposure, which was

40

regulated by the interplay of KKS activation. The data provided solid evidences on

41

hematological effects of airborne PM through inducing the activation of the KKS,

42

complement and coagulation systems, which would be valuable in the risk assessment

43

on

air

pollution-related

cardiovascular

2


diseases.

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Table of Contents

45

3



47

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INTRODUCTION

48

With the acceleration of industrialization and urbanization, dust-haze air

49

pollution outbreak frequently occurs in China, and is now becoming a global

50

concern.1 High concentration of airborne fine particulate matter (PM, aerodynamic

51

diameter less than 2.5 µm) has been extensively recognized as the main reason for the

52

formation and deterioration of haze weather. These fine particles are readily inhaled

53

by human body, deposit in the respiratory system, penetrate the air-blood barrier, and

54

reach the blood circulation system as well as the other body organs, thus posing a

55

serious threat to human health.2-4 Particularly, the accumulating epidemiological data

56

revealed the positive association between PM exposure and cardiovascular disease’s

57

morbidity and mortality.5-7

58

The currently available information on the pathophysiological events

59

precipitating PM-induced cardiovascular diseases generally involved inflammation,

60

oxidative stress, and imbalance of autonomic nervous system.8,9 However,

61

PM-mediated biological processes in blood circulation system are rarely known.

62

There are abundant zymogen systems in plasma, which can trigger a series of cascade

63

activation in response to endogenous or exogenous stimulations, thus regulating the

64

downstream physiological functions.10 The kallikrein-kinin system (KKS), also

65

known as the contact system, is one of the pivotal plasma protease systems, and it

66

undergoes the proteolytic activation cascade initiated by the auto-activation of

67

Hageman factor XII (FXII) on negatively charged surfaces, causing the conversion of

68

plasma prekallikrein (PPK) to plasma kallikrein (PK), and subsequent cleavage of

69

high-molecular-weight kininogen (HK) to liberate a biologically active peptide,

70

bradykinin.11 The role of the KKS is widely implicated in many physiological

71

processes,12,13

72

conditions,14-16 Despite of the limited data showing acute exposure to airborne PM

73

could induce the transcriptional expression of bradykinin-related genes in rat lung and

74

heart tissues,17 how they directly influence plasma KKS components remains unclear.

75

Given PM-induced deleterious effects on the cardiovascular system and the critical

76

pathophysiological effects of the KKS involved in cardiovascular diseases, the

and

also

extensively

correlated

4


with

diverse

pathological

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77

question whether airborne fine particles could cause the activation of the KKS or not

78

is worthy of being studied.

79

Considering the extensive crosstalk of plasma KKS with some other zymogen

80

cascades, such as the complement, and coagulation systems, their coexistence and

81

interplay in plasma maintain the hematological homeostasis, and ensure a successful

82

host defense in compromised barrier settings.18,19 Dysregulation of one or some

83

zymogens may trigger diverse cascade activation, resulting in clinical manifestations

84

of cardiovascular diseases involved with critical thrombotic and/or inflammatory

85

complications.19

86

thrombogenicity and inflammation are important pathophysiologic events in

87

PM-caused cardiovascular effects though,20,21 the direct association of fine particle

88

exposure with the multiple cascade systems, including the KKS, complement and

89

coagulation systems remain to be elucidated.

Accumulating

epidemiological

data

has

manifested

that

90

In the present study, two kinds of airborne fine particles collected by quartz fiber

91

filter (QFF) and polyprogylene filter (PPF) in Beijing were submitted to the screening

92

of their potential hematological effects, based on the evaluation of the cascade

93

activation of the KKS, complement and coagulation systems using the in vitro, ex vivo

94

and in vivo assays. The findings provided substantial evidences on the deleterious

95

effects of haze exposure on hematological homeostasis, which would be valuable in

96

PM-induced cardiovascular disease risk assessment.

97

MATERIALS AND METHODS

98

PM Sampling and Characterization. The details about airborne PM sampling

99

and preparation were provided in Supporting Information (SI). The morphology of the

100

collected fine particle samples was analyzed by scanning electron microscopy (SEM)

101

(Hitachi, S-3000N, Japan). Their hydrodynamic diameters and surface charges, when

102

suspended in Milli-Q water or plasma, were detected by a Zeta Sizer Nano ZS

103

(Malvern Nano ZS, Nalvem, UK). The component characterization was described in

104

SI. The exposure doses used in the following experiments were carefully designed

105

considering the environment relevance and previous toxicological studies involved 5



106

with haze exposure.22-24

107

Ex vivo KKS Activation Assay. The mouse plasmas (C57BL/6, Vital River

108

Laboratory Animal Technology Co. Ltd.) containing platelets were used for ex vivo

109

studies. The preparation protocol was as follows. 900 μL of intracardial blood was

110

collected from each mouse using the syringe prefilled with 100 μL of 3.6% sodium

111

citrate, immediately after the animal sacrifice by CO2 inhalation. After blood

112

collection, 20-min concentration (300 g, room temperature) was performed for all

113

samples, and the supernatant plasmas were transferred to new tubes for the

114

subsequent protease analysis. The freshly prepared mouse plasma was treated with a

115

series of concentrations of QFF-PM1, PPF-PM1, QFF-PM2.5 or PPF-PM2.5 (5, 10, 25,

116

50, 100, and 200 μg/mL) at 37 °C for 2 h. The blank membrane extracts from QFFs

117

and PPFs were used as the concentration points of 0 for the corresponding treatments,

118

which were adapted to the similar condition in the following experiments, unless

119

otherwise stated. The negative control (NC) was prepared by adding Milli-Q water in

120

plasma at the volume ratio of 1:9, and Kaolin treatment (1 mg/mL, Sinopharm, China)

121

was used as the positive control. After incubation, the plasma samples were

122

subsequently submitted to Western blot for characterizing the cascade activation of

123

the contact system, including the cleavages of PPK (75 kDa), FXII (80 kDa) and HK

124

(120 kDa), and PK formation (52 kDa), according to the protocol reported

125

previously.25,26 The primary antibodies used included anti-PPK (1:1000, R&D,

126

AF2498), anti-FXII (1:1000, Cedarlane laboratories, CL20055AP), and anti-HK

127

(1:500, Abcam, ab175386), and the corresponding second antibodies were from

128

ZSGB-BIO (1:500, Beijing, China). The development reagent was from Pierce ECL

129

(Thermo Scientific, USA). The specific target protein bands, whose responses were

130

confirmed by the treatment of positive control (Kaolin), were selectively presented for

131

all mouse Western blot results, considering the compact display of the related

132

immunoblot figures. The time-course experiment and plasma kallikrein-like activity

133

test were explained in SI.

134

The KKS activation effects of PM components, including particles and

135

endotoxin, were investigated by simulation experiments using the substituents of 6


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136

amorphous silica particles (SiNPs, nanoComposix, San Diego, USA, Figure S1) and

137

l../../../Youdao/Dict/7.5.2.0/resultui/dict/?keyword=ipopolysaccharide

138

Sigma-Aldrich, USA), respectively. Namely, the mouse plasma was incubated with

139

series of concentrations of SiNPs (10, 25 and 50 μg/mL), LPS (10, 25 and 50 μg/mL),

140

and the mixtures of SiNPs (10, 25 and 50 μg/mL) plus LPS (10 μg/mL) at 37 °C for 2

141

h. The concentrations of LPS were selected based on the minimal effective level in

142

inducing PPK activation to test its potential influence on particle-induced KKS

143

activation. The stimulated plasma was subsequently submitted to Western blot for

144

PPK activation as described above. The quantitative results in different treatments

145

were expressed in PK/PPK ratio according to their pixel densities of Western blot

146

results evaluated by Image J software (Fiji-win32).

(LPS,

147

In vitro KKS Activation Experiment. Human purified proteases of FXII,

148

activated FXII (FXIIa), PPK, PK, and HK (Enzyme Research Laboratories, Inc) were

149

used for in vitro KKS activation assay. In brief, 250 ng QFF-PM2.5 or PPF-PM2.5 was

150

incubated with 46.4 μg/mL FXII, a mixture of 46.4 μg/mL FXII and 80 μg/mL PPK,

151

or a mixture of 46.4 μg/mL FXII, 80 μg/mL PPK, and 42.4 μg/mL HK in 25 μL of 10

152

mM PBS containing 50 μM ZnSO4 (pH 7.4) for 2 h. Considering the substantial

153

knowledge on the cascade activation of the KKS,11 the treatments of zymogen

154

combinations with PM were neatly designed. The activation of FXII and PPK was

155

also characterized by the comparison with Western blot bands of FXIIa (46 μg/mL)

156

and PK (80 μg/mL), respectively. The stimulation in each group was stopped by the

157

addition of Laemmli buffer, and the samples were subsequently submitted to Western

158

blot. The primary antibodies for HK (1:2000) and PPK/PK (1:1000) were from R&D

159

(AF1569) and Abcam (ab1006), respectively, and the other antibodies were the same

160

as those used in ex vivo experiments for mouse plasma contact system activation

161

described above. The activation of human protease zymogens in the KKS was

162

characterized by the decreases of FXII (80 kDa), PPK (75 kDa) and HK (120 kDa),

163

and the increases of FXIIa (52 kDa), PK (52 kDa) and cleaved HK (56 kDa).

164 165

FXIIa

Activity

Assay.

The

chromogenic

peptide

substrate

(H-D-CHA-Gly-Arg-pNA2AcOH, Pefachrome, Pentapharm, Switzerland) was used 7



166

for the determination of FXIIa activity. Milli-Q water (NC), Kaolin (1 mg/mL,

167

positive control), QFF-PM2.5 (0, 100 μg/mL) or PPF-PM2.5 (0, 100 μg/mL) were

168

incubated with 46 μg/mL FXII purified protein (Enzyme Research Laboratories, Inc)

169

in 10 μL of 10 mM PBS containing 50 μM ZnSO4 for 1 h. After centrifugation (5000

170

g, 3 min), 5 μL of the supernatant sample was gently mixed with 5 μL of 2 mM

171

substrate, and the mixture was added in 90 μL of reaction buffer containing 50 mM

172

tris-imidazole and 150 mM NaCl. The optical density value at the wavelength of 405

173

nm was immediately recorded at the interval of 30 s for 30 min, using a microplate

174

reader (Thermo Fisher Scientific, USA). The activity of FXIIa in each treatment was

175

calculated relative to the negative control.

176

Native Gel Analysis for the Binding of FXII with PM2.5. A series of

177

concentrations of QFF-PM2.5 or PPF-PM2.5 (0, 50, 100, 200 μg/mL) were incubated

178

with 46 μg/mL FXII purified zymogen in 10 μL of PBS buffer system (50 μM ZnSO4)

179

at 37 ℃ for 2 h, respectively. The pure FXII without any treatments was used as the

180

NC. The prepared samples were subsequently submitted to the separation in 8%

181

native gel. The unbound FXII could perform normal electrophoresis on the gel, while

182

the complex of FXII-PM2.5 would be restrained. The protein bands from different

183

treatments were visualized by immunoblotting using the antibodies described above

184

or silver staining (Beyotime Institute of Biotechnology, China).

185

Spectroscopic Analysis for the Conformational Change of FXII. According

186

to the spectroscopic properties of FXII,25 its conformational change due to the binding

187

with PM2.5 could be evaluated. Briefly, 100 μg/mL QFF-PM2.5 or PPF-PM2.5 was

188

incubated with 46.4 μg/mL FXII in 100 μL of PBS buffer system containing 50 μM

189

ZnSO4, at 37 °C for 2 h. The corresponding blank filter extracts (QFF-Ctr and

190

PPF-Ctr) were tested for the comparative study. The UV absorbance at 280 nm was

191

determined for each sample using a spectrophotometer (DR5000, HACH, USA), and

192

the fluorescence spectra was monitored using a spectrofluorimeter (Horiba,

193

Fluoromax-4, Edison NJ, USA) with λex of 280 nm and λem in the range of 300-450

194

nm.

195

Ex vivo Assays for Complement System Activation. The stimulation of plasma 8


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196

using PM2.5 samples was performed using the similar protocol of ex vivo KKS

197

activation assay. The samples prepared were subsequently submitted to Western blot.

198

The

199

anti-complement 5 (anti-C5, ab11898) were from abcam (1:1000, Boston, USA), and

200

the corresponding second antibodies were from ZSGB-BIO (1:500, Beijing, China).

201

The pixel density was evaluated using ImageJ software (Fiji-win32) for the

202

quantitative analysis, and the result was presented by the value of each treatment

203

relative to the NC.

primary

antibodies

of

anti-complement

3

(anti-C3,

ab11887)

and

204

The levels of activated complement 3 (C3a) and activated complement 5 (C5a) in

205

PM2.5-treated plasma were measured using the quantitative ELISA kits according to

206

the manufacture's instruction (R&D, USA). Briefly, 100 μL of standards or the

207

plasma samples from different treatments were added into the antibody coated

208

96-well plate, and incubated at 37 °C for 40 min. The plate was subsequently

209

incubated with 100 μL of enzyme conjugate at 37 °C for 10 min after 5-time wash

210

with the buffer, and the reaction was terminated with the stop solution. Finally, the

211

absorbance at 450 nm in each well was immediately measured by a microplate reader

212

(VARIOSKAN FLASH, Thermo Scientific, USA), and the concentrations of C3a and

213

C5a in plasma samples were calculated according to the standard curves. The final

214

result was depicted by the value of each treatment relative to the NC.

215

Ex vivo Assay for Coagulation System Activation. The freshly collected

216

mouse plasma (total 100 μL) with or without 0.05 g/mL lipid bilayer (TECO Medical

217

Instruments, Germany) and 25 mM calcium ion, was incubated with 100 μg/mL

218

QFF-PM2.5 or PPF-PM2.5 at 37 °C for 2 min. The plasma with the addition of equal

219

volume of Milli-Q water was used as the NC. Kaolin treatment (1 mg/mL) was used

220

as the positive control. The reaction was terminated on ice, and the supernatant was

221

submitted to the analysis of activated thrombin level using a commercial kit

222

(BioVision, USA). This assay was based on thrombin activation-caused proteolytic

223

cleavage of a synthetic substrate and the subsequent release of a fluorophore, which

224

was measured by the fluorescence reader (VARIOSKAN FLASH, Thermo Scientific,

225

USA). Briefly, 20 μL of the treated sample was loaded in a 96-well white plate, and 9



226

mixed with 30 μL of thrombin assay buffer. After adding 50 μL of thrombin substrate

227

mixture into each well, the fluorescence (Ex/Em: 350/450 nm) was immediately

228

monitored in a kinetic mode for 30 min at the interval of 30 s. The activated thrombin

229

level in each treatment was quantitatively calculated using the standard curve

230

according to the kit specification, and the final result was expressed in thrombin level

231

or the value relative to the NC.

232

KKS Inhibitor Assays. To verify the role of FXII or the KKS in PM-induced

233

effects on plasma protease zymogens, two kinds of inhibitors were selected, i.e. corn

234

trypsin inhibitor (CTI, Merk, Germany) specific for FXII, and aprotinin (Sigma, USA)

235

for plasma protease, like FXIIa and PK etc. As for ex vivo experiments, the freshly

236

collected mouse plasma supplemented with 100 μg/mL CTI or 300 μM aprotinin was

237

treated by 100 μg/mL QFF- or PPF-PM2.5 at 37 °C for 2 h. The plasma with the

238

addition of equal volume of Milli-Q water was used as the NC. After the incubation,

239

the plasma samples were submitted to Western blot and ELISA analysis, respectively,

240

according to the protocols described above. Regarding in vitro experiments, a series

241

of concentrations of CPI (50, 100, 200 μg/mL) and aprotinin (100, 200, 300 μM) were

242

tested for their inhibition effects on PM2.5 induced FXII activation, using FXIIa

243

activity assay.

244

In vivo Experiments for the Crosstalk of Plasma Proteases. Twenty

245

pathogen-free male C57BL/6 mice (5 wks, 22 ± 2 g) were commercially purchased

246

from the Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China), and

247

randomly divided into four groups (n = 5), i.e. the control, QFF-PM2.5, CTI and

248

QFF-PM2.5 plus CTI groups. The exposure doses of QFF-PM2.5 and CTI were both

249

2.25 mg/kg body weight (b.w.), and the administration was performed by tail vein

250

injection at the ratio of 5 μL/g b.w. The control group was treated with the same

251

volume of PBS. The blood samples (60 μL) were collected after the circulation time

252

of 30 min and 60 min through the tail tip excision. After 90 min, the mice were

253

sacrificed by CO2 inhalation, and the intracardial blood samples (600 μL) were

254

immediately collected for plasma preparation, which was the same as the protocol

255

used for ex vivo experiments. The animal experiment strictly followed the requirement 10


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256

of the Animal Care and Use Committee of Research Center for Eco-Environmental

257

Sciences, Chinese Academy of Sciences, and all the in vivo treatments were

258

performed under the sterile condition. No animal death was observed during the

259

exposure experiments. Western blot was performed to reveal the time courses for PPK

260

activation, C3 and C5 cleave, and the changes in activated thrombin levels in the

261

control and QFF-PM2.5 exposure groups. The inhibition effect of CTI was evaluated

262

by PPK activation assay at the monitoring time points of 30, 60 and 90 min, and its

263

effects on C3, C5 and activated thrombin levels were tested at the time point of 90

264

min. All the antibodies and the kit used for plasma protease analysis were the same as

265

those described above.

266

Statistical Analysis. Statistical analysis was performed using the software of

267

SPSS 17.0. Each assay was independently carried out for three times or more, and the

268

quantitative data was presented as the mean ± standard deviation (SD). The

269

significant difference among different groups was evaluated by one-way analysis of

270

variance (ANOVA) followed by Tukey’s post hoc test or Student T test.

271

RESULTS AND DISCUSSION

272

Characterization of Airborne Fine Particles and Their Component Analysis.

273

The airborne fine particles, PM1 and PM2.5 were collected by both inorganic and

274

organic filters, i.e. QFFs and PPFs, in Beijing during winter heating season. The PM

275

weight recovered from each filter and the corresponding recovery were shown in

276

Table S1. Apparently, more PM2.5 could be extracted from the filters than did PM1,

277

and its recovery was relatively higher than that of PM1 for both kinds of filters. When

278

the sampling filters were compared, PM recoveries of QFFs (41.21% for PM1, and

279

56.72% for PM2.5, respectively) were much higher than those of PPFs (24.56% for

280

PM1, and 34.35% for PM2.5, respectively), due to the stronger adsorbability of organic

281

filter.27 Altogether, the PM recoveries followed the order of PPF-PM1 < QFF-PM1
0.05). The results suggested the activation of the KKS triggered by PM2.5

529

could regulate the coagulation system as well.

530

In biological system, the KKS cascade can activate the complement cascade via

531

the crosstalk of several plasma proteases, like FXIIa and PK etc. On one hand, FXIIa

532

can trigger the KKS and the complement system cascade through the activation of

533

PPK and C1 component.52 On the other hand, PK can also directly cleave complement

534

components, including C3 and C5, as well as their fragments.53 FXII activation can

535

also initiate the intrinsic pathway of coagulation via FXIIa-mediated activation of FXI

536

accumulated in the clot.54,55 Additionally, the complement system can facilitate the

537

coagulation cascade activation by both direct manner and inflammation-mediated

538

indirect manner. For example, mannan-binding lectin serine protease 2 (MASP2) of

539

the lectin complement-activation pathway can trigger the coagulation by converting

540

prothrombin to thrombin.55 Inflammation, due to complement system activation, can

541

also facilitate the coagulation through the activation of extrinsic coagulation

542

pathway.56 Therefore, the disturbance of airborne fine particles on FXII

543

auto-activation could trigger a series of cascade activation of the KKS, complement

544

and coagulation systems, thus causing diverse hematological effects and the related

545

cardiovascular outcomes.

546

Altogether, the present study firstly revealed airborne fine particles induced

547

hematological effects through regulating the interplay of the KKS, complement and

548

coagulation systems, which would be a promise in elucidating the increasing

549

cardiovascular diseases due to haze exposure. Considering the high complexity of the

550

compositions in airborne fine particles, more extensive studies are still urgently

551

required to clarify more specific component-correlated hematological effects and

552

other potential hazards to target organs, like pulmonary toxicity or lung damage.

553 554

ASSOCIATED CONTENT 20


Page 20 of 34

Page 21 of 34


555

Supporting Information

556

The Supporting Information is available free of charge on the ACS Publications

557

website.

558

Methods: PM sampling and preparation, Characterization of the components in PM

559

samples, Time-course of ex vivo KKS activation, Plasma kallikrein-like activity test.

560

Tables and Figures: Table S1. The recoveries of PM1 and PM2.5 collected by QFF and

561

PPF filters; Table S2. The components measured in QFF- and PPF-PM2.5 samples;

562

Figure S1. Characterization of SiNPs; Figure S2. Time courses for the cascade

563

activation of the KKS induced by 100 μg/mL QFF- or PPF-PM2.5; Figure S3. PM2.5

564

treatments altered kallikrein-like activities in plasma based on ex vivo experiments;

565

Figure S4. PM2.5 treatments increased FXIIa activities in vitro; Figure S5. CTI

566

inhibited QFF-PM2.5 induced KKS activation in vivo; Figure S6. The unbound FXII

567

upon QFF- or PPF-PM2.5 treatments based on native PAGE separation coupled with

568

silver staining; Figure S7. The time course for plasma C3 (A) and C5 (B) expressions

569

in vivo upon the treatment of 2.25 mg/kg b.w. QFF-PM2.5; Figure S8. The alteration of

570

thrombin levels in plasma upon Kaolin treatment ex vivo.

571 572

AUTHOR INFORMATION

573

Corresponding Author

574

* Phone/fax: 86 10-62849334.

575

*E-mail address: [email protected].

576

ORCID

577

Qunfang Zhou: 0000-0003-2521-100X

578

Notes

579

The authors declare no competing financial interest.

580

ACKNOWLEDGMENTS

581

This study was financially supported by the National Natural Science Foundation of

582

China (21477153, 21461142001, 21621064, and 21522706), and the Chinese

583

Academy of Science (14040302, QYZDJ-SSW-DQC017). 21



584

ABBREVIATIONS

585

Complement 3 (C3), Complement 5 (C5), Activated complement 3 (C3a), Activated

586

complement 5 (C5a), Corn trypsin inhibitor (CTI), Hageman factor XII (FXII),

587

Activated FXII (FXIIa), High-molecular-weight kininogen (HK), Kallikrein-kinin

588

system

589

(LPS), Negative control (NC), Particulate matter (PM), Plasma kallikrein (PK),

590

Polyprogylene filter (PPF), PPF control (PPF-Ctr), Plasma prekallikrein (PPK),

591

Quartz fiber filter (QFF), QFF control (QFF-Ctr), Scanning electron microscopy

592

(SEM), Silica particles (SiNPs).

593

REFERENCES

594

(1) Leung, P. Y.; Wan, H. T.; Billah, M. B.; Cao, J. J.; Ho, K. F.; Wong, C. K. Chemical and biological

595

characterization of air particulate matter2.5, collected from five cities in China. Environ. Pollut. 2014, 194,

596

188-195.

597

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733

FIGURE LEGENDS

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Figure 1. Characterization of airborne fine particles. Representative SEM images of

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(A) PM1 and (B) PM2.5 collected by QFF and PPF filters. (C) Hydrodynamic

736

diameters and (D) zeta potentials of PM1 and PM2.5 in water and plasma (n = 4). *p

Airborne Fine Particles Induce Hematological Effects through

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