We examined the effects of the proinflammatory cytokine, tumor necrosis factora (TNF,), on the expression of proteolytically activated thrombin receptor (PATR) in human umbilical vein endothelial cells (HUVEC). PATR mRNA and protein levels were measured in confluent HUVEC monolayers after challenge with TNF,. Northern analysis indicated that TNF, treatment resulted in 2-to 3-fold decrease in PATR mRNA in a timeand dose-dependent manner. PATR mRNA level returned to the control level within 6 hr. The nuclear run-on assay indicated that the decreased mRNA signal was due to reduction in the transcription rate. Immunoblotting experiments indicated that the decrease in expression of PATR protein followed in time the decrease in mRNA; the lowest level of protein expression was achieved at 22 hr after TNF, treatment. PATR protein returned to basal value within 40 hr after TNF, challenge. To assess alterations in endothelial cell function after TNF, treatment, we measured thrombin-induced increase in cytosolic Ca2+ ([Ca"],) and the cell shape change (measured by decrease in electrical impedance of endothelial cell monolayer). In HUVEC treated with TNF, (100 U/ml for 22 hr), the rise in [Caz+Ii after thrombin challenge was -2-fold less than in control thrombin-treated cells. The decrease in electrical impedance of HUVEC monolayers in response to thrombin after TNF,treatment was also significantly reduced. However, the rise in [Ca2+li in response to histamine was not altered by TNF, pretreatment. In conclusion, TNF, exposure of endothelial cells decreased both mRNA and protein expression of PATR, which explain the decreased activation of thrombin generated signals after the TNF, exposure.