Abstract
Here we describe a chemical biology approach for elucidating potential toxicity mechanisms for thrombosis-related side effects. This work takes advantage of a large chemical biology data set comprising the effects of known, well-characterized reference agents on the cell surface levels of tissue factor (TF) in a primary human endothelial cell-based model of vascular inflammation, the BioMAP® 3C system. In previous work with the Environmental Protection Agency (EPA) for the ToxCast™ program, aryl hydrocarbon receptor (AhR) agonists and estrogen receptor (ER) antagonists were found to share an usual activity, that of increasing TF levels in this system. Since human exposure to compounds in both chemical classes is associated with increased incidence of thrombosis-related side effects, we expanded this analysis with a large number of well-characterized reference compounds in order to better understand the underlying mechanisms. As a result, mechanisms for increasing (AhR, histamine H1 receptor, histone deacetylase or HDAC, hsp90, nuclear factor kappa B or NFκB, MEK, oncostatin M receptor, Jak kinase, and p38 MAPK) and decreasing (vacuolar ATPase or V-ATPase) and mTOR) TF expression levels were uncovered. These data identify the nutrient, lipid, bacterial, and hypoxia sensing functions of autophagy as potential key regulatory points controlling cell surface TF levels in endothelial cells and support the mechanistic hypothesis that these functions are associated with thrombosis-related side effects in vivo.
Highlights
Tissue factor, thromboplastin or CD142, is the cell-surface receptor for the serine protease factor VIIa and, as part of the factor VIIa-TF enzymatic complex, functions as the primary cellular initiator of blood coagulation [1,2,3,4]
Thrombosis is restricted to sites of tissue damage where the vessel has been denuded of endothelial cells and tissue factor is expressed on sub-endothelial smooth muscle cells or fibroblasts, or to sites of inflammation or stress where expression of tissue factor has been induced on endothelial cells and monocytes recruited from the blood
The BioMAP 3C system consists of human primary endothelial cells stimulated for 24 h with a cocktail of proinflammatory cytokines (IL-1β + TNFα + IFNγ), followed by measurement of cell surface levels of MCP-1, VCAM-1, TF, TM, IL-8, MIG, HLA-DR, E-selectin and uPAR; total protein levels (measured by sulforhodamine B (SRB) staining, see Experimental Section below and a more detailed description in Supplemental Text, Materials and Methods); and cell proliferation
Summary
Thromboplastin or CD142, is the cell-surface receptor for the serine protease factor VIIa and, as part of the factor VIIa-TF enzymatic complex, functions as the primary cellular initiator of blood coagulation [1,2,3,4]. Thrombosis is a normal component of wound healing, as the platelet-fibrin clots produced by coagulation recruit a variety of growth factors directly to the injured site, promoting angiogenesis and tissue repair. Thrombosis is restricted to sites of tissue damage where the vessel has been denuded of endothelial cells and tissue factor is expressed on sub-endothelial smooth muscle cells or fibroblasts, or to sites of inflammation or stress where expression of tissue factor has been induced on endothelial cells and monocytes recruited from the blood. Increased levels of TF are associated with a number of disease settings: cardiovascular disease conditions, such as atherosclerosis and acute coronary artery syndrome; cancer; diabetes; liver injury; and infectious diseases, such as human immunodeficiency virus (HIV), herpes simplex virus-1 (HSV-1) and Ebola virus hemorrhagic fever, which is a consumptive coagulopathy [4,5]. Increased TF levels have been correlated with risk of thrombosis [10]
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