Inhibition of the endothelial cell activation by antithrombin in vitro

We examined whether antithrombin (AT) inhibits tumor necrosis factor (TNF)-alpha-induced endothelial cell activation to elucidate molecular mechanism(s) of the anti-inflammatory activity of AT. AT inhibited the increase in E-selectin expression in cultured human umbilical vein endothelial cells (HUVECs) stimulated with TNF-alpha. In contrast, chemically modified AT that lacks affinity for heparin did not. AT inhibited the TNF-alpha-induced interaction of NF-kappaB p65 with p300, a homologue of cAMP-responsive element binding protein (CREB)-binding protein (CBP). AT increased both intracellular levels of cAMP and binding of phosphorylated-CREB to DNA in HUVECs. Forskolin showed the inhibitory effect similar to that of AT and pretreatment of HUVECs with KT-5720, an inhibitor of protein kinase A, reversed the inhibitory effect of AT. These observations suggested that AT inhibited the TNF-alpha-induced increase in E-selectin expression in HUVECs by inhibiting the interaction of NF-kappaB with CBP/p300 through cAMP-dependent protein kinase A-induced CREB activation. This inhibitory activity of AT might depend on its binding to heparin-like substances on the endothelial cell. Such an inhibitory effect of AT on TNF-alpha-induced endothelial cell activation might at least partly contribute to its anti-inflammatory activity.