Endothelial cells from human umbilical veins produce a procoagulant identified as thromboplastin (tissue factor, factor III) when stimulated with the phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA), phytohaemagglutinin (PHA) or endotoxin, Inducible thromboplastin synthesis (i.e. synthesis of the protein component of thromboplastin, apoprotein III) was totally inhibited by cycloheximide and actinomycin D, indicating that de novo protein and RNA syntheses are necessary. Serum enhanced the induced apoprotein synthesis. Of the total thromboplastin activity in homogenates of stimulated endothelial cells, about 50--70% was available on the cell surface for interaction with other coagulation factors, inactivation by trypsin and neutralization with antiserum against apoprotein III. Induced synthesis of thromboplastin in endothelial cells was 2--7-fold enhanced by the presence of several other cell types in optimal ratio 4--10 cells per endothelial cell. Some of these cell types were themselves thromboplstin producers (U-937, U-937-4), some were not inducible (lymphocytes, granulocytes and the lymphoblast lines Daudi and Molt 4). This enhancing effect was also seen with cell-free culture supernatants, but these were generally somewhat less effective than the intact cells. Supernatants derived from cells cultured in the presence of TPA, PHA or endotoxin were in most cases more effective than supernatants from unstimulated cells.

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.

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SummaryHuman umbilical vein endothelial cells in culture synthesize thromboplastin upon stimulation with phytohaemagglutinin (PHA) or the tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The thromboplastin activity is further strongly enhanced in a time dependent reaction by the presence of gel-filtered platelets or platelet aggregates. This effect was demonstrable at platelet concentrations lower than those normally found in plasma, it may thus be of pathophysiological relevance. The thromboplastin activity increased with increasing number of platelets added. Cycloheximide inhibited the increase, suggesting that de novo synthesis of the protein component of thromboplastin, apoprotein III, is necessary.When care was taken to remove monocytes no thromboplastin activity and no apoprotein HI antigen could be demonstrated in suspensions of gel-filtered platelets, platelets aggregated with thrombin or homogenized platelets when studied with a coagulation assay and an antibody neutralization technique.

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