Endothelial protease-activated receptor-2 induces tissue factor expressionand von Willebrand factor release

A second protease-activated receptor (PAR-2) that could be activated by try psin or more physiologically by mast cell tryptase has been recently cloned . Both the structure and activation mechanism of PAR-2 was similar to the f unctional thrombin receptor (PAR-1). Although many effects of the coagulati on protease thrombin on the vascular endothelium could be attributed to PAR -1 activation, very little is known about the physiological and pathophysio logical role of PAR-2. We investigated whether stimulation of PAR-2 on endo thelial cells induced two cellular responses that play a central role in pr imary and secondary haemostasis: the release of high molecular weight von W illebrand factor (hmw-VWF) from Weibel-Palade bodies and the de novo synthe sis of tissue factor (TF) mRNA and protein. Human umbilical vein endothelia l cells (HUVEC) were incubated with agonists for PAR-2 at 37 degrees C. Bot h trypsin and SLIGKV increased TF mRNA and activity and induced the release of hmw-VWF due to elevated levels of cytosolic Ca2+. Trypsin (10 nM) induc ed a 6-fold increase of TF mRNA and reduced time until fibrin clot formatio n to 37%, indicating trebling of the cell surface located TF activity, Stim ulation of HUVEC with the PAR-2 agonist peptide SLIGKV induced a dose-depen dent increase of TF mRNA up to 6 times and TF activity up to 3 times. Relea se of hmw-VWF was achieved both after incubation of HUVEC with trypsin and SLIGKV and was directly depending on intracellular Ca2+ mobilization, To ma ke results comparable to the functional thrombin receptor, homologous exper iments were carried out using the PAR-1 agonists thrombin and SFLLRN.