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

Abstract

Summary 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.

Objectives: 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.

Figure 1

Effect of thrombin on the expression of Ang/Tie2 signaling molecules. EA.hy926 cells were incubated with indicated concentrations of thrombin for 12h either before (panels A, C, and E) or after (panels B, D, and F) treating monolayers with PC-S195A (50 nM, 30 min). Protein levels in cell lysates for Tie2 (A and B), Ang1 (C and D) and Ang2 (E and F) were measured by ELISA. * p< 0.05 as compared to 0 nM Th. Time course of the thrombin-mediated expression of Ang1 (G) and Ang2 (H) in the PC-S195A-pretreated cells were measured after incubation with 2 nM thrombin followed by measuring protein levels in cell lysates by ELISA. Symbols are: ○, PC-S195A; ●, thrombin + PC-S195A; □, thrombin alone. *p< 0.05 as compared to 0 nM Th.

Figure 1

Effect of thrombin on the expression of Ang/Tie2 signaling molecules. EA.hy926 cells were incubated with indicated concentrations of thrombin for 12h either before (panels A, C, and E) or after (panels B, D, and F) treating monolayers with PC-S195A (50 nM, 30 min). Protein levels in cell lysates for Tie2 (A and B), Ang1 (C and D) and Ang2 (E and F) were measured by ELISA. * p< 0.05 as compared to 0 nM Th. Time course of the thrombin-mediated expression of Ang1 (G) and Ang2 (H) in the PC-S195A-pretreated cells were measured after incubation with 2 nM thrombin followed by measuring protein levels in cell lysates by ELISA. Symbols are: ○, PC-S195A; ●, thrombin + PC-S195A; □, thrombin alone. *p< 0.05 as compared to 0 nM Th.

Figure 2

The occupancy of EPCR by its ligand switches the PAR-1-dependent effect of thrombin on the expression of Tie2, Ang1, and Ang2. Primary HUVECs were incubated with APC (20 nM) or thrombin (2 nM) for 16h before or after treating cells with PC-S195A followed by measuring Ang1 and Ang2 levels in supernatant (A) and cell lysate (B) by a regular ELISA and cell surface (C) by a cell-based ELISA. *p< 0.05 as compared to thrombin. (D) Time course of phosphorylation of Tie2 in lysates of primary HUVECs was analyzed by ELISA after treating cells with APC (20 nM) or thrombin (2 nM) ± PC-S195A (50 nM, 30 min) for 16h. The ratio of phosphor-Tie2 to total Tie2 is shown in y-axis. Symbols are: ○, control buffer; ●, thrombin alone; □, PC-S195A alone; ■, thrombin + PC-S195A; △, APC.

Figure 3

EPCR- and PAR-1-dependent effect of thrombin on the expression of Tie2, Ang1, and Ang2. EA.hy926 cells were preincubated with blocking antibodies to EPCR and PAR-1 (25 μg/mL, 30min) followed by treatment with PC-S195A (50 nM, 30min) and stimulation by thrombin (2 nM for 12h). Protein levels of Tie2 (A) Ang1 (B) and Ang2 (C) were measured by ELISA. *p< 0.05 as compared to 2 nM Th with PC-S195A (A and B) or compared to PC-S195A alone (C). Cont, not treated control; B, blocking; NB, nonblocking

Figure 4

Effects of different proteases on the expression of Tie2, Ang1, and Ang2 proteins. Primary HUVECs were incubated with indicated proteases (2 nM thrombin, 20 nM APC or GD-APC) for 16h with (black bars) or without (white bars) sequential treatment with MβCD (10 mM, 1h) and PC-S195A (50 nM, 30 min) followed by measuring expression levels of Tie2 (A) Ang1 (B) and Ang2 (C) by ELISA. *p< 0.05 as compared to without MβCD.

Figure 5

S1P₁- and PI3K-dependent thrombin regulation of the expression of Tie2, Ang1, and Ang2. The PC-S195A pretreated primary HUVECs were incubated with thrombin (2 nM, 12h) before or after transfection with control or specific siRNA for S1P₁ (10 nM, 24h) or treatment with LY294002 (10 μM, 1h). Expression levels of Tie2 (A) Ang1 (B) and Ang2 (C) were measured by ELISA. .*p< 0.05 as compared to 2 nM Th + PC-S195A. (D) Primary HUVECs were preincubated with blocking antibodies to EPCR, PAR-1, or Tie2 (25 μg/mL, 30min) or transfected with siRNA for Tie2 (10 nM, 24h) followed by incubation with PC-S195A and stimulation by thrombin (2 nM for 12h). The cell permeability was measured as described under “Materials and Methods”. *p< 0.05 as compared to PC-S195A alone. NS, non-specific

Figure 6

PC-S195A inhibits the mobilization of P-selectin and Ang2 onto endothelial cell surface. EA.hy926 cells (A and B) or primary HUVECs (C and D) were incubated with thrombin (2 nM) with or without pretreatment of cells with PC-S195A. The cell surface P-selectin (A and C) or Ang2 (B and D) was measured by an ELISA described under “Materials and Methods”. Symbols are: ○, control buffer; ●, thrombin alone; □, PC-S195A alone; ■, thrombin + PC-S195A. *p< 0.05 as compared to Th + PC-S195A.

Figure 7

Hypothetical model of crosstalks between EPCR, PAR-1, S1P₁, and Tie2. EPCR is associated with caveolin-1 (Cav-1) in lipid-rafts of endothelial cells when the receptor is not occupied by Gla-domain of protein C/APC. Thrombin cleavage of PAR-1 elicits disruptive responses through signaling via G_q and/or G_12/13, thereby activating the NF-κB pathway. However, the occupancy of EPCR by protein C (PC) results in the dissociation of EPCR from caveolin-1, thereby switching the specificity of PAR-1 signaling by coupling it to the G_i-protein and/or transactivation of the G_i-protein coupled receptor, S1P₁, and mediating the phosphorylation of the receptor by the PI3K/Akt pathway. The EPCR-dependent PAR-1 cleavage by thrombin also increases the expression levels of Ang1 and Tie2, thus initiating/amplifying the PI3K/Akt-dependent protective pathway through the Ang1-mediated phosphorylation of Tie2.