SummaryCultured human umbilical vein endothelial cells responded to thrombin (10−2 – 10 NIH u/ml) with a 2-5 fold increase in thromboplastin activity. The maximum response was reached after 4 hr in serum-free medium. The effect of thrombin was fully inhibited by the presence of 50% (v/v) fetal calf serum or more in the medium, by preincubation of thrombin with hirudin or by treatment of thrombin with N-bromosuccinimide or phenylmethylsulfonyl fluoride. The thrombin-induced thromboplastin activity was inhibited by incubation of the cells with cycloheximide (2 μg/ml) or actinomycin D (2 μg/ml) showing that the response depended on de novo protein and RNA synthesis. It was also suppressed by exposure of the cells to two different phosphodiesterase inhibitors, 3-butyl-l-methyl-xanthine (5 · 10−4 M) and rac-4 (3-butoxy-4-methoxybenzyl)-2-imidazole (5 · 10−4 M), to the transmethylation inhibitors 3-deazaadenosine (10−5 M) and 1-homocysteine thiolactone (2 · 10−5 M) in combination and to the intracellular calcium antagonist 8-(N,N-diethylamino)-octyl 3,4,5,-tri-methoxybenzoate hydrochloride (8 · 10−5 M). Our results suggest that small amounts of thrombin can induce thromboplastin synthesis in endothelial cells in vitro and that this synthesis probably is regulated by the intracellular level of cAMP, by cytoplasmic Ca2+ and possibly also by transmethylation reactions.
Human umbilical vein endothelial cells (HUVEC) in primary confluent cultures lost their normal polygonal shape and assumed a 'contracted' appearance as judged by phase contrast microscopy when exposed to highly purified bovine thrombin (2 N.I.H. u/ml). Total actin in thrombin-exposed cells did not differ from that of control cells, as measured by the deoxyribonuclease I inhibition assay. However, the monomeric actin pool (unpolymerized actin) in thrombin-treated HUVEC was c. 15% smaller (P less than 0.01) than in control HUVEC (in which it represented approximately 50% of total actin). Transmission electron microscopy showed that thrombin-stimulated HUVEC contained more and thicker bundles of filamentous actin than control cells. Polymerization of actin and reorganization of actin microfilaments may contribute to the shape changes of HUVEC induced by thrombin.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.