Physical and Functional Interaction of Diesel Exhaust Particles with Platelets

A Solomon1, E Smyth2, N Mitha2, S Pitchford0,2, A Vydyanath2, P Luther2, AJ Thorley2, TD Tetley2, M Emerson2. 1Imperial College London, London, UK, 2Kings College London, London, UK

A growing number of epidemiological and clinical studies support the concept that components of the cardiovascular system are affected by exposure to air pollutants, especially particulate matter with a diameter ≤2.5 μm (PM 2.5). Additionally, inhalation of nanosized particles (≤ 100 nm) increases cardiovascular risk via toxicological and inflammatory processes and translocation of nanoparticles into the bloodstream has been shown in experimental models. It has been suggested that traffic derived particles, such as diesel exhaust particles (DEP) are a major factor in pollution-associated cardiovascular incidences. As the inappropriate activation of platelets underlies many cardiovascular events, we investigated the ability of DEP to interact physically with platelets and modulate their function.

The interaction of DEP with platelets was examined by transmission electron microscopy, where platelets were fixed, stained, sectioned and visualized on a A/JEOL 1200 EX electron microscope. The functional consequences of exposure to DEP were assessed by measuring in vitro (human) and in vivo (wild type c57bl/6 male mice (20-25g)) platelet aggregation (Tymvios et al., Real-time measurement of non-lethal platelet thromboembolic responses in the anaesthetized mouse. Thrombosis Haemostasis, 2008 Feb;99(2):435-40.) along with measurements of cytotoxicity, dense granule secretion, intracellular calcium concentration([Ca2+]i) and ex vivo histological analysis of murine lungs. Groups were compared using a Kruskal-Wallis test or student’s t-test and all data presented as mean ± S.E.M.

DEP (50 μg ml-1) were internalized and seen in proximity to the open canalicular system of platelets. When in proximity to DEP, platelets adopted an activated conformation. DEP (0.2, 3, 6 and 12 μg ml-1) induced in vitro platelet aggregation in a concentration-dependent manner (3.5, 10.2, 40.5 and 45.9%) where 12 μg ml-1 was significantly different to equivalent concentrations of carbon black (P<0.0003) and gold nanoparticles (P<0.0001). Sub-threshold concentrations of DEP (1 μg ml-1) also enhanced platelet aggregation to an approximate EC50 concentration of thrombin (0.06 U ml-1). DEP-induced platelet aggregation was associated with changes in [Ca2+]i , dense granule release, surface P-selectin expression but not cell cytotoxicity assessed as lactate dehydrogenase release. Sub-threshold concentrations of DEP (1.2 μg mouse-1 administered i.v) also potentiated collagen (50 μg kg-1 i.v) induced platelet aggregation in vivo compared to controls (389±57 control versus 726.9±12 treated, area under the curve, P<0.05, n=6) and post mortem examination of murine lungs revealed diesel agglomerates along with platelets within the lung vasculature after histological staining.

In conclusion, this study demonstrates that DEP physically interact with human platelets and are able to induce and enhance platelet aggregation both in vitro and in vivo. The reactivity of DEP is not shared by inert nanoparticles of similar size (gold) or chemical composition (carbon black) indicating a distinct effect of DEP. Our data provide a potential mechanism for the increased thrombotic risk associated with exposure to ambient fine particle emissions.