The ligand occupancy of endothelial protein C receptor switches the protease-activated receptor 1-dependent signaling specificity of thrombin from a permeability-enhancing to a barrier-protective response in endothelial cells

Abstract

Recent studies have indicated that activated protein C (APC) may exert its cytoprotective and anti-inflammatory activities through the endothelial protein C receptor (EPCR)-dependent cleavage of protease-activated receptor 1 (PAR-1) on vascular endothelial cells. Noting that (1) the activation of protein C on endothelial cells requires thrombin, (2) relative to APC, thrombin cleaves PAR-1 with approximately 3 to 4 orders of magnitude higher catalytic efficiency, and (3) PAR-1 is a target for the proinflammatory activity of thrombin, it is not understood how APC can elicit a protective signaling response through the cleavage of PAR-1 when thrombin is present. In this study, we demonstrate that EPCR is associated with caveolin-1 in lipid rafts of endothelial cells and that its occupancy by the gamma-carboxyglutamic acid (Gla) domain of protein C/APC leads to its dissociation from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway through coupling of PAR-1 to the pertussis toxin-sensitive G(i)-protein. Thus, when EPCR is bound by protein C, the PAR-1 cleavage-dependent protective signaling responses in endothelial cells can be mediated by either thrombin or APC. These results provide a new paradigm for understanding how PAR-1 and EPCR participate in protective signaling events in endothelial cells.

Figure 1

Binding of protein C and PC-Gla/meizothrombin to soluble EPCR and the cleavage of PAR-1 exodomain by the proteases. (A) An ELISA-based assay using the monoclonal antibody HPC4 (1 μg/mL) was used to measure the affinity of either protein C (○) or PC-Gla/meizothrombin (■) for interaction with soluble EPCR (0.5 μmol/L). (B) EA.hy926 cells were transiently transfected with ALP-PAR-1 reporter plasmid and incubated with increasing concentrations of thrombin (○), meizothrombin (□), PC-Gla/meizothrombin (■) and APC (▵) for 1 hour. The activity of soluble alkaline phosphatase (ALP) was measured as described under “PAR-1 cleavage assay.” The activity of 10 nmol/L thrombin is presented as 100%. OD, optical density. Error bars represent SD.

Figure 2

The permeability barrier enhancing and proapoptotic effects of thrombin are reversed by the ligand occupancy of EPCR. (A) EA.hy926 cells were incubated with increasing concentrations of thrombin (○), thrombin + anti-PAR-1 antibody (●), meizothrombin (□), PC-Gla/meizothrombin (■), thrombin + PC-S195A (50 nmol/L) (▴), and APC (▵) for 3 hours before inducing permeability with 5 nmol/L thrombin for 10 minutes. (B) The same as (A) except that after incubation of endothelial cells with increasing concentrations of thrombin (○), thrombin + PC-S195A (▴), and APC (▵), the permeability was induced with LPS (10 ng/mL) for 4 hour. (C) EA.hy926 cells were treated with increasing concentrations of thrombin (○), thrombin + PC-S195A (▴), and APC (▵) for 24 hours, followed by induction of apoptosis with staurosporine (5 μmol/L) for 4 hours. The number of apoptotic cells is expressed as the percentage of TUNEL-positive cells of the total number of nuclei. The number of TUNEL-positive cells in the absence of staurosporine was 10%-15%. (D) The same as (A) except that the concentration dependence of the protective effect of PC-S195A in the presence of 2 nmol/L thrombin (○) is measured in the permeability assay. The extent of the protective effect of 10 nmol/L APC (▵) is also presented. OD, optical density. Error bars represent SD.

Figure 3

The barrier-enhancing effect of TRAP and its reversal by the ligand occupancy of EPCR. (A) EA.hy926 cells were incubated with increasing concentrations of thrombin (○), thrombin + thrombomodulin fragments 456 (TM456) (●), TRAP (□), TRAP + wild-type protein C (■), TRAP + PC-S195A (▵), TRAP + wild-type protein C + blocking anti-PAR-1 (H-111) (▴), and TRAP + PC-S195A + anti-PAR-1 (H-111) (▿) for 3 hours before inducing permeability with 5 nmol/L thrombin for 10 minutes. (B) The same as (A) except that the permeability was directly monitored as a function increasing concentrations of thrombin (○), thrombin + PC-S195A (▴), TRAP (□), and TRAP + PC-S195A (■) by incubation of EA.hy926 cells with different agonists for 3 hours (the 5 nmol/L thrombin step for inducing permeability has been omitted in this assay). The first data point on the y-axis represents the baseline permeability level. OD, optical density. Error bars represent SD.

Figure 4

The effect of thrombin on the permeability of endothelial cells in the presence of protein C S195A and siRNA for PAR-4. (A) EA.hy926 cells were transiently transfected with siRNA for PAR-4 or control siRNA (1 μg each). After 3 days of incubation at 37°C, total cellular proteins were extracted, separated on 10% SDS-PAGE, and Western-blotted with antibodies directed to either PAR-4 or PAR-1. An antibody against actin was used as internal control. (B) EA.hy926 cells were incubated with increasing concentrations of thrombin (○), thrombin with prior treatment of cells for 3 days with control siRNA (●), thrombin with prior treatment of cells with siRNA for PAR-4 (□), thrombin + PC-S195A (■), thrombin + PC-S195A with prior treatment of cells with control siRNA (▵), and thrombin + PC-S195A with prior treatment of cells with siRNA for PAR-4 (▴) for 3 hours before inducing permeability with 5 nmol/L thrombin for 10 minutes. (C) The same as (B) except that the barrier-protective effect of APC was monitored in the absence (▵) and presence of control siRNA (●) or siRNA for PAR-4 (□). (D) The barrier-protective effect of thrombin + PC-S195A in endothelial cells is sensitive to PTX. EA.hy926 cells were incubated with increasing concentrations of thrombin (○), thrombin with prior treatment of cells with 100 ng/mL PTX for 16 hours (●), APC (□), APC with prior treatment of cells with 100 ng/mL PTX for 16 hours (■), thrombin + PC-S195A (▵), and thrombin + PC-S195A with prior treatment of cells with PTX (▴) for 3 hours before inducing permeability with 5 nmol/L thrombin. Error bars represent SD.

Figure 5

SDS-PAGE, immunoblotting, and coimmunoprecipitation of lipid raft/caveolae preparations derived from EA.hy926 cells with and without treatment with PC-S195A. (A) After SDS-PAGE of membrane fractions prepared by discontinuous sucrose gradient ultracentrifugation, they were immunoblotted with antibodies directed to PAR-1, EPCR, and caveolin-1. (B) The same as (A) except that the fractions were derived from EA.hy926 cells treated with 50 nmol/L PC-S195A. (C) Total cellular proteins from nontreated and PC-S195A-treated EA.hy926 cells were immunoprecipitated with anti-caveolin-1, anti-PAR-1, and anti-EPCR antibodies, separated on SDS-PAGE and immunoblotted with different pairs of the same antibodies.

Figure 6

The permeability barrier-enhancing effect of thrombin is reversed by the ligand occupancy of EPCR with PC-S195A, but not with factor VIIa. EA.hy926 cells were incubated with APC (10 nmol/L) and thrombin (2 nmol/L) in the absence and presence of either PC-S195A or factor VIIa (50 nmol/L) for 3 hours before inducing permeability with 5 nmol/L thrombin for 10 minutes. B-αEPCR is blocking anti-EPCR (clone RCR-252), B-αPAR-1 (H-111) is blocking anti-PAR-1, and NB-αPAR-1 (S-19) is nonblocking anti-PAR-1, which have been incubated with endothelial cells for 30 minutes before treatment with APC. Baseline represents the permeability of untreated control cells not stimulated with thrombin. Error bars represent SD.