Inhibition of the thromboplastin response of endothelial cells in vitro

Confluent monolayers of human umbilical vein endothelial cells in culture responded with a 5-fold increase in thromboplastin (tissue factor) synthesis when exposed to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (50 ng/ml) or endotoxin (ETX) (25 micrograms/ml) for 16 hr. This induced thromboplastin synthesis was markedly inhibited by exposure of the cells to two different phosphodiesterase inhibitors, methylisobutylxanthine (MIX) and rac-4(3-butoxy-4-methoxybenzyl)-2-imidazole idinone (RO-20-1724) and to the transmethylation inhibitors 3-deazaadenosine (DZA) and 1-homocysteine thiolactone (Hcy) in combination. It was slightly (TPA) or not at all (ETX) inhibited upon exposure of the cells to the intracellular calcium antagonist 8-(N,N-diethylamino)-octyl 3,4, 5-trimethoxybenzoate hydrochloride (TMB-8). However, in the presence of MIX TMB-8 had a moderate additional inhibitory effect on TPA-induced thromboplastin response. The thromboplastin response of endothelial cells in vitro thus probably depends on transmethylation events for its full expression. It is also strongly modulated by the intracellular level of cAMP.