150 Development of an in vitro thrombosis model to assess endothelial regulation of thrombus formation

BackgroundArterial thrombosis culminating in myocardial infarction and stroke is one of the leading causes of death in the western world. There are currently a number of in vitro models to investigate the complex mechanisms regulating thrombosis however they fail to consider the role of the endothelium, and the effects of endothelial dysfunction. Recent evidence suggests that the efficacy of P2Y12 inhibitors may partially be dependent on a healthy endothelium. The aim of this study is to develop a novel in vitro model of arterial thrombosis, which incorporates endothelial cells allowing their contribution to thrombosis and antithrombotic efficacy to be assessed.MethodsHuman umbilical vein endothelial cells (HUVECs) were cultured on Ibidi μ-slides (VI0.4) and alternate chambers coated with collagen (100 μg/ml). Whole blood was perfused through a HUVEC lined channel before passing through a collagen-coated channel, at a shear rate of 1000-s. Platelets were fluorescently labelled with DIOC6 and collagen-mediated adhesion and thrombus generation analysed using confocal microscopy. To assess the effects of endothelial activation on thrombus formation, cells were exposed to tumour necrosis factor alpha (TNF-alpha; 10 ng/ml 24 hour). Expression levels of VCAM-1, ICAM-1, E-Selectin and P-selectin were assessed using immunofluorescence and Western blotting to verify endothelial cell activation.ResultsExogenous addition of endothelial derived platelet inhibitors, CD39, PGE1 and nitric oxide significantly reduced thrombus size and surface coverage in collagen coated chambers (p