Thrombin upregulates the angiopoietin-Tie2 Axis: endothelial protein C receptor occupancy prevents the thrombin mobilization of angiopoietin 2 and P-selectin from Weibel-Palade bodies.
BACKGROUND
Activated protein C (APC) in complex with endothelial protein C receptor (EPCR) can reverse the barrier-disruptive and cytotoxic effects of proinflammatory cytokines by cleaving protease-activated receptor 1 (PAR-1). Recently, it was reported that the PAR-1-dependent vascular barrier-protective effect of APC is mediated through transactivation of the angiopoietin (Ang)-Tie2 signaling pathway. The antagonist of this pathway, Ang2, is stored in Weibel-Palade bodies within endothelial cells.


OBJECTIVE
To determine whether the occupancy of EPCR by its ligand can switch the PAR-1-dependent signaling specificity of thrombin through the Ang-Tie2 axis.


METHODS
We activated endothelial cells with thrombin before and after treating them with the catalytically inactive Ser195-->Ala substitution mutant of protein C. The expression levels of Ang1, Ang2 and Tie2 in response to thrombin were measured by both an enzyme-linked immunosorbent assay and a cell permeability assay in the absence and presence of small interfering RNA and a blocking antibody to Tie2.


RESULTS
Thrombin upregulated the expression of both Ang1 and Tie2 but downregulated the expression of Ang2 when EPCR was occupied by its ligand. The Ang1-Tie2-dependent protective effect of thrombin was initiated through protein C inhibiting the rapid mobilization of Ang2 from Weibel-Palade bodies. Interestingly, the protein C mutant also inhibited the thrombin mobilization of P-selectin.


CONCLUSIONS
These results suggest a physiologic role for the low concentration of thrombin in maintaining the integrity of the EPCR-containing vasculature through the PAR-1-dependent inhibition of Ang2 and P-selectin release from Weibel-Palade bodies.
Bae
J-S
JS
Department of Herbal Pharmaceutical Engineering, College of Herbal Bio-Industry, Daegu Haany University, Gyeongsan, Korea. BaeJS@dhu.ac.kr
Rezaie
A R
AR
D042702:Angiopoietin-2 / Q000502:physiology*; D002461:Cell Line, Transformed; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D019007:P-Selectin / Q000502:physiology*; D010766:Phosphorylation; D042787:Receptor, TIE-2 / Q000502:physiology*; D013917:Thrombin / Q000502:physiology*; D015854:Up-Regulation / Q000502:physiology*; D020302:Weibel-Palade Bodies / Q000502:physiology*
D042702:Angiopoietin-2; D019007:P-Selectin; D042787:Receptor, TIE-2; D013917:Thrombin