Air pollution has recently become a major concern worldwide. It has been proposed that exposure to high ambient pollutant particles leads to adverse health impacts which may contribute to as many as three million premature deaths per year ^[1][2][1,2]. There is extensive evidence to confirm the various adverse outcomes of the small particles on our health after both acute and long-term exposure [3]. These fine particles enter the blood circulation through the respiratory tract and quickly activate the pulmonary and systemic inflammatory responses. Activation of the inflammatory cytokines, oxidative stress, and adhesion molecules results in atherosclerosis ^[4][5][6][4,5,6]. Previous reports revealed a significant association between high ambient particulate matter and an increased incidence of cardiovascular disease including ischemic stroke ^[7][8][7,8], acute coronary syndrome ^[9][10][11][9,10,11], and thrombosis ^[12][13][14][12,13,14].

Particulate matter (PM) can consist of a variety of components depending on the source ^[15][16][15,16]. PM is defined according to particle size. Coarse particulate matter, PM₁₀ has an aerodynamic diameter of between 2.5 to 10 microns. Fine particulate matter, PM_2.5 has a particle size of more than 0.1 micron, but less than 2.5 microns in aerodynamic diameter. Ultrafine particulate matter (UFP) has an aerodynamic diameter of less than 0.1 micron ^[17][18][17,18]. Diesel exhaust particles (DEP) are the particles released from the combustion of fuel in diesel-fuelled vehicles and consist of a mixture of polycyclic aromatic hydrocarbon, organic compounds, sulfate, nitrate, other trace elements, and metals such as copper, iron, nickel, vanadium, and zinc [19]. These particles enter the body via inhalation. The coarse particles are mostly trapped in the upper airway, whereas smaller particles can pass beyond the lower airway, passing into the blood, causing adverse effects throughout the body ^[17][18][17,18]. Past evidence has demonstrated that PM could induce oxidative stress ^{[20][21][22][23][24][25][26][27]}[20,21,22,23,24,25,26,27], result in DNA damage ^[25][28][29][25,28,29], and activate local and systemic inflammatory response ^{[22][23][25][26][30]}[22,23,25,26,30]. Furthermore, PM has also been shown to impair vascular function ^[30][31][30,31], increase the expression of vascular inflammatory biomarkers, including intercellular adhesion molecules (ICAM-1), vascular cell adhesion molecules (VCAM-1), and P-selectin ^[24][32][24,32]. VCAM-1 and ICAM-1 and p-selectin are vascular adhesion molecules that play an important role in thrombus formation by promoting leukocyte-endothelial and leukocyte-platelet interaction during the inflammatory response ^[24][32][24,32]. Increased levels of these adhesion molecules instigate the recruitment of inflammatory cells into vascular endothelium resulting in the release of microparticles and the activation of platelet adhesion and platelet aggregation. Blood microparticles are small parts of cell membrane, secreted from various cell types, including endothelial cells, monocytes, and platelets, and containing both phosphatidylserine and tissue factor, the potent procoagulants, which further activate the down-stream coagulation cascade ^[33][34][33,34].

Normal hemostasis is a complex system mainly maintaining stable physiology in the body and protecting against pathological processes. The generation of thrombin is the key mechanism that links blood clot formation and the fibrinolytic system, the counterbalance that controls the thrombotic process ^[33][35][33,35]. In general, vascular endothelial injury induces platelet adhesion, platelet activation, and the release of the von Willebrand factor (vWF), tissue factor (TF), cytokines and chemokines. Primary hemostasis occurs when platelets adhere to the injured site, activating platelet aggregation, which is promoted by the vWF. TF is the main coagulation factor that initiates blood coagulation by activation of Factor VII in the extrinsic pathway, resulting in the generation of thrombin. This would further stimulate elements of the intrinsic pathway including Factors IX and VIII and then the downstream common coagulation pathway including Factors X and V, resulting in production of the prothrombinase complex (FVa-Xa), which generates excessive thrombin, leading to fibrin clot formation ^[33][35][33,35]. In the fibrinolytic pathway, tissue plasminogen activator (tPA) and plasminogen-activator inhibitor-1 (PAI-1) are the main regulators that modulate fibrinolysis. tPA activates and causes the cleavage of plasminogen into plasmin, resulting in the degradation of fibrin clots. PAI-1 is a serine protease inhibitor, which inhibits tPA, preventing clot lysis. An imbalance of tPA and PAI-1 levels therefore has an impact on fibrin clot formation [35]. Hemostasis and fibrinolytic pathways are summarized in Figure 1.

Inhalation exposure to PM induces pulmonary and systemic inflammation and oxidative stress. It increases the expression of adhesion molecules resulting in the recruitment of inflammatory cells and the activation of the vascular endothelium, platelets, and coagulation cascade, causing fibrin clots, while hampering fibrinolytic activity, and eventually contributing to thrombosis. β₂AR: beta-2 adrenergic receptor, DNA: deoxyribonucleic acid, ICAM-1: intercellular adhesion molecules-1, IFN-γ. interferon- γ, IL-1β: interleukin-1β, IL-6: interleukin-6, MCP-1: monocyte chemoattractant protein-1, NOS: nitric oxide synthase, PAI-1: plasminogen activator inhibitor-1, ROS: reactive oxygen species, SOD: superoxide dismutase, TF: tissue factor, TNF-α: tumor necrosis factor-α, tPA: tissue plasminogen activator, VCAM-1: vascular adhesion molecule-1.

Currently, knowledge surrounding the outcomes of PM-mediated coagulation and fibrinolysis is still inconclusive. In theis next sectionsreview, the effects of PM on the inflammatory responses, oxidative stress, adhesion molecules, and coagulation factors related to thrombosis from in vitro, in vivo, and clinical studies are comprehensively summarized and discussed. The understanding accrued herefrom this overview of the pathological process of PM-mediated thrombosis will help in limiting or preventing the damaging effects of PM exposure on our health.

Over the past decade, it has been shown that polluted air particles can activate inflammation, and oxidative stress and cause cell death ^[36][37][36,37]. In vitro reports indicate that exposure to PM could enhance the inflammatory response and oxidative stress, activating the coagulation cascade and inducing cell death, leading to a prothrombotic state [38]. Vanadium pentoxide (V₂O₅) is one of the toxic substances, a consequence of burning fuel oil and fly ash. An in vitro study showed that V₂O₅ could directly affect the human umbilical vein endothelial cells (HUVECs), by enhancing oxidative stress, and increasing expression of adhesion molecules, which resulted in shape changes, decreased cell proliferation, and increased apoptosis [38]. A report revealed that murine peritoneal macrophages incubated with urban PM could induce inflammatory cytokines release, resulting in phenotype changes (M1/M2 polarization), cell injury, and decreased engulfment function [39]. Other reports had shown that PM exposure induced lung macrophage differentiation into a more pro-inflammatory subtype (M1-phenotype) rather than an anti-inflammatory subpopulation (M2-phenotype) ^[40][41][40,41], which attenuates the phagocytic activity against bacterial invasion, and probably led in more susceptible to pulmonary infection ^{[39][40][41][42][43]}[39,40,41,42,43].

PM could also induce cAMP secretion and activate further downstream pathways, resulting in the enhancement of PM-mediated IL-6 release in murine alveolar macrophages (MH-S) and human alveolar macrophages [44]. In addition, administration of albuterol, a β₂AR agonist, enhanced PM-induced IL-6 release in human alveolar macrophages and MH-S cell lines, while the alveolar macrophages from Adrb2^−/− mice incubated with albuterol showed a decreased PM-mediated IL-6 response [44]. These findings suggest that PM-mediated IL-6 release was dependent on the activation of β₂AR, encoded by the Adrb2 gene. Moreover, PM was also shown to activate the microparticles and cause intracellular calcium release, and to enhance tissue factor function in HUVECs and peripheral blood mononuclear cells (PBMCs) [45]. The activation of tissue factors would further trigger blood coagulation via the TF-FVII complex, leading to thrombus formation.

In in vitro studies of venous blood from rodent models, incubation with DEP rapidly induced platelet activation and platelet aggregation in a dose-dependent manner ^{[46][47][48][49]}[46,47,48,49]. This effect was emphasized in diabetic mice, which were more vulnerable to thrombotic complications [49]. In addition, incubation of HUVECs with DEP revealed decreasing tPA and PAI-1 activity [50]. These findings suggested that both DEP and PM induced thrombosis by activating the tissue factor pathway and enhancing platelet aggregation, as well as inhibiting the fibrinolytic process, thus promoting blood clot formation and thrombosis.

The different types of particles also had differing effects on outcomes. Positively-charged amine-particles could enhance platelet function, as indicated by the shortening of PFA100 closure time in both 60-nm UFP and 400-nm amine-polystyrene particles. In contrast, negatively-charged carboxylated UFP and unmodified UFP did not affect platelet function [51]. This means that the character of each particle also plays a role in PM-induced platelet aggregation. A summary of these in vitro reports on the effects of PM on inflammation, oxidative stress, adhesion molecules, coagulation, and cell proliferation is shown in Table 1.

Consistent with in vitro reports, in vivo studies showed that the β₂-adrenergic receptor (β₂AR) in the macrophages encoded for by the Adrb2 gene was the key receptor that modulates PM-induced inflammation and thrombosis [44]. Adrb2-knockout mice had increased the PM-mediated IL-6 release, whereas this effect was blunted in specific Lyms-Cre Adrb2^flox/flox mice together with a decrease in thrombus formation and tissue factors [44]. In addition, Lyms-Cre Adrb2^flox/flox mice pretreated with a β₂AR agonist, Formoterol, showed that these mice which lack the Adrb2 gene in the macrophages had blunted IL-6 and TF response, in comparison to Adrb2^flox/flox mice after PM exposure [44]. Furthermore, PM exposure in mice with the depleted alveolar macrophages resulted in a decrease in both IL-6 release and thrombus formation [52]. These findings supported the results from in vitro studies which investigated PM induced inflammation through activation of the sympathetic nervous system via β₂AR signaling in the macrophages. This further activated the release of IL-6, TNF-α, and TF leading to the thrombus formation ^{[44][52][53][54][55][56][57]}[44,52,53,54,55,56,57].

PM may also induce oxidative stress, resulting in DNA damage ^[55][57][55,57]. Sirtuin 1 (Sirt1), the NF-ĸB regulatory gene, plays a key role in controlling the effects of PM-mediated inflammation [53]. Sirt1-knockout mice showed a higher level of inflammatory cytokines such as IL-6 and TNF-α after being exposed to PM_2.5 [53], whereas a blunted inflammatory response was observed in the IL-6 knockout mice ^[52][56][52,56]. These findings emphasized the mechanistic links between the inhalation of PM and the stimulation of the sympathetic nervous system via β₂AR, resulting in IL-6 release, systemic inflammation, and thrombosis, these processes being regulated by Sirt1. In addition, PM also induced oxidative stress in the rodent models. An increase in nitric oxide synthase (NOS) and heme oxygenase-1 (HO-1), and a decrease in catalase function in response to PM exposure were observed, together with an increase in the antioxidative response indicated by increase in glutathione (GSH) and a decrease in ascorbate level ^[55][57][55,57]. This increased anti-oxidative effect could be due to the compensation of the PM-mediated oxidative effects in those models.

PM_2.5 may induce alveolar wall thickening and also enhance adhesion molecule and TF function [54]. PM_2.5 also damaged vascular endothelial cells, resulting in TF release, which further activated the coagulation cascade and enhanced thrombus formation ^{[53][54][55][56]}[53,54,55,56]. Intratracheal instillation of road tunnel dust in C57BL/6 mice could trigger TF release more extensively than in the mice exposed to urban dust, which was more pronounced at 48 h than at four hours after exposure [58]. These findings indicated that PM induced vascular injury and activated the TF pathway, with this process dependent on the composition of the PM and occurring in a time-dependent manner.

A study in hamsters revealed that PM_2.5 exposure showed lower vWF levels, despite higher markers of vascular injury and vascular adhesion molecules [54]. This condition is characteristic of disseminated intravascular coagulation (DIC) which demonstrated obvious effects of PM_2.5 on the prothrombotic state including evidence of extensive microvascular thrombi, decreased vWF, decreased coagulation factor levels, and prolonged clotting time, which resulted from a combination of the action of many clotting factors and vWF [54]. However, there are inconsistent reports. In a study in mice exposed to PM_2.5 and PM₁₀, no significant changes in vWF secretion and white blood cell (WBC) influx in the lungs and plasma were observed [59]. Although these two studies performed similar repetitive PM exposure for the same duration, these inconsistent findings could result from the differences in dose of PM exposure and in the species used. In the case of PM-mediated thrombosis, it has been shown that PM exposure could induce platelet activation, platelet function ^[59][60][59,60], and coagulation factors such as TF, and Factors II, VIII, and X ^[52][54][52,54]. Activation of platelets, TF, and coagulation cascades would further accelerate thrombin generation and induce a prothrombotic state ^{[44][51][52][53][54][56][58][60]}[44,51,52,53,54,56,58,60]. One report revealed more thrombus formation at 48 h than at four hours after exposure, indicating that PM-accelerated thrombosis could be time-dependent [58].

The effect of PM on the fibrinolytic system remains controversial. Most studies reported that PM exposure alleviated fibrinolysis as a result of increased PAI-1 and decreased tPA mRNA expression, which caused the suppression of fibrinolytic activity and ultimately promoted a prothrombotic state ^[53][55][56][53,55,56]. A study which reported outcomes contradictory to these found that repetitive PM exposure increased tPA, reflecting the enhancement of fibrinolytic function [54]. It is possible that prolonged or chronic repetitive exposure to PM might induce extensive blood clots, leading to increased fibrinolytic activity as a compensatory response. However, further studies focusing on the fibrinolytic pathway are needed to enable us to understand the balance of each fibrinolytic factor and the ultimate effects of PM on fibrinolysis.

The effect of PM exposure on the blood cell count was inconclusive due to the conflicting data. Although PM was shown to increase the number of red blood cells (RBC) and hemoglobin (Hb) levels in mice [59], another study showed no significant impact [55]. The discordant results could be due to the difference in the duration of PM exposure, or the repetitive or prolonged duration of exposure, which might have more impact on PM-mediated changes than a single exposure or for a shorter duration. No WBC count changes were associated with PM exposure ^[55][59][55,59]. The effects of PM on platelet count were also uncertain since there were conflicting data among reports ^{[52][54][55][59]}[52,54,55,59]. The possible explanation is that there were differences in particle type, exposure dose and/or duration, and in the severity of systemic activation of the coagulation pathway, especially the induction of DIC in those studies. Nevertheless, these in vivo studies emphasized that PM could induce lung injury via the stimulation of inflammation and oxidative stress, which further activates platelets and the coagulation system, leading to a hypercoagulable state. A summary of in vivo reports on the effects of PM on inflammation, oxidative stress, and the coagulation system is shown in Table 2.