© 2000 American Heart Association, Inc.
Thrombosis |
Factor Xa Activates Endothelial Cells by a Receptor Cascade Between EPR-1 and PAR-2
From Sanofi-Synthélabo Recherche, Toulouse, France.
Correspondence to J.M. Herbert, Cardiovascular/Thrombosis Research Department, Sanofi-Synthélabo Recherche, 195 route d’Espagne, 31036 Toulouse, France. E-mail jean-marc.herbert{at}sanofi.com
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Abstract |
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Abstract—In addition to its pivotal role in hemostasis, factor Xa binds to human umbilical vein endothelial cells through the recognition of a protein called effector cell protease receptor (EPR-1). This interaction is associated with signal transduction, generation of intracellular second messengers, and modulation of cytokine gene expression. Inhibitors of factor Xa catalytic activity block these responses, thus indicating that the factor Xa–dependent event of local proteolysis is absolutely required for cell activation. Because EPR-1 does not contain proteolysis-sensitive sites, we investigated the possibility that signal transduction by factor Xa requires proteolytic activation of a member of the protease-activated receptor (PAR) gene family. Catalytic inactivation of factor Xa by DX9065 suppressed factor Xa–induced increase in cytosolic free Ca2+ in endothelial cells (IC50=0.23 µmol/L) but failed to reduce ligand binding to EPR-1. In desensitization experiments, trypsin or the PAR-2–specific activator peptide, SLIGKV, ablated the Ca2+ signaling response induced by factor Xa. Conversely, pretreatment of endothelial cells with factor Xa blocked the PAR-2–dependent increase in cytosolic Ca2+ signaling, whereas PAR-1–dependent responses were unaffected. Direct cleavage of PAR-2 by factor Xa on endothelial cells was demonstrated by cleavage of a synthetic peptide duplicating the PAR-2 cleavage site and by immunofluorescence with an antibody to a peptide containing the 40–amino acid PAR-2 extracellular extension. These data suggest that factor Xa induces endothelial cell activation via a novel cascade of receptor activation involving docking to EPR-1 and local proteolytic cleavage of PAR-2.
Key Words: EPR-1 • PAR-2 • factor Xa • cells, endothelial
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Introduction |
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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+.3 4 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,7 8 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.
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Methods |
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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 (3x104 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
(3x105 cells/well) as previously
described.3 6 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 (3x105 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.
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Results and Discussion |
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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,1 5 6 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).
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), factor Xa (
),
trypsin (
), or SLIGKV (
) were added to fura 2–loaded HUVECs, and
intracellular free Ca2+ was measured. Results are means of
3 experiments performed in triplicate. B, Confluent cells were
incubated for 120 minutes at 4°C with increasing concentrations of
125I–factor Xa in the presence of saline (
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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-19 14 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.
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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.
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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).
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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).
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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.
Received March 1, 2000; accepted May 17, 2000.
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