Protease-activated receptors in cancer: A systematic review.

Abstract

The traditional view of the role of proteases in tumor growth, progression and metastasis has significantly changed. Apart from their contribution to cancer progression, it is evident that a subclass of proteases, such as thrombin, serves as signal molecules controlling cell functions through the protease-activated receptors (PARs). Among the four types of PAR (PAR1-4; cloned and named in order of their discovery), PAR1, PAR3 and PAR4 are activated by thrombin, unlike PAR2, which is activated by trypsin-like serine proteases. Thrombin has been proven to be a significant factor in both the behavior of cancer in its involvement in hemostasis and blood coagulation. Thrombin is a key supporter of various cellular effects relevant to tumor growth and metastasis, as well as a potent activator of angiogenesis, which is essential for the growth and development of all solid tumor types. This review presents an overview of the role of PAR-mediated thrombin in angiogenesis and cancer, focusing on the ability of PAR1- and PAR4-mediated thrombin to affect tumorigenesis and angiogenesis.

Full text

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ONCOLOGY LETTERS 2: 599-608, 2011
Abstract. The traditional view of the role of proteases in tumor
growth, progression and metastasis has significantly changed.
Apart from their contribution to cancer progression, it is
evident that a subclass of proteases, such as thrombin, serves
as signal molecules controlling cell functions through the
protease-activated receptors (PARs). Among the four types of
PAR (PAR1-4; cloned and named in order of their discovery),
PAR1, PAR3 and PAR4 are activated by thrombin, unlike
PAR2, which is activated by trypsin-like serine proteases.
Thrombin has been proven to be a significant factor in both
the behavior of cancer in its involvement in hemostasis and
blood coagulation. Thrombin is a key supporter of various
cellular effects relevant to tumor growth and metastasis, as
well as a potent activator of angiogenesis, which is essential
for the growth and development of all solid tumor types. This
review presents an overview of the role of PAR-mediated
thrombin in angiogenesis and cancer, focusing on the ability
of PAR1- and PAR4-mediated thrombin to affect tumorigen-
esis and angiogenesis.
Contents
1. Introduction
2. Thrombin level in cancer and PAR activation
3. Thrombin and PAR in angiogenesis
4. Thrombin and PAR in tumorigenesis and metastasis
5. Therapeutic implications in cancer
6. Conclusions
1. Introduction
The G-protein-coupled receptor (GPCR) superfamily
comprises the largest and most functionally diverse group of
signaling molecules. These receptors play essential roles in
the normal regulation of the majority of biological processes.
They are also of great significance in human diseases, such as
cancer. GPCRs are able to interact with a variety of agonists,
such as peptides, lipids and ions. Proteases are one of the most
noteworthy agonists of GPCRs.
Proteases have previously been associated with tumor
progression due to their ability to degrade extracellular
matrices, facilitating tumor cell invasion and metastasis
(1). However, studies have shown that these enzymes target
diverse substrates and promote tumor evolution (2), a fact that
significantly altered the traditional view of the role of prote-
ases in tumor growth and progression. It is well known that
a subclass of proteases serves as signal molecules controlling
cell functions through specific GPCRs, the protease-activated
receptors (PARs) (3,4). The PARs are activated by low
concentrations of certain extracellular serine proteases.
Four types of PAR (PAR1-4) have been cloned and named
in order of their discovery. These PARs share the same
basic mechanism of activation in that proteases cleave at a
specific site within the extracellular N-terminus to expose a
new N-terminal tethered ligand domain, which binds to and
activates the cleaved receptor. PAR1, PAR3 and PAR4 are
activated by thrombin, one of the important extracellular
serine proteases (5,6). On the other hand, PAR2 is activated by
trypsin-like serine proteases, including trypsin, tryptase and
coagulation proteases upstream of thrombin, tissue factors
(TFs) VIIa and Xa, but not by thrombin (4,7-12).
Thrombin has been proven to be crucial in the behavior of
cancer and in its involvement in hemostasis and blood coagu-
lation. Thrombin is a key supporter of various cellular effects
relevant to tumor growth and metastasis as well as a potent
Protease-activated receptors in cancer: A systematic review
NA HAN1, KETAO JIN2,3, KUIFENG HE2, JIANG CAO1,4 and LISONG TENG2
1Sir Run Run Shaw Institute of Clinical Medicine, Zhejiang University: Key Laboratory of Biotherapy of Zhejiang Province,
Zhejiang University, Hangzhou, Zhejiang 310016; 2Department of Surgical Oncology, First Affiliated Hospital,
College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003; 3Department of Surgery,
Zhuji Hospital, Wenzhou Medical College, Zhuji, Zhejiang 311800; 4Clinical Research Center,
Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, P.R. China
Received January 12, 2011; Accepted April 6, 2011
DOI: 10.3892/ol.2011.291
Correspondence to: Dr Jiang Cao, Sir Run Run Shaw Institute
of Clinical Medicine, Zhejiang University: Key Laboratory of
Biotherapy of Zhejiang Province, and Clinical Research Center,
Second Affiliated Hospital, College of Medicine, Zhejiang
University, 88 Jiefang Road, Hangzhou, Zhejiang 310003, P.R. China
E-mail: jinketao2001@zju.edu.cn
Dr Lisong Teng, Department of Surgical Oncology, First Affiliated
Hospital, College of Medicine, Zhejiang University, 79 Qingchun
Road, Hangzhou, Zhejiang 310003, P.R. China
E-mail: lsteng@zju.edu.cn
Key words: angiogenesis, cancer, protease-activated receptors,
tumorigenesis

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HAN et al: PARs IN CANCER
600
activator of angiogenesis, which is essential for the growth
and development of all solid tumor types. Thrombin actions in
cancer are PAR-mediated.
This review provides an overview of the role of PAR-
mediated thrombin actions in cancer, focusing on the involve-
ment of PAR1- and PAR4-mediated thrombin in tumorigenesis
and angiogenesis.
2. Thrombin level in cancer and PAR activation
Thrombin generated in the circulation during activation of the
coagulation cascade has multiple cellular effects, including
the induction of cell proliferation and motility, enhancement
of vascular permeability, deposition of matrix fibrin, promo-
tion of tumor cell seeding, adhesion to endothelium and
extracellular matrix, induction of platelet aggregation, and
enhancement of the metastatic capacity of tumors (13,14).
A common feature in cancer patients is the high level of
thrombin formation in tumor cells. It has been shown that
numerous tumor cell types express the transmembrane protein
tissue factor, which, when exposed to circulating factor VII,
activates factor X, leading to the generation of thrombin (15).
Thrombin generation is possibly a direct result of the over-
activation of the coagulation system (hypercoagulability), a
widely described abnormality in various cancer patients (16).
Thrombin activates PAR1, PAR3 and PAR4. Thrombin
activates PAR1 in two stages (9). Firstly, it binds to PAR1
on either side of the cleavage site. Secondly, it cleaves PAR1
between Arg41 and Ser42 to expose a new N-terminal tethered
ligand domain, SFLLRN. The tethered ligand interacts with
domains in extracellular loop 2, which presumably alters the
conformation of the receptor to permit coupling to G-proteins.
PAR3 also contains the thrombin binding sites, whereas PAR4
lacks thrombin binding sites and only responds to higher
concentrations of thrombin (5,11). The differences in the
mechanism of activation mentioned above exhibit functional
consequences in tumorigenesis and angiogenesis.
3. Thrombin and PARs in angiogenesis
Action of PAR-mediated thrombin on endothelial cells.
Although thrombin is best known for its direct role in clot
formation via platelet activation and fibrin deposition, a
number of the effects of thrombin in cancer may be mediated
by promoting angiogenesis in vivo (17-19). Numerous cellular
effects of thrombin on endothelial cells contribute to the
angiogenic action of thrombin (Table I) (17-30).
Angiogenesis involves the activation and invasion of the
endothelial cells through their basement membrane and
migration to distal sites. Studies have demonstrated that
thrombin contributes to each of these events (18,19,23-25).
This cellular action of thrombin on endothelial cell adhe-
sion may indicate a significant role in the activation of the
normally quiescent endothelial cells in the initiation of the
angiogenic cascade. A key event in early angiogenesis is the
local dissolution of the basement membrane of the parent
vessel. Endothelial cells need to overcome the barrier of their
anchorage to basement membrane components in order to
migrate to distal sites, proliferate and form the lumen of the
new vessel. Exposure of endothelial cells to thrombin causes a
time- and dose-dependent decrease in the attachment of these
cells to basement membrane components, with a concomitant
increase in matrix metalloproteinase 2 (MMP-2) activation
(24,25). This decrease not only allows for the migration of
endothelial cells, but releases other angiogenic factors that are
sequestered in the extracellular matrix.
Integrin αvβ3 was identified as a marker of the angio-
genic phenotype of endothelial cells in vascular tissue (31).
Antibodies or peptide antagonists of this integrin inhibited
angiogenesis induced by basic fibroblast growth factor in
the rabbit cornea model (26). Furthermore, integrin αvβ3
antagonists inhibit tumor-induced angiogenesis by inducing
apoptosis in angiogenic blood vessels without affecting
mature vessels, which express minimal αvβ3. Endothelial
cells attach to thrombin via the angiogenic integrin αvβ3,
which is up-regulated by thrombin (19,25). This attachment
provides endothelial cells with survival signals during their
anchorage-independent migration (19). More importantly,
both integrin αvβ3 and MMP-2 functionally coexist on the
surface of angiogenic capillaries (26).
Thrombin also has chemotactic and aptotactic effects on
endothelial cells in that it up-regulates the expression of the
vascular endothelial growth factor (VEGF) receptors (KDR
and Flt1) and synergizes with the key angiogenic factor VEGF
in endothelial cell proliferation (27). It has been shown that
8-12 h after exposure of endothelial cells to thrombin, cells
are sensitized to the action of VEGF. The mitogenic activity
is increased by more than 100% over the level expected
from the additive effects of thrombin and VEGF alone. The
thrombin-treated cells respond to VEGF-induced DNA
synthesis in a synergistic manner (27). This synergistic effect
Table I. Effects of PAR-mediated thrombin on endothelial
cells.
PAR-mediated Refs.
Effects of thrombin
on ECs
Decrease in the attachment
of ECs to BMC
Increase in MMP-2 activation
Up-regulation of integrin αvβ3

PAR1-, PAR3- and
PAR4-mediated





17,
20-22
18,19,
23,24
25
19,25,
26
Chemotactic and aptotactic
effects on ECs
Up-regulation of VEGF
receptors (KDR and Flt1)
Up-regulation of VEGF
Synergy with VEGF
in EC proliferation
Vascular smooth
muscle cell migration
27
27


28
27
29,30
ECs, endothelial cells; BMC, basement membrane components;
MMP-2, matrix metalloproteinase 2; VEGF, vascular endothelial
growth factor.

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ONCOLOGY LETTERS 2: 599-608, 2011
601
of thrombin with VEGF can be explained by the finding that
thrombin increases the level of VEGF receptors KDR and
Flt-1 as mentioned above.
The PAR family members, PAR1, PAR2, PAR3 and PAR4,
are expressed in arterial and/or venous endothelial cells
(20,22,32-34). Endothelial PARs serve as sensors of extracel-
lular proteases and transmit signals after cleavage by proteases
such as thrombin (35,36). Previous studies indicated that
coagulation factors upstream and downstream of thrombin
mediate the activation of PAR1 in endothelial cells (35-37).
Thrombin activation of PAR1 generates cytoskeletal rear-
rangements in endothelial cells and induces cell contraction
and rounding (38,39). Endothelial cell contraction destabilizes
cell-cell contacts, causing a subsequent increase in vascular
permeability that facilitates the passage of molecules and cells
from the blood into subendothelial compartments. Activation
of PAR1 in the vascular endothelium also leads to increased
surface expression of the adhesion molecules, such as intercel-
lular adhesion molecule-1, vascular cell adhesion molecule-1,
P-selectin and E-selectin (40).
Action of PAR-mediated thrombin on platelets. The process
of postnatal angiogenesis is regulated by a continuous inter-
play of stimulators and inhibitors of angiogenesis, and their
imbalance contributes to numerous inflammatory, malignant,
ischemic and immune disorders (41). A renewed interest in the
overlap between angiogenesis and platelets has been observed
(42) with findings of various clinical trials showing that
anticoagulation improves cancer survival (43,44) beyond the
benefit derived from the treatment of deep vein thrombosis
alone. Platelets act as the initial responder to vascular change
and provide a flexible delivery system for angiogenesis-related
molecules (45-48).
It is known that platelets stimulate endothelial cells in
culture and promote the assembly of capillary-like structures
in vitro (49). Platelets may modulate angiogenesis by releasing
promoters, such as VEGF, basic fibroblast growth factor
(bFGF), epidermal growth factor (EGF), platelet-derived
growth factor (PDGF) and matrix metalloproteinases (MMPs)
(Table II) (42,45,48,50-58). Platelets comprise a wide range of
angiogenesis inhibitors including endostatin, platelet factor-4,
thrombospondin-1, 2-macroglobulin, plasminogen activator
inhibitor-1 and angiostatin (Table II) (48,59,60). Although
platelets contain three types of secretory granules (α-granules,
dense granules and lysosomes), most angiogenic regula-
tory proteins have been localized to α-granules. α-granules
comprise proteins that enhance the adhesive process, promote
cell-cell interactions and stimulate vascular repair. By
adhering to the endothelium of injured organs and tissues and
then secreting the contents of their α-granules, platelets may
be capable of depositing high concentrations of angiogenesis
regulatory proteins in a localized manner (50).
Platelets are regulated through numerous agonist-induced
signaling pathways, the most potent of which is thrombin
(61,62). Human platelets express two functional thrombin recep-
tors, PAR1 and PAR4 (63-65). Thrombin acts through PAR1
and PAR4 on human platelets to signal activation responses,
such as calcium mobilization, release of procoagulant
molecules (e.g., P-selectin) from α-granules, release of small
molecules (e.g., ADP) from dense granules, activation of glyco-
protein IIbIIIa/integrin αIIbβ3 (GPIIbIIIa) and aggregation
(65-72). Thrombin activates PAR1 at concentrations 10-fold
less than PAR4, but activation of PAR4 provides a longer
stimulus (61,73).
Numerous experimental data and clinical investiga-
tions have suggested that platelets are major regulators of
angiogenesis. However, since platelets contain both pro- and
anti-angiogenic regulatory proteins and because it has been
assumed that the contents of α-granules are homogeneous, it is
unclear how platelets either stimulate or inhibit angiogenesis.
Italiano et al (50) provided new details about the organiza-
tion of angiogenesis regulatory proteins in the α-granules
of platelets and addressed the mechanism of how the selec-
tive release of these granules leads to the regulation of
angiogenesis. Using double immunofluorescence and immu-
noelectron microscopy, these authors showed that pro- and
anti-angiogenic proteins are divided into distinct subpopula-
tions of α-granules in platelets and megakaryocytes. The
double immunofluorescence labeling of VEGF and endostatin,
or that for thrombospondin-1 and bFGF, confirms the segrega-
tion of stimulators and inhibitors into separate and distinct
α-granules. These observations motivated the hypothesis that
distinct populations of α-granules undergo selective release.
Furthermore, the treatment of human platelets with a selec-
tive PAR4 agonist (AYPGKF-NH2) resulted in the release of
endostatin-containing, but not VEGF-containing granules,
whereas the selective PAR1 agonist (TFLLR-NH2) released
VEGF, but not endostatin-containing granules. Results of this
Table II. Dual regulation of platelets on angiogenesis.
Dual regulation of platelets
on angiogenesis
PAR-mediated Refs.
Pro-angiogenic regulation
of platelets by releasing
angiogenic promoters:
VEGF

bFGF
EGF
PDGF
MMPs
PAR1-mediated 50


42,45,48,
51-58
Anti-angiogenic regulation
of platelets by releasing
angiogenic inhibitors:
Endostatin
Platelet factor-4
Thrombospondin-1
2-macroglobulin
Plasminogen activator
inhibitor-1
Angiostatin
PAR4-mediated 50
48,59,60
VEGF, vascular endothelial growth factor; bFGF, basic fibroblast
growth factor; EGF, epidermal growth factor; PDGF, platelet derived
growth factor; MMPs, matrix metalloproteinases.

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HAN et al: PARs IN CANCER
602
study (50) demonstrated the separate packaging of angiogen-
esis regulators into pharmacologically and morphologically
distinct populations of α-granules in platelets and may provide
a mechanism by which platelets locally stimulate or inhibit
angiogenesis. Tumors may hijack the angiogenic properties of
platelets to generate new blood vessel growth by manipulating
the PARs on platelets and triggering the selective release of
predominantly proangiogenic factors.
4. Thrombin and PARs in tumorigenesis and metastasis
Action of PAR-mediated thrombin in tumorigenesis. Thrombin
markedly increases the growth potential of tumor cells
(Table III) (74-81), although these effects may be partially
attributed to its pro-angiogenic effects (27,82). By mobi-
lizing adhesion molecules, such as the αIIbβ3 integrin (83-85),
P-selectin (86,87) and CD40 ligand (88) to the cell surface,
thrombin enhances adhesion between tumor cells, platelets,
endothelial cells and the extracellular matrix, and contributes
to tumor progression. Thrombin also triggers the release of
growth factors (89), chemokines and extracellular proteins (90)
that promote the proliferation and migration of tumor cells.
The microenvironment of tumors is replete with thrombin,
which activates PARs, and tumor cells, which also express
PARs. Malignant cells secrete thrombin, which affects prolif-
eration and mediates metastatic processes, such as cellular
invasion, extracellular matrix degradation, angiogenesis
and tissue remodeling. In the setting of cancer, the ability of
thrombin to act via PARs was highlighted by the demonstra-
tion of PAR expression in carcinosarcoma (91). Additionally,
mounting evidence showed that the PAR family is involved
in neoplasia (92). In particular, PAR1 is expressed by a wide
range of tumor cells (91,93-96). The expression of PAR1 has
been correlated with the malignant phenotype. For PAR1, a
role in the progression of epithelial tumors, including breast
(75,82,94,97,98), colon (76,99), kidney (100), pulmonary
tumor (101), melanoma (102) and hepatocellular carcinoma
(80,103) has been shown.
The effects of thrombin in human colon cancer cells have
been found to be mediated by functional PAR4. Firstly, the
Table III. Activity of PAR-mediated thrombin in tumorigenesis.
Tumor types PAR-mediated Activity In vivo/In vitro Refs.
Colon carcinoma
Melanoma
Melanoma
Melanoma
Colon carcinoma
Colon carcinoma
Colon carcinoma
Colon carcinoma
Colon carcinoma
Human chondrosarcoma
Hepatocellular carcinoma
Gastric carcinoma
Not available
Not available
Not available
Not available
Not available
Not available
PAR1-mediated
PAR1-mediated
Not available
PAR1-, PAR4-mediated
PAR1-, PAR4-mediated
PAR2-mediated
Amplifying tumor-platelet adhesion
Amplifying tumor-platelet adhesion
Increasing adhesion to platelets
Promoting cell proliferation
Increasing adhesion to platelets
Promoting cell proliferation
Inducing cell proliferation and motility
Promoting cell proliferation
Promoting adhesion and migration
Promoting cell proliferation
Promoting cell migration
Promoting cell proliferation
In vitro
In vitro
In vivo
In vivo
In vivo
In vivo
In vitro
In vitro
In vitro
In vitro
In vitro
In vitro
74
74
75
75
75
75
76
77
78
79
80
81
Table IV. Activity of PAR-mediated thrombin in tumor invasion and metastasis.
Tumor types PAR-mediated Activity In vivo/In vitro Refs.
Colon carcinoma
Colon carcinoma
Melanoma
Melanoma
Melanoma
Melanoma
Melanoma
Melanoma
Melanoma
Renal cancer
Breast cancer
Breast cancer
Not available
Not available
Not available
Not available
Not available
PAR1-mediated
PAR1-mediated
PAR1-, PAR2-mediated
PAR1-mediated
PAR1-mediated
PAR1-mediated
Not available
Promoting metastases
Promoting metastases
Promoting metastases
Promoting metastases
Promoting metastases
Promoting metastases
Promoting metastases
Promoting metastases
Promoting cell invasion
Promoting cell invasion
Promoting cell invasion
Promoting cell invasion
In vivo
In vivo
In vivo
In vivo
In vivo
In vivo
In vivo
In vivo
In vitro
In vitro
In vitro
In vitro
74
75
74
75
84
98
110
111
112
113
82
97

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ONCOLOGY LETTERS 2: 599-608, 2011
603
PAR4 agonist AP4 mimics the effects of thrombin on cell
proliferation. However, AP4 does not activate other PARs.
Secondly, the challenge of CHO-PAR4-expressing cells with
AP4 has the same impact on calcium transients as that in
HT-29 cells. Thirdly, no effect on calcium transients is noted
following the challenge of HT-29 cells with reverse peptide.
AP4 was capable of promoting colon cancer cell proliferation
since, at maximally active concentrations, its effect exhibited
up to a 250% increase in the cell number in HT-29 cells.
PAR4 should thus be regarded as a crucial receptor by which
thrombin modulates colon carcinogenesis (104).
Action of PAR-mediated thrombin in tumor metastasis.
Tumor cell metastasis or the dissemination of a tumor from
its original site to distant organs and tissues is an inherently
inefficient process. The ability of tumor cells to activate the
coagulation system and to generate thrombin has been shown
to enhance metastatic efficiency (105-108), while antico-
agulant therapies interfere with metastatic disease in animal
models and in humans (109). The prometastatic activity of
thrombin in cancer has been well demonstrated (Table IV)
(74,75,82,84,97,98,110-113). The principal thrombin receptor,
PAR1, has been implicated in the promotion of these effects
(Table IV).
PAR1 expression is correlated with metastatic potential.
In human breast cancer, PAR1 expression is associated with
tumor progression (82), and in prostate cancer it plays a role
in bone metastasis (114). Metastatic human melanoma cell
lines express PAR1 (102,110). The overexpression of PAR1
in murine and human melanoma cells results in enhanced
metastasis in mice (98,115). The overexpression of PAR1
also increases matrigel invasion by melanoma cells (112) and
thrombin stimulates the motility of colon carcinoma cells in
a PAR1-dependent manner (76,78). PAR1 antisense markedly
reduces the invasion of a metastatic breast cancer cell line
through a matrigel barrier (82). Pre-treatment of tumor cells
with PAR1 agonist peptides alters their adhesive behavior and
increases pulmonary metastasis (98). Thrombin-dependent
PAR1 signaling induces the proliferation of metastatic tumor
cells (116) and can be anti-apoptotic (117). PAR1 enhances
the αvβ5 integrin-dependent migration of tumor cells (118).
PAR1 has also been proposed to play a role in the pathological
invasion processes of breast cancer (82,97).
The prometastatic effects of thrombin on tumor cells may
involve the receptor cross-activation of PAR2, due to the
fact that the metastasis was enhanced by PAR2 stimulation
(111). Although the precise mechanism of PAR2 signaling
in metastasis remains to be determined, it is known that the
tethered ligand of PAR1 activates PAR2. PAR2 may thus
be activated by thrombin-cleaved PAR1, and PAR2 may act
as a relevant receptor for thrombin signaling under certain
conditions (119).
5. Therapeutic implications in cancer
The angiogenic and tumor-promoting effects of thrombin
provide the basis for the development of thrombin receptor
antagonists for therapeutic application in cancer. A number
Table V. PAR1 and PAR4 antagonists.
PAR1 antagonists Characteristic Selectivity Refs.
AFLARAA


Anti-PAR1 pepducin (P1pal-12)

FR-171113
RWJ-56110
RWJ-58259
SCH-328725

SCH-205831

SCH-79797

BMS-200261
BMS-200661
BMS-200260
tc-Y-NH2
Anti-PAR4 pepducin (P4pal-10)

YD-3
P20.1


Peptide with multiple alanine substitutions
in both critical and non-critical
residues of SFLLRN
Palmitoylated peptide based on
the human PAR1 i3 loop
Non-peptide PAR1 antagonist
Peptide-mimetic PAR1 antagonists
Indole-based SFLLR peptide mimetic
Non-peptide PAR1 antagonist based on
the natural product himbacine
Non-peptide PAR1 antagonist based on
the natural product himbacine
Non-peptide PAR1 antagonist based on
the natural product himbacine
Peptide-mimetic PAR1 antagonists
Peptide-mimetic PAR1 antagonists
Peptide-mimetic PAR1 antagonists
Trans-cinnamoyl (tc-) PAR4 peptide analogue
Palmitoylated peptide based on
the human PAR4 i3 loop
Non-peptide PAR4 antagonist
Monoclonal antibody directed against
the N-terminal 20 amino acid portion
of human PAR4
Not available 134
PAR1 selective 135,136
PAR1 selective
PAR1 selective
PAR1 selective
PAR1 selective
137
138-140
140-142
137
PAR1 selective 143
PAR1 selective 144
PAR1 selective
PAR1 selective
PAR1 selective
PAR4 selective
Higher selectivity for
PAR4 over PAR1
PAR4 selective
PAR4 selective
145,146
146
146
147-149
135,136,
147,150
138,151
152

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HAN et al: PARs IN CANCER
604
of promising targets may be utilitzed for drug discovery for
cancer therapeutics within the clotting cascade. Therapeutic
approaches to down-regulating thrombin generation in cancer
may accomplish three goals: anticoagulation, prevention of
angiogenesis and prevention of tumor growth and metastasis.
Thrombin directly affects signaling pathways that mediate
cell functions and clot formation, which provide a growth
medium for tumor cells. Therefore, anticoagulant drugs may
prove efficacious in cancer treatment as they are capable of
reducing the hypercoagulability of cancer. Thrombin-targeted
anticoagulant strategies designed to affect both the prothrom-
botic properties of tumors and their growth and metastatic
potential, have been evaluated in a number of pre-clinical
and clinical studies (120-122). However, studies providing
convincing evidence that this approach predictably improves
survival in cancer are limited (43,44).
Since it is believed that all tumors require angiogenesis
for tumor growth and metastasis, targeting tumor vascula-
ture with anti-angiogenic agents has developed into a novel
strategy for treating a number of solid tumors (123,124).
Anti-angiogenic agents generally elicit few toxic side effects
in contrast to standard chemotherapeutic agents. Nevertheless,
patients treated with both anti-angiogenic agents and standard
chemotherapy resulted in an unexpected high incidence of
both arterial and venous thrombosis, as reported in a number
of clinical trials (123,125). This serious complication has
been observed with various promising anti-angiogenic agents,
including VEGF Trap (124,125). It is plausible that synergistic
vascular toxicity occurs between anti-angiogenic agents and
chemotherapy drugs since virtually all chemotherapeutic
agents injected intravenously stimulated increased thrombin
generation (126). Therefore, adding thrombin-targeted antico-
agulants to combination drug regimens, including agents that
interact with the endothelium, may aid in the prevention of
some of these thrombotic complications by blocking thrombin
generation (126,127). Further stimulation for the addition
of anticoagulants to cancer treatment regimens is evident
in recent experimental studies in which non-anticoagulant
properties of anticoagulant drugs have been exploited to
reduce angiogenesis, tumor growth and metastasis (128-131).
Combination regimens of standard chemotherapeutics with
anticoagulants may provide added benefit for control of tumor
progression and simultaneously reduce the risks for serious
thrombotic complications. However, such a hypothesis should
be confirmed by prospective randomized controlled clinical
trials of anticoagulant drugs in cancer.
Inhibitors of thrombin have achieved success in antico-
agulant therapy, but are also accompanied with the risk of
bleeding (132,133). PARs themselves were considered to be
attractive targets for therapeutic drug development. Efforts to
develop receptor inhibitors as compared to targeting thrombin
are currently regarded as a priority. As outlined in Table V
(134-152), substantial success has been achieved in the develop-
ment of PAR1 and PAR4 antagonists.
PAR antagonists act by blocking the interaction of the
newly exposed tethered ligand with binding sites on the
extracellular surface of the receptor, but do not inhibit
thrombin binding or receptor cleavage. Small molecule PAR1
antagonists have been generated based on the structure of the
peptide ligand for PAR1 (4). Bradykinin-derived blocking
peptides appear to directly bind and suppress PAR1 activa-
tion and do not act as thrombin inhibitors (137). However,
a number of these molecules lack PAR1 selectivity due to
structural similarities to activating peptides of other PARs.
More selective and potent non-peptide PAR1 antagonists
for both experimental studies and pharmaceutical use in
humans are also currently available (7,137,139-143). In addi-
tion to increased PAR1 selectivity, these compounds exhibit
relatively potent inhibitory actions against both thrombin
and agonist/peptide-stimulated responses. The orally active
PAR1 antagonist developed by Schering (SCH-205831), which
suppresses PAR1 by competitively inhibiting the TL-binding
site (143), has been found to be effective as an antithrombotic
agent in humans. A number of other PAR1 antagonists and
monoclonal antibodies generated against the cleavage site of
PAR1 have also been used to block the activation of PAR1,
but their use is limited by relatively low efficacies (134,146,
153-155). The development of effective PAR1 antagonists is in
the early stages. However, further description and classifica-
tion of the recently developed compounds is likely to yield
crucial data towards generating novel effective antagonists for
PAR1, as well as for other PARs.
PAR4 antagonism has also been shown using the peptide
trans-cinnamoyl-YPGKF-NH2 in human platelets (148),
although its use in vivo was limited to its non-PAR actions
(156). A novel approach to receptor inhibition, through
targeting the receptor intracellular loops with palmitoylated
membrane-penetrating peptides termed pepducins, has
succeeded in developing a relatively high potency PAR4
antagonist (135,150,157). Pepducin, P4pal-10, has been proven
to be of use in blocking PAR4 activation both in vivo and
in vitro (135,157), although it is not completely selective for
PAR4 (156).
6. Conclusions
Evidence of PAR-mediated thrombin functions in angio-
genesis, tumorigenesis and metastasis is well established, as
mentioned above. PAR-mediated thrombin exerts its effects in
cancer indirectly by promoting angiogenesis, which is essential
for the growth and development of all solid tumor types, and
directly by promoting tumor growth and metastasis. The key
objective of investigating the role of PAR-mediated thrombin
in cancer is to develop thrombin-targeted drugs and PAR
antagonists for therapeutic application in cancer treatment.
Thrombin-targeted anticoagulant strategies designed to affect
both the prothrombotic properties of tumors and their growth
and metastatic potential have been evaluated in a number of
pre-clinical and clinical studies. However, studies providing
evidence that this approach may predictably improve survival
in cancer are limited. Therapeutic approaches that target PARs
themselves were considered to be attractive targets for thera-
peutic drug development. The development of effective PAR
antagonists, however, remains in the early stages.
Acknowledgements
This study was supported by the State Key Basic Research
and Development Program of China (973 Program, Grant
No. 2009CB521704), the National High-tech Research and

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ONCOLOGY LETTERS 2: 599-608, 2011
605
Development Program of China (863 Program, Grant No.
2006AA02A245), the National Natural Science Foundation of
China (Grant No. 30271450 and 30672365) and the Zhejiang
Provincial Science and Technology Project (Grant No.
2009C13021).
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