Changes to the structure of blood clots formed in the presence of fine particulate matter

Both long-term and short-term exposure (one to two hours) to particulate matter are associated with morbidity and mortality caused by cardiovascular diseases. The underlying mechanisms leading to cardiovascular events are unclear, however, changes to blood coagulability upon exposure to ultrafine particulate matter (UFPM, the smallest of which can enter the circulation) is a plausible mechanism. Objectives: This study aims to investigate the direct effects of particulate matter on fibrin polymerization, lateral aggregation and the formation of fibrin network structure. Methods: Standard Urban Particulate Matter (PM) was suspended in Tris buffer centrifuged and filtered with <200nm filter to obtain ultrafine PM or their water-soluble components. Purified normal fibrinogen was made to clot by adding thrombin and calcium chloride in the presence of varying concentrations of PM. Permeation properties (Darcy constant [Ks]) and turbidity of clots were measured to investigate the effects on flow-rate, pore size, and fibrin polymerization. In addition, confocal microscopy was performed to study detailed clot structure. Results: Total PM increased the Ks of clots in a dose dependant manner (Ks = 4.4, 6.9 and 13.2 x 10-9 cm2 for 0, 50 and 100 g/ml total PM concentrations, respectively). Filtered PM also produced a significant increase in Ks at PM concentration of 17 g/ml. Final turbidity measurements at 20min were obtained for varying concentrations of PM. Maximum optical density (OD) for 1 mg/ml fibrinogen at 0, 50, 100 and 200 g/ml total PM concentrations were 0.39, 0.42, 0.45 and 0.46, respectively. The maximum OD for 0, 17, 34 and 68 g/ml filtered PM concentrations were 0.39, 0.42 0.47 and 0.51, respectively, suggesting an increase in fibre diameter with increasing particulate concentration. The lag phase was significantly shorter and the rate of polymerisation was significantly faster in the presence of 68 g/ml filtered PM. Confocal microscopy results showed decrease in fibre density without a significant increase in fibre diameter in the presence of total PM and filtered PM. Conclusion. The results indicate that total PM and filtered PM are capable of causing alterations to the fibrin polymerization and network structure as shown by the changes in permeation properties, the turbidity experiments as well as by confocal microscopy. 1. Background and objectives Epidemiological studies have shown that exposure to air pollution is associated with increased hospitalisation and mortality due to cardiovascular disease [1-3]. Peters et al. showed that recent Inhaled Particles X, (23–25 September 2008, Manchester) IOP Publishing Journal of Physics: Conference Series 151 (2009) 012029 doi:10.1088/1742-6596/151/1/012029 c © 2009 IOP Publishing Ltd 1 exposure to air pollution was associated with myocardial infarction with an odds ratio of 2.92 (95% Confidence Interval (95% CI), 2.22 to 3.83, p<0.001) [4]. Air pollution constitutes gaseous pollutants and particulate matter (PM) of varying sizes. There is increasing evidence that suggests that exposure to varying concentrations of PM is associated with myocardial infarction (MI), coronary heart disease (CHD), arrhythmia and cerebrovascular disease [5, 6]. The main source of traffic related PM is from diesel and petrol combustion engines, although road surface, tyre and brake wear all contribute to PM. PM has been classified according to size. PM10, PM2.5 and PM0.1 are particles less than 10 μm, 2.5 μm and 0.1 μm in aerodynamic diameter, respectively. The particles consist mainly of elemental carbon (carbon black or soot), with small amounts of trace metals (such as lead, zinc, iron and aluminium), sulphate salts and nitrate salts. There may also be traces of varying compositions of polycyclic aromatic hydrocarbons. The size of the particles determines their ability to penetrate deep into the lungs, finer particles reaching the bronchioles and the alveoli. In order to enter the blood circulation, particles have to pass through the air-blood barrier, which is only 0.5 μm thick, and is where gaseous exchange takes place. Ultrafine particles (UFP; <0.2 μm) have been found capable of crossing the cellular membrane of the alveoli without the need for endocytosis or any form of receptor-mediated, actin-based process. In the lungs, the particles have been found in the tissue compartments, cells and within capillaries [7]. The presence of UFP in blood has been shown in vivo [8, 9]. One of the most important plasma proteins during the coagulation process is fibrinogen. The final step in the formation of clot is the conversion of fibrinogen to fibrin which polymerizes into a threedimensional network. The polymerization of fibrinogen is initiated by the action of thrombin to cause the conversion of fibrinogen to fibrin. The fibrin clot is broken down by the process of fibrinolysis through the action of plasmin. During clot formation, plasminogen is bound to fibrin. Tissue plasminogen activator (t-PA) converts plasminogen to plasmin, initiating fibrinolysis. The rate of fibrinolysis may depend on fibrin fibre diameter and fibrin clot structure. Fibrin clot with a dense structure made of thin fibres dissolved more slowly than coarse fibrin clot structure made of thicker fibres. The formation of abnormal structure of fibrin clot has been linked to the thrombosis[10-12]. People with myocardial infarction are more likely to have tighter and more rigid fibrin clot network with lower permeability compared to normal subjects [13]. According to confocal microscopy data, plasma clots from coronary artery disease (CAD) patients were made of fibrin fibres that were shorter, thinner and more numerous than clots from normal control subjects. Furthermore, the fibres from the CAD patients were more resistant to lysis with a slower rate of lysis than that of control subjects [14]. In this study we have studied the characteristics of blood clots formed in vitro in the presence of PM and UFP. 2. Study description 2.1. Particulate matter The PM used was a Standard Reference Material (SRM) 1648 purchased from National Institute of Standards and Technology (NIST). The SRM was collected from St Louis, MO, United States, accumulated over a period of over 12 months. The SRM mainly consists of carbon particles with various elements such as aluminium, iron, potassium, lead, sodium and zinc. Two portions of the SRM were suspended in Tris buffer (0.05M Tris-HCl, 0.1M NaCl, pH7.5) at concentration of 5mg/ml. One portion was kept aside as total particulate matter (TPM). Another portion was centrifuged at 13,400 rpm for 30 minutes and the supernatant filtered with a serological 0.22 m filter with a hydrophilic polyethersulfone membrane (Millex GP, Millipore). This portion was called filtered particulate matter (FPM). 2.2. Fibrin permeation Purified fibrinogen (100 l) was made to clot by adding 10 l of activation mixture (thrombin and calcium chloride). The final concentrations were 1 mg/ml, 1 U/ml and 10 mM for fibrinogen, Inhaled Particles X, (23–25 September 2008, Manchester) IOP Publishing Journal of Physics: Conference Series 151 (2009) 012029 doi:10.1088/1742-6596/151/1/012029