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THROMBOSIS AND HEMOSTASIS
Biased agonism of protease-activated receptor 1 by activated protein C caused by
noncanonical cleavage at Arg46
Laurent O. Mosnier,1 Ranjeet K. Sinha,1 Laurent Burnier,1 Eveline A. Bouwens,1 and John H. Griffin1
1Department
of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA
Activated protein C (APC) exerts endothelial cytoprotective actions that require
protease-activated receptor 1 (PAR1),
whereas thrombin acting via PAR1 causes
endothelial disruptive, proinflammatory
actions. APC’s activities, but not thrombin’s, require PAR1 located in caveolae.
PAR1 is a biased 7-transmembrane receptor because G proteins mediate thrombin’s signaling, whereas ␤-arrestin 2 mediates APC’s signaling. Here we elucidate
novel mechanisms for APC’s initiation of
signaling. Biochemical studies of APC’s
protease specificity showed that APC
cleaved PAR1 sequences at both Arg41
and Arg46. That PAR1 cleavage at Arg46
can occur on cells was supported by
APC’s cleavage of N-terminal-SEAPtagged R41Q-PAR1 but not R41Q/R46QPAR1 mutants transfected into cells and
by anti-PAR1 epitope mapping of APCtreated endothelial cells. A synthetic peptide composing PAR1 residues 47-66,
TR47, stimulated protective signaling in
endothelial cells as reflected in Akt and
glycogen synthase kinase 3␤ phosphory-
lation, Ras-related C3 botulinum toxin
substrate 1 activation, and barrier stabilization effects. In mice, the TR47 peptide
reduced VEGF-induced vascular leakage.
These in vitro and in vivo data imply that
the novel PAR1 N-terminus beginning at
residue Asn47, which is generated by
APC cleavage at Arg46, mediates APC’s
cytoprotective signaling and that this
unique APC-generated N-terminal peptide tail is a novel biased agonist for
PAR1. (Blood. 2012;120(26):5237-5246)
Introduction
Activated protein C (APC) is a homeostatic serine protease that
provides beneficial effects via antithrombotic activities and also via
cytoprotective actions that are based on its cell-signaling properties.1 Wild-type APC and cytoprotective-selective APC mutants
reduce damage in vitro to cultured cells under stress and reduce
mortality and organ injury in multiple in vivo murine injury
models. Moreover, many reports, but not all studies, of APC’s
protective actions show that protease-activated receptor 1 (PAR1)
and the endothelial protein C receptor (EPCR) are required for
APC’s protective actions.1-5 PAR1, a G protein coupled receptor
(GPCR), which is also known as a 7-transmembrane receptor, is a
primary thrombin receptor on human platelets, and thrombintriggered signaling arises because of cleavage at the PAR1 canonical Arg41 site to generate the agonist N-terminal tethered ligand
that begins with residue Ser42, which causes PAR1-initiated
signaling.6-8 PAR1 as well as the other 3 known human and murine
PARs are activated on many cell types by many proteases with a
panoply of biologic activities.8,9
PAR1 mediates thrombin’s proinflammatory and endothelial
barrier disruptive actions.8,10 The conundrum then arose of how
PAR1 signaling resulting from cleavage at Arg41 could mediate
thrombin’s proinflammatory and endothelial barrier disruptive
actions as well as the opposite anti-inflammatory and barrier
stabilizing effects of APC.10-12 Some insights came from demonstrations that membrane localization of PAR1 with EPCR in caveolae,
or caveolin-1-rich microdomains, could influence PAR1-selective
signaling and that APC-PAR1-dependent transactivation of other
receptors, S1P1, PAR2, and Tie2, can arise.11,13-16 The selective
nature of thrombin and APC for PAR1-dependent effects on
endothelial barrier function is striking, as the former promotes Ras
homolog gene family member A (RhoA) activation whereas the
latter promotes Ras-related C3 botulinum toxin substrate 1 (Rac1)
activation—RhoA promotes endothelial barrier leakage and Rac1
promotes barrier stabilization.10,11,17 Thrombin and APC are biased
activators of PAR1 because thrombin requires G protein-dependent
signaling for RhoA activation, whereas APC requires ␤-arrestin
2-dependent signaling for Rac1 activation.18 PAR1 can be allosterically influenced by retention of thrombin on activated PAR1
because of thrombin’s binding to the hirudin-like sequence of
residues 51-56 and by potential association of PAR1 with EPCRprotein C/APC, which appears to alter the functional selectivity of
thrombin.19-23 PAR1 can be cleaved by MMP1 at Asp39 to generate
an N-terminus differing from that due to cleavage at Arg41, but this
alternative cleavage is not associated with remarkable differences
in PAR1 activities.24 Nonetheless, these multiple insights, which
show PAR1 to be a biased GPCR with multiple allosteric sites, fail
to define a clear mechanism for biased activation of PAR1 by
2 different proteases, namely, thrombin and APC, with their
remarkable differences in functional selectivity.
To explain the biased activation of PAR1 by APC, we hypothesized that PAR1-dependent signaling by APC involves a novel
cleavage of the receptor’s N-terminal domain, differing from that
of thrombin, which then reveals a novel cryptic intramolecular
pharmacophore that causes APC’s cytoprotective, biased signaling.
To test this hypothesis, we used mass spectroscopy, cells transfected with PAR1 constructs, synthetic peptides, cell-signaling
Submitted August 24, 2012; accepted October 24, 2012. Prepublished online
as Blood First Edition paper, November 13, 2012; DOI 10.1182/blood-2012-08452169.
The online version of this article contains a data supplement.
Presented in abstract form at the 53rd Annual Meeting of the American Society
of Hematology, San Diego, CA, December 10 and 12, 2011.33,34
BLOOD, 20 DECEMBER 2013 䡠 VOLUME 120, NUMBER 26
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2012 by The American Society of Hematology
5237
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5238
MOSNIER et al
assays, cytoprotective assays, and a murine vascular leakage injury
model. The extensive in vitro and in vivo data in this report provide
strong support for this hypothesis that provides a resolution for the
thrombin-APC-PAR1-signaling conundrum.
Methods
Materials
SCH79797 (Tocris), thrombin receptor activating peptide that is a synthetic
peptide (TFLLRNPNDK) resembling residues 42-51 of human PAR1
(TRAP; Anaspec), thrombin (ERL), and recombinant wild-type APC
(Xigris, Eli Lilly) were from commercial sources. Mouse anti-ERK1/2
(3A7), rabbit anti-pThr202/Tyr204-ERK1/2 (197G2), mouse anti-Akt
(40D4), rabbit anti-pSer473Akt (D9E), mouse anti–glycogen synthase
kinase 3␤ (GSK3␤; 3D10), and rabbit anti–pSer9-GSK3␤ (D3A4) were
from Cell Signaling. Anti-PAR1 mouse monoclonal antibodies SPAN12b,
ATAP2, and WEDE-15b (note the “b” denotes that this is a subcloned
version of the original published antibody)25 were kindly provided by
Dr L. Brass (University of Pennsylvania, Philadelphia, PA). Peptides (see
supplemental Figure 2 for sequences; available on the Blood Web site; see
the Supplemental Materials link at the top of the online article) were
synthesized and purified to ⬎ 95% purity (Biosynthesis).
Constructs
SEAP-PAR1 and wt-EPCR constructs were made as reported with a notable
modification.26 Three Arg residues at positions 25 (⫺1 of mature PAR1),
27 (2), and 28 (3) were mutated to GQQ to prevent significant APC and
thrombin-mediated cleavages at these sites in the N-terminal SEAP fusion
constructs. The linker sequence now contains (… PGYSG-1A1QQP …)
with the SEAP C-terminus underlined, the mature PAR1 N-terminus in
italics and the GQQ mutations in bold. GQQ-SEAP-PAR1 is herein referred
to as wt-SEAP-PAR1 and was used to introduce the R41Q, R46Q, and
R41Q/R46Q mutations using Quickchange mutagenesis (Stratagene). SEAPPAR1s, either in the presence or absence of wt-EPCR, were transfected into
HEK-293 cells to obtain stable cells expressing the various SEAP-PAR1
constructs.
Cells
HEK-293 (ATCC) and EA.hy.926 (Dr C. J. S. Edgell, University of North
Carolina, Chapel Hill, NC) were grown and maintained as described.26
PAR1 cleavage assays
Cleavage of the PAR1 synthetic peptide representing residues 33-62 of the
PAR1 N-terminal tail by APC and thrombin was performed as described.26
All reactions contained 50␮M TR33-62 peptide (supplemental Figure 2),
500nM APC or 10nM thrombin in HEPES-buffered saline (25mM HEPES,
pH 7.4, 147mM NaCl, and 4mM KCl) with 4mM CaCl2 and 0.6mM MgCl2
unless stated otherwise. Molecular masses of isolated peptide fragments
were determined by the Scripps Research Institute Mass Spectrometry and
Metabolomics facility. Analysis of PAR1 cleavage by APC or thrombin
using the SEAP-PAR1 cleavage reporters was done in 96-well plates as
described.26 After correction for background activity in the absence of
protease, values were expressed as percentage of the total SEAP activity
present on the cells.
Mapping of the canonical and noncanonical cleavage site
sensitive epitopes of anti-PAR1 antibodies
To determine the specific binding of antibodies WEDE15, ATAP2, and
SPAN12 to PAR-1 peptides (TR24-41, TR24-46, TR42-66, TR47-66, and
TR33-62), peptides were coated at 10␮M on Maxisorp 96-well plates,
followed by 2-hour incubation with TBS/3% (weight/volume) BSA to block
the plates. Antibodies were diluted to 2 ␮g/mL in TBS/3% (weight/volume)
BSA, and incubated for 1 hour at room temperature. Anti–mouse HRP-
BLOOD, 20 DECEMBER 2013 䡠 VOLUME 120, NUMBER 26
conjugated secondary antibody (Dako North America) was used to detect
bound primary antibodies at A490nm.
On Cell Western (OCW) assays for anti-PAR1 antibodies
EA.hy.926 cells were grown in black clear-bottom 96-well plates (Corning)
until confluent. Cells were washed and incubated at 37°C with thrombin
(0.25nM) or APC (100nM) in HMM2 buffer (HBSS; Invitrogen) supplemented with 1.3mM CaCl2, 0.6mM MgCl2, and 0.1% endotoxin free BSA
(Calbiochem) for 3 hours. After a quick wash with ice-cold HMM2, cells
were fixed in methanol free 4% paraformaldehyde (Pierce Chemical) for
20 minutes at room temperature after which plates were blocked in Odyssey
blocking buffer supplemented with 3% BSA overnight. Subsequently,
plates were incubated with mouse anti-PAR1 antibodies SPAN12b, ATAP2,
or WEDE-15b (all 10 ␮g/mL) in Odyssey blocking buffer, followed by
1/1000 biotinylated goat antimouse and 1/4000 IRDye 800CW Streptavidin
(LI-COR) with 1/10 000 Draq5 (Biostatus). PAR1 on the cell surface was
detected using the In Cell Western module of Odyssey Imager with Image
Studio Software Version 2.0. Fluorescence signals were corrected for cell
number and background staining, and signals were normalized to buffer
only controls at the corresponding time point.
Western blotting
EA.hy.926 cells were grown in 6-cm dishes (3 ⫻ 106 cells per plate) for
48 hours and serum starved overnight. Peptides (50␮M), APC (70nM), or
thrombin (54nM) in serum-free media were added to the cells for different
time points as indicated. In some experiments, cells were pretreated with
SCH79797 (1␮M) or vehicle control (DMSO) in serum-free media for
20 minutes. Cells were washed with cold Dulbecco PBS (Invitrogen), and
cell lysates were made with 200 ␮L of 1.5% NP-40 lysis buffer containing
1 times protease and phosphatase inhibitor cocktail (Pierce Chemical).
After clarification (30 minutes, 14 000 rpm), lysates (100 ␮g) were mixed
with reducing SDS sample buffer (LI-COR) and separated on 12%
SDS-PAGE (Bio-Rad). Proteins were transferred to Immobilon-FL PVDF
membrane (Millipore), blocked with Odyssey Blocking Buffer (LI-COR),
and incubated with a mouse-rabbit combination of pan and phosphospecific antibodies against either Akt, GSK3␤ and ERK1/2. Blots were
developed with IRDye 680 and IRDye 800CW donkey antimouse or
donkey antirabbit secondary antibodies (LI-COR) and scanned on the
Odyssey Imager (LI-COR). Quantification of integrated fluorescence
intensity (K counts) was done using Odyssey Application Version 3.0
software (LI-COR).
Rac1 activation
The pGEXTK-PAK1 70-117 construct encoding a GST fusion to p21activated kinase (PAK-1)–binding domain was kindly made available by Dr
J. Chernoff (Fox Chase Cancer Center, Philadelphia, PA; Addgene plasmid
12217). GST-PAK1was purified from transformed BL21 (DE3) Escherichia coli using B-Per lysis buffer (Pierce Chemical) with lysozyme,
DNAse I, and Halt EDTA-free protease inhibitor cocktail (Pierce Chemical)
on glutathione-agarose according to the manufacturer’s recommendation.
Pull-down of active Rac1 was performed as described.27 Briefly, endothelial
EA.hy.926 cells (5 ⫻ 106 per plate) were grown in 100-mm dishes for
48 hours and serum starved overnight before addition of peptides (50␮M)
for 30 or 180 minutes. Lysates (2 mg) were mixed with GST-PAK1
glutathione-agarose (150 ␮g) and after washing active GTP-Rac1 was
eluted from GST-PAK1 glutathione-agarose by boiling in reducing SDS
sample buffer (LI-COR). Active GTP-Rac1 was resolved on 12% SDSPAGE, transferred to Immobilon-FL PVDF membrane, and immunoblotted
with a mouse anti-Rac1 antibody (BD Biosciences) and IRDye 800CW
donkey anti–mouse secondary antibodies (LI-COR). Immunoblots were
scanned on the Odyssey Imager (LI-COR). Quantification of integrated
fluorescence intensity (K counts) was done using Odyssey Application
Version 3.0 software (LI-COR).
Endothelial barrier protection
Permeability of endothelial cell barrier function was determined as
described with minor modifications. Briefly, EA.hy.926 endothelial cells
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BLOOD, 20 DECEMBER 2013 䡠 VOLUME 120, NUMBER 26
BIASED AGONISM OF PAR1 BY ACTIVATED PROTEIN C
5239
Table 1. PAR1 peptide fragments generated by APC or thrombin proteolysis
Fragment
Retention time, min
Cleaved by
Exp mass, Da
Calculated mass, Da
A
8.2
IIa/APC
1001
1000
B
9.7
APC
635
635
C
11.0
APC
2053
2053
D
12.6
IIa/APC
2670
E
13.8
—
3651
PAR1 peptide (33-62)
ATNATLDPR-41
Cleavage site
R41
42-SFLLR-46
R46
47-NPNDKYEPFWEDEEKN
R46
2670
42-SFLLRNPNDKYEPFWEDEEKN
R41
3652
ATNATLDPRSFLLRNPNDKYEPFWEDEEKN
—
— indicates not applicable.
(5 ⫻ 104 cells/well) were grown on polycarbonate membrane Transwell
inserts (Costar, 3-␮m pore size, 12-mm diameter). When confluent, cells
were incubated with APC (20nM), TR47 (50␮M), or a synthetic peptide
containing a scrambled, randomized sequence composed of amino acid
residues 47-66 of human PAR1 (scrTR47; 50␮M) for 4 hours in serum-free
media with 0.1% BSA (fatty acid poor and endotoxin free fraction V,
Calbiochem). After incubation with thrombin (10nM) for 10 minutes, the
media in the inner chamber was replaced with complete medium containing
4% BSA and 0.67 mg/mL Evans blue. Endothelial cell permeability was
determined by absorbance of Evans blue in the outer chamber at 650 nm.
Permeability was expressed as the fold change in absorbance compared
with that in the absence of thrombin (normalized to 1).
In vivo vascular permeability assay
The study was approved by the Institutional Animal Care and Use
Committee of the Scripps Research Institute and complied with National
Institutes of Health guidelines. SKH1-E hairless male (6-8 weeks old) were
from Charles River Laboratories. Vascular permeability was determined
using a VEGF-induced leakage model with some modifications.28,29 Briefly,
100 ␮L of a sterile-filtered solution containing 0.5% (weight/volume)
Evans blue (Sigma-Aldrich) in 0.9% NaCl (Sigma-Aldrich) was injected in
isoflurane-anesthetized mice. After 30 minutes, 50 ␮L of peptides (125 ␮g;
TR47 or scrTR47) or PBS was injected intravenously in the retro-orbital
sinus of mice anesthetized with ketamine-xylazine (100 and 10 mg/kg,
respectively). After 5 minutes, mice received subcutaneously 15 ␮L of
75 ng/injection recombinant mouse VEGF165 (BioVision) in 0.1% BSAPBS (3 sites on the right side of the abdomen) or vehicle (2 sites on the left
side). After 30 minutes, mice were killed, photographed, and Evans blue
extravasation in the skin was determined using the Odyssey Imager
(LI-COR) in the 700-nm channel with 4-mm offset. Quantification of the
intensity of the Evans blue dye signal was done using the Odyssey
Application Software Version 3.0. A mean value for 3 data points (VEGF)
or 2 data points (BSA; injection sites) was made for each mouse and
normalized to the VEGF sites in PBS-injected mice. In total, 5 independent
experiments were performed using a total of 23 mice (n ⫽ 11 for PBS,
n ⫽ 6 for TR47, and n ⫽ 6 for scrTR47).
Statistical analysis
Statistical significance (P ⬍ .05) was determined using Student t test or
1-way ANOVA and Bonferroni multiple comparison posttests as appropriate (Prism Version 5.0 software, GraphPad Software).
APC in the presence of CaCl2 and MgCl2, it became evident that
initially APC generated 2 fragments (retention time 8.2 and
12.6 minutes compared with 13.8 minutes for the parental TR33-62
peptide; Table 1) that were identical to the 2 fragments generated
by thrombin (Figure 1A). In time, APC, but not thrombin,
generated 2 additional fragments (retention time 9.7 and 11.0 minutes; Table 1) that at least were partially derived from the initial
12.6 minutes cleavage fragment. The fact that no additional
fragments were observed after more than 5 days of TR33-62
incubation with APC suggest that these second APC-derived
fragments were derived from a specific cleavage rather than from
nonspecific proteolysis. To identify the second APC cleavage site,
peptide fragments were isolated and their molecular mass was
determined by mass spectrometry (supplemental Figure 1). As
anticipated, molecular mass of the initial APC and thrombin
generated fragments (peak A, 1001 Da; and peak D, 2670 Da)
corresponded to peptide fragments TR33-41 and TR42-62, respectively, consistent with cleavage at Arg41 (Table 1). The APCspecific fragments (peak B, 635 Da; and peak C, 2053 Da) were
consistent with cleavage at Arg46 and the generation of TR42-46
and TR47-62 peptide fragments. Thrombin cleaved TR33-62
(Figure 1B) at Arg41 completely within 60 minutes with the
concomitant generation of equimolar concentrations of TR3341(Figure 1C) and TR42-62 (Figure 1D). In contrast, complete
TR33-62 cleavage by APC required ⬎ 30 hours; and although
TR33-41 was generated at the expected equimolar concentration
(Figure 1C), TR42-62 never reached more than ⬃ 40% of its
potential maximum molar concentration (Figure 1D). Instead,
similar rates were observed for generation of the TR42-62 fragment
and generation of the TR42-46 and TR47-62 fragments derived
from cleavage at Arg46 (Figure 1E). The formation of TR42-62
seemed not to be the rate-limiting step for the generation of
TR42-46 and TR47-62 fragments as APC produced these fragments at approximately similar rates from a synthetic TR42-66
peptide (Figure 1F). Thrombin did not cleave the TR42-66
fragment. Thus, in a purified system, APC cleaved a synthetic
PAR1 N-terminal tail peptide, TR33-62, at both Arg41 and Arg46,
whereas thrombin cleaved TR33-62 only at Arg41 (Table 1).
Cleavage of PAR1 at Arg46 by APC on cells
Results
Identification of an APC-specific noncanonical cleavage in
PAR1
Previously, we have shown that APC cleaved a synthetic PAR1
N-terminal tail peptide (TR33-62), although proteolysis by APC
was slow and required prolonged incubation with the peptide.26 It
was noted at the time that additional proteolytic fragments were
generated by APC that became apparent after 20 hours and that the
appearance of these fragments were less obvious in the presence of
EDTA. On additional characterization of TR33-62 cleavage by
To determine whether APC cleaved PAR1 at Arg46 on cells, a
SEAP-PAR1 fusion construct was used that readily detects PAR1
cleavage when transfected into HEK-293 cells by the release of
alkaline phosphatase activity (SEAP) in the medium. Previously,
we have used this system to demonstrate the important cofactor
function of the EPCR for the cleavage of PAR1 by APC and the
characterization of PAR1 cleavage by cytoprotective-selective and
anticoagulant selective APC mutants.30 However, we discovered
that such PAR1 reporter constructs are subject to potential artifacts
when PAR1 cleavage site mutations at Arg41, Arg46, or both
residues were made because thrombin and APC could still readily
release SEAP into the media despite these cleavage site mutations,
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A
BLOOD, 20 DECEMBER 2013 䡠 VOLUME 120, NUMBER 26
MOSNIER et al
B
A214 (mAU)
2000
0 hr
16 hr
1000
46 hr
500
68 hr
6
8
10
12
Time (min)
14
30
20
0
16
C
40
10
122 hr
0
TR33-62
APC
IIa
50
APC
1500
TR33-62 (µM)
5240
0
5
10
15
20
Time (hr)
25
30
D
50
40
30
20
TR33-41
APC
IIa
10
0
0
5
10
15
20
Time (hr)
25
TR42-62 (µM)
TR33-41 (µM)
50
TR42-62
APC
IIa
30
20
10
0
30
E
40
Figure 1. APC cleaves a synthetic PAR1 N-terminal
peptide at Arg41 and Arg46. Cleavage of a PAR1
N-terminal tail peptide (TR33-62) by APC (500nM) or
thrombin (10nM) in the presence of CaCl2 (4mM) and
MgCl2 (0.6mM) and resolution of cleavage fragments
by HPLC using C18 reverse phase chromatography.
(A) Chromatograms of APC-mediated TR33-62 cleavage over time. (B) Time course of TR33-62 cleavage by
APC (䡺) or thrombin (f). (C) Generation of the
N-terminal cleavage fragment derived from cleavage at
Arg41 (TR33-41) by APC (ƒ) or thrombin (). (D) Generation of the C-terminal cleavage fragment derived
from cleavage at Arg41 (TR42-62) by APC (⌬) or
thrombin (Œ). (E) Generation of the N-terminal TR42-46
(〫, ⽧) and C-terminal TR47-62 fragments (E, F)
derived from cleavage at Arg46 by APC (open symbols)
or by thrombin (closed symbols). (F) Time courses for
cleavage of peptide TR42-66 (䡺, f) and generation of
TR42-46 (〫, ⽧) and TR47-66 (E, F) cleavage fragments by APC (open symbols) or thrombin (closed
symbols). Representative chromatograms (A) and
(B-F) data points represent the mean ⫾ SD (N ⱖ 3).
0
5
10
15
20
Time (hr)
10
20
Time (hr)
25
30
TR42-66 fragments (µM)
TR42-62 fragments (µM)
F
50 TR42-46
APC
40
IIa
TR47-62
APC
IIa
30
20
10
0
0
5
10
15
20
Time (hr)
25
30
50
40
30
20
10
0
0
especially at higher concentrations of enzyme. We showed that Arg
residues in the SEAP-PAR1 linker or either Arg27 or Arg28
(residues 2 and 3 of mature PAR1) were the sites for undesired
cleavages because mutation of these 3 Arg residues to Gly/Gln/Gln
abolished cleavage of R41Q-SEAP-PAR1 by thrombin. Thus, to
avoid misleading or erroneous conclusions from SEAP-PAR1
cleavage studies that use constructs that have these 3 Arg residues
intact, constructs lacking these 3 Arg residues are needed. Consistent with previous reports, APC cleaved GQQ-wt-SEAP-PAR1
(hereafter referred to as wild-type [wt]) in the presence of wt-EPCR
at concentrations ranging between 1nM and 100nM that are
typically required to convey PAR1-dependent cytoprotective effects on cells in vitro (Figure 2A). Half-maximal cleavage of
wt-SEAP-PAR1 in the presence of wt-EPCR required 23nM APC
compared with 0.31nM thrombin (Figures 2A-B). Mutation of the
canonical thrombin cleavage site Arg41 to Gln resulted in a 5-fold
shift of the APC dose-response curve (R41Q EC50 133nM),
whereas mutation of noncanonical APC cleavage site in PAR1 at
Arg46 almost completely ablated APC-mediated PAR1 cleavage
(extrapolated R46Q EC50 961nM; Figure 2A). Thus, APC could
cleave PAR1 on cells in the presence of EPCR at both Arg41 and
Arg46. In contrast to APC, thrombin cleaved R46Q-SEAP-PAR1
like wt (R46Q EC50 0.66nM), whereas no cleavage by thrombin
could be observed for R41Q-SEAP-PAR1, confirming that thrombin cleaved PAR1 at Arg41 and not at Arg46 (Figure 2B). No
APC-mediated cleavage of PAR1 was observed when both Arg41
and Arg46 were mutated, indicating that Arg41 and Arg46 compose
30
the only PAR1 cleavage sites for APC in the presence of EPCR
(Figure 2A). EPCR was identified as an essential cofactor to APC
for the cleavage and activation of PAR1 as no cleavage of
R41Q-SEAP-PAR1 or R46Q-SEAP-PAR1 was observed in the
absence of EPCR, indicating that cleavage at Arg46, like Arg41, by
APC required EPCR (Figure 2C). Consistent with the results in the
presence of EPCR, when EPCR was absent, thrombin cleaved
wt-SEAP-PAR1 and R46Q-SEAP-PAR1 similarly, but no cleavage
of R41Q-SEAP-PAR1 by thrombin could be observed (Figure 2C).
To determine the PAR1 proteolysis profile by APC on endothelial cells expressing endogenous PAR1 and EPCR, an OCW assay
was set up using different anti-PAR1 antibodies whose epitopes are
differentially affected by cleavage at either Arg41 or Arg46.
SPAN12 is a cleavage-sensitive antibody (raised against residues
35-NATLDPRSFLLR-46)25 that bound to intact PAR1 (TR33-62)
but not to PAR1 cleaved at either Arg41 (TR42-66) or Arg46
(TR47-66); this antibody recognized the N-terminal part of PAR1
after cleavage at Arg46 (TR24-46) but not at Arg41 (TR24-41;
Figure 2D). The epitope of ATAP2 was mapped to residues
43-SFLLR-46 located just downstream of the cleavage site at
residue Arg41.31 ATAP2 recognized intact PAR1 (TR33-62) and
PAR1 cleaved at Arg41 (TR42-66) but not PAR1 cleaved at Arg46
(TR47-66) or the N-terminal part of PAR1 after cleavage at either
Arg41 (TR24-41) or Arg46 (TR24-46; Figure 2D). WEDE15
(raised against residues 51-KYEPFWEDEEKNES-64)25 bound
intact PAR1 (TR33-62) and cleaved PAR1 equally well regardless
of whether cleavage occurred at Arg41 (TR42-66) or Arg46
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wt
R41Q
R46Q
R41Q/R46Q
100
80
60
40
20
0
0 0.1
1
10
APC (nM)
SEAP-PAR1 cleavage (%)
B
100
5241
wt
R41Q
R46Q
R41Q/R46Q
100
80
60
40
20
0
0 0.01
0.1
1
thrombin (nM)
10
D
APC
R41Q
R46Q
40
20
0.25
0.00
100
no
ne
1
10
Enzyme (nM)
0.50
33
-6
2
0.1
0.75
47
-6
6
0
1.00
42
-6
6
0
SPAN12
ATAP2
WEDE15
1.25
24
-4
6
thrombin
WT
R41Q
80
R46Q
60
100
24
-4
1
SEAP-PAR1 cleavage (%)
C
Antibody binding (A490)
A
BIASED AGONISM OF PAR1 BY ACTIVATED PROTEIN C
PAR1 peptide
F
G
me
APC
thrombin
(TR47-66; Figure 2D). Using a novel OCW assay, which proved to
be a greatly improved cellular ELISA, allowed for careful normalization of the antibody-binding signal to the total cell number
(Figure 2E). Incubation of EA.hy.926 endothelial cells with APC
resulted in a rapid decease of SPAN12 reactivity (⬃ 15% after
1 hour and ⬍ 5% after 3 hours compared with 100% at t ⫽ 0),
whereas WEDE15 reactivity remained relatively constant (85%90%), indicating cleavage of PAR1 by APC (Figure 2F). Interestingly, a significant fraction of PAR1 molecules on cells was cleaved
by APC at Arg46, as is evident from the reduction of ATAP2
reactivity (70% at 60 minutes and ⬃ 50% at 180 minutes). In
contrast, incubation of EA.hy.926 endothelial cells with thrombin
resulted in a rapid decease of SPAN12 reactivity to ⬍ 5% after
15 minutes, whereas ATAP2 and WEDE15 reactivity remained
relatively constant (90% and 80% at 15 minutes and 75% and 65%
at 180 minutes, respectively), consistent with cleavage of PAR1 by
thrombin at Arg41 but not at Arg46 (Figure 2G). Thus, both data
derived from SEAP-PAR1 mutant cleavage constructs on transfected cells as well as data from cleavage site sensitive antibodies
on endothelial cells were consistent with noncanonical cleavage of
PAR1 by APC at Arg46. These data provide strong evidence
leading to a novel paradigm in which the opposite PAR1-dependent
cellular effects of APC versus thrombin are because of differential
activation of PAR1 caused by cleavage at Arg46 by APC versus
cleavage at Arg41 by thrombin.
Surface PAR1 (fold)
ATAP
rge
Dra
q5
WE
DE
me
rge
Dra
q5
AT
AP
WEDE
1.2 APC
WEDE15
1.0
0.8
ns
*
0.6
0.4
*
0.2
0.0
0
60
ATAP2
SPAN12
*
120
Time (min)
180
Surface PAR1 (fold)
E
NONE
Figure 2. Noncanonical cleavage of PAR1 at Arg46
by APC on cells. Cleavage of wt-SEAP-PAR1 and
SEAP-PAR1 cleavage site mutants by (A) APC and
(B) thrombin in the presence of wt-EPCR and in the
absence of EPCR (C). (䡺, f) represent wt-SEAPPAR1; (⌬, Œ), R41Q-SEAP-PAR1; (E, F), R46QSEAP-PAR1; and (〫, ⽧), R41Q/R46Q-SEAP-PAR1.
(C) Closed symbols indicate thrombin; and open
symbols, APC. (D) Mapping of the canonical and
noncanonical cleavage site sensitive epitopes of antiPAR1 antibodies SPAN12, ATAP2, and WEDE15
using synthetic peptides coated onto microtiter plate
wells. Peptides represent the N-terminal (TR24-41) or
C-terminal fragment (TR42-66) of PAR1 after cleavage at Arg41 or the N-terminal (TR24-47) or C-terminal
fragment (TR47-66) of PAR1 after cleavage at Arg46
or the entire cleavage site region of the PAR1
N-terminal tail (TR33-62). (E) OCW of anti-PAR1
antibodies WEDE15 and ATAP2 on EA.hy.926 endothelial cells after incubation with control buffer (NONE),
APC (100nM), or thrombin (0.25nM) for 3 hours.
Cell-bound anti-PAR1 antibodies were detected with
biotinylated goat anti–mouse secondary antibodies
and IRDye 800CW streptavidin in the 800-nm channel
of the Odyssey Imager (green). Draq5 was used for
cell number normalization and detected in the 700-nm
channel (red). Overlay of the Draq5 700 channel and
anti-PAR1 800-nm channel is indicated in yellow
(merge). The presence and loss of PAR1 epitopes on
endothelial cell surfaces were determined using OCW
quantification of cleavage site sensitive and insensitive PAR1 antibodies on EA.hy926 endothelial cells
versus time for incubation of cells with (F) APC
(100nM) or (G) thrombin (0.25nM). The cell surface
was probed for PAR-1 with (E) WEDE15 (detecting all
PAR1 regardless of cleavage at either Arg41 or
Arg46), (䡺) ATAP2 (detecting uncleaved and PAR1
cleaved only at Arg41), or (〫) SPAN12 (detecting
only uncleaved PAR1). (D-E) Representative experiment in triplicates. (A-C,F-G) Data points represent
the mean ⫾ SEM (N ⱖ 3). (F-G) *P ⬍ .001, compared with WEDE15. ns indicates not significant.
SEAP-PAR1 cleavage (%)
BLOOD, 20 DECEMBER 2013 䡠 VOLUME 120, NUMBER 26
1.2 thrombin
1.0
ATAP2
WEDE15
0.8
ns
0.6
ns
0.4
0.2
0.0
SPAN12
*
0
*
60
120
180
Time (min)
APC-like signaling by an agonist peptide derived from the
novel tethered ligand sequence that arises from cleavage of
PAR1 at Arg46
Cleavage of PAR1 by APC at Arg46 destroys the classic TRAP
tethered ligand sequence (S42FLLRNPNDKY …) and creates a
novel PAR1 N-terminus (N47PNDKY …). To test the hypothesis
that this newly generated PAR1 N-terminus can act as a tethered
ligand and mediate APC-specific signaling, a peptide (TR47, PAR1
residues 47-66; see supplemental Figure 2) was synthesized and
tested on EA.hy.926 endothelial cells for activation of the cytoprotective Akt signaling cassette. The TR47 peptide induced robust
and sustained activation of Akt as determined by phosphorylation
of Ser473, starting as early as 15 minutes after addition of TR47
and lasting ⬎ 180 minutes (Figure 3A,C). To confirm activation of
Akt by TR47, Akt-mediated inactivation of GSK3␤ via phosphorylation at Ser9 was determined, a well-known downstream substrate
for Akt.32 TR47 induced significant Ser9-GSK3␤ phosphorylation
starting at 30 minutes with a time course that fell well within the
time course of TR47-mediated Akt activation (Figure 3B,D). No
phosphorylation of Akt at Ser473 (Figure 3C) or GSK3␤ at Ser9
(Figure 3D) was observed for cells treated with a scrambled control
peptide (scrTR47, supplemental Figure 2), indicating that the
effects of TR47 were specific for the newly created N-tethered
ligand sequence of PAR1 after cleavage at Arg46. Concentrations
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5242
A
B
TR47
0
1
5
15
30 60
90 180
min
TR47
0
1
5
15
GSK3ß
D
TR47
scrTR47
3
2
1
3
GSK3β
TR47
scrTR47
2
1
2
1
TR47 (µM)
90
18
0
60
30
5
1
Time (min)
of TR47 (EC50 ⫽ 1.2␮M) that were required to induce Akt
phosphorylation were comparable with those reported for TRAPmediated induction of signaling in endothelial cells with noticeable
TR47-mediated Akt phosphorylation in the high nanomolar range
and maximal effects achieved at 10␮M TR47 (Figure 3E). Activation of Akt by TR47 was dependent on PAR1 because TR47 failed
to induce Akt phosphorylation at Ser473 in the presence of the
PAR1 inhibitor SCH79797 (Figure 3F). Similarly, induction of
Ser9-GSK3␤ phosphorylation by TR47 required PAR1 because
this effect was inhibited by the PAR1 antagonist SCH79797
(Figure 3G). Thus, a peptide with the sequence of the new
N-terminus of PAR1 generated on cleavage at Arg46 induced
PAR1-dependent activation of Akt and PAR1-dependent phosphorylation of GSK3␤, strongly suggesting that activation of PAR1 at
Arg46 creates a novel functional tethered ligand that is capable of
PAR1-dependent activation of specific cell-signaling pathways.
The TR47 peptide mimics APC-induced signaling but does not
mimic thrombin-induced or TRAP-induced cell signaling
Classic activation of PAR1 by thrombin or TRAP results in the
activation of the MAPK pathway as typically demonstrated by
rapid phosphorylation of ERK1/2 at Thr202 and Tyr204 or Thr185
and Tyr187, respectively. Consistent with earlier reports, APC
induced a transient but modest activation of ERK1/2 (Figure
4A-B).14 Interestingly, TR47 failed to induce any noticeable
activation of ERK1/2 under conditions where TRAP and APC did
so (Figure 4B). Thus, TR47 does not activate the classic PAR1
MAPK pathway that is activated by thrombin and TRAP. Remarkably, TR47 induced activation of Akt (Figure 3C) with a time
course that mimicked APC’s robust activation of Akt (Figure
60
18
0
60
1
15
0
0
50
10
0
2
10
4
0.
0
0.
08
0
GSK3β
vehicle
SCH79797
2
18
0
0
vehicle
SCH79797
3
15
1
Akt
1
2
G
0
3
3
pSer9-GSKβ (fold)
pSer473-Akt (fold)
4
Figure 3. The novel tethered ligand TR47 peptide
induces PAR1-dependent signaling in endothelial
cells. Phosphorylation of pSer473-Akt and pSer9GSK3␤ by TR47 peptide was monitored in endothelial
cells. (A) Time course of Akt phosphorylation at Ser473
by TR47 (50␮M). (B) Time course of GSK3␤ phosphorylation at Ser9 by TR47 (50␮M). (C) Phosphorylation of
Akt at Ser473 by TR47 (50␮M) or a control scrTR47
peptide (50␮M) over time. (D) Phosphorylation of GSK3␤
at Ser9 by TR47 (50␮M) and a control scrTR47 peptide
(50␮M) over time. (E) TR47 peptide dose dependence
for phosphorylation of Akt at Ser473 at 60 minutes.
(F) Time course for phosphorylation of Akt at Ser473 by
TR47 (50␮M) in the presence of the PAR1 inhibitor,
SCH79797, or vehicle control (DMSO). (G) Time course
for phosphorylation of GSK3␤ at Ser9 by TR47 (50␮M) in
the presence of the PAR1 inhibitor, SCH79797, or
vehicle control (DMSO). (A-B) Representative experiments. (C-G) Data points represent the mean ⫾ SEM
(N ⱖ 3).
Time (min)
F
Akt
15
1
0
18
0
90
60
30
5
15
1
0
0
Time (min)
pSer473-Akt (fold)
min
Akt
Akt
4
5
90 180
pGSK3ß
0
E
30 60
pAkt
pSer9-GSK3β (fold)
C5
pSer473-Akt (fold)
BLOOD, 20 DECEMBER 2013 䡠 VOLUME 120, NUMBER 26
MOSNIER et al
Time (min)
4C-D). In contrast and reflecting the striking functional selectivity
of TR47 and APC, neither thrombin nor TRAP induced Ser473-Akt
phosphorylation under the experimental conditions used. Similar to
activation of Akt, APC induced a sustained phosphorylation of
GSK3␤ at Ser9 (Figure 4E-F), mimicking the effect of TR47
(Figure 3D). Unlike this effect of APC and TR47, incubation of
endothelial cells with TRAP did not result in Ser9-GSK3␤
phosphorylation (Figure 4F). Together, these signaling data indicated that the N-terminus generated by cleavage of PAR1 at Arg41
causes activation of the MAPK pathway, whereas the N-terminus
generated by cleavage of PAR1 at Arg46 causes activation of the
Akt pathway. Thus, the different N-termini that arise from different
cleavages of PAR1 by APC are biased agonists with remarkable
functional selectivity.
TR47-induced vascular-endothelial protective effects in vitro
and in vivo
Using ␤-arrestin-mediated signaling versus G protein-dependent
signaling is a hallmark of biased signaling by GPCRs. Recently,
PAR1 was shown to exhibit biased signaling because activation of
Rac1 by APC and APC-mediated endothelial barrier protection
requires ␤-arrestin-2 and dishevelled-2 scaffolding, whereas
thrombin-induced vascular leakage and RhoA activation requires
G proteins but not ␤-arrestins.18 Accordingly, the TR47 peptide, but
not the scrTR47 control peptide, induced APC-like activation of
Rac1 (Figure 5A). In addition, mimicking the well-known vasculoprotective activity of APC, TR47 protected confluent endothelial
barriers against thrombin-induced permeability, whereas the control scrTR47 peptide was without effect (Figure 5B).
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BLOOD, 20 DECEMBER 2013 䡠 VOLUME 120, NUMBER 26
TR47
0
1
5
15
30
60
90
min
pERK1/2
3
2
1
0
0
ERK1/2
TRAP
TR47
scrTR47
APC
4
C
0
1
5
15
30
60
90
4
min
pAkt
Akt
thrombin
0
1
5
15
Time (min)
D
APC
30
60
90
min
pAkt
APC
thrombin
TRAP
3
2
1
0
0
Akt
0
F
APC
1
5
15
30
60 90 180
min
pGSK3ß
GSK3ß
TRAP
0
Time (min)
1
15
60 180
min
pGSK3ß
pSer9-GSK3β (fold)
E
0
ERK1/2
5
18
min
pERK1/2
0
90
5243
18
60
60
30
60
15
15
3
15
1
1
0
1
B
APC
pT202/Y204-ERK1/2 (fold)
A
pSer473-Akt (fold)
Figure 4. The TR47 peptide and TRAP are PAR1
biased agonists. Differential phosphorylation was determined for pThr202/Tyr204-ERK1/2, pSer473-Akt, and
pSer9-GSK3␤ that was induced by TRAP, TR47 peptide,
control scrTR47 peptide, APC, and thrombin. (A) Time
course of ERK1/2 phosphorylation at Thr202/Tyr204 by
APC (70nM) and TR47 (50␮M). (B) Phosphorylation of
ERK1/2 at Thr202/Tyr204 by TRAP (50␮M), TR47 (50␮M),
scrTR47 (50␮M), and APC (70nM) over time. (C) Time
course of Akt phosphorylation at Ser473 by APC (70nM)
and thrombin (54nM). (D) Phosphorylation of Akt at
Ser473 by APC (70nM), thrombin (54nM), and TRAP
(50␮M) over time. (E) Time course of GSK3␤ phosphorylation at Ser9 by APC (70nM) and TRAP (50␮M). (F) Phosphorylation of GSK3␤ at Ser9 by APC (70nM) and TRAP
(50␮M) over time. (A,C,E) Representative experiments.
(B,D,F) Data points represent the mean ⫾ SEM (N ⱖ 3).
BIASED AGONISM OF PAR1 BY ACTIVATED PROTEIN C
APC
TRAP
3
2
1
0
18
60
15
1
0
0
GSK3ß
Time (min)
To probe whether TR47 could induce vascular protective effects
in vivo, a modification of the modified Miles assay was used in
which vascular permeability is measured by the extravasation of
Evans blue dye in the skin induced by local subcutaneous injection
of VEGF165. Immunocompetent SKH1 hairless mice were used to
avoid the need for hair removal that often can result in artifactual
leakage resulting from inflammation of the skin. Evans blue
extravasation in the skin was quantified using the Odyssey
near-infrared fluorescent imager at 700 nm. TR47 or scrTR47 were
injected intravenously in the retro-orbital sinus 5 minutes before
local subcutaneous injection of recombinant murine VEGF or BSA
control and 30 minutes after intravenous administration of Evans
blue (Figure 5C). In the absence of TR47 (PBS control), VEGF
induced clearly distinguishable areas of Evans blue extravasation,
whereas after injection of BSA vehicle control only the needle
points marking the injection site could be observed (Figure 5D).
Quantification of Evans blue extravasation by near-infrared fluorescence at 700 nm eliminated the time-consuming need for Evans
blue extraction from punch biopsy and provided reliable and
reproducible results (Figure 5E). The TR47 peptide significantly
decreased vascular leakage by 45% compared with PBS control,
whereas vascular leakage in the presence of the scrTR47 control
peptide was indiscriminately from PBS control (Figure 5F).
Neither TR47 nor scrTR47 affected vascular leakage in the absence
of VEGF. Thus, like APC, the TR47 peptide causes activation of
Rac1, stabilizes endothelial barriers in vitro, and in vivo can
markedly reduce vascular leakage.
Discussion
This study provides in vitro and in vivo experimentation to assess
the hypothesis that PAR1-dependent signaling by APC involves a
noncanonical cleavage of the receptor’s N-terminal domain that
reveals a novel cryptic intramolecular pharmacophore, which
causes APC’s cytoprotective, biased signaling. The results show
that APC can cleave a synthetic PAR1 N-terminal polypeptide at
both Arg41 and Arg46. Cleavage by APC of transfected R41QSEAP-PAR1 but not R41Q/R46Q-SEAP-PAR1 constructs supports
the notion that APC can cleave PAR1 at Arg46 on cells, a concept
that is consistent with epitope mapping of PAR1 on endothelial
cells after APC cleavage of PAR1. Thus, data support the hypothesis that APC can cleave PAR1 on endothelial cells at Arg46.
After our initial report last year33,34 of the novel concept that
APC cleaves PAR1 at Arg46 to generate a unique biased agonist
N-terminal peptide, but before this present paper was submitted, a
recent report provided data consistent with this concept.35 That
recent report and our studies coincide in advancing the paradigm
that APC’s PAR1-dependent protective actions are based on Arg46
cleavage. This present paper provides a number of novel aspects for
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BLOOD, 20 DECEMBER 2013 䡠 VOLUME 120, NUMBER 26
MOSNIER et al
Rac1 lysate
*
ns
5
4
*
*
3
2
1
0.5
sc
rT
R
47
0
1.0
TR
47
*
1.5
180 min
no
ne
GTP-Rac1 (Fold)
GTP-Rac1
30
PC
180
A
30
B
TR47
no
ne
TR47
Permeability (fold)
no
ne
A
no
ne
5244
thrombin
PBS
D
0.0
none
C
+ Evans blue
APC
TR47
+ VEGF
+ peptides
TR47
scrTR47
analysis
experimental time line
0 min.
PBS
BSA
VEGF
BSA
F
TR47
Vascular leakage (fold)
E
VEGF
65 min.
30 35
Figure 5. The TR47 peptide inhibits endothelial permeability in vitro and in vivo vascular leakage in mice.
(A) Activation of Rac1 by TR47 peptide, control peptide,
and APC was determined and is shown as pull-down of
active Rac1 (GTP-Rac1) with PAK1 (top left), total Rac1
(top right panel), and quantification of Rac1 activation at
180 minutes (bottom panel) using APC (70nM), TR47
(50␮M), and scrTR47 (50␮M). *P ⬍ .05. ns indicates not
significant. (B) In vitro protection against thrombininduced endothelial permeability by APC (25nM), TR47
(50␮M), and scrTR47 (50␮M). (C) Experimental time line
for the in vivo VEGF-induced vascular leakage model.
(D) Photographs are seen for Evans blue extravasation in
the skin of TR47-treated mice (right) or of control PBStreated mice (left) injected subcutaneously with VEGF
(3 left arrows) or with BSA control (2 right arrows).
(E) Heat map display of Evans blue extravasation quantified by the Odyssey near-infrared imager at 700 nm.
TR47-treated mice (right) or control PBS-treated (left)
mice were injected subcutaneously with VEGF (3 left
arrows) or with BSA control (2 right arrows). (F) In vivo
VEGF-induced vascular leakage in mice is shown for
mice treated with PBS control, TR47 peptide (125 ␮g), or
control scrTR47 peptide (125 ␮g). Data are also shown
for BSA-treated control mice that received PBS, TR47, or
scrTR47. (A top panels, D-E) Representative experiments. (A bottom panel, B,F) Data points represent the
mean ⫾ SEM (N ⱖ 3). *P ⬍ .05.
*
1.5
*
1.0
0.5
this new paradigm. Our studies uniquely provide direct evidence
that APC can cleave the PAR1 polypeptide at Arg46, that the
unique N-terminal peptide generated by this cleavage (TR47)
triggers signaling via the Akt pathway and via Rac1 activation,
that TR47 signaling requires PAR1, and an in vivo proof of
concept showing that the TR47 peptide protects against vascular
leakage in vivo.
What are the functional consequences of APC’s cleavage at
Arg46 in PAR1? After Arg46 cleavage, the known PAR1 agonist
sequence containing SFLLR is no longer tethered to PAR1. Our
studies showed that the TR47 peptide representing the sequence of
the novel N-terminus that is generated by cleavage at Arg46 exerts
remarkable biologic activities, including signaling reflected in
phosphorylation of Akt and GSK3␤ and endothelial barrier stabilization. These cytoprotective activities caused by TR47 peptide
reflect the general activity profile of APC but not that of thrombin
or of the thrombin-generated N-terminal sequence represented in
TRAP. Distinctions between the 2 peptides are plentiful, including
the rapid phosphorylation of ERK1/2 caused by TRAP but not
TR47 and the slower phosphorylation of Akt and GSK3␤ caused by
TR47 but not TRAP. The PAR1 inhibitor SCH79797 ablates the
phosphorylation of Akt and GSK3␤ by TR47, showing that these
actions of TR47 require PAR1. Thus, data support the hypothesis
that APC’s cleavage of PAR1 at Arg46 generates a tethered ligand
pharmacophore composing at least part of the amino acid sequence
S
TR
47
sc
rT
R
47
47
47
VEGF
PB
BSA
TR
VEGF
TR
BSA
sc
r
VEGF
PB
S
0.0
BSA
of PAR1 residues 47-66, which in vitro initiates a pattern of
cytoprotective signaling that resembles the PAR1-dependent cytoprotective functional activities described for APC. However, whether
another PAR or even a PAR-unrelated receptor can contribute to
TR47-mediated signaling cannot be excluded.
What are the in vivo functional consequences of cleavage at
Arg46 by APC? Our studies showed that, when the TR47 peptide is
given to mice that are subjected to VEGF-induced vascular
leakage, this APC-mimetic peptide significantly reduces vascular
leakage. The in vivo data for TR47’s barrier stabilization in a
vascular leakage murine injury model resemble data for the ability
of TR47 to stabilize endothelial cell barrier function in vitro. Thus,
all data are consistent with the hypothesis that the novel PAR1
N-terminus generated by APC’s cleavage at Arg46 functions as a
biased tethered ligand, which causes PAR1 signaling that promotes
cytoprotective functions.
This study provokes a shift in the PAR1 paradigm. Recent
insights into mechanisms for regulating signaling by GPCRs
emphasize the complexities associated with biased agonism and
allosteric modulation.36-39 Emerging structural and pharmacologic
information supports the notion that GPCRs exist in dynamic
conformational ensembles and that agonists, biased agonists, or
allosteric ligands stabilize 1 or more conformational subsets at the
expense of others with functional consequences and highly selective signaling outcomes.36,40-45 Proteolysis at Arg41 versus at Arg46
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BLOOD, 20 DECEMBER 2013 䡠 VOLUME 120, NUMBER 26
PAR1 cleavage at Arg41
TRAP
BIASED AGONISM OF PAR1 BY ACTIVATED PROTEIN C
PAR1 cleavage at Arg46
Thrombin
APC
TR47
AGONIST BIAS
G-protein
BIASED
BALANCED
ß-Arrestin
BIASED
Signaling BIAS
G-protein
ERK1/2
RhoA
Signaling BIAS
ß-Arrestin
PI3K/Akt
Rac1
PROINFLAMMATORY
EFFECTS
CYTOPROTECTIVE
EFFECTS
Figure 6. Biased agonism of PAR1 resulting from canonical cleavage at Arg41
by thrombin or by noncanonical cleavage at Arg46 by APC. PAR1 is a biased
receptor with biased agonists.18 Four different PAR1 agonists are shown along the
top bar that can vary in their ability and efficacy to activate different signaling
pathways in cells, thus depicting a signaling phenomenon that has been labeled
“functional selectivity” or “biased agonism.” The receptor’s bias is directly based on
the spectrum of signaling that is mediated either via 1 or more G proteins or via
␤-arrestin-2, whereas the agonist bias is directly related to the sites of cleavage in the
extracellular N-terminus (ie, resulting from cleavage either at Arg41 or at Arg46). The
TRAP peptide(s) represent the N-terminal sequence of PAR1 that exists after
cleavage at Arg41, whereas TR47 represents the N-terminal sequence of PAR1 that
exists after cleavage at Arg46, as described in this report. Each agonist can have
distinctly different properties because each one can stabilize a different subset of the
dynamic conformational ensembles of PAR1. The conformer subsets that are
stabilized by TRAP and thrombin promote signaling via different G proteins, whereas
conformer subsets stabilized by APC’s action, and presumably TR47, promote
signaling via ␤-arrestins, especially ␤-arrestin-2, and dishevelled-2. However, one
must note that TRAP is similar but not functionally equivalent to thrombin; and it is
probable, but not yet shown, that the TR47 peptide does not have all the same
activities and efficacy as APC. Not depicted are the effects of MMP1 cleavage at
Asp39 in PAR1 or potential allosteric PAR1 modulatory factors, including binding of
thrombin to the hirudin-like sequence of PAR1, localization of PAR1 in caveolae with
caveolin-1, association with EPCR containing bound protein C or APC, dimerization
with other PARs, or small molecular allosteric modulators.41,46
de facto alters the spectrum of conformations of PAR1 because of
differences in sequence and N-terminus after cleavage at either
Arg41 or Arg46. Consequently, cleavage by APC at Arg46 changes
the readily accessible subsets of conformations of PAR1 with
functional consequences (Figure 6). PAR1 is a biased receptor that
can have multiple covalently linked cryptic tethered ligands that
begin at Pro40, Ser42 or Asn47Othe first 2 tethered ligands
generate a very similar spectrum of signaling, whereas the
N-terminal Asn47 tethered ligand stimulates different and very
distinctive signal transduction pathways.6,18,24 Thus, PAR1 can
understandably mediate the often opposite effects of thrombin and
APC because each protease can generate very different tethered
peptide agonists that mimic TRAP or TR47, which are intrinsically
biased agonists.
Allosteric modulation of PAR1 by thrombin after cleavage at
Arg41 can potentially arise because of the binding of thrombin’s
exosite I to the hirudin-like PAR1 residues 51-55. The binding of an
EPCR-protein C complex before any PAR1 cleavage might allosterically alter PAR1 responses caused by thrombin’s cleavage at
5245
Arg 41, thereby resulting in cytoprotective effects of thrombin.23,47
Allosteric modulation of PAR1 may also involve its association
with 1 or more proteins that are located in caveolin 1-containing
microdomains. Small molecular weight allosteric antagonists of
PAR1 have recently been described.41 Much remains to be learned
about PAR1’s biased ligands and the allosteric regulation of PAR1
signaling.
This new mechanism for PAR1 cytoprotective actions involving
APC’s cleavage at Arg46 may be clinically relevant when considering potential explanations for the unfortunate serious side effect of
hemorrhagic stroke caused by the PAR1 inhibitor, vorapaxar, in
recent large phase 3 clinical trials.48,49 APC stabilizes endothelial
barriers via ␤-arrestin–dependent signaling, whereas thrombin
disrupts endothelial barriers via G proteins.10,18 Thus, it is possible
that vorapaxar, as a PAR1 general antagonist, inhibits the beneficial
effects of the endogenous protein C system on the blood-brainbarrier in addition to inhibiting thrombin’s prothrombotic effects
on platelets, thereby increasing risk for blood-brain-barrier leakage.5 Although studies are needed to clarify this potential mechanism for vorapaxar, it is clear that drug discovery efforts to identify
compounds that antagonize PAR1’s G protein-dependent signaling
but not ␤-arrestin 2-dependent signaling may prove fruitful.
Similarly, APC-mimetic compounds that promote cytoprotective
PAR1-dependent signaling while avoiding thrombin-mimetic signaling would be very interesting biased PAR1 agonists.
In conclusion, an extensive combination of in vitro and in vivo
data supports the novel paradigm for the biochemical mechanisms
mediating APC’s cytoprotective signaling via PAR1 in which
APC’s cleavage at Arg46 in PAR1 generates a new N-terminal
tethered ligand beginning with Asn47, which is a biased agonist
that initiates cytoprotective signaling pathways.
Acknowledgments
The authors thank Dr L. Brass (University of Pennsylvania,
Philadelphia, PA) for the gift of PAR1 antibodies; Dr C. J. S.
Edgell (University of North Carolina, Chapel Hill, NC) for the
EA.hy.926 endothelial cells; and M. Mathias and D. Rozenshteyn
for excellent technical assistance.
This work was supported in part by the National Heart, Lung,
and Blood Institute (grants HL087618 and HL104165, L.O.M.; and
grants HL31950 and HL52246, J.H.G.), postdoctoral fellowships
from the Swiss National Science Foundation, the Fondation Suisse
pour les Bourses en Médecine et Biologie, and Novartis (PBGEP3134242 and PASMP3_140065; L.B.), and the American Heart
Association Western States Affiliate (postdoctoral fellowship;
E.A.B.).
Authorship
Contribution: L.O.M., R.K.S., L.B., and E.A.B. performed experiments; L.O.M., R.K.S., L.B., E.A.B., and J.H.G. designed the
research and analyzed results; and L.O.M. and J.H.G. wrote the
manuscript.
Conflict-of-interest disclosure: L.O.M. and J.H.G. are inventors
on a pending patent application concerning PAR1 peptides that is
the property of the Scripps Research Institute. The remaining
authors declare no competing financial interests.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
5246
BLOOD, 20 DECEMBER 2013 䡠 VOLUME 120, NUMBER 26
MOSNIER et al
Correspondence: Laurent O. Mosnier, Department of Molecular
and Experimental Medicine (MEM-180), Scripps Research Insti-
tute, 10550 North Torrey Pines Rd, La Jolla, CA 92037; e-mail:
[email protected].
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2012 120: 5237-5246
doi:10.1182/blood-2012-08-452169 originally published
online November 13, 2012
Biased agonism of protease-activated receptor 1 by activated protein C
caused by noncanonical cleavage at Arg46
Laurent O. Mosnier, Ranjeet K. Sinha, Laurent Burnier, Eveline A. Bouwens and John H. Griffin
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