Factor Xa Activates Endothelial Cells by a Receptor Cascade Between EPR-1 and PAR-2

Altieri and Edgington¹ have reported recently that a membrane protein similar to the light chain of factor Va might be the membrane receptor of factor Xa on monocytes. This protein, called effector protease receptor-1 (EPR-1), behaves as a cofactor for factor Xa to catalyze prothrombin activation in the absence of added factor Va.² Recently, we and others demonstrated the existence of such a population of high-affinity, functional, factor Xa–binding sites in human vascular endothelial cells (HUVECs) and showed that exposure of HUVECs to factor Xa induced phosphoinositide turnover and an increase in intracellular free Ca²⁺.³⁴ Most important, through binding to this receptor, factor Xa was also a potent mitogen for endothelial cells.³ Moreover, a recent article by Papapetropoulos et al⁵ showed increased interleukin-6 release by HUVECs after treatment with factor Xa. These activities of factor Xa are affected by selective factor Xa inhibitors, which suggests that occupancy of EPR-1 alone by factor Xa is not sufficient for cell activation and that protease activity is required for factor Xa to induce signal transduction and subsequent activation of vascular endothelial cells.⁶ In this respect, factor Xa behaves like thrombin or trypsin, both of which require full catalytic activity to exhibit cellular effects,⁷⁸ but differs from them by the fact that both thrombin and trypsin receptors (respectively, PAR-1 [protease-activated receptor-1] and PAR-2 [protease-activated receptor-2]) need to be specifically cleaved by these proteases to be activated.⁹ Here we show that EPR-1 is necessary to localize factor Xa in close proximity to the cellular membrane where it then selectively cleaves and activates PAR-2, thus representing a novel mode of cascade receptor activation.

Methods

Cell Culture and Free [Ca²⁺]_i Measurements

HUVECs (Clonetics) were routinely cultured in 75-cm² flasks coated with human fibronectin (5 μg/cm²) in RPMI 1640 medium containing 10% fetal bovine serum, 100 IU penicillin, 100 μg/mL streptomycin, 2 mmol/L glutamine, heparin (100 μg/mL), and 30 μg/mL endothelial cell growth supplement (ECGS, Sigma Chemical Co). HUVECs, used from the third to the sixth passage, were detached with a nonenzymatic cell dissociation solution (Sigma) and resuspended in physiological salt solution (composition in mmol/L: NaCl 145, KCl 5, MgCl₂ 1, CaCl₂ 10, glucose 5.6, and HEPES/NaOH 5, pH 7.4.) containing fura 2-acetoxymethylester (1 μmol/L) and incubated at 37°C for 30 minutes as previously described.⁶ For the preincubation experiments, HUVECs were incubated for 10 minutes at 37°C with the various proteases at the indicated concentrations. After 2 washes in physiological salt solution, cells were resuspended (3×10⁴ cells/mL), and the experiments were performed at 37°C under constant stirring in a spectrofluorometer.

Binding Experiments

¹²⁵I-labeled factor Xa binding experiments were performed on cell monolayers cultured in 24-well cluster plates (3×10⁵ cells/well) as previously described.³⁶ Medium was aspirated, and the cells were washed twice with buffer containing (in mmol/L) NaCl 137, KCl 4, glucose 11, EDTA 10, and HEPES 10, pH 7.45. Cells were then incubated at 4°C with 200 μL of the same buffer without EDTA containing 0.5% bovine serum albumin and 5 mmol/L CaCl₂ in the presence of human ¹²⁵I–factor Xa (1 nmol/L; 2000 Ci/mmol, Amersham) and the tested compounds. After 120 minutes, the buffer was aspirated and the cells washed 3 times with ice-cold binding buffer. Cells were then digested with 1 mL of 0.2N NaOH, 1% SDS, and 10 mmol/L EDTA for 5 minutes, and the resulting solution was counted in a gamma counter. Results for equilibrium binding experiments were analyzed as described.¹⁰

Hydrolysis of PAR Peptides

The PAR-1 peptide (biotinyl-LDPRSFLLRNPNDKYEPFWED-EEE-Edans), PAR-2 peptide (biotinylaminocaproyl-RSSKGRSLIGKVDGTSHVTGKE-Edans), or mutated PAR-2 peptide (biotinylaminocaproyl-RSSKGASLIGKVDGTSHVTGKE-Edans; Neosystem) was solubilized at a final concentration of 10 μmol/L in HEPES buffer containing (in mmol/L) NaCl 137, KCl 4, glucose 11, and CaCl₂ 5,pH 7.45, and incubated for 30 minutes at 37°C in the presence of 100 nmol/L factor Xa, trypsin, or thrombin. The reaction was stopped by the addition of 10 μmol/L DX9065 (Daiichi Pharmaceuticals), 100 μg/mL soybean trypsin inhibitor (SBTI), or 200 nmol/L hirudin (Sanofi∼Synthélabo), respectively. The solution was then incubated for 30 minutes at 37°C under constant stirring in the presence of streptavidin-coated beads (Sigma). The samples were centrifuged, and the optical density at 360 nm was measured on the supernatants.

To determine the level of hydrolysis of the PAR peptides by HUVEC-bound factor Xa, confluent HUVECs (3×10⁵ cells/well) cultured in 24-well cluster plates were rinsed and preincubated for 120 minutes at 4°C with factor Xa (10 nmol/L) diluted in buffer (HEPES buffer containing 137 mmol/L NaCl, 4 mmol/L KCl, 11 mmol/L glucose, and 5 mmol/L CaCl₂, pH 7.45) in the absence or presence of the anti–EPR-1 monoclonal antibody B6 (100 μg/mL; a kind gift of Dr D. Altieri, Yale University, New Haven Conn), L₈₃-L₈₈-G (10 μmol/L), or DX9065 (1 μmol/L). Unbound factor Xa was removed by washing 6 times with binding buffer, and the cells were incubated for 30 minutes at 37°C with 10 nmol/L ¹²⁵I–PAR-1 and ¹²⁵I–PAR-2 peptides (Amersham). The reaction was stopped by adding 10 μmol/L DX9065, and 100 μL of the supernatant was incubated for 30 minutes under constant stirring in the presence of streptavidin-coated beads. The samples were centrifuged and rinsed, and the radioactivity was measured by scintillation counting. The same experiments without factor Xa were performed as controls.

Phosphoinositide Turnover in Transfected HEK293 Cells

Human embryonic kidney (HEK) 293 cells expressing PAR-1 and PAR-2 were generated by transfecting wild-type HEK293 cells by calcium phosphate precipitation and collecting the cells 72 hours later for transient expression. Expression vectors for PAR-1 and PAR-2 were kind gifts of Dr J.C. Chambard (URM 6543, CNRS, Nice, France). Phosphoinositide turnover was measured on confluent cell monolayers as described by Berridge et al.¹¹ In brief, cells were incubated for 72 hours in normal-culture medium containing myo-[³H]inositol (5 μCi/mL). The cell monolayers were then washed twice with phosphate-buffered saline and incubated for 30 minutes with phosphate-buffered saline containing 20 mmol/L LiCl. The cells were then stimulated in the same medium with 100 nmol/L factor Xa, 100 μmol/L TFLLRNPNDK, 10 nmol/L thrombin, or 100 μmol/L SLIGKV for an additional 30 minutes at 37°C. At the end of the incubation period, buffer was aspirated and the cells were extracted with an ice-cold methanol/0.1N HCl (50/50, vol/vol) solution for 30 minutes. Extracts were then neutralized with 1 mol/L Na₂CO₃, and [³H]inositol monophosphate was separated on columns containing 1 mL of AG1-X8 resin.

Immunohistochemical Detection of Cleaved PAR-1 and PAR-2 on HUVECs

PAR-1 and PAR-2 immunohistochemistry was performed on cultured HUVECs that were fixed with 4% formaldehyde. Cell monolayers were reacted with an anti–PAR-1 monoclonal antibody recognizing the N-terminal peptide 35-46 of the receptor (Immunotech) or with an anti–PAR-2 monoclonal antibody recognizing the N-terminal peptide 23-32 of the receptor (a kind gift of Dr D. Altieri, Yale University, New Haven, Conn). The cells were then incubated for 30 minutes at 4°C with a biotinylated secondary anti-IgG monoclonal antibody (Sigma) in blocking buffer. Cells were rinsed and incubated with avidin–horseradish peroxidase complex (Vector Laboratories) for 30 minutes and developed for 10 minutes with a fluorescein-tyramine complex (NEN Life Science). The stained cells were mounted by using the ProLong Antifade kit (Molecular Probes). For quantification of the cleaved PARs, a similar method was used, except that a ¹²⁵I–anti-IgG monoclonal antibody (Amersham) was used to detect the presence of the primary antibodies. Triplicate incubations were performed and terminated by the addition of 3 mL of ice-cold assay buffer. Cells were then rinsed and incubated for 30 minutes in 0.2 mL of 0.5N NaOH, 1% SDS, and 10 mmol/L EDTA, followed by rapid vacuum filtration over glass-fiber filters (Skatron Instruments Inc). Filters were then washed twice with 5 mL of ice-cold incubation buffer and dried, and the radioactivity was measured by scintillation counting. Nonspecific binding was defined as the binding of the ¹²⁵I–anti-IgG antibody measured in the absence of the primary antibody, and specific binding was defined as the difference between total binding and nonspecific binding.

Results and Discussion

To determine whether factor Xa could activate HUVECs in such a PAR-dependent, EPR1-mediated manner, we measured the effect of factor Xa on intracellular Ca²⁺ increase in HUVECs. This parameter has been previously found to be highly sensitive not only to this protease but also to others, such as thrombin, trypsin, SFLLRN, and SLIGKV, the latter 2 peptides that activate PAR-1 and PAR-2, respectively, on vascular endothelial cells.⁹ These agonists induced an intracellular Ca²⁺ increase in HUVECs in a dose-dependent manner (Figure 1A). In this cell system, in which EPR-1 was detected by Western blotting (not shown), factor Xa induced an intracellular Ca²⁺ increase in an EPR-1–dependent manner, as demonstrated by the inhibitory effect of B6, a monoclonal anti–EPR-1 antibody that has been described several times as an inhibitor of binding of factor Xa to vascular endothelial and smooth muscle cells,¹⁵⁶ or with Leu₈₃-Leu₈₈-G, a peptide representing the interconnecting EGF sequence in factor Xa, both of which blocked the binding of factor Xa to EPR-1 on HUVECs and inhibited its effect on intracellular Ca²⁺ (Figures 1B and 1C and Table 1). Comparative dose-response curves for L₈₃-L₈₈-G and B6 demonstrated a good correlation between their effect on the binding of ¹²⁵I–factor Xa to HUVECs and their effect on the calcium response evoked by factor Xa on these cells (Figure 1C). It is noteworthy that at maximal factor Xa binding–inhibitory concentrations (10 μmol/L and 100 μg/mL, respectively), neither L₈₃-L₈₈-G nor B6 affected the catalytic activity of factor Xa (not shown). Moreover, a nonrelevant monoclonal antibody (anti–ELAM-1), as well as a “scrambled” peptide of the Leu₈₃-Leu₈₈-G sequence, hardly affected the effect of factor Xa on intracellular calcium (not shown).

The first evidence of an original mode of receptor activation by factor Xa came from the observation of an inhibitory effect of DX9065, a direct inhibitor of factor Xa¹² that strongly affected the factor Xa–induced intracellular Ca²⁺ increase, with an IC₅₀ of 0.23 μmol/L, but did not interfere with the binding of factor Xa to EPR-1 (Figure 1B and Table 1). This effect of factor Xa was not affected by high doses of aprotinin or hirudin, which shows that it was not due to thrombin or trypsin generation that might have occurred at the cell surface (Table 1). In this cell system, we also found that preincubation of the cells with pertussis toxin (50 ng/mL) strongly affected (86% inhibition, P<0.001) 100 nmol/L factor Xa–induced intracellular Ca²⁺ increase (not shown), indicating that factor Xa not only binds to EPR-1 (which is not coupled to G proteins) but also interacts with and activates a specific G protein–coupled receptor. This represents further evidence for an interaction with a PAR receptor, shown to be coupled to G proteins.⁹ To gain further insight into the nature of this receptor (ie, whether it is thrombin- or trypsin-sensitive), we performed desensitization experiments. As shown in Figure 2, when HUVECs were first challenged with thrombin or with the PAR-1 agonist SFLLRN, a second challenge with these compounds was ineffective, thus showing complete desensitization of the PAR-1 receptor. Under these conditions, factor Xa, the PAR-2 agonist peptide SLIGKV, or trypsin still elicited a marked intracellular Ca²⁺ increase (Figure 2 and Table 2) on these thrombin-desensitized cells, therefore showing that factor Xa does not activate HUVECs by means of selective activation of PAR-1. The same results were found with TFLLRNPNDK, a peptide that has been recently shown to activate PAR-1 selectively with no effect on PAR-2¹³ (Figure 2). On the contrary, on HUVEC in which the calcium response to trypsin or SLIGKV was desensitized, the response to factor Xa was abolished (Figure 2 and Table 2), therefore showing that desensitization of HUVECs for the PAR-2 response hinders factor Xa activation of the cells. Under these conditions, preincubation of the cells with trypsin (100 nmol/L, 10 minutes, 37°C) did not affect the binding of ¹²⁵I–factor Xa to HUVECs, thus showing that desensitization of the factor Xa response did not occur as a result of nonspecific digestion of EPR-1 but rather at the level of PAR-2 that was desensitized in HUVECs after incubation with trypsin.¹⁴ This observation of an effect of factor Xa on PAR-2 was further confirmed on cells preincubated with factor Xa that, in this case, were desensitized not only for factor Xa itself (Figure 2) but also for the selective PAR-2 activator, SLIGKV, whereas selective PAR-1 activation was unaffected (Figure 2). Under these latter experimental conditions, the effect of trypsin was not totally reduced (50% inhibition, P<0.05), an effect that could have been due to a nonspecific effect of this protease on PAR-1⁹¹⁴ but that could also be explained by a possible “recycling” of PAR-2 at the cell surface during the experiment performed at 37°C, thus allowing partial resensitization of the cells to this protease. In an attempt to further clarify this point, we performed the same experiments in the presence of brefeldin A, a compound that has been shown, by disrupting Golgi stores of PAR-2, to strongly attenuate resensitization of the calcium response of HUVECs to trypsin.¹⁵ Under these conditions (incubation with 10 μg/mL brefeldin A), we found that low concentrations of trypsin (10 nmol/L) totally desensitized the cells for additional stimulation with trypsin or factor Xa (10 nmol/L) but were without effect on the activity of thrombin (10 nmol/L), thus confirming our earlier observation of an effect of factor Xa via PAR-2 only.

At this stage, although we have shown that factor Xa acted in an EPR-1–dependent manner via selective activation of PAR-2, we prepared to demonstrate that this protease cleaved PAR-2 in vitro and on the cells. For this purpose, we incubated factor Xa in the presence of synthetic peptides representing the N-terminal extracellular domains of PAR-1 and PAR-2, cleaved after incubation of the cells with thrombin and trypsin, respectively. As shown in Figure 3A, factor Xa, either alone or in the presence of calcium or factor Va, did not hydrolyze the synthetic 23-mer peptide corresponding to the sequence present in PAR-1. This peptide was optimally cleaved by thrombin (33% hydrolysis) and slightly hydrolyzed by trypsin (8% hydrolysis). On the contrary, trypsin and factor Xa cleaved the synthetic 22-mer PAR-2 peptide (Figure 3B) at a site that corresponded to the expected cleavage site of trypsin and tryptase (Arg-9; not shown). This peptide was not cleaved by thrombin (3% hydrolysis). Proteolytic cleavage of the N-terminal extracellular domain of the PAR-2 receptor by factor Xa was highly selective, as shown by the lack of efficacy of this protease for a “mutated” PAR-2 peptide, in which Arg-9 was replaced by Ala-9 (Figure 3C). It is noticeable that cleavage of the PAR-2 peptide by factor Xa occurred only in the presence of calcium but independently of factor Va, thus explaining why other authors¹⁶ who performed similar experiments, but without calcium, did not find any cleavage of PAR-2 by factor Xa.

Similarly, the radiolabeled PAR-1 peptide was not cleaved by factor Xa preincubated with HUVECs under conditions where increased prothrombin activation by factor Xa bound to EPR-1 was already demonstrated,⁶ whereas under the same experimental conditions, the radiolabeled PAR-2 peptide was almost totally hydrolyzed by HUVEC-bound factor Xa (Figure 3E). This hydrolysis by factor Xa was strongly reduced in the presence of DX9065 or after preincubation of the cells with the L₈₃-L₈₈-G peptide or B6, 2 compounds that have been shown to inhibit the binding of factor Xa to EPR-1 on HUVECs³ (Figure 1B). When a higher concentration of DX9065 (10 μmol/L) was used, total inhibition of the activity of HUVEC-bound factor Xa was observed (98% inhibition, P<0.001). It is notable that hydrolysis of the PAR-2 peptide by HUVEC-bound factor Xa occurred at much lower enzyme and substrate concentrations than those used in the purified systems, which suggests that EPR-1 acts as a cofactor for factor Xa to cleave PAR-2. In this respect, the role of EPR-1 might be very similar to that reported for factor V/Va that, in the presence of anionic phospholipids and calcium, strongly enhances thrombin generation by factor Xa.³

The requirement of PAR-2 for factor Xa to activate the cells was further demonstrated in HEK293 cells (which constitutively express EPR-1, as assessed by Western blotting; not shown). Factor Xa could only increase the production of inositol monophosphate when PAR-2 was present and showed no effect on wild-type cells or when PAR-1 was transfected (Figure 4).

To further prove that factor Xa cleaves PAR-2 at the surface of HUVECs and “unmasks” a new amino terminus that then serves as a tethered peptide ligand, we have an antibody directed against the 40-mer peptide cleaved by proteases on PAR-2. This antibody allowed us to selectively detect, both qualitatively and quantitatively, the presence of PAR-2 in the nonactivated state (ie, not cleaved by proteases) at the surface of the cells. As shown in Figure 5, after incubation of HUVECs with factor Xa or trypsin (Figures 5c and 5d), the amount of uncleaved PAR-2 detected by the antibody strongly decreased (98% and 95% decrease, respectively; P<0.001). Incubation of the cells with thrombin, however, only slightly decreased the amount of uncleaved PAR-2 (Figure Vb). On the contrary, immunodetection of uncleaved PAR-1 by a similar method (Figures 5e through 5g) confirmed that PAR-1 was highly activated after incubation with thrombin (93% decrease, P<0.001), whereas it was not affected by factor Xa (Figure 5g).

In conclusion, we now report that factor Xa, in cooperation with its receptor on endothelial cells, EPR-1, cleaves PAR-2 by a novel mode of cascade receptor activation.

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Footnotes