Regulation of the thrombin receptor response in human endothelial cells.

At present only little information is available about the regulation of the thrombin receptor activity in human endothelial cells. The study presented was performed to clarify the problem of thrombin receptor regulation in human endothelial cells. Endothelial cells were isolated from the vein or artery of human umbilical cords. These cells were stimulated with thrombin or with the thrombin receptor activation peptides (TRAP) SFLLRN or SFLLRNPNDKYEPF and subsequently the von Willebrand factor (vWf) release was measured as a physiologically significant response regarding the thrombin receptor activation. Shortly after the first activation by thrombin or SFLLRN, the cells were desensitised to a second stimulation. After a recovery phase of 10 min, merely 35% of the receptor response was obtained by subsequent stimulation. Expanding the recovery time to 90 min resulted in a vWf release of nearly 100% of the amount measured after the first stimulation. The resensitisation rate was similar for thrombin and SFLLRN stimulated cells. Adding RNA synthesis inhibitors or protein synthesis inhibitors had no effect on the recovery of the receptor response. Therefore, a de novo synthesis of receptor protein must be excluded as a means of resensitising endothelial cells to thrombin. It was shown that the dephosphorylation of tyrosine kinase inactivated receptors is also not responsible for receptor regeneration. These results prove that endothelial cells are capable of developing full thrombin receptor activity after a relatively short recovery phase of only 60-90 min as compared to the recovery phase of 16-24 h in megacaryoblastic cell lines. Desensitisation is similar in thrombin and TRAP stimulated cells. We assumed a transport mechanism for intracellular stored vesicles for receptor resensitisation; a co-localisation with vWf in the Weibel-Palade bodies is improbable.