REGULATION OF THE THROMBIN RECEPTOR RESPONSE IN HUMAN ENDOTHELIAL-CELLS

At present only little information is available about the regulation o f the thrombin receptor activity in human endothelial cells. The study presented was performed to clarify the problem of thrombin receptor r egulation in human endothelial cells. Endothelial cells were isolated from the vein or artery of human umbilical cords. These cells were sti mulated with thrombin or with the thrombin receptor activation peptide s (TRAP) SFLLRN or SFLLRNPNDKYEPF and subsequently the von Willebrand factor (vWf) release was measured as a physiologically significant res ponse regarding the thrombin receptor activation. Shortly after the fi rst activation by thrombin or SFLLRN, the cells were desensitised to a second stimulation. After a recovery phase of 10 min, merely 35% of t he 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 resensitisatio n rate was similar for thrombin and SFLLRN stimulated cells. Adding RN A synthesis inhibitors or protein synthesis inhibitors had no effect o n the recovery of the receptor response. Therefore, a de novo synthesi s of receptor protein must be excluded as a means of resensitising end othelial cells to thrombin. It was shown that the dephosphorylation of tyrosine kinase inactivated receptors is also not responsible for rec eptor regeneration. These results prove that endothelial cells are cap able of developing full thrombin receptor activity after a relatively short recovery phase of only 60-90 min as compared to the recovery pha se of 16-24 h in megacaryoblastic cell lines. Desensitisation is simil ar in thrombin and TRAP stimulated cells. We assumed a transport mecha nism for intracellular stored vesicles for receptor resensitisation; a co-localisation with vWf in the Weibel-Palade bodies is improbable.