Particulate Matter Induces Tissue Factor and Attenuates Thrombomodulin in Rat Lungs.
Authors: Frederix Kim , Kooter Ingeborg M. , Van Oerle Rene , Hamulyak Karly , Spronk Henri M.H. , Cate Hugo Ten 
Published: October 2019

Abstract
Exposure to particulate matter (PM) is an environmental determinant of chronic lung- and cardiovascular disease (CVD). Gene array expression analysis in rats exposed to PM showed a marked increase in tissue factor in the lung (Kooter et al, Inhal Toxicol.2005;17(1):53–65). We hypothesised that pro-inflammatory effects of PM would affect the cardiovascular system, in part through a prothrombotic action. To functionally characterize such effects we determined tissue factor (TF) activity and effects on thrombin generation in tissues from animals challenged with PM. Spontaneous hypertensive rats (SHR) rats were exposed via intratracheal instillation to different concentrations of PM collected from a highway tunnel (road tunnel dust: 0.3–1–3–10 mg PM/kg bodyweight (BW)) for 4 and 48 hours. For comparison we also tested organ- homogenates of mice that received 2 mg/kg endotoxin i.p. for 6 hours, as a well characterized inflammation driven prothrombotic animal model. Tissue factor activities show an increase with increasing concentrations of road tunnel dust. For 10mg/kg BW there is a significant difference both after 4 and 48 hours of exposure compared to controls (1044.5 ± 471.6, 1020.8 ± 61.6 vs. 397 ± 231.1 pM, p<0.02 ). For comparison, tissue factor activity in lungs of endotoxemic mice (25.8 ± 5.28 pM) was significantly higher than in lungs of control mice (12.7 ± 1.73 pM, p<0.05), whereas the activity in other organs (brain, heart, spleen, liver, kidney) was comparable between both groups. TF in the lung is known to increase under inflammatory conditions. We adapted the Calibrated Automated Thrombogram (CAT) that measures the thrombin generating potential in human platelet poor plasma (PPP) so that we were able to add tissue homogenates and measure the effect on thrombin generation. There is an 8-fold reduction in the endogenous thrombin potential (ETP) upon addition of lung homogenate from untreated rats and mice compared to the ETP of pooled PPP from 80 healthy volunteers (174.5 ± 61 vs. 1436.6 ± 112 nM min). We established that this inhibition is primarily due to the presence of thrombomodulin in the lungs, becausethe inhibitory effect was absent in protein C deficient human plasma andlungs from TM pro/pro mice that have a 100-fold reduction in thrombomodulin cofactor activity show a complete recovery of the ETP (1685.5 ± 208.8 nM min).
With increasing concentrations of road tunnel dust the ETP increases with significant differences at 10mg/kg BW after 4 hours (389.5 ± 147.7 vs. 174.5 ± 61 for saline, p<0.05). After 48 hours the ETP augments further for the highest concentrations (1440.7 ± 288.8 nM min vs. saline: 163.9 ± 64 nM min, p<0.0001). This indicates a concentration- and time dependent reduction in thrombomodulin activity after PM exposure. Endotoxin challenge caused an increase in ETP (940.8 ± 523.1 vs. 194.3 ± 158.7nM min) for lung homogenates, whereas no changes were observed for brain (1794.8 ± 49.6 vs. 1801.8 ± 42.8 nM min) and heart (784.6±90 vs. 873.8 ± 74.3 nM min) compared to control tissues. The combination of these measurements indicates that the procoagulant state in the lungs after exposure to PM is related to inflammation and can be (partially) explained by the ratio of tissue factor and thrombomodulin activities in these organs.