Brefeldin A inhibits thrombin receptor regeneration in human endothelial cells.

Endothelial cells, once stimulated with thrombin, are resistant to subsequent stimulation. After a recovery period of about 60 min the cells are sensitized again for activation by thrombin. The resensitization is independent of the receptor de novo synthesis. Therefore an intracellular pool of thrombin receptors that is possibly co-localized with the Golgi apparatus has been assumed. Brefeldin A (BFA) has been used extensively to investigate the intracellular sorting of proteins because of its dramatic alteration of the structural and functional organization of the Golgi apparatus. Accordingly we have examined the effects of BFA on the regeneration of the thrombin receptor response in human umbilical vein and artery cells. The von Willebrand factor (VWF) release from Weibel-Palade vesicles and the intracellular calcium mobilization were used as physiological parameters of thrombin receptor activation. The addition of BFA (2 microg/ml) to endothelial cells or the reduction of the incubation temperature from 37 degrees C to 16 degrees C blocked the receptor response regeneration almost completely.

Trypsin induced von Willebrand factor release from human endothelial cells in mediated by PAR-2 activation.

Nystedt and co-workers cloned in 1994 a second protease activatable receptor (PAR-2) that could be activated by trypsin but not by thrombin (1). In this study, we investigated whether trypsin induced stimulation of endothelial cells is linked to PAR-2 activation. We have found by mRNA analysis that endothelial cells of venous and arterial origin express both protease activatable receptors. The functional thrombin receptor and the protease activated receptor-2 (PAR-2) mediate apparently the same effects in human vascular endothelial cells. Both, the activation of the thrombin receptor with thrombin or SFLLRN and the activation of the PAR-2 with trypsin or SLIGRL induced intracellular calcium mobilisation and a subsequent release of von Willebrand factor (vWf) from Weibel-Palade bodies. As a consequence, it can be concluded that endothelial cells have two different receptors mediating the same cellular responses after activation.