Advertisement

Thrombosis Journal

, 17:4 | Cite as

Protease-activated receptors (PARs): mechanisms of action and potential therapeutic modulators in PAR-driven inflammatory diseases

  • Dorothea M. Heuberger
  • Reto A. SchuepbachEmail author
Open Access
Review

Abstract

Inflammatory diseases have become increasingly prevalent with industrialization. To address this, numerous anti-inflammatory agents and molecular targets have been considered in clinical trials. Among molecular targets, protease-activated receptors (PARs) are abundantly recognized for their roles in the development of chronic inflammatory diseases. In particular, several inflammatory effects are directly mediated by the sensing of proteolytic activity by PARs.

PARs belong to the seven transmembrane domain G protein-coupled receptor family, but are unique in their lack of physiologically soluble ligands. In contrast with classical receptors, PARs are activated by N-terminal proteolytic cleavage. Upon removal of specific N-terminal peptides, the resulting N-termini serve as tethered activation ligands that interact with the extracellular loop 2 domain and initiate receptor signaling. In the classical pathway, activated receptors mediate signaling by recruiting G proteins. However, activation of PARs alternatively lead to the transactivation of and signaling through receptors such as co-localized PARs, ion channels, and toll-like receptors.

In this review we consider PARs and their modulators as potential therapeutic agents, and summarize the current understanding of PAR functions from clinical and in vitro studies of PAR-related inflammation.

Abbreviations

AC

Adenylyl Cyclase

AKT

Protein kinase B

aPC

Activated Protein C

COPD

Chronic Obstructive Pulmonary Syndrome

COX

Cyclooxygenase

ECL

Extracellular Loop

EGFR

Epidermal Growth Factor Receptor

EPCR

Endothelial Protein C Receptor

ERK

Extracellular Signal-regulated Kinase

F2R

Coagulation Factor 2 Receptor

F2RL1

Coagulation Factor 2 Receptor-Like 1

F2RL2

Coagulation Factor 2 Receptor-Like 2

F2RL3

Coagulation Factor 2 Receptor-Like 3

FVIIa

Activated Coagulation Factor VII

FXa

Activated Coagulation Factor X

GM-CSF

Granulocyte Macrophage Colony-stimulating Factor

GPCR

G Protein-Coupled Receptor

IBD

Inflammatory Bowel Disease

IBS

Irritable Bowel Syndrome

ICAM

Intercellular Adhesion Molecule

IL

Interleukin

LepA

Pseudomonas aeruginosa-Derived Large Extracellular Protease

LPS

Lipopolysaccharides

MAPK

Mitogen-Activated Protein Kinase

MMP

Matrix Metalloprotease

NFκB

Nuclear Factor kappa B

PAR

Protease-Activated Receptor

penC

Penicillium citrinum-Derived Alkaline Serine Protease C

PGE2

Prostaglandin E2

PI3K

Phosphatidylinositol-3-Kinase

PLCβ

Phospholipase C beta

Rac1

Ras-Related C3 Botulinum Toxin Substrate 1

RhoA

Ras homolog gene family, member A

RSTK

Receptor Serine/Threonine Kinase

RTK

Receptor Tyrosine Kinase

SpeB

Streptococcal Pyrogenic Exotoxin B

TF

Tissue Factor

TLR

Toll-Like Receptor

TM

Thrombomodulin

TRPV

Transient Receptor Potential Channels Vanilloid Subtype

VCAM

Vascular Cell Adhesion Molecule

VEGF

Vascular Endothelial Growth Factor

VEGFR

Vascular Endothelial Growth Factor Receptor

Introduction

The four mammalian members of the protease-activated receptor (PAR) family PAR1, PAR2, PAR3, and PAR4 are encoded by the genes F2R [1], F2RL1 [2], F2RL2 [3], and F2RL3 [4], respectively. Human PAR1 was discovered in 1991 as a key thrombin receptor on platelets [5, 6]. Although human and mouse PAR2 genes are homologous to PAR1 genes, PAR2 is not responsive to thrombin [2, 7, 8]. Unexpected responses of platelets to thrombin in PAR1 knockout mice lead to the discovery of the thrombin receptors PAR3 and PAR4 [4, 9, 10]. PAR regulation varies between species and tissues, with differing expression levels, protease cleaving activities, dimerization with other receptors, compartimentalization, trafficking, posttranslational modifications, and co-localization with co-receptors, as shown in Fig. 1.
Fig. 1

Mechanisms of PAR activation. PAR activation is regulated by a direct proteolytic cleavage at the N-terminus, b homo- or heterodimerization with other PARs and transactivation through the cleaved tethered ligand, c compartmentalization on the cell surface, d degradation or recycling by endosomal trafficking, e posttranslational modifications such as glycosylation, phosphorylation, and ubiquitination, and f co-localization with other receptors and cofactors

Studies of PAR activation under physiological conditions are crucial for the understanding of the pathophysiological roles of PARs, such as those in inflammatory disorders.

Cleavage and activation of PARs and signal transduction

PARs are specifically cleaved and irreversibly activated by various endogenous proteases, and by exogenous proteases from bacteria, plants, fungi, and insects. Proteases, soluble or cell membrane associated (bound to co-receptors or specific membrane compartments), cleave specific N-terminal peptides of PARs, resulting in exposure of new N-terminal peptides that serve as tethered activation ligands, which bind a conserved region on extracellular loop 2 (ECL2) [5, 11]. This interaction initiates conformational changes and alters affinity for intracellular G proteins [12]. Various N-terminal cleavage sites have been described, and these have various active conformations with specific G protein preferences. Multiple cleavage site-specific cellular responses are generally referred to as biased signaling, and the ensuing models describe how distinct proteases with distinct cleavage sites induce protease-specific responses via the same PAR [13, 14].

In contrast with PAR-activating proteases, other proteases cleave PARs at cleavage sites that are not related to signaling. Under these conditions, shedding of the PAR1 terminus, which removes the thrombin activation site, was first recognized as a mechanism for rendering platelets irresponsive to thrombin [15]. These truncated PARs can no longer be proteolyticaly activated, but remain activated by ligands from adjacent PARs [16]. Alternatively, truncated PARs bind soluble peptides with affinity for ECL2 by mimicking the tethered ligand. Both mechanisms result in receptor activation [17, 18]. Multiple ECL2-binding agonist peptides have been described and shown to induce signaling from truncated and uncleaved PARs (see agonist peptides in Tables 5, 6, 7).

PAR activation by proteolytical cleavage

PAR-cleaving proteases are a focus of many current studies. Whereas some PAR-cleaving proteases produce N-terminal components with regulatory roles, others render the receptors irresponsive to further protease exposure as shown in Fig. 2 and summarized in Tables 1, 2, 3 and 4. Important proteases are discussed below.
Fig. 2

Proteolytic PAR cleavage. a N-terminal sequences of human PARs (PAR1–4) containing potential cleavage sites. b Proteolytic cleavage of PARs by soluble exogenous proteases exposes new N-terminal sequences that serve as tethered ligands for G protein dependent activation of receptors. Alternatively, proteolytic cleavage at other sites destroys the function of the receptor to prevent intracellular signal transduction

Table 1

PAR1 cleaving proteases

 

Protease

Major cleavage site

Additional cleavage sites

Mammalian proteases

Thrombin

R41S42

 

aPC

R46N47

R41S42

FVIIa

unknown

 

FXa

R41S42

 

Trypsin

R41S42

 

Chymase

unknown

 

MMP-1

D39P40, L44L45, F87I88

N47P48, R70L71,K82Q83

MMP-2

L38D39

 

MMP-3,-8,-9

R41S42

 

MMP-12

unknown

 

MMP-13

S42F43

L38T39, mouse

Cathepsin G

R41S42, F55W56, Y69R70

 

Neutrophil elastase

A36T37, V72S73, A86F87

 

Proteinase-3

A36T37, P48N49, V72S73, A92S93

 

Plasmin

K32A33, R41S42, R70 L71, K76 S77, K82 Q83

 

Kallikrein-4,-5,-6

unknown

 

Kallikrein-14

R46N47

 

Granzyme A,B, K

unknown

 

Calpain-1

K32A33, S76K77

 

Non-mammalian proteases

PA-BJ

R41S42, R46N47

 

Thrombocytin

R41S42, R46N47

 

DerP1

unknown

 

Gingipain R

R41S42

 

SpeB

L44L45

 

LepA

unknown

 

S.pneumoniae proteases

unknown

 

Thermolysin

F43L44, L44L45

 

penC

R41S42

 
Table 2

PAR2 cleaving proteases

 

Protease

Major cleavage site

Additional cleavage sites

Mammalian proteases

Thrombin

R36S37

 

aPC

unknown

 

FXa

R36S37

 

Trypsin

R36S37

K34G35, K51G52, K72L73

Tryptase

R36S37

 

Chymase

G35R36

L38I39, mouse

Matriptase

R36S37

 

Cathepsin G

F65S66

F59S60, F64S65

Cathepsin S

G40K41

E56P57, mouse

Neutrophil elastase

A66S67, S67V68

V42D43,V48T49,V53T54,V58T59,T74T75,V76F77

Proteinase-3

D62E63

V48T49,V55E56,T57V58 V61D62,K72L73,T74T75,T75V76,V76F77

Plasmin

R36S37

K34G35

Testisin

unknown

 

Kallikrein-4,

unknown

 

Kallikrein-5,-6,-14

R36S37

 

Calpain-2

unknown

 

Non-mammalian proteases

Der-P1,-P2,-P3,-P9

unknown

 

Cockroach E1-E3

R36S37

 

Gingipain R

unknown

 

LepA

unknown

 

EPa

S37L38

S38L39, rat

S.pneumoniae proteases

unknown

 

Thermolysin

unknown

 

Serralysin

unknown

 

P.acnes proteases

unknown

 

aPA

unknown

 

Bromelain

unknown

 

Ficin

unknown

 

Papain

unknown

 

penC

R36S37

 
Table 3

PAR3 cleaving proteases

 

Protease

Major cleavage site

Additional cleavage sites

Mammalian proteases

Thrombin

K38T39

mouse PAR3 at K37S38

aPC

R41G42

 

FXa

R41G42

 

Trypsin

unknown

 
Table 4

PAR4 cleaving proteases

 

Protease

Major cleavage site

Additional cleavage sites

Mammalian proteases

Thrombin

R47G48

 

Trypsin

R47G48

 

Cathepsin G

R47G48

 

Kallikrein-14

unknown

 

Non-mammalian proteases

PA-BJ

R47G48

 

Thrombocytin

R47G48

 

Der-P3

unknown

 

Gingipain R

R47G48

 

LepA

unknown

 

S.pneumoniae proteases

unknown

 

Bromelain

unknown

 

Ficin

unknown

 

Papain

unknown

 

Mammalian proteases

Serine proteases

Thrombin, the key protease of coagulation, is generated by proteolytic cleavage of zymogen prothrombin. Although thrombin production predominantly occurs on platelets and subendothelial vascular walls, extravascular thrombin has been detected in synovial fluid [19] and around tumors [20]. Thrombin has long been known to activate platelets, and the discovery of PAR1 initiated research into the underlying molecular mechanisms. PAR1 contains a hirudin-like domain, which has a high affinity thrombin binding site and recruits thrombin via exosite I. This interaction enables thrombin to specifically and efficiently activate PAR1 [6]. Similarly, PAR3 contains a hirudin-like thrombin recruitment site, which results in cleavage [9, 21]. In other studies, mouse PAR3 maintained thrombin recruitment activity but lost its receptor function, as discussed above [22, 23, 24]. Thrombin also cleaves and activates PAR4, which, in contrast with PAR1, lacks a hirudin-like domain. Thus, higher concentrations of thrombin activate PAR4 and initiate intracellular signaling [10]. PAR2 is considered the only PAR that resists cleavage or activation by thrombin [4, 25], although emerging evidence suggests that at very high concentrations (100–500 nM), thrombin may directly cleave and activate PAR2 [26, 27].

In contrast with thrombin, the anticoagulant protease activated protein C (aPC) binds to the co-receptor endothelial protein C receptor (EPCR) to promote the cleavage and activation of co-localized PAR1 [28, 29] and induce anti-apoptotic and protective effects on endothelial barrier permeability [29, 30, 31, 32, 33]. Compartmentalization of PAR1 and co-localization with EPCR in calveolae is crucial for efficient cleavage by aPC [13]. Moreover, aPC cleaves PAR3 in humans and mice [21, 34, 35] and acts as a PAR3 shedding protease that prevents thrombin-induced barrier disruption [21]. However, the dependency of aPC cleavage of PAR3 on EPCR remains controversial [21, 35]. Similar to aPC, coagulation factor Xa binds EPCR and mediates proteolytic activation of PAR1 and PAR3 [21, 28, 36, 37, 38, 39]. In addition, EPCR-bound factor Xa reportedly cleaves PAR2 and initiates inflammatory signaling [40]. PAR2 was also shown to be activated by tissue factor (TF)-bound coagulation factor VIIa [40, 41, 42]. Yet recent studies suggest that the TF-VIIa complex does not directly activate PAR2, and rather activates matriptase, which cleaves and activates PAR2 [42, 43, 44]. Anti-inflammatory signaling was also previously related to PAR1 cleavage by EPCR-bound VIIa [45, 46]. Taken together, these studies indicate that TF-Xa–VIIa complexes activate PAR1 and PAR2 [47].

Trypsins are PAR-activating proteases with roles as major digestive enzymes in the duodenum [48]. Trypsin is also secreted by epithelial cells, nervous system cells [49], and tumor cells [50, 51]. Trypsins may also be involved in cell growth and coagulation, as suggested by secretion from human vascular endothelial cells [52]. Trypsin cleaves human PAR1 and PAR4 at putative protease cleavage sites, and thereby prevents thrombin signaling in endothelial cells and platelets [4, 53]. Trypsin is the major PAR2 cleaving protease that initiates inflammatory signaling [2, 7].

Tryptase is the main protease of mast cells, and activates PAR2 by proteolytic cleavage to induce calcium signaling and proliferation [54, 55, 56, 57]. The source tissue of tryptase reportedly plays an important role in the cleavage and induction of tryptase-activated PAR signaling, reflecting differences in posttranslational modifications, such as glycosylation and sialic acid modifications [54, 58]. Tryptase induces calcium signaling via PAR1 when PAR2 is co-expressed, but cannot activate human platelets, suggesting that tryptase does not directly cleave PAR1 [54, 55, 56, 57]. Chymase is a mast cell serine protease that also cleaves PAR1 in human keratinocytes and fibroblasts, and thus prevents thrombin sensitivity [59]. Moreover, the epithelial serine protease matriptase cleaves and initiates inflammatory responses in human and mouse keratinocytes and in Xenopus oocytes overexpressing human PAR2 [44, 60, 61, 62, 63].

PARs have been identified as substrates of kallikreins, which are serine proteases that have been related to various inflammatory and tumorigenic processes [64]. Kallikrein-4 increases intracellular calcium levels via PAR1 and PAR2, but activates PAR1 most efficiently [65]. Kallikrein-14 induces calcium signaling via PAR1, PAR2, and PAR4, but can also shed PAR1 to prevent signaling. Rat platelets are activated by kallikrein-14 via the proteolytic cleavage of PAR4, but are not activated by kallikrein-5 and kallikrein-6 [66]. Instead, neurotoxic effects of kallikrein-6 were inhibited by blocking PAR1 and PAR2, indicating a direct proteolytic role in PAR activation [67].

Neutrophils are mobilized to sites of inflammation and infection, where they modulate inflammatory signaling, in part by secreting PAR-cleaving proteases. The neutrophil serine protease cathepsin G prevents thrombin-induced effects by cleaving PAR1 into non-functional parts [68, 69]. In contrast, cathepsin G reportedly induced chemoattractant signaling via PAR1, further supporting the role of cathepsin G in PAR1 activation [70]. Another unexpected observation of cathepsin G was that cleavage sites differ between recombinant and native human PAR2 [26, 71, 72]. These discrepancies may reflect the influence of cell types and posttranslational modifications on PAR cleavage. Studies in mice and humans show that platelet activation by cathepsin G is dependent on PAR3 and PAR4 [71, 73, 74]. Cathepsin G also cleaves and activates PAR4 on endothelial cells [75]. The neutrophil proteases elastase and proteinase-3 cleave recombinant PAR1 and PAR2 at various sites [26, 72]. Recently, rat elastase was shown to cleave and activate PAR1, although sequences of rat and human PAR1 have low homology [76]. In contradiction with neutrophil proteases that prevent PAR signaling at sites of inflammation, monocytes secret the protease cathepsin S, which initiates inflammatory signaling by cleaving PAR2 [72, 77, 78]. Low concentrations of the fibrinolytic protease plasmin prevent platelet activation by cleaving PAR1, whereas high concentrations of plasmin lead to the cleavage and activation of PAR1 [79]. Plasmin also cleaves PAR2 and prevents subsequent activation by trypsin [26, 80].

The serine proteases granzyme A and granzyme B induce intracellular signaling pathways that lead to neuronal death via PAR1 [81, 82]. Recently, granzyme K was also shown to activate PAR1 and promote inflammatory endothelial signaling [83, 84]. Few studies show activation of PAR1 by proteases of the granzyme family, and the details of this interaction remain poorly characterized.

Cysteine proteases

Calpain-1 is a calcium-dependent cysteine protease that has been associated with inflammatory disorders, and initiates calcium signaling pathways by activating PAR1 [26]. At very high concentrations, calpain-2 was also shown to cleave PAR2, and the authors suggested that this cleavage event inactivated PAR2 [26]. Recently, calpain-1 was shown to be induced by thrombin-activated PAR1, and subsequently regulated the internalization of PAR1 [85].

Metalloproteases

Matrix metalloproteases (MMPs) are known to be involved in various inflammatory- and cancer-related conditions. MMP-1 cleaves human PAR1 and initiates platelet activation [86, 87, 88, 89]. MMP-1 also regulates cancer cell activities depending on PAR1 availability [90]. Similarly, MMP-2 cleaves human PAR1 and enhances platelet activation [91], and MMP-3, MMP-8, and MMP-9 were shown to induce platelet activation via PAR1 [92]. Whether these three MMPs cleave PAR2 is not clear, although PAR2 activation by trypsin induced secretion of MMP-9 in human airways, suggesting that MMP-9 is a PAR2-activating protease [93]. In mice, PAR1 expression was regulated by MMP-12, and activated PAR1 increased MMP-12 secretion [94, 95]. A similar feedback loop involving MMP-12 and PAR2 has been reported in mice [96]. Moreover, MMP-13 was shown to activate PAR1 and induce intracellular signaling [87], and thrombin-induced activation of PAR1 and PAR3 was associated with increased levels of MMP-13 in human chondrocytes [24].

In addition to coagulation and inflammation, PAR activation may play roles in human germ cells, where the serine protease testisin activates PAR2 and induces calcium signaling and ERK1/2 activation. This interaction may play roles in the regulation of ovarian and testicular cancer, as suggested previously [97, 98].

Non-mammalian proteases

Exogenous proteases from various species that modulate PAR activation are disscues in the following section and are summarized in Fig. 3.
Fig. 3

Non-mammalian exogenous proteases induce PAR-driven pathological effects. Various proteases are secreted from bacteria, amoebae, insects, plants, fungi, and snakes, and can cleave PARs and modulate signal transduction, leading to inflammation, thrombosis, or pain

Bacterial proteases

Endogenous mammalian proteases are not the only regulators of PAR activation. Indeed, both pathogenic and commensal bacteria secret various proteases that cleave PARs and act as inflammatory modulators [99]. In this section, we describe bacterial proteases that either activate PARs, and thus allow bacteria to penetrate host barriers, or inactivate PARs to prevent inflammatory signaling by the host.

The human pathogen Pseudomonas aeruginosa secrets two PAR-cleaving proteases with contrasting effects. The exoprotease LepA cleaves and activates PAR1, PAR2, and PAR4, and subsequently induces nuclear factor kappa B (NFκB) promoter activity [100], whereas cleavage by elastase EPa inactivates PAR2 to prevent inflammation in lungs [101].

The streptococcal pyrogenic exotoxin B (SpeB) of Group A Streptococcus also inactivates PAR1 by cleaving it, and thereby renders human platelets unresponsive to thrombin [102]. In mice, proteases of Streptococcus pneumoniae cleaved PAR2 and facilitated the spread of the pathogen from the airways into the blood stream [103]. PAR1 has also been associated with S. pneumonia-mediated sepsis in mice, although direct cleavage of PAR1 was not shown [104, 105]. Pulmonary inflammation from S. pneumoniae infections is reduced in PAR4 knockout mice [106], further supporting this causal link.

Inflammation-associated periodontal diseases are predominantly induced by the Porphyromonas gingivalis cysteine protease gingipain R, which activates PAR2 [107, 108]. Subsequently, gingipain R activates PAR1 and PAR4, and thereby, human platelets [109, 110, 111]. This mechanism may also explain associations between periodontitis and cardiovascular events [112].

In addition, supernatants from Propionibacterium acnes cultures initiated inflammatory signaling in human keratinocytes via PAR2 [92]. The virulence of P. acnes was also reduced in PAR2 knockout mice [113], further suggesting that PAR2 is involved in bacterial infections.

Serralysin is a matrix metalloprotease expressed by Serratia marcescens, and induced inflammation in human airway cells via PAR2 in vitro [114].

Finally, Bacillus thermoproteolyticus rokko secretes the metalloprotease thermolysin, which cleaves and inactivates PAR1 to prevent thrombin-induced signaling in rat astrocytes [115, 116]. The in vitro effects of PAR2-cleavage by thermolysin, however, vary between cell lines [116].

Amoeba proteases

In acanthamoebic keratitis, PAR2 triggers inflammation following secretion of the plasminogen activator (aPA) by Acanthamoeba strains, leading to induction of IL-8 in human corneal epithelial cells [117].

Reptile proteases

Following snakebites, coagulation disorders in humans and mice occur due to the presence of venom proteases. In Proatheris superciliaris bites, venom proteases activate platelets by activating PAR1 and PAR4 [118]. Bothrops atrox and B. jararaca are snake species of the family viperidae. These snakes secrete the serine proteases PA-BJ and thrombocytin, which activate human platelets via PAR1 and PAR4 [119].

Insect proteases

Several cysteine and serine proteases from insects induce inflammation-associated diseases such as asthma. For example, dust mite allergens contain the serine proteases DerP2, DerP3, and DerP9 [120] and the cysteine protease DerP1. DerP1 induces PAR2-dependent signaling, whereas thrombin-induced PAR1-signaling is prevented by these proteases in human epithelial cells [121]. DerP3 was also recently shown to activate PAR4, and this process was associated with allergies to dust mites [122].

Similar to proteases from house dust mites, three serine proteases (E1–E3) from cockroach extracts activate PAR2 and induce inflammatory signaling in mice and humans [123, 124, 125].

Fungal proteases

Pen C is a serine protease from Penicillium citrinum that induces IL-8 in human airway cells by activating PAR1 and PAR2 [126]. Proteases from Aspergillus fumigatus have also been shown to prevent PAR2-dependent inflammation [127]. Moreover, serine proteases from Alternaria alternate induced calcium signaling in human bronchial cells and induced inflammation in mice by secreting IL-33 following PAR2 activation [128, 129, 130].

Plant proteases

Bromelain is a mixture of cysteine proteases that is extracted from pineapple which is used as a PAR-independent anti-inflammatory agent [131]. Bromelain cleaves PAR2 and thereby prevents the associated inflammatory signaling [132]. In another study, however, bromelain, ficin, and papain activated PAR2 and PAR4 by proteolytic cleavage, leading to increased intracellular calcium levels [133]. Thus, further studies are required to further clarify the modes of action of pineapple proteases.

Cleavage-independent PAR activation by agonist peptides

Independent of proteolytic cleavage, PARs can be activated by synthetic soluble ligands corresponding with cleaved N-terminal sequences, or can be transactivated by cleavage-generated N-terminal regions of homo- or heterodimer partners.

Synthetic peptides that mimic the first six amino acids of tethered N-terminal ligands can act as agonist peptides that activate PARs in the absence of cleavage events [11, 18, 134]. Specific activation of PARs by a soluble agonist peptide was first shown for human PAR1 with the peptide SFLLRN [6, 18]. However, this peptide also activated PAR2 [135, 136, 137] and therefore various peptides were tested for specific PAR1 activation. Yet, PAR1 was the most specifically and efficiently activated by TFLLRN [138]. In addition to thrombin agonist peptides, other PAR1 agonist peptides have been identified. In particular, the peptide NPNDKYEPF reproduced the effects of aPC [28], and PRSFFLRN corresponds with the N-terminal peptide generated by MMP-1 [86]. SLIGKV corresponds with the trypsin cleaved N-terminal region of human PAR2. However, the corresponding rat N-terminus SLIGRL is a more specific and efficient PAR2 agonist in rodents and humans [136, 139], and only the synthetic peptide LIGRLO achieved this effect more efficiently than SLIGRL in humans [140]. The roles of ECL-2 in specific PAR activation have been shown using labeled PAR2 agonist peptides [141, 142]. Because the thrombin generated PAR3 peptide does not activate the G protein autonomously, no such agonist peptides have been identified to date [9, 143]. GYPGKF corresponds with the thrombin-cleaved human PAR4 and has weak activity as an agonist [144]. But replacement of the first amino acid glycine (G) with alanine (A) induced PAR4 by 10-fold. This peptide may be suitable as a platelet activator in humans and mice [145].

Several models of PAR–PAR interactions have been proposed and extensively studied based on PAR transactivation by agonist peptides [146]. When PAR1 is blocked on endothelial cells, however, thrombin, and not the PAR1-specific agonist peptide TFLLRN, induces signaling, reportedly by facilitating the heterodimerization of PAR1 and PAR2 [147]. Thrombin activation of the PAR1–PAR2 heterodimer leads to constitutive internalization and activation of β-arrestin by the PAR1 C-tail [146]. Accordingly, the required co-localization of PAR1 and PAR2 was shown in a human overexpression system, in mice studies of sepsis, and in PAR1–PAR2-driven cancer growth in a xenograft mouse model [148, 149]. In other studies, stable heterodimerization of human PAR1 and PAR4 was shown in platelet cells, and thrombin accelerated platelet activation under these conditions [150, 151]. Similar studies of mouse platelets showed efficient activation of platelets by thrombin in the presence of PAR3–PAR4 heterodimers [143]. Consistent with the thrombin-cleaved PAR3 peptide, which is not self-activating, PAR3 signaling was observed in the presence of PAR1 or PAR2 [22, 23, 34, 152]. Yet, heterodimerization influenced signal transduction and PAR membrane delivery due to enhanced glycosylation [153].

In addition to activation by heterodimerization, PARs interact with other receptors, such as ion channels, other G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs), receptor serine/threonine kinases (RSTKs), NOD-like receptors, and TLRs [154]. In particular, PAR2 initiated inflammatory signaling pathways, resulting in pain due to transactivation of the ion channels TRPV1 and TRPV4 in humans and mice [155, 156, 157, 158, 159]. Similar inflammatory effects follow transactivation of the RTKs EGFR and VEGFR by PAR2 and PAR4 [160, 161, 162, 163]. Bacterial interactions with PARs suggest important roles of PARs in infectious disease. In agreement, TLRs recognize bacteria-derived molecules and contribute to innate immunity [164, 165]. Moreover, direct interactions of PAR2 with TLR3 and TLR4 were necessary for inflammatory responses to LPS in human cell lines and knockout mice and rats [166, 167, 168, 169, 170, 171].

PAR signaling

Activation pathways

PARs belong to a large family of GPCRs and induce multiple signaling pathways after coupling with heterodimeric G proteins. Activation of the Gα-subunit due to the exchange of a guanine from GDP to GTP results in dissociation of the Gβγ-dimer and activation of downstream pathways [172, 173].

Following proteolytic cleavage or induction of agonist peptides, the engaged signaling pathways vary between tissues, cell lines, and the availability of co-receptors for transactivation. Depending on the ligand, specific α-subunits are activated, and these regulate subsequent cellular functions as summarized in Fig. 4. For example, thrombin-stimulated PAR1 activates the small GTPase protein RhoA via ERK1/2 kinases, but not via Rac1, whereas aPC-stimulated PAR1 induces Rac1 via Akt kinase, but not via RhoA [13, 174, 175, 176]. Moreover, in accordance with PAR1 cleavage sites, aPC prevents thrombin-induced RhoA signaling [16]. However, in contrast with thrombin-induced RhoA activation on platelets and endothelial cells, PAR1-agonist peptides and thrombin activated the inhibitory G protein Gi which leads to the inhibition of adenylyl cyclase in human fibroblasts [177, 178]. Other studies indicate that PAR2 activation is less tissue specific than PAR1 activation, and trypsin and VIIa cleaved PAR2 and activated Gαq and Gi, resulting in calcium influx, MAPK activation, and inflammatory signaling [8, 179].
Fig. 4

G protein-coupled signaling induced by PAR activation. Depending on the tethered ligand, activated PAR couples with G protein α-subtypes. Gαq activates phospholipase Cβ (PLCβ), which mobilizes calcium. This further activates MAPKs (ERK1/2) and induces Ras signaling. Primarily, Gα12/12 and Gaq activate the Rho pathway. Gαi inhibits the activation of adenylyl cyclase, which leads to reduced production of cAMP. In contrast, the βγ-subunit functions as a negative regulator when bound to the α-subunit. After receptor activation, subunits separate, and the βγ-subunit interacts with other proteins, thereby activating or inhibiting signaling

Signaling by tethered ligands can differ from that generated by corresponding soluble agonist peptides. For example, thrombin-cleaved PAR1 activated Gα12/13 and Gαq and induced Rho and Ca2+ signaling, whereas the PAR1-agonist peptide activated only Ga12/13 and downstream RhoA-dependent pathways that affected endothelial barrier permeability [180]. Similar observations of human platelets suggested that platelet activation followed coupling of thrombin-activated PAR1 with multiple heterotrimeric G protein subtypes, including Gα12/13 and Gαq [181, 182, 183]. Moreover, trypsin and the PAR2-agonist peptide induced ERK1/2 signaling and inflammation by activating PAR2 [29, 180, 184, 185, 186]. β-arrestins also play major roles in PAR-induced signaling independently of G protein activation. For instance, aPC-activated PAR1 induces cytoprotective effects by recruiting β-arrestin in endothelial cells. Thus, aPC cleavage fails to protect β-arrestin deficient cells from the effects of thrombin [187, 188]. In addition, multiple studies show that activated PAR2 co-localizes with β-arrestin-1 and arrestin-2 and induces ERK1/2 signaling [77, 189, 190, 191].

Desensitization and termination

PAR activation is regulated by internalization and proteolytic desensitization, which limits the duration of signaling. For instance, PAR1 is constitutively internalized and recycled or agonist-induced internalized and degraded as described in [192, 193] and shown in the scheme of Fig. 5. As discussed above, some PAR-cleaving proteases abolish receptor responses by removing (shedding) or destroying the tethered ligands. For example, PAR1 is inactivated following cleavage by cathepsin G, and thrombin activation is hence prevented, allowing the formation of clotting under inflammatory conditions.
Fig. 5

PAR trafficking. Activation-independent constitutive or agonist-induced internalization regulates PAR1 signaling

Depending upon proteolytic cleavage, PAR1 rapidly internalizes or accumulates on the cell surface [194, 195]. Activated PAR1 is internalized via clathrin- and dynamin-dependent mechanisms, and is sorted from early endosomes to lysosomes for degradation [196, 197, 198, 199]. Although the mechanisms that terminate PAR1 signaling are not clearly understood, this process is known to involve phosphorylation, ubiquitination, and recruitment of β-arrestin [200, 201, 202, 203, 204]. In contrast with PAR1, activated PAR2 is not constitutively internalized [205]. Thus, to prevent persistent signaling upon activation, PAR2 is phosphorylated and ubiquitinated and then binds β-arrestin before being internalized and degraded [206, 207, 208]. Under these conditions, the activated and internalized PAR2 is not recycled and instead induces β-arrestin-dependent endosomal ERK1/2 signaling in the cytoplasm [189, 191, 209]. Thus, large cytoplasmic stores of newly generated PAR2 are required for rapid externalization and activation on cell membranes [210]. Although less is known about how PAR4 signaling is terminated, recent observations suggest that PAR4 internalization is independent of β-arrestin and slowly occurs via clathrin- and dynamin-dependent pathways [211]. In agreement, human platelets internalized PAR4 much slower than PAR1, and exhibited prolonged PAR4 signaling activity [212]. Moreover, growing evidence indicates that PAR–PAR heterodimerization is important for internalization, and that the underlying mechanisms include PAR2-dependent glycosylation of PAR4, thus affecting membrane transport [153]. Upon internalization, endosomal PAR4 dimerizes with the purinergic receptor P2Y12 and induces Akt signaling by recruiting β-arrestin within endosomes [213].

Depending on stimuli, PAR expression patterns are regulated by complex combinations of cell surface presentation, endocytosis, vesicle born or recycled (i.e., re-exocytosed) receptors, and trafficking modes that are linked to posttranslational modifications of PAR.

Role of PARs in inflammation

With the current increases in the prevalence of inflammatory diseases, published in in vitro and in vivo studies of the roles of PARs in inflammation have become more numerous. These are reviewed below.

Systemic inflammation and inflammatory cells in the cardiovascular system

PARs are critical for the interplay between clotting proteases of platelets, endothelial cells, and vascular smooth muscle cells that regulate hemostasis, vascular barrier function, vascular tone, vascular homeostasis, cell adhesion, and inflammatory responses [150]. The roles of PARs in these processes vary significantly between species. Specifically, whereas functional PAR1 and PAR4 are expressed in human platelets [214], PAR1, PAR3, and PAR4 have been found in guinea pig platelets [215]. Whereas mouse and rat platelets lack PAR1, they are activated at low concentrations of thrombin, which is recruited by PAR3 onto the surface of platelets and then efficiently activates PAR4 [4]. Due to interspecies differences in PAR expression, mouse and rat studies of PARs are difficult to translate to humans. PARs in endothelial cells contribute positive regulatory signals for endothelial adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin [216, 217], all of which promote vascular barrier function. As a counterpart of intravascular cells, PAR4 induces leukocyte migration [75], and PAR2 expressed on macrophages promotes inflammatory modulators such as interleukin-8 (IL-8) [218]. These modes of signaling all contribute to a complex PAR-mediated interplay of endothelial cells that is orchestrated by intravascular cells and cytokine secretion. In addition, PARs, particularly PAR1, regulate vascular barrier function, and hence, extravasation of macromolecules such as complement proteins and antibodies. In addition, thrombin-mediated activation of PAR1 increases endothelial barrier permeability by activating mitogen-activated protein kinases (MAPKs) [219]. Although this effect is reversed by activated protein C (aPC)-mediated activation of PAR1 [28, 174, 175, 220]. Thrombin further promotes prostaglandin 2 (PGE2) secretion, and consequent endothelial barrier permeability [221]. Similarly, PAR1 activation increased vascular leakage in a murine model [222]. Inflammatory mediators, such as tumor necrosis factor alpha (TNFα), were shown to regulate the expression of endothelial PAR2, and the authors suggested that these data were indicative of barrier protective effects of PAR2 [223]. Several other studies show that PAR2 activation induces endothelium-dependent relaxation in blood vessels of mice and in arteries of rats [224, 225, 226, 227, 228]. In contrast, dual activities of PAR2 on blood vessels were reported in a study of rats [229]. In this line, thrombin-activated PAR1 induced the expression of vascular endothelial growth factor in smooth muscle cells [230], thus revealing the relationship between coagulation and vascular growth. Although the roles of PARs in the development of arteriosclerosis are yet to be elucidated, PAR2 and PAR4 were induced in human arteries under inflammatory conditions [223], suggesting important roles of PARs in vascular inflammation.

Chronic inflammation of the gastrointestinal tract

In the gut lumen, human and bacterial proteases are both present at high concentrations. Similar to endothelial barriers, proteases regulate intestinal barrier permeability via PARs, all four of which are expressed by cells of the gastrointestinal tract [9, 224, 231, 232]. Trypsins and tryptases are prominent intestinal proteases, suggesting likely involvement of PAR2 as a major receptor of intestinal inflammation. In accordance, intestinal tight junctions are disrupted by PAR2-activating proteases, leading to inflammatory signaling in humans and rats [139, 206, 233, 234]. Although the roles of PARs in irritable bowel syndrome (IBS) and inflammatory bowel diseases remain unclear, roles of PARs in intestinal barrier function have been described. Specifically, PAR1 and PAR2 regulated permeability and chloride secretion, which are involved in diarrhea and constipation in IBS patients [234, 235, 236]. In addition, activated endosomal PAR2 caused persistent pain in a mouse model of IBS [209].

Inflammatory diseases of the respiratory system

It has long been suggested that PARs are involved in the pathophysiology of respiratory disorders, reflecting observations of elevated levels of PAR-activating proteases, such as thrombin and tryptase, in bronchoalveolar lavage fluid from patients with pulmonary inflammation [237, 238]. In a sheep asthma model and in asthmatic patients, tryptase inhibitors reduced inflammation [239, 240], further indicating important roles of PAR2 in respiratory disease. These roles of PARs are also suggested by the prominence of a variety of non-mammalian PAR-activating proteases, such as those of house dust mites and cockroaches [120, 123, 124]. Expression of PAR1, PAR2, and PAR4 on bronchial epithelial and smooth muscle cells induced inflammatory signaling in multiple studies [55, 121, 241, 242, 243, 244, 245]. PAR2 is also upregulated in epithelial cells of patients with asthma and chronic obstructive pulmonary syndrome (COPD) [246, 247]. Whether PAR2 activation results in bronchoconstriction or dilatation remains controversial, in part owing to interspecies differences and tissue dependencies [242, 248, 249]. In humans, however, PAR1-agonist peptides with thrombin, and a PAR2-agonist peptide with trypsin and tryptase, induced bronchoconstriction by inducing Ca2+ signaling in airway smooth muscle cells [241, 244]. Moreover, the long-term activation of PAR1 and PAR2 led to pulmonary fibrosis in mice models [250].

Inflammatory skin diseases

High concentrations of exogenous proteases are present on the skin of various species, and these may activate PARs to regulate epidermal permeability and barrier function [251]. Indeed, epidermal inflammation has been linked to PAR1 and PAR2 activation in keratinocytes, which comprise the epidermal barrier with sub-epidermal skin fibroblasts [179, 252, 253]. Subsequent release of IL-8, IL-6, and granulocyte macrophage colony-stimulating factor (GM-CSF) was also observed previously [254], potentially involving NFκB activation [255]. In addition, the inflammatory roles of PAR2 have been demonstrated in mice models of atopic dermatitis due to elevated tryptase and PAR2 expression levels [256, 257]. Similar to studies in mouse models, PAR2 was upregulated in patients with atopic dermatitis, and PAR2 agonists increased itch, causing irresponsiveness of sensory nerves to therapy with antihistamines [258].

Rheumatic disease

“Rheumatic disease” is a common term for autoimmune diseases that affect joints, bones, and muscles. Although rheumatic disorders are numerous, some of the common underlying symptoms include chronic joint inflammation, stiffness, and pain [259]. Currently, PAR2 is the only PAR that has been associated with the development of rheumatic diseases [260]. Direct roles of PAR2 in rheumatic diseases were first indicated in 2003 in a mouse study by Ferrell et al. [261]. In their study, a PAR2-agonist peptide induced strong inflammatory effects in wt mice, causing joint swelling and synovial hyperemia, whereas joint swelling was absent in PAR2 deficient mice [261]. Similarly, in patients with rheumatoid arthritis, PAR2 is upregulated in inflamed tissues [262]. Further increases in PAR2 expression were noted in monocytes, and the PAR2-agonist peptide upregulated IL-6. In contrast, PAR2 expression was decreased after treatments with antirheumatic drugs [263], further supporting the role of PAR2 in rheumatic disease.

PAR modulators as targets for therapy

The complexity of PAR regulation is indicated by the culmination of specific proteolytic cleavage modes (inactivating or activating), protease inhibitors, and cofactors, and with the effects of PAR glycosylation and dimerization (Fig. 1). In this section we discuss classes of agonists and antagonists that have been tested as PAR modulators for use as therapeutic agents as summarized in Fig. 6 and Tables 5, 6 and 7.
Fig. 6

PAR modulators. Pharmacological substances, such as 1) peptides and peptidomimetics, 2) blocking antibodies, 3) small molecules, 4) pepducins, and 5) parmodulins are used as therapeutic agents that affect PAR activities

Table 5

PAR1 signaling modulators

Class

Agonist/ Antagonist

Name

Receptor/Cell/Tissue type

Cellular response

Peptide

Agonist

SFLLRN/−NH2

Human

Induces platelet activation [6, 138, 265, 278, 279]

TFLLRN/−NH2

Human

Induces platelet activation, enhances endothelial barrier permeability [137, 138, 265]

NPNDKYEPF/−NH2

Human

Induces cytoprotective signaling [28, 187]

PRSFLLRN/−NH2

Human

Induces platelet activation [86]

Human

Induces ERK1/2 activation [280]

Antagonist

YFLLRN

Human

Compets with thrombin and PAR1-AP and prevents platelet activation [278, 279]

Peptidomimetic

Antagonist

RWJ-56110

Human

Blunts thrombin and PAR1-AP effects on platelets and vascular endothelial cells [264, 281, 282]

Human

Blocks MMP-1 activaiton in SMCs [87]

RWJ-58259

Guinea pig

Blocks thrombin and PAR1-AP platelet activation [215, 283]

Rat

Blocks thrombin induced calcium release in AoSMC Inhibits intimal thickening [111, 215, 264, 273]

Mouse

Prevents destruction of intestinal barrier [62, 284]

Non-peptide small molecule

Antagonist

FR17113

Human

Blocks PAR1-AP induced platelet activation [285, 286]

Human

Inhibits thrombin and PAR1-AP induced ERK1/2 activation [287]

ER129614–06

Human

Blocks thrombin and PAR1-AP induced platelet activation [288]

Guinea pig

Shows antithrombotic effects [289]

F16357, F16618

Human

Blocks PAR1-AP induced platelet activation [290]

Rat

Shows antithrombotic effects [291]

SCH79797

Human

Blocks thrombin and PAR1-AP induced calcium release and platelet activation [292]

Human, Mouse

Induces NETs formation and increases bacterial killing capacity [293]

SCH203009

Human

Blocks thrombin and PAR1-AP induced platelet activation [292]

SCH530348 (vorapaxar)

Human, Monkey

Blocks thrombin and PAR1-AP induced platelet activation [266]

E5555 (atopaxar)

Human

Blocks thrombin and PAR1-AP induced platelet activation and inhibits thrombus formation [267]

Guinea pig

Bleeding time not affected [267, 294]

Q94

Human

Blocks thrombin induced calcium release [295]

Mouse

Blocks thrombin induced ERK1/2 activation [296]

Pepducin

Antagonist

P1pal-12

Human

Blocks thrombin induced platelet activation [268]

Human

Blocks platelet activation [86]

Human

Blocks MMP-1 induced endothelial damage [297]

Mouse

Reduces lung vascular damage and sepsis lethality [297, 298]

P1-pal7

(PZ-128)

Human

Blocks MMP-1 induced Akt signaling in cancer cells [150]

Human

Blocks platelet activation [86]

Mouse

Inhibits tumor growth [280]

Guinea pig

Prevents from systemic platelet activation [86]

Parmodulin

Antagonist

ML161 (Parmodulin-2)

Human

Blocks thrombin and PAR1-AP induced platelet activation [299]

Human

Blocks thrombin induced inflammatory signaling on endothelial cells [269]

Mouse

Blocks thrombus formation [300]

Antibiotic

Antagonist

Doxycycline

Human

Inhibits thrombin induced cancer cell migration [301, 302]

Human

Blocks MMP-1 cleavage [303]

Antibody

Antagonist

ATAP-2

WEDE

Human

Blocks thrombin cleavage of PAR1 and thrombin induced calcium release [147]

Table 6

PAR2 signaling modulators

Class

Agonist/ Antagonist

Name

Receptor/Cell/Tissue type

Cellular response

Peptide

Agonist

SLIGRL/−NH2

Human, Rat

Induces calcium release [2, 8, 136, 139]

SLIGKV/−NH2

Human

Induces calcium release [136]

2f-LIGRLO/−NH2

Human, Rat

Induces calcium release [140]

Antagonist

FSLLRY-NH2

Human

Blocks trypsin, not SLIGRL activation, reduces proinflammatory IL-8 and TNFα [82]

Rat

Inhibits neuropathic pain [304]

LSIGRL-NH2

Human

Blocks trypsin, not SLIGRL induced calcium release [305]

Peptidomimetic

Antagonist

K14585,

K12940

Human

Reduces SLIGKV induced calcium release [306]

Human

Inhibits SLIGRL induced NFkB activation [307]

C391a

Human, Mouse

Blocks calcium release and MAPK activation [308]

Non-peptide small molecule

Agonist

GB110

Human

Induces calcium release [309]

AC-5541,

AC-264613

Human

Induces calcium release [310]

Rat

Induces edema and hyperalgesia [310]

Antagonist

ENMD-1068

Human

Blocks p.acnes induced calcium release and induction of IL-1a, IL-8 and TNFα [92]

Human

Inhibited FVIIa induced cancer cell migration [311]

Mouse

Reduces joint inflammation [260]

Mouse

Blocks calcium release and reduces liver fibrosis [312]

GB83

Human

Inhibits trypsin and PAR2-AP calcium release [313]

GB88

Human

Blocks PAR2 induced calcium release [309]

Rat

Reduces acute paw edema, inhibits PAR2-AP induced inflammation [309, 314]

AZ8838

AZ3451

Human

Blocks PAR2-AP induced calcium release and β-arrestin recruitment [315]

Pepducin

Antagonist

P2pal-18S

Human

Blocks PAR2 induced calcium release [316]

Mouse

Decreases risk for developing severe biliary pancreatitis [317]

P2pal-14GQ

Human

Blocks PAR2 induced calcium release [316]

Antibiotic

Antagonist

Tetracyclines

(Tetracycline,

Doxycycline,

Minocycline)

Human

Inhibits SLIGRL induced IL-8 release [318]

Mouse

Topical application of tetracycline decreases PAR2 induced skin inflammation [319]

Rat

Subantimicrobial doses of doxycycline inhibit PAR2 induced inflammation [320]

Antibody

 

SAM-11

Mouse

Reduces joint inflammation [260]

Mouse

Prevents allergic inflammation [124]

B5

Mouse

Reduces joint inflammation [260]

Mouse

Inhibits allergic airway inflammation [124]

MAB3949

Human

Blocks trypsin induced PAR2 activation [315]

Table 7

PAR4 signaling modulators

Class

Agonist/ Antagonist

Name

Receptor/Cell/Tissue type

Cellular response

Peptide

Agonist

GYPGQV/−NH2

Human, Rat

Induces platelet activation [144]

GYPGKF/−NH2

Human, Rat

Induces platelet activation [144]

AYPGKF/−NH2

Human, Mouse

Induces platelet activation [145]

Peptidomimetic

Antagonist

tc-YGPKF

Rat

Blocks thrombin and PAR4-AP induced platelets aggregation [321]

Non-peptide small molecule

Antagonist

YD-3

Human

Blocks thrombin induced platelet activation [282, 322, 323, 324, 325]

Mouse, Rat, Rabbit

Blocks thrombin and PAR4-AP induced platelets activation [323, 324, 325]

ML-354

Human

Blocks PAR4-AP induced platelet activation [326, 327, 328]

BMS-986120

Human

Blocks PAR4-AP induced calcium release and platelet activation [329]

Human

Blocks thrombus formation at high shear stress [277]

Monkey

Blocks platelet activation [329]

Pepducin

Antagonist

P4pal-10

Human, Mouse

Blocks thrombin and PAR4-AP induced platelet activation [268]

Rat

Blocks thrombin and PAR4-AP induced platelets activation [330]

P4pal-i1

Human

Blocks PAR4 induced platelets activation [150]

Peptide agonists and antagonists are short synthetic peptides that mimick the PAR-tethered ligand that is liberated by proteolytic cleavage, as described above. These peptides either induce signal transduction or prevent cleavage-dependent signaling following PAR rapid internalization, and some C- or N-terminal modifications of soluble ligand sequences have resulted in increased activation efficiency [18]. Peptidomimetic antagonists are small protein-like chains that mimick the tethered ligands of PARs, and were recently used as PAR modulators for the first time [264].Soon after PARs were discovered, PAR1 blocking antibodies were reported [265], and these blocked protease binding and or the cleavage site of the receptor. Non-peptide small molecules, such as the PAR1 antagonists vorapaxar [266] and atopaxar [267], also interact with PARs, mainly via ECL2.Only two classes of intracellular PAR antagonists have been developed to date. Pepducins are cell penetrating palmitoylated peptides that were derived from the intracellular loop of PAR, and these interfere with G protein binding [268]. Parmodulins, in contrast, are small molecules that bind PARs at the G protein binding pocket of the C-tail to compete with Gαq subunits, but not with other Gα subunits [269].

Examination of agonists and antagonists in vitro and in preclinical studies (Tables 5, 6 and 7)

Clinical studies

Despite the importance of PARs in various pathophysiological conditions, few PAR modulating tools have been tested in clinical studies, and even fewer have been established for treatment. Since the identification of PAR1 as a platelet thrombin receptor, an abundance of research has been conducted to identify PAR1 antagonists that can block platelet activation and prevent thrombotic cardiovascular events. The first clinically approved PAR1 antagonist was the small-molecule antagonist vorapaxar [266]. Phase II clinical trials of this agent showed reduced risks for myocardial infarction in patients treated with vorapaxar in combination with standard antiplatelet therapy. Moreover, the risks of bleeding complications were not significantly increased [270]. Subsequently, two large-scale phase III multicenter, randomized, double-blind, placebo-controlled studies of vorapaxar (ZONTIVITY, SCH530348) were performed. In the Thrombin Receptor Antagonist in Secondary Prevention of Atherothrombotic Ischemic Events–Thrombolysis in Myocardial Infarction 50 (TRA 2°P-TIMI 50; details at www.ClinicalTrials.gov; NCT00526474) study, the rate of cardiovascular events at the second efficacy endpoint were significantly reduced by vorapaxar in combination with standard antiplatelet therapy [271]. Furthermore, in the Thrombin Receptor Antagonist for Clinical Event Reduction in Acute Coronary Syndrome (TRACER; details at www.ClinicalTrials.gov; NCT00527943) study, vorapaxar reduced the hazard of first myocardial infarction of any type in patients who were treated within 24 h of having symptoms of a cardiovascular event. However, in the TRACER study, vorapaxar failed to prevent secondary ischemic events [272]. Because vorapaxar increased bleeding complications in the clinical setting, the alternative PAR1 antagonist atopaxar (E5555) [267] was tested in a phase II clinical trial called (Lessons From Antagonizing the Cellular Effects of Thrombin-Acute Coronary Syndromes (LANCELOT-ACS; details at www.ClinicalTrials.gov; NCT00548587) study [273]. Atopaxar inhibited platelet aggregation in ACS patients in a dose-dependent manner, and caused no side effects of abnormal platelet activation, such as bleeding [274, 275]. Yet, patients receiving atopaxar had dose-dependent increases in liver abnormalities [273].

To prevent the bleeding problems that arise from treatments with PAR1 antagonists, a new class of PAR1 antagonist was designed, and the member pepducin PZ-128 (P1-pal7) was tested in a phase I trial [276]. This study showed no reduction in platelet aggregation, but the platelet blocking effect of PZ-128 was reversible ex vivo in the presence of saturating concentrations of the PAR1 agonist peptide SFLLRN. Based on these promising findings, the new PAR1 blocking agent PZ-128 was considered in the coronary artery disease study Thrombin Receptor Inhibitory Pepducin-Percutaneous Coronary Intervention (TRIP-PCI). Data from this phase II trial are not yet available (details at www.ClinicalTrials.gov; NCT02561000).

As an alternative to PAR1 targeted antithrombotic drugs, the PAR4 small-peptide antagonist BMS-986120 reduced reversible thrombus formation ex vivo in a phase I trial [277]. Consequently, this promising anticoagulant PAR4 antagonist is currently being compared with a standard anticoagulant drug in a phase II study of stroke recurrence (details at www.ClinicalTrials.gov; NCT02671461).

Conclusion

Since the identification of PARs in the 1990s, studies of the complex mechanisms of PAR activation have been abundant, and these have clarified the roles of PARs in inflammatory disease. Various mammalian and non-mammalian proteases have also been recognized as PAR-mediated regulators of physiological and pathophysiological processes. Despite the development of various PAR modulators, few have been approved for therapeutic use. Obstacles to this therapeutic strategy include species differences in PAR expression and limited bioavailability of modulators in vivo and in clinical studies. Further research is needed to identify specific and efficient anti-inflammatory PAR modulators.

Notes

Acknowledgements

We would like to thank Hermenegild K. Heuberger for the drawing of the figures.

We thank Enago (http://www.enago.com) for the english language review.

Funding

We would like to express our sincere gratitude for the support given to RAS by the Swiss National Science Foundation grant #PZ00P3_136639 for the salary of DMH.

Availability of data and materials

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

Authors’ contributions

Manuscript preparation by DMH and RAS. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.
    Bahou WF, Nierman WC, Durkin AS, Potter CL, Demetrick DJ. Chromosomal assignment of the human thrombin receptor gene: localization to region q13 of chromosome 5. Blood. 1993;82:1532–7.PubMedPubMedCentralGoogle Scholar
  2. 2.
    Nystedt S, Emilsson K, Wahlestedt C, Sundelin J. Molecular cloning of a potential proteinase activated receptor. Proc Natl Acad Sci U S A. 1994;91:9208–12.PubMedPubMedCentralCrossRefGoogle Scholar
  3. 3.
    Schmidt VA, Nierman WC, Maglott DR, Cupit LD, Moskowitz KA, Wainer JA, Bahou WF. The human proteinase-activated receptor-3 (PAR-3) gene. Identification within a par gene cluster and characterization in vascular endothelial cells and platelets. J Biol Chem. 1998;273:15061–8.PubMedCrossRefPubMedCentralGoogle Scholar
  4. 4.
    Kahn ML, Zheng YW, Huang W, Bigornia V, Zeng D, Moff S, Farese RV Jr, Tam C, Coughlin SR. A dual thrombin receptor system for platelet activation. Nature. 1998;394:690–4.PubMedCrossRefPubMedCentralGoogle Scholar
  5. 5.
    Vu TK, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 1991;64:1057–68.PubMedCrossRefPubMedCentralGoogle Scholar
  6. 6.
    Vu TK, Wheaton VI, Hung DT, Charo I, Coughlin SR. Domains specifying thrombin-receptor interaction. Nature. 1991;353:674–7.PubMedCrossRefPubMedCentralGoogle Scholar
  7. 7.
    Nystedt S, Larsson AK, Aberg H, Sundelin J. The mouse proteinase-activated receptor-2 cDNA and gene. Molecular cloning and functional expression. J Biol Chem. 1995;270:5950–5.PubMedCrossRefPubMedCentralGoogle Scholar
  8. 8.
    Nystedt S, Emilsson K, Larsson AK, Strombeck B, Sundelin J. Molecular cloning and functional expression of the gene encoding the human proteinase-activated receptor 2. Eur J Biochem. 1995;232:84–9.PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Ishihara H, Connolly AJ, Zeng D, Kahn ML, Zheng YW, Timmons C, Tram T, Coughlin SR. Protease-activated receptor 3 is a second thrombin receptor in humans. Nature. 1997;386:502–6.PubMedCrossRefPubMedCentralGoogle Scholar
  10. 10.
    Xu WF, Andersen H, Whitmore TE, Presnell SR, Yee DP, Ching A, Gilbert T, Davie EW, Foster DC. Cloning and characterization of human protease-activated receptor 4. Proc Natl Acad Sci U S A. 1998;95:6642–6.PubMedPubMedCentralCrossRefGoogle Scholar
  11. 11.
    Gerszten RE, Chen J, Ishii M, Ishii K, Wang L, Nanevicz T, Turck CW, Vu TK, Coughlin SR. Specificity of the thrombin receptor for agonist peptide is defined by its extracellular surface. Nature. 1994;368:648–51.PubMedCrossRefPubMedCentralGoogle Scholar
  12. 12.
    Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, Plevin R. Proteinase-activated receptors. Pharmacol Rev. 2001;53:245–82.PubMedPubMedCentralGoogle Scholar
  13. 13.
    Russo A, Soh UJ, Paing MM, Arora P, Trejo J. Caveolae are required for protease-selective signaling by protease-activated receptor-1. Proc Natl Acad Sci U S A. 2009;106:6393–7.PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    Zhao P, Metcalf M, Bunnett NW. Biased signaling of protease-activated receptors. Front Endocrinol (Lausanne). 2014;5:67.CrossRefGoogle Scholar
  15. 15.
    Renesto P, Si-Tahar M, Moniatte M, Balloy V, Van Dorsselaer A, Pidard D, Chignard M. Specific inhibition of thrombin-induced cell activation by the neutrophil proteinases elastase, cathepsin G, and proteinase 3: evidence for distinct cleavage sites within the aminoterminal domain of the thrombin receptor. Blood. 1997;89:1944–53.PubMedPubMedCentralGoogle Scholar
  16. 16.
    Ludeman MJ, Kataoka H, Srinivasan Y, Esmon NL, Esmon CT, Coughlin SR. PAR1 cleavage and signaling in response to activated protein C and thrombin. J Biol Chem. 2005;280:13122–8.PubMedCrossRefPubMedCentralGoogle Scholar
  17. 17.
    Chen J, Ishii M, Wang L, Ishii K, Coughlin SR. Thrombin receptor activation. Confirmation of the intramolecular tethered liganding hypothesis and discovery of an alternative intermolecular liganding mode. J Biol Chem. 1994;269:16041–5.PubMedPubMedCentralGoogle Scholar
  18. 18.
    Scarborough RM, Naughton MA, Teng W, Hung DT, Rose J, Vu TK, Wheaton VI, Turck CW, Coughlin SR. Tethered ligand agonist peptides. Structural requirements for thrombin receptor activation reveal mechanism of proteolytic unmasking of agonist function. J Biol Chem. 1992;267:13146–9.PubMedPubMedCentralGoogle Scholar
  19. 19.
    Chang MC, Lan WH, Chan CP, Lin CP, Hsieh CC, Jeng JH. Serine protease activity is essential for thrombin-induced protein synthesis in cultured human dental pulp cells: modulation roles of prostaglandin E2. J Oral Pathol Med. 1998;27:23–9.PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    Kohli M, Williams K, Yao JL, Dennis RA, Huang J, Reeder J, Ricke WA. Thrombin expression in prostate: a novel finding. Cancer Investig. 2011;29:62–7.CrossRefGoogle Scholar
  21. 21.
    Burnier L, Mosnier LO. Novel mechanisms for activated protein C cytoprotective activities involving noncanonical activation of protease-activated receptor 3. Blood. 2013;122:807–16.PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    Kaufmann R, Schulze B, Krause G, Mayr LM, Settmacher U, Henklein P. Proteinase-activated receptors (PARs)--the PAR3 neo-N-terminal peptide TFRGAP interacts with PAR1. Regul Pept. 2005;125:61–6.PubMedCrossRefPubMedCentralGoogle Scholar
  23. 23.
    McLaughlin JN, Patterson MM, Malik AB. Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization. Proc Natl Acad Sci U S A. 2007;104:5662–7.PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    Huang CY, Lin HJ, Chen HS, Cheng SY, Hsu HC, Tang CH. Thrombin promotes matrix metalloproteinase-13 expression through the PKCdelta c-Src/EGFR/PI3K/Akt/AP-1 signaling pathway in human chondrocytes. Mediat Inflamm. 2013;2013:326041.CrossRefGoogle Scholar
  25. 25.
    Coughlin SR. How the protease thrombin talks to cells. Proc Natl Acad Sci U S A. 1999;96:11023–7.PubMedPubMedCentralCrossRefGoogle Scholar
  26. 26.
    Loew D, Perrault C, Morales M, Moog S, Ravanat C, Schuhler S, Arcone R, Pietropaolo C, Cazenave JP, van Dorsselaer A, Lanza F. Proteolysis of the exodomain of recombinant protease-activated receptors: prediction of receptor activation or inactivation by MALDI mass spectrometry. Biochemistry. 2000;39:10812–22.PubMedCrossRefPubMedCentralGoogle Scholar
  27. 27.
    Mihara K, Ramachandran R, Saifeddine M, Hansen KK, Renaux B, Polley D, Gibson S, Vanderboor C, Hollenberg MD. Thrombin-mediated direct activation of proteinase-activated receptor-2 (PAR2): another target for thrombin signaling. Mol Pharmacol. 2016;89(5):606–14.PubMedCrossRefPubMedCentralGoogle Scholar
  28. 28.
    Schuepbach RA, Madon J, Ender M, Galli P, Riewald M. Protease activated receptor-1 cleaved at R46 mediates cytoprotective effects. J Thromb Haemost. 2012;10(8):1675–84.PubMedPubMedCentralCrossRefGoogle Scholar
  29. 29.
    Riewald M, Petrovan RJ, Donner A, Mueller BM, Ruf W. Activation of endothelial cell protease activated receptor 1 by the protein C pathway. Science. 2002;296:1880–2.PubMedCrossRefPubMedCentralGoogle Scholar
  30. 30.
    Cheng T, Liu D, Griffin JH, Fernandez JA, Castellino F, Rosen ED, Fukudome K, Zlokovic BV. Activated protein C blocks p53-mediated apoptosis in ischemic human brain endothelium and is neuroprotective. Nat Med. 2003;9:338–42.PubMedCrossRefPubMedCentralGoogle Scholar
  31. 31.
    Domotor E, Benzakour O, Griffin JH, Yule D, Fukudome K, Zlokovic BV. Activated protein C alters cytosolic calcium flux in human brain endothelium via binding to endothelial protein C receptor and activation of protease activated receptor-1. Blood. 2003;101:4797–801.PubMedCrossRefPubMedCentralGoogle Scholar
  32. 32.
    Mosnier LO, Griffin JH. Inhibition of staurosporine-induced apoptosis of endothelial cells by activated protein C requires protease-activated receptor-1 and endothelial cell protein C receptor. Biochem J. 2003;373:65–70.PubMedPubMedCentralCrossRefGoogle Scholar
  33. 33.
    Ruf W, Riewald M. Tissue factor-dependent coagulation protease signaling in acute lung injury. Crit Care Med. 2003;31:S231–7.PubMedCrossRefPubMedCentralGoogle Scholar
  34. 34.
    Ranjan S, Goihl A, Kohli S, Gadi I, Pierau M, Shahzad K, Gupta D, Bock F, Wang HJ, Shaikh H, et al. Activated protein C protects from GvHD via PAR2/PAR3 signalling in regulatory T-cells. Nat Commun. 2017;8(1):311.PubMedPubMedCentralCrossRefGoogle Scholar
  35. 35.
    Madhusudhan T, Wang H, Straub BK, Grone E, Zhou Q, Shahzad K, Muller-Krebs S, Schwenger V, Gerlitz B, Grinnell BW, et al. Cytoprotective signaling by activated protein C requires protease-activated receptor-3 in podocytes. Blood. 2012;119:874–83.PubMedPubMedCentralCrossRefGoogle Scholar
  36. 36.
    Riewald M, Kravchenko VV, Petrovan RJ, O'Brien PJ, Brass LF, Ulevitch RJ, Ruf W. Gene induction by coagulation factor Xa is mediated by activation of protease-activated receptor 1. Blood. 2001;97:3109–16.PubMedCrossRefPubMedCentralGoogle Scholar
  37. 37.
    Guo H, Liu D, Gelbard H, Cheng T, Insalaco R, Fernandez JA, Griffin JH, Zlokovic BV. Activated protein C prevents neuronal apoptosis via protease activated receptors 1 and 3. Neuron. 2004;41:563–72.PubMedCrossRefPubMedCentralGoogle Scholar
  38. 38.
    Schuepbach RA, Riewald M. Coagulation factor Xa cleaves protease-activated receptor-1 and mediates signaling dependent on binding to the endothelial protein C receptor. J Thromb Haemost. 2010;8:379–88.PubMedCrossRefPubMedCentralGoogle Scholar
  39. 39.
    Stavenuiter F, Mosnier LO. Noncanonical PAR3 activation by factor Xa identifies a novel pathway for Tie2 activation and stabilization of vascular integrity. Blood. 2014;124:3480–9.PubMedPubMedCentralCrossRefGoogle Scholar
  40. 40.
    Camerer E, Gjernes E, Wiiger M, Pringle S, Prydz H. Binding of factor VIIa to tissue factor on keratinocytes induces gene expression. J Biol Chem. 2000;275:6580–5.PubMedCrossRefPubMedCentralGoogle Scholar
  41. 41.
    Morris DR, Ding Y, Ricks TK, Gullapalli A, Wolfe BL, Trejo J. Protease-activated receptor-2 is essential for factor VIIa and Xa-induced signaling, migration, and invasion of breast cancer cells. Cancer Res. 2006;66:307–14.PubMedCrossRefPubMedCentralGoogle Scholar
  42. 42.
    Rothmeier AS, Liu E, Chakrabarty S, Disse J, Mueller BM, Ostergaard H, Ruf W. Identification of the integrin-binding site on coagulation factor VIIa required for proangiogenic PAR2 signaling. Blood. 2018;131:674–85.PubMedPubMedCentralCrossRefGoogle Scholar
  43. 43.
    Rothmeier AS, Disse J, Mueller BM, Liu EB, Ostergaard H, Ruf W. Proangiogenic TF-FVIIa-PAR2 signaling requires Matriptase-independent integrin interaction. Blood. 2016;128:3756.Google Scholar
  44. 44.
    Le Gall SM, Szabo R, Lee M, Kirchhofer D, Craik CS, Bugge TH, Camerer E. Matriptase activation connects tissue factor-dependent coagulation initiation to epithelial proteolysis and signaling. Blood. 2016;127:3260–9.PubMedPubMedCentralCrossRefGoogle Scholar
  45. 45.
    Sen P, Gopalakrishnan R, Kothari H, Keshava S, Clark CA, Esmon CT, Pendurthi UR, Rao LV. Factor VIIa bound to endothelial cell protein C receptor activates protease activated receptor-1 and mediates cell signaling and barrier protection. Blood. 2011;117:3199–208.PubMedPubMedCentralCrossRefGoogle Scholar
  46. 46.
    Kondreddy V, Wang J, Keshava S, Esmon CT, Rao LVM, Pendurthi UR. Factor VIIa induces anti-inflammatory signaling via EPCR and PAR1. Blood. 2018;131:2379–92.PubMedCrossRefPubMedCentralGoogle Scholar
  47. 47.
    Riewald M, Ruf W. Mechanistic coupling of protease signaling and initiation of coagulation by tissue factor. Proc Natl Acad Sci U S A. 2001;98:7742–7.PubMedPubMedCentralCrossRefGoogle Scholar
  48. 48.
    Rinderknecht H. Activation of pancreatic zymogens. Normal activation, premature intrapancreatic activation, protective mechanisms against inappropriate activation. Dig Dis Sci. 1986;31:314–21.PubMedCrossRefPubMedCentralGoogle Scholar
  49. 49.
    Koshikawa N, Hasegawa S, Nagashima Y, Mitsuhashi K, Tsubota Y, Miyata S, Miyagi Y, Yasumitsu H, Miyazaki K. Expression of trypsin by epithelial cells of various tissues, leukocytes, and neurons in human and mouse. Am J Pathol. 1998;153:937–44.PubMedPubMedCentralCrossRefGoogle Scholar
  50. 50.
    Koivunen E, Huhtala ML, Stenman UH. Human ovarian tumor-associated trypsin. Its purification and characterization from mucinous cyst fluid and identification as an activator of pro-urokinase. J Biol Chem. 1989;264:14095–9.PubMedPubMedCentralGoogle Scholar
  51. 51.
    Koivunen E, Ristimaki A, Itkonen O, Osman S, Vuento M, Stenman UH. Tumor-associated trypsin participates in cancer cell-mediated degradation of extracellular matrix. Cancer Res. 1991;51:2107–12.PubMedPubMedCentralGoogle Scholar
  52. 52.
    Koshikawa N, Nagashima Y, Miyagi Y, Mizushima H, Yanoma S, Yasumitsu H, Miyazaki K. Expression of trypsin in vascular endothelial cells. FEBS Lett. 1997;409:442–8.PubMedCrossRefPubMedCentralGoogle Scholar
  53. 53.
    Nakayama T, Hirano K, Shintani Y, Nishimura J, Nakatsuka A, Kuga H, Takahashi S, Kanaide H. Unproductive cleavage and the inactivation of protease-activated receptor-1 by trypsin in vascular endothelial cells. Br J Pharmacol. 2003;138:121–30.PubMedPubMedCentralCrossRefGoogle Scholar
  54. 54.
    Molino M, Woolkalis MJ, Reavey-Cantwell J, Pratico D, Andrade-Gordon P, Barnathan ES, Brass LF. Endothelial cell thrombin receptors and PAR-2. Two protease-activated receptors located in a single cellular environment. J Biol Chem. 1997;272:11133–41.PubMedCrossRefPubMedCentralGoogle Scholar
  55. 55.
    Berger P, Tunon-De-Lara JM, Savineau JP, Marthan R. Selected contribution: tryptase-induced PAR-2-mediated Ca(2+) signaling in human airway smooth muscle cells. J Appl Physiol (1985). 2001;91:995–1003.CrossRefGoogle Scholar
  56. 56.
    Berger P, Perng DW, Thabrew H, Compton SJ, Cairns JA, McEuen AR, Marthan R, Tunon De Lara JM, Walls AF. Tryptase and agonists of PAR-2 induce the proliferation of human airway smooth muscle cells. J Appl Physiol (1985). 2001;91:1372–9.CrossRefGoogle Scholar
  57. 57.
    Akers IA, Parsons M, Hill MR, Hollenberg MD, Sanjar S, Laurent GJ, McAnulty RJ. Mast cell tryptase stimulates human lung fibroblast proliferation via protease-activated receptor-2. Am J Physiol Lung Cell Mol Physiol. 2000;278:L193–201.PubMedCrossRefPubMedCentralGoogle Scholar
  58. 58.
    Compton SJ, Renaux B, Wijesuriya SJ, Hollenberg MD. Glycosylation and the activation of proteinase-activated receptor 2 (PAR(2)) by human mast cell tryptase. Br J Pharmacol. 2001;134:705–18.PubMedPubMedCentralCrossRefGoogle Scholar
  59. 59.
    Schechter NM, Brass LF, Lavker RM, Jensen PJ. Reaction of mast cell proteases tryptase and chymase with protease activated receptors (PARs) on keratinocytes and fibroblasts. J Cell Physiol. 1998;176:365–73.PubMedCrossRefPubMedCentralGoogle Scholar
  60. 60.
    Seitz I, Hess S, Schulz H, Eckl R, Busch G, Montens HP, Brandl R, Seidl S, Schomig A, Ott I. Membrane-type serine protease-1/matriptase induces interleukin-6 and -8 in endothelial cells by activation of protease-activated receptor-2: potential implications in atherosclerosis. Arterioscler Thromb Vasc Biol. 2007;27:769–75.PubMedCrossRefPubMedCentralGoogle Scholar
  61. 61.
    Bocheva G, Rattenholl A, Kempkes C, Goerge T, Lin CY, D'Andrea MR, Stander S, Steinhoff M. Role of matriptase and proteinase-activated receptor-2 in nonmelanoma skin cancer. J Invest Dermatol. 2009;129:1816–23.PubMedCrossRefPubMedCentralGoogle Scholar
  62. 62.
    Camerer E, Barker A, Duong DN, Ganesan R, Kataoka H, Cornelissen I, Darragh MR, Hussain A, Zheng YW, Srinivasan Y, et al. Local protease signaling contributes to neural tube closure in the mouse embryo. Dev Cell. 2010;18:25–38.PubMedPubMedCentralCrossRefGoogle Scholar
  63. 63.
    Sales KU, Friis S, Konkel JE, Godiksen S, Hatakeyama M, Hansen KK, Rogatto SR, Szabo R, Vogel LK, Chen W, et al. Non-hematopoietic PAR-2 is essential for matriptase-driven pre-malignant progression and potentiation of ras-mediated squamous cell carcinogenesis. Oncogene. 2015;34:346–56.PubMedCrossRefPubMedCentralGoogle Scholar
  64. 64.
    Caliendo G, Santagada V, Perissutti E, Severino B, Fiorino F, Frecentese F, Juliano L. Kallikrein protease activated receptor (PAR) axis: an attractive target for drug development. J Med Chem. 2012;55:6669–86.PubMedCrossRefPubMedCentralGoogle Scholar
  65. 65.
    Ramsay AJ, Dong Y, Hunt ML, Linn M, Samaratunga H, Clements JA, Hooper JD. Kallikrein-related peptidase 4 (KLK4) initiates intracellular signaling via protease-activated receptors (PARs). KLK4 and PAR-2 are co-expressed during prostate cancer progression. J Biol Chem. 2008;283:12293–304.PubMedCrossRefPubMedCentralGoogle Scholar
  66. 66.
    Oikonomopoulou K, Hansen KK, Saifeddine M, Vergnolle N, Tea I, Blaber M, Blaber SI, Scarisbrick I, Diamandis EP, Hollenberg MD. Kallikrein-mediated cell signalling: targeting proteinase-activated receptors (PARs). Biol Chem. 2006;387:817–24.PubMedPubMedCentralGoogle Scholar
  67. 67.
    Yoon H, Radulovic M, Wu J, Blaber SI, Blaber M, Fehlings MG, Scarisbrick IA. Kallikrein 6 signals through PAR1 and PAR2 to promote neuron injury and exacerbate glutamate neurotoxicity. J Neurochem. 2013;127:283–98.PubMedPubMedCentralCrossRefGoogle Scholar
  68. 68.
    Molino M, Blanchard N, Belmonte E, Tarver AP, Abrams C, Hoxie JA, Cerletti C, Brass LF. Proteolysis of the human platelet and endothelial-cell thrombin receptor by neutrophil-derived Cathepsin-G. J Biol Chem. 1995;270:11168–75.PubMedCrossRefPubMedCentralGoogle Scholar
  69. 69.
    Molino M, Blanchard N, Belmonte E, Tarver AP, Abrams C, Hoxie JA, Cerletti C, Brass LF. Cathepsin-G cleaves the human thrombin receptor. Thromb Haemost. 1995;73:923.Google Scholar
  70. 70.
    Wilson TJ, Nannuru KC, Singh RK. Cathepsin G recruits osteoclast precursors via proteolytic activation of protease-activated Receptor-1. Cancer Res. 2009;69:3188–95.PubMedCrossRefPubMedCentralGoogle Scholar
  71. 71.
    Ramachandran R, Sadofsky LR, Xiao YP, Botham A, Cowen M, Morice AH, Compton SJ. Inflammatory mediators modulate thrombin and cathepsin-G signaling in human bronchial fibroblasts by inducing expression of proteinase-activated receptor-4. Am J Phys Lung Cell Mol Phys. 2007;292:L788–98.Google Scholar
  72. 72.
    Ramachandran R, Mihara K, Chung H, Renaux B, Lau CS, Muruve DA, DeFea KA, Bouvier M, Hollenberg MD. Neutrophil elastase acts as a biased agonist for proteinase-activated receptor-2 (PAR2). J Biol Chem. 2011;286:24638–48.PubMedPubMedCentralCrossRefGoogle Scholar
  73. 73.
    Cumashi A, Ansuini H, Celli N, De Blasi A, O'Brien PJ, Brass LF, Molino M. Neutrophil proteases can inactivate human PAR3 and abolish the co-receptor function of PAR3 on murine platelets. Thromb Haemost. 2001;85:533–8.PubMedCrossRefPubMedCentralGoogle Scholar
  74. 74.
    Sambrano GR, Huang W, Faruqi T, Mahrus S, Craik C, Coughlin SR. Cathepsin G activates protease-activated receptor-4 in human platelets. J Biol Chem. 2000;275:6819–23.PubMedPubMedCentralCrossRefGoogle Scholar
  75. 75.
    Vergnolle N, Derian CK, D'Andrea MR, Steinhoff M, Andrade-Gordon P. Characterization of thrombin-induced leukocyte rolling and adherence: a potential proinflammatory role for proteinase-activated receptor-4. J Immunol. 2002;169:1467–73.PubMedCrossRefPubMedCentralGoogle Scholar
  76. 76.
    Walsh SW, Nugent WH, Solotskaya AV, Anderson CD, Grider JR, Strauss JF 3rd. Matrix metalloprotease-1 and elastase are novel uterotonic agents acting through protease-activated receptor 1. Reprod Sci. 2017.  https://doi.org/10.1177/1933719117732162.
  77. 77.
    Kumar VRS, Darisipudi MN, Steiger S, Devarapu SK, Tato M, Kukarni OP, Mulay SR, Thomasova D, Popper B, Demleitner J, et al. Cathepsin S cleavage of protease-activated Receptor-2 on endothelial cells promotes microvascular diabetes complications. J Am Soc Nephrol. 2016;27:1635–49.CrossRefGoogle Scholar
  78. 78.
    Elmariah SB, Reddy VB, Lerner EA. Cathepsin S signals via PAR2 and generates a novel tethered ligand receptor agonist. PLoS One. 2014;9(6):e99702.PubMedPubMedCentralCrossRefGoogle Scholar
  79. 79.
    Kuliopulos A, Covic L, Seeley SK, Sheridan PJ, Helin J, Costello CE. Plasmin desensitization of the PAR1 thrombin receptor: kinetics, sites of truncation, and implications for thrombolytic therapy. Biochemistry. 1999;38:4572–85.PubMedCrossRefPubMedCentralGoogle Scholar
  80. 80.
    Domotor E, Bartha K, Machovich R, Adam-Vizi V. Protease-activated receptor-2 (PAR-2) in brain microvascular endothelium and its regulation by plasmin and elastase. J Neurochem. 2002;80:746–54.PubMedCrossRefPubMedCentralGoogle Scholar
  81. 81.
    Wang T, Lee MH, Choi E, Pardo-Villamizar CA, Lee SB, Yang IH, Calabresi PA, Nath A. Granzyme B-induced neurotoxicity is mediated via activation of PAR-1 receptor and Kv1.3 channel. PLoS One. 2012;7:e43950.PubMedPubMedCentralCrossRefGoogle Scholar
  82. 82.
    Lee PR, Johnson TP, Gnanapavan S, Giovannoni G, Wang T, Steiner JP, Medynets M, Vaal MJ, Gartner V, Nath A. Protease-activated receptor-1 activation by granzyme B causes neurotoxicity that is augmented by interleukin-1beta. J Neuroinflammation. 2017;14:131.PubMedPubMedCentralCrossRefGoogle Scholar
  83. 83.
    Cooper DM, Pechkovsky DV, Hackett TL, Knight DA, Granville DJ. Granzyme K activates protease-activated receptor-1. PLoS One. 2011;6:e21484.PubMedPubMedCentralCrossRefGoogle Scholar
  84. 84.
    Sharma M, Merkulova Y, Raithatha S, Parkinson LG, Shen Y, Cooper D, Granville DJ. Extracellular granzyme K mediates endothelial activation through the cleavage of protease-activated receptor-1. FEBS J. 2016;283:1734–47.PubMedCrossRefPubMedCentralGoogle Scholar
  85. 85.
    Alvarez-Arce A, Lee-Rivera I, Lopez E, Hernandez-Cruz A, Lopez-Colome AM. Thrombin-induced Calpain activation promotes protease-activated receptor 1 internalization. Int J Cell Biol. 2017;2017:1908310.PubMedPubMedCentralCrossRefGoogle Scholar
  86. 86.
    Trivedi V, Boire A, Tchernychev B, Kaneider NC, Leger AJ, O'Callaghan K, Covic L, Kuliopulos A. Platelet matrix metalloprotease-1 mediates thrombogenesis by activating PAR1 at a cryptic ligand site. Cell. 2009;137:332–43.PubMedPubMedCentralCrossRefGoogle Scholar
  87. 87.
    Austin KM, Covic L, Kuliopulos A. Matrix metalloproteases and PAR1 activation. Blood. 2013;121:431–9.PubMedPubMedCentralCrossRefGoogle Scholar
  88. 88.
    Austin KM, Nguyen N, Javid G, Covic L, Kuliopulos A. Noncanonical matrix metalloprotease-1-protease-activated receptor-1 signaling triggers vascular smooth muscle cell dedifferentiation and arterial stenosis. J Biol Chem. 2013;288:23105–15.PubMedPubMedCentralCrossRefGoogle Scholar
  89. 89.
    Allen M, Ghosh S, Ahern GP, Villapol S, Maguire-Zeiss KA, Conant K. Protease induced plasticity: matrix metalloproteinase-1 promotes neurostructural changes through activation of protease activated receptor 1. Sci Rep. 2016;6:35497.PubMedPubMedCentralCrossRefGoogle Scholar
  90. 90.
    Boire A, Covic L, Agarwal A, Jacques S, Sherifi S, Kuliopulos A. PAR1 is a matrix metalloprotease-1 receptor that promotes invasion and tumorigenesis of breast cancer cells. Cell. 2005;120:303–13.PubMedCrossRefPubMedCentralGoogle Scholar
  91. 91.
    Sebastiano M, Momi S, Falcinelli E, Bury L, Hoylaerts MF, Gresele P. A novel mechanism regulating human platelet activation by MMP-2-mediated PAR1 biased signaling. Blood. 2017;129:883–95.PubMedCrossRefPubMedCentralGoogle Scholar
  92. 92.
    Lee SE, Kim JM, Jeong SK, Jeon JE, Yoon HJ, Jeong MK, Lee SH. Protease-activated receptor-2 mediates the expression of inflammatory cytokines, antimicrobial peptides, and matrix metalloproteinases in keratinocytes in response to Propionibacterium acnes. Arch Dermatol Res. 2010;302:745–56.PubMedPubMedCentralCrossRefGoogle Scholar
  93. 93.
    Vliagoftis H, Schwingshackl A, Milne CD, Duszyk M, Hollenberg MD, Wallace JL, Befus AD, Moqbel R. Proteinase-activated receptor-2-mediated matrix metalloproteinase-9 release from airway epithelial cells. J Allergy Clin Immunol. 2000;106:537–45.PubMedCrossRefPubMedCentralGoogle Scholar
  94. 94.
    Hou HH, Wang HC, Cheng SL, Chen YF, Lu KZ, Yu CJ. MMP-12 activates protease activated receptor (PAR)-1, upregulates placenta growth factor and leads to pulmonary emphysema. Am J Physiol Lung Cell Mol Physiol. 2018;315(3):L432–42.PubMedCrossRefPubMedCentralGoogle Scholar
  95. 95.
    Raza SL, Nehring LC, Shapiro SD, Cornelius LA. Proteinase-activated receptor-1 regulation of macrophage elastase (MMP-12) secretion by serine proteinases. J Biol Chem. 2000;275:41243–50.PubMedCrossRefPubMedCentralGoogle Scholar
  96. 96.
    Zang N, Zhuang JG, Deng Y, Yang ZM, Ye ZX, Xie XH, Ren L, Fu Z, Luo ZX, Xu FD, Liu EM. Pulmonary C fibers modulate MMP-12 production via PAR2 and are involved in the long-term airway inflammation and airway Hyperresponsiveness induced by respiratory syncytial virus infection. J Virol. 2016;90:2536–43.PubMedCentralCrossRefGoogle Scholar
  97. 97.
    Hooper JD, Nicol DL, Dickinson JL, Eyre HJ, Scarman AL, Normyle JF, Stuttgen MA, Douglas ML, Loveland KA, Sutherland GR, Antalis TM. Testisin, a new human serine proteinase expressed by premeiotic testicular germ cells and lost in testicular germ cell tumors. Cancer Res. 1999;59:3199–205.PubMedPubMedCentralGoogle Scholar
  98. 98.
    Driesbaugh KH, Buzza MS, Martin EW, Conway GD, Kao JP, Antalis TM. Proteolytic activation of the protease-activated receptor (PAR)-2 by the glycosylphosphatidylinositol-anchored serine protease testisin. J Biol Chem. 2015;290:3529–41.PubMedCrossRefPubMedCentralGoogle Scholar
  99. 99.
    Potempa J, Pike RN. Corruption of innate immunity by bacterial proteases. J Innate Immun. 2009;1:70–87.PubMedPubMedCentralCrossRefGoogle Scholar
  100. 100.
    Kida Y, Higashimoto Y, Inoue H, Shimizu T, Kuwano K. A novel secreted protease from Pseudomonas aeruginosa activates NF-kappaB through protease-activated receptors. Cell Microbiol. 2008;10:1491–504.PubMedCrossRefPubMedCentralGoogle Scholar
  101. 101.
    Dulon S, Leduc D, Cottrell GS, D'Alayer J, Hansen KK, Bunnett NW, Hollenberg MD, Pidard D, Chignard M. Pseudomonas aeruginosa elastase disables proteinase-activated receptor 2 in respiratory epithelial cells. Am J Respir Cell Mol Biol. 2005;32:411–9.PubMedCrossRefPubMedCentralGoogle Scholar
  102. 102.
    Ender M, Andreoni F, Zinkernagel AS, Schuepbach RA. Streptococcal SpeB cleaved PAR-1 suppresses ERK phosphorylation and blunts thrombin-induced platelet aggregation. PLoS One. 2013;8:e81298.PubMedPubMedCentralCrossRefGoogle Scholar
  103. 103.
    van den Boogaard FE, Brands X, Duitman J, de Stoppelaar SF, Borensztajn KS, Roelofs J, Hollenberg MD, Spek CA, Schultz MJ, van ‘t Veer C, van der Poll T. Protease-activated receptor 2 facilitates bacterial dissemination in pneumococcal pneumonia. J Infect Dis. 2018;217:1462–71.PubMedCrossRefPubMedCentralGoogle Scholar
  104. 104.
    Schouten M, Van't Veer C, Roelofs JJ, Levi M, van der Poll T. Protease-activated receptor-1 impairs host defense in murine pneumococcal pneumonia: a controlled laboratory study. Crit Care. 2012;16:R238.PubMedPubMedCentralCrossRefGoogle Scholar
  105. 105.
    Asehnoune K, Moine P. Protease-activated receptor-1: key player in the sepsis coagulation-inflammation crosstalk. Crit Care. 2013;17:119.PubMedPubMedCentralCrossRefGoogle Scholar
  106. 106.
    de Stoppelaar SF, Van't Veer C, van den Boogaard FE, Nieuwland R, Hoogendijk AJ, de Boer OJ, Roelofs JJ, van der Poll T. Protease activated receptor 4 limits bacterial growth and lung pathology during late stage Streptococcus pneumoniae induced pneumonia in mice. Thromb Haemost. 2013;110:582–92.PubMedCrossRefPubMedCentralGoogle Scholar
  107. 107.
    Holzhausen M, Spolidorio LC, Ellen RP, Jobin MC, Steinhoff M, Andrade-Gordon P, Vergnolle N. Protease-activated receptor-2 activation: a major role in the pathogenesis of Porphyromonas gingivalis infection. Am J Pathol. 2006;168:1189–99.PubMedPubMedCentralCrossRefGoogle Scholar
  108. 108.
    Francis N, Ayodele BA, O'Brien-Simpson NM, Birchmeier W, Pike RN, Pagel CN, Mackie EJ. Keratinocyte-specific ablation of protease-activated receptor-2 prevents gingival inflammation and bone loss in a mouse model of periodontal disease. Cell Microbiol. 2018;20(11):e12891.PubMedCrossRefPubMedCentralGoogle Scholar
  109. 109.
    Lourbakos A, Chinni C, Thompson P, Potempa J, Travis J, Mackie EJ, Pike RN. Cleavage and activation of proteinase-activated receptor-2 on human neutrophils by gingipain-R from Porphyromonas gingivalis. FEBS Lett. 1998;435:45–8.PubMedCrossRefPubMedCentralGoogle Scholar
  110. 110.
    Lourbakos A, Potempa J, Travis J, D'Andrea MR, Andrade-Gordon P, Santulli R, Mackie EJ, Pike RN. Arginine-specific protease from Porphyromonas gingivalis activates protease-activated receptors on human oral epithelial cells and induces interleukin-6 secretion. Infect Immun. 2001;69:5121–30.PubMedPubMedCentralCrossRefGoogle Scholar
  111. 111.
    Lourbakos A, Yuan YP, Jenkins AL, Travis J, Andrade-Gordon P, Santulli R, Potempa J, Pike RN. Activation of protease-activated receptors by gingipains from Porphyromonas gingivalis leads to platelet aggregation: a new trait in microbial pathogenicity. Blood. 2001;97:3790–7.PubMedCrossRefPubMedCentralGoogle Scholar
  112. 112.
    Papapanagiotou D, Nicu EA, Bizzarro S, Gerdes VE, Meijers JC, Nieuwland R, van der Velden U, Loos BG. Periodontitis is associated with platelet activation. Atherosclerosis. 2009;202:605–11.PubMedCrossRefPubMedCentralGoogle Scholar
  113. 113.
    Ikawa K, Nishioka T, Yu Z, Sugawara Y, Kawagoe J, Takizawa T, Primo V, Nikolic B, Kuroishi T, Sasano T, et al. Involvement of neutrophil recruitment and protease-activated receptor 2 activation in the induction of IL-18 in mice. J Leukoc Biol. 2005;78:1118–26.PubMedCrossRefPubMedCentralGoogle Scholar
  114. 114.
    Kida Y, Inoue H, Shimizu T, Kuwano K. Serratia marcescens serralysin induces inflammatory responses through protease-activated receptor 2. Infect Immun. 2007;75:164–74.PubMedCrossRefPubMedCentralGoogle Scholar
  115. 115.
    Chen X, Earley K, Luo W, Lin SH, Schilling WP. Functional expression of a human thrombin receptor in Sf9 insect cells: evidence for an active tethered ligand. Biochem J. 1996;314(Pt 2):603–11.PubMedPubMedCentralCrossRefGoogle Scholar
  116. 116.
    Ubl JJ, Sergeeva M, Reiser G. Desensitisation of protease-activated receptor-1 (PAR-1) in rat astrocytes: evidence for a novel mechanism for terminating Ca2+ signalling evoked by the tethered ligand. J Physiol. 2000;525(Pt 2):319–30.PubMedPubMedCentralCrossRefGoogle Scholar
  117. 117.
    Tripathi T, Abdi M, Alizadeh H. Protease-activated receptor 2 (PAR2) is upregulated by Acanthamoeba plasminogen activator (aPA) and induces proinflammatory cytokine in human corneal epithelial cells. Invest Ophthalmol Vis Sci. 2014;55:3912–21.PubMedPubMedCentralCrossRefGoogle Scholar
  118. 118.
    Laing GD, Compton SJ, Ramachandran R, Fuller GL, Wilkinson MC, Wagstaff SC, Watson SP, Kamiguti AS, Theakston RD, Senis YA. Characterization of a novel protein from Proatheris superciliaris venom: proatherocytin, a 34-kDa platelet receptor PAR1 agonist. Toxicon. 2005;46:490–9.PubMedCrossRefPubMedCentralGoogle Scholar
  119. 119.
    Santos BF, Serrano SM, Kuliopulos A, Niewiarowski S. Interaction of viper venom serine peptidases with thrombin receptors on human platelets. FEBS Lett. 2000;477:199–202.PubMedCrossRefPubMedCentralGoogle Scholar
  120. 120.
    Sun G, Stacey MA, Schmidt M, Mori L, Mattoli S. Interaction of mite allergens Der p3 and Der p9 with protease-activated receptor-2 expressed by lung epithelial cells. J Immunol. 2001;167:1014–21.PubMedCrossRefPubMedCentralGoogle Scholar
  121. 121.
    Asokananthan N, Graham PT, Fink J, Knight DA, Bakker AJ, McWilliam AS, Thompson PJ, Stewart GA. Activation of protease-activated receptor (PAR)-1, PAR-2, and PAR-4 stimulates IL-6, IL-8, and prostaglandin E2 release from human respiratory epithelial cells. J Immunol. 2002;168:3577–85.PubMedCrossRefPubMedCentralGoogle Scholar
  122. 122.
    Lin YP, Nelson C, Kramer H, Parekh AB. The allergen Der p3 from house dust mite stimulates store-operated ca(2+) channels and mast cell migration through PAR4 receptors. Mol Cell. 2018;70:228–241.e225.PubMedCrossRefPubMedCentralGoogle Scholar
  123. 123.
    Kondo S, Helin H, Shichijo M, Bacon KB. Cockroach allergen extract stimulates protease-activated receptor-2 (PAR-2) expressed in mouse lung fibroblast. Inflamm Res. 2004;53:489–96.PubMedCrossRefPubMedCentralGoogle Scholar
  124. 124.
    Asaduzzaman M, Nadeem A, Arizmendi N, Davidson C, Nichols HL, Abel M, Ionescu LI, Puttagunta L, Thebaud B, Gordon J, et al. Functional inhibition of PAR2 alleviates allergen-induced airway hyperresponsiveness and inflammation. Clin Exp Allergy. 2015;45:1844–55.PubMedCrossRefPubMedCentralGoogle Scholar
  125. 125.
    Polley DJ, Mihara K, Ramachandran R, Vliagoftis H, Renaux B, Saifeddine M, Daines MO, Boitano S, Hollenberg MD. Cockroach allergen serine proteinases: isolation, sequencing and signalling via proteinase-activated receptor-2. Clin Exp Allergy. 2017;47:946–60.PubMedPubMedCentralCrossRefGoogle Scholar
  126. 126.
    Chiu LL, Perng DW, Yu CH, Su SN, Chow LP. Mold allergen, pen C 13, induces IL-8 expression in human airway epithelial cells by activating protease-activated receptor 1 and 2. J Immunol. 2007;178:5237–44.PubMedCrossRefPubMedCentralGoogle Scholar
  127. 127.
    Kauffman HF, Tomee JF, van de Riet MA, Timmerman AJ, Borger P. Protease-dependent activation of epithelial cells by fungal allergens leads to morphologic changes and cytokine production. J Allergy Clin Immunol. 2000;105:1185–93.PubMedCrossRefPubMedCentralGoogle Scholar
  128. 128.
    Boitano S, Flynn AN, Schulz SM, Hoffman J, Price TJ, Vagner J. Potent agonists of the protease activated receptor 2 (PAR2). J Med Chem. 2011;54:1308–13.PubMedPubMedCentralCrossRefGoogle Scholar
  129. 129.
    Boitano S, Flynn AN, Sherwood CL, Schulz SM, Hoffman J, Gruzinova I, Daines MO. Alternaria alternata serine proteases induce lung inflammation and airway epithelial cell activation via PAR2. Am J Physiol Lung Cell Mol Physiol. 2011;300:L605–14.PubMedPubMedCentralCrossRefGoogle Scholar
  130. 130.
    Snelgrove RJ, Gregory LG, Peiro T, Akthar S, Campbell GA, Walker SA, Lloyd CM. Alternaria-derived serine protease activity drives IL-33-mediated asthma exacerbations. J Allergy Clin Immunol. 2014;134:583–592.e586.PubMedPubMedCentralCrossRefGoogle Scholar
  131. 131.
    Rathnavelu V, Alitheen NB, Sohila S, Kanagesan S, Ramesh R. Potential role of bromelain in clinical and therapeutic applications. Biomed Rep. 2016;5:283–8.PubMedPubMedCentralCrossRefGoogle Scholar
  132. 132.
    Borrelli F, Capasso R, Severino B, Fiorino F, Aviello G, De Rosa G, Mazzella M, Romano B, Capasso F, Fasolino I, Izzo AA. Inhibitory effects of bromelain, a cysteine protease derived from pineapple stem (Ananas comosus), on intestinal motility in mice. Neurogastroenterol Motil. 2011;23:745–e331.PubMedCrossRefPubMedCentralGoogle Scholar
  133. 133.
    Reddy VB, Lerner EA. Plant cysteine proteases that evoke itch activate protease-activated receptors. Br J Dermatol. 2010;163:532–5.PubMedPubMedCentralCrossRefGoogle Scholar
  134. 134.
    Lerner DJ, Chen M, Tram T, Coughlin SR. Agonist recognition by proteinase-activated receptor 2 and thrombin receptor. Importance of extracellular loop interactions for receptor function. J Biol Chem. 1996;271:13943–7.PubMedCrossRefPubMedCentralGoogle Scholar
  135. 135.
    Hollenberg MD, Yang SG, Laniyonu AA, Moore GJ, Saifeddine M. Action of thrombin receptor polypeptide in gastric smooth muscle: identification of a core pentapeptide retaining full thrombin-mimetic intrinsic activity. Mol Pharmacol. 1992;42:186–91.PubMedPubMedCentralGoogle Scholar
  136. 136.
    Blackhart BD, Emilsson K, Nguyen D, Teng W, Martelli AJ, Nystedt S, Sundelin J, Scarborough RM. Ligand cross-reactivity within the protease-activated receptor family. J Biol Chem. 1996;271:16466–71.PubMedCrossRefPubMedCentralGoogle Scholar
  137. 137.
    Hollenberg MD, Saifeddine M, al-Ani B, Kawabata A. Proteinase-activated receptors: structural requirements for activity, receptor cross-reactivity, and receptor selectivity of receptor-activating peptides. Can J Physiol Pharmacol. 1997;75:832–41.PubMedCrossRefPubMedCentralGoogle Scholar
  138. 138.
    Vassallo RR Jr, Kieber-Emmons T, Cichowski K, Brass LF. Structure-function relationships in the activation of platelet thrombin receptors by receptor-derived peptides. J Biol Chem. 1992;267:6081–5.PubMedPubMedCentralGoogle Scholar
  139. 139.
    al-Ani B, Saifeddine M, Hollenberg MD. Detection of functional receptors for the proteinase-activated-receptor-2-activating polypeptide, SLIGRL-NH2, in rat vascular and gastric smooth muscle. Can J Physiol Pharmacol. 1995;73:1203–7.PubMedCrossRefPubMedCentralGoogle Scholar
  140. 140.
    McGuire JJ, Saifeddine M, Triggle CR, Sun K, Hollenberg MD. 2-furoyl-LIGRLO-amide: a potent and selective proteinase-activated receptor 2 agonist. J Pharmacol Exp Ther. 2004;309:1124–31.PubMedCrossRefPubMedCentralGoogle Scholar
  141. 141.
    Al-Ani B, Saifeddine M, Kawabata A, Hollenberg MD. Proteinase activated receptor 2: role of extracellular loop 2 for ligand-mediated activation. Br J Pharmacol. 1999;128:1105–13.PubMedPubMedCentralCrossRefGoogle Scholar
  142. 142.
    Al-Ani B, Saifeddine M, Kawabata A, Renaux B, Mokashi S, Hollenberg MD. Proteinase-activated receptor 2 (PAR(2)): development of a ligand-binding assay correlating with activation of PAR(2) by PAR(1)- and PAR(2)-derived peptide ligands. J Pharmacol Exp Ther. 1999;290:753–60.PubMedPubMedCentralGoogle Scholar
  143. 143.
    Nakanishi-Matsui M, Zheng YW, Sulciner DJ, Weiss EJ, Ludeman MJ, Coughlin SR. PAR3 is a cofactor for PAR4 activation by thrombin. Nature. 2000;404:609–13.PubMedCrossRefPubMedCentralGoogle Scholar
  144. 144.
    Hollenberg MD, Saifeddine M, Al-Ani B, Gui Y. Proteinase-activated receptor 4 (PAR4): action of PAR4-activating peptides in vascular and gastric tissue and lack of cross-reactivity with PAR1 and PAR2. Can J Physiol Pharmacol. 1999;77:458–64.PubMedCrossRefPubMedCentralGoogle Scholar
  145. 145.
    Faruqi TR, Weiss EJ, Shapiro MJ, Huang W, Coughlin SR. Structure-function analysis of protease-activated receptor 4 tethered ligand peptides. Determinants of specificity and utility in assays of receptor function [In process citation]. J Biol Chem. 2000;275:19728–34.PubMedCrossRefPubMedCentralGoogle Scholar
  146. 146.
    Lin H, Trejo J. Transactivation of the PAR1-PAR2 heterodimer by thrombin elicits beta-arrestin-mediated endosomal signaling. J Biol Chem. 2013;288:11203–15.PubMedPubMedCentralCrossRefGoogle Scholar
  147. 147.
    O'Brien PJ, Prevost N, Molino M, Hollinger MK, Woolkalis MJ, Woulfe DS, Brass LF. Thrombin responses in human endothelial cells. Contributions from receptors other than PAR1 include the transactivation of PAR2 by thrombin-cleaved PAR1. J Biol Chem. 2000;275:13502–9.PubMedCrossRefPubMedCentralGoogle Scholar
  148. 148.
    Shi X, Gangadharan B, Brass LF, Ruf W, Mueller BM. Protease-activated receptors (PAR1 and PAR2) contribute to tumor cell motility and metastasis. Mol Cancer Res. 2004;2:395–402.PubMedPubMedCentralGoogle Scholar
  149. 149.
    Jaber M, Maoz M, Kancharla A, Agranovich D, Peretz T, Grisaru-Granovsky S, Uziely B, Bar-Shavit R. Protease-activated-receptor-2 affects protease-activated-receptor-1-driven breast cancer. Cell Mol Life Sci. 2014;71:2517–33.PubMedCrossRefPubMedCentralGoogle Scholar
  150. 150.
    Leger AJ, Jacques SL, Badar J, Kaneider NC, Derian CK, Andrade-Gordon P, Covic L, Kuliopulos A. Blocking the protease-activated receptor 1-4 heterodimer in platelet-mediated thrombosis. Circulation. 2006;113:1244–54.PubMedCrossRefPubMedCentralGoogle Scholar
  151. 151.
    Sveshnikova AN, Balatskiy AV, Demianova AS, Shepelyuk TO, Shakhidzhanov SS, Balatskaya MN, Pichugin AV, Ataullakhanov FI, Panteleev MA. Systems biology insights into the meaning of the platelet's dual-receptor thrombin signaling. J Thromb Haemost. 2016;14:2045–57.PubMedCrossRefPubMedCentralGoogle Scholar
  152. 152.
    Hansen KK, Saifeddine M, Hollenberg MD. Tethered ligand-derived peptides of proteinase-activated receptor 3 (PAR3) activate PAR1 and PAR2 in Jurkat T cells. Immunology. 2004;112:183–90.PubMedPubMedCentralCrossRefGoogle Scholar
  153. 153.
    Cunningham MR, McIntosh KA, Pediani JD, Robben J, Cooke AE, Nilsson M, Gould GW, Mundell S, Milligan G, Plevin R. Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4). J Biol Chem. 2012;287:16656–69.PubMedPubMedCentralCrossRefGoogle Scholar
  154. 154.
    Gieseler F, Ungefroren H, Settmacher U, Hollenberg MD, Kaufmann R. Proteinase-activated receptors (PARs) - focus on receptor-receptor-interactions and their physiological and pathophysiological impact. Cell Commun Signal. 2013;11:86.PubMedPubMedCentralCrossRefGoogle Scholar
  155. 155.
    Amadesi S, Cottrell GS, Divino L, Chapman K, Grady EF, Bautista F, Karanjia R, Barajas-Lopez C, Vanner S, Vergnolle N, Bunnett NW. Protease-activated receptor 2 sensitizes TRPV1 by protein kinase Cepsilon- and A-dependent mechanisms in rats and mice. J Physiol. 2006;575:555–71.PubMedPubMedCentralCrossRefGoogle Scholar
  156. 156.
    Grant AD, Cottrell GS, Amadesi S, Trevisani M, Nicoletti P, Materazzi S, Altier C, Cenac N, Zamponi GW, Bautista-Cruz F, et al. Protease-activated receptor 2 sensitizes the transient receptor potential vanilloid 4 ion channel to cause mechanical hyperalgesia in mice. J Physiol. 2007;578:715–33.PubMedCrossRefPubMedCentralGoogle Scholar
  157. 157.
    Zhao P, Lieu T, Barlow N, Sostegni S, Haerteis S, Korbmacher C, Liedtke W, Jimenez-Vargas NN, Vanner SJ, Bunnett NW. Neutrophil elastase activates protease-activated Receptor-2 (PAR2) and transient receptor potential Vanilloid 4 (TRPV4) to cause inflammation and pain. J Biol Chem. 2015;290:13875–87.PubMedPubMedCentralCrossRefGoogle Scholar
  158. 158.
    Saifeddine M, El-Daly M, Mihara K, Bunnett NW, McIntyre P, Altier C, Hollenberg MD, Ramachandran R. GPCR-mediated EGF receptor transactivation regulates TRPV4 action in the vasculature. Br J Pharmacol. 2015;172:2493–506.PubMedPubMedCentralCrossRefGoogle Scholar
  159. 159.
    Tsubota M, Ozaki T, Hayashi Y, Okawa Y, Fujimura A, Sekiguchi F, Nishikawa H, Kawabata A. Prostanoid-dependent bladder pain caused by proteinase-activated receptor-2 activation in mice: involvement of TRPV1 and T-type ca(2+) channels. J Pharmacol Sci. 2018;136:46–9.PubMedCrossRefPubMedCentralGoogle Scholar
  160. 160.
    Chung H, Ramachandran R, Hollenberg MD, Muruve DA. Proteinase-activated receptor-2 transactivation of epidermal growth factor receptor and transforming growth factor-beta receptor signaling pathways contributes to renal fibrosis. J Biol Chem. 2013;288:37319–31.PubMedPubMedCentralCrossRefGoogle Scholar
  161. 161.
    Sabri A, Guo J, Elouardighi H, Darrow AL, Andrade-Gordon P, Steinberg SF. Mechanisms of protease-activated receptor-4 actions in cardiomyocytes. Role of Src tyrosine kinase. J Biol Chem. 2003;278:11714–20.PubMedCrossRefPubMedCentralGoogle Scholar
  162. 162.
    Fasano A. Zonulin, regulation of tight junctions, and autoimmune diseases. Ann N Y Acad Sci. 2012;1258:25–33.PubMedPubMedCentralCrossRefGoogle Scholar
  163. 163.
    Al-Ani B, Hewett PW, Cudmore MJ, Fujisawa T, Saifeddine M, Williams H, Ramma W, Sissaoui S, Jayaraman PS, Ohba M, et al. Activation of proteinase-activated receptor 2 stimulates soluble vascular endothelial growth factor receptor 1 release via epidermal growth factor receptor transactivation in endothelial cells. Hypertension. 2010;55:689–97.PubMedCrossRefPubMedCentralGoogle Scholar
  164. 164.
    Palm E, Demirel I, Bengtsson T, Khalaf H. The role of toll-like and protease-activated receptors in the expression of cytokines by gingival fibroblasts stimulated with the periodontal pathogen Porphyromonas gingivalis. Cytokine. 2015;76:424–32.PubMedCrossRefPubMedCentralGoogle Scholar
  165. 165.
    Damien P, Cognasse F, Payrastre B, Spinelli SL, Blumberg N, Arthaud CA, Eyraud MA, Phipps RP, McNicol A, Pozzetto B, et al. NF-kappaB links TLR2 and PAR1 to soluble Immunomodulator factor secretion in human platelets. Front Immunol. 2017;8:85.PubMedPubMedCentralCrossRefGoogle Scholar
  166. 166.
    Rallabhandi P, Nhu QM, Toshchakov VY, Piao W, Medvedev AE, Hollenberg MD, Fasano A, Vogel SN. Analysis of proteinase-activated receptor 2 and TLR4 signal transduction: a novel paradigm for receptor cooperativity. J Biol Chem. 2008;283:24314–25.PubMedPubMedCentralCrossRefGoogle Scholar
  167. 167.
    Nhu QM, Shirey K, Teijaro JR, Farber DL, Netzel-Arnett S, Antalis TM, Fasano A, Vogel SN. Novel signaling interactions between proteinase-activated receptor 2 and toll-like receptors in vitro and in vivo. Mucosal Immunol. 2010;3:29–39.PubMedCrossRefPubMedCentralGoogle Scholar
  168. 168.
    Williams JC, Lee RD, Doerschuk CM, Mackman N. Effect of PAR-2 deficiency in mice on KC expression after Intratracheal LPS administration. J Signal Transduct. 2011;2011:415195.PubMedPubMedCentralCrossRefGoogle Scholar
  169. 169.
    Zhou B, Zhou H, Ling S, Guo D, Yan Y, Zhou F, Wu Y. Activation of PAR2 or/and TLR4 promotes SW620 cell proliferation and migration via phosphorylation of ERK1/2. Oncol Rep. 2011;25:503–11.PubMedCrossRefPubMedCentralGoogle Scholar
  170. 170.
    Bucci M, Vellecco V, Harrington L, Brancaleone V, Roviezzo F, Mattace Raso G, Ianaro A, Lungarella G, De Palma R, Meli R, Cirino G. Cross-talk between toll-like receptor 4 (TLR4) and proteinase-activated receptor 2 (PAR(2) ) is involved in vascular function. Br J Pharmacol. 2013;168:411–20.PubMedCrossRefPubMedCentralGoogle Scholar
  171. 171.
    Weithauser A, Bobbert P, Antoniak S, Bohm A, Rauch BH, Klingel K, Savvatis K, Kroemer HK, Tschope C, Stroux A, et al. Protease-activated receptor-2 regulates the innate immune response to viral infection in a coxsackievirus B3-induced myocarditis. J Am Coll Cardiol. 2013;62:1737–45.PubMedPubMedCentralCrossRefGoogle Scholar
  172. 172.
    Oldham WM, Hamm HE. How do receptors activate G proteins? Adv Protein Chem. 2007;74:67–93.PubMedCrossRefPubMedCentralGoogle Scholar
  173. 173.
    Oldham WM, Hamm HE. Heterotrimeric G protein activation by G-protein-coupled receptors. Nat Rev Mol Cell Biol. 2008;9:60–71.PubMedCrossRefPubMedCentralGoogle Scholar
  174. 174.
    Feistritzer C, Riewald M. Endothelial barrier protection by activated protein C through PAR1-dependent sphingosine 1-phosphate receptor-1 crossactivation. Blood. 2005;105:3178–84.PubMedCrossRefPubMedCentralGoogle Scholar
  175. 175.
    Finigan JH, Dudek SM, Singleton PA, Chiang ET, Jacobson JR, Camp SM, Ye SQ, Garcia JG. Activated protein C mediates novel lung endothelial barrier enhancement: role of sphingosine 1-phosphate receptor transactivation. J Biol Chem. 2005;280:17286–93.PubMedCrossRefPubMedCentralGoogle Scholar
  176. 176.
    Mosnier LO, Sinha RK, Burnier L, Bouwens EA, Griffin JH. Biased agonism of protease-activated receptor 1 by activated protein C caused by noncanonical cleavage at Arg46. Blood. 2012;120:5237–46.PubMedPubMedCentralCrossRefGoogle Scholar
  177. 177.
    Hung DT, Wong YH, Vu TK, Coughlin SR. The cloned platelet thrombin receptor couples to at least two distinct effectors to stimulate phosphoinositide hydrolysis and inhibit adenylyl cyclase. J Biol Chem. 1992;267:20831–4.PubMedPubMedCentralGoogle Scholar
  178. 178.
    Hung DT, Vu TK, Wheaton VI, Ishii K, Coughlin SR. Cloned platelet thrombin receptor is necessary for thrombin-induced platelet activation. J Clin Invest. 1992;89:1350–3.PubMedPubMedCentralCrossRefGoogle Scholar
  179. 179.
    Santulli RJ, Derian CK, Darrow AL, Tomko KA, Eckardt AJ, Seiberg M, Scarborough RM, Andrade-Gordon P. Evidence for the presence of a protease-activated receptor distinct from the thrombin receptor in human keratinocytes. Proc Natl Acad Sci U S A. 1995;92:9151–5.PubMedPubMedCentralCrossRefGoogle Scholar
  180. 180.
    McLaughlin JN, Shen L, Holinstat M, Brooks JD, Dibenedetto E, Hamm HE. Functional selectivity of G protein signaling by agonist peptides and thrombin for the protease-activated receptor-1. J Biol Chem. 2005;280:25048–59.PubMedCrossRefPubMedCentralGoogle Scholar
  181. 181.
    Offermanns S, Laugwitz KL, Spicher K, Schultz G. G proteins of the G12 family are activated via thromboxane A2 and thrombin receptors in human platelets. Proc Natl Acad Sci U S A. 1994;91:504–8.PubMedPubMedCentralCrossRefGoogle Scholar
  182. 182.
    Klages B, Brandt U, Simon MI, Schultz G, Offermanns S. Activation of G12/G13 results in shape change and rho/rho-kinase- mediated myosin light chain phosphorylation in mouse platelets. J Cell Biol. 1999;144:745–54.PubMedPubMedCentralCrossRefGoogle Scholar
  183. 183.
    Moers A, Nieswandt B, Massberg S, Wettschureck N, Gruner S, Konrad I, Schulte V, Aktas B, Gratacap MP, Simon MI, et al. G13 is an essential mediator of platelet activation in hemostasis and thrombosis. Nat Med. 2003;9:1418–22.PubMedCrossRefPubMedCentralGoogle Scholar
  184. 184.
    Klarenbach SW, Chipiuk A, Nelson RC, Hollenberg MD, Murray AG. Differential actions of PAR2 and PAR1 in stimulating human endothelial cell exocytosis and permeability: the role of Rho-GTPases. Circ Res. 2003;92:272–8.PubMedCrossRefPubMedCentralGoogle Scholar
  185. 185.
    Fortunato TM, Vara DS, Wheeler-Jones CP, Pula G. Expression of protease-activated receptor 1 and 2 and anti-tubulogenic activity of protease-activated receptor 1 in human endothelial colony-forming cells. PLoS One. 2014;9:e109375.PubMedPubMedCentralCrossRefGoogle Scholar
  186. 186.
    Kim YV, Di Cello F, Hillaire CS, Kim KS. Differential Ca2+ signaling by thrombin and protease-activated receptor-1-activating peptide in human brain microvascular endothelial cells. Am J Physiol Cell Physiol. 2004;286:C31–42.PubMedCrossRefPubMedCentralGoogle Scholar
  187. 187.
    Soh UJ, Trejo J. Activated protein C promotes protease-activated receptor-1 cytoprotective signaling through beta-arrestin and dishevelled-2 scaffolds. Proc Natl Acad Sci U S A. 2011;108:E1372–80.PubMedPubMedCentralCrossRefGoogle Scholar
  188. 188.
    Roy RV, Ardeshirylajimi A, Dinarvand P, Yang L, Rezaie AR. Occupancy of human EPCR by protein C induces beta-arrestin-2 biased PAR1 signaling by both APC and thrombin. Blood. 2016;128:1884–93.PubMedPubMedCentralCrossRefGoogle Scholar
  189. 189.
    DeFea KA, Zalevsky J, Thoma MS, Dery O, Mullins RD, Bunnett NW. Beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2. J Cell Biol. 2000;148:1267–81.PubMedPubMedCentralCrossRefGoogle Scholar
  190. 190.
    Ge L, Shenoy SK, Lefkowitz RJ, DeFea K. Constitutive protease-activated receptor-2-mediated migration of MDA MB-231 breast cancer cells requires both beta-arrestin-1 and -2. J Biol Chem. 2004;279:55419–24.CrossRefGoogle Scholar
  191. 191.
    Stalheim L, Ding Y, Gullapalli A, Paing MM, Wolfe BL, Morris DR, Trejo J. Multiple independent functions of arrestins in the regulation of protease-activated receptor-2 signaling and trafficking. Mol Pharmacol. 2005;67:78–87.PubMedCrossRefPubMedCentralGoogle Scholar
  192. 192.
    Hein L, Ishii K, Coughlin SR, Kobilka BK. Intracellular targeting and trafficking of thrombin receptors. A novel mechanism for resensitization of a G protein-coupled receptor. J Biol Chem. 1994;269:27719–26.PubMedPubMedCentralGoogle Scholar
  193. 193.
    Takafuta T, Wu G, Murphy GF, Shapiro SS. Human beta-filamin is a new protein that interacts with the cytoplasmic tail of glycoprotein Ibalpha. J Biol Chem. 1998;273:17531–8.PubMedCrossRefPubMedCentralGoogle Scholar
  194. 194.
    Vouret-Craviari V, Grall D, Chambard JC, Rasmussen UB, Pouyssegur J, Van Obberghen-Schilling E. Post-translational and activation-dependent modifications of the G protein-coupled thrombin receptor. J Biol Chem. 1995;270:8367–72.PubMedCrossRefPubMedCentralGoogle Scholar
  195. 195.
    Schuepbach RA, Feistritzer C, Brass LF, Riewald M. Activated protein C-cleaved protease activated receptor-1 is retained on the endothelial cell surface even in the presence of thrombin. Blood. 2008;111:2667–73.PubMedPubMedCentralCrossRefGoogle Scholar
  196. 196.
    Trejo J, Hammes SR, Coughlin SR. Termination of signaling by protease-activated receptor-1 is linked to lysosomal sorting. Proc Natl Acad Sci U S A. 1998;95:13698–702.PubMedPubMedCentralCrossRefGoogle Scholar
  197. 197.
    Paing MM, Johnston CA, Siderovski DP, Trejo J. Clathrin adaptor AP2 regulates thrombin receptor constitutive internalization and endothelial cell resensitization. Mol Cell Biol. 2006;26:3231–42.PubMedPubMedCentralCrossRefGoogle Scholar
  198. 198.
    Dores MR, Chen B, Lin H, Soh UJ, Paing MM, Montagne WA, Meerloo T, Trejo J. ALIX binds a YPX(3)L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/MVB sorting. J Cell Biol. 2012;197:407–19.PubMedPubMedCentralCrossRefGoogle Scholar
  199. 199.
    Grimsey NJ, Coronel LJ, Cordova IC, Trejo J. Recycling and endosomal sorting of protease-activated Receptor-1 is distinctly regulated by Rab11A and Rab11B proteins. J Biol Chem. 2016;291:2223–36.PubMedCrossRefPubMedCentralGoogle Scholar
  200. 200.
    Trejo J, Coughlin SR. The cytoplasmic tails of protease-activated receptor-1 and substance P receptor specify sorting to lysosomes versus recycling. J Biol Chem. 1999;274:2216–24.PubMedCrossRefPubMedCentralGoogle Scholar
  201. 201.
    Ishii K, Chen J, Ishii M, Koch WJ, Freedman NJ, Lefkowitz RJ, Coughlin SR. Inhibition of thrombin receptor signaling by a G-protein coupled receptor kinase. J Biol Chem. 1994;269:1125–30.PubMedPubMedCentralGoogle Scholar
  202. 202.
    Paing MM, Stutts AB, Kohout TA, Lefkowitz RJ, Trejo J. Beta -Arrestins regulate protease-activated receptor-1 desensitization but not internalization or down-regulation. J Biol Chem. 2002;277:1292–300.PubMedCrossRefPubMedCentralGoogle Scholar
  203. 203.
    Chen J, De S, Damron DS, Chen WS, Hay N, Byzova TV. Impaired platelet responses to thrombin and collagen in AKT-1-deficient mice. Blood. 2004;104:1703–10.PubMedPubMedCentralCrossRefGoogle Scholar
  204. 204.
    Wolfe BL, Marchese A, Trejo J. Ubiquitination differentially regulates clathrin-dependent internalization of protease-activated receptor-1. J Cell Biol. 2007;177:905–16.PubMedPubMedCentralCrossRefGoogle Scholar
  205. 205.
    Soh UJ, Dores MR, Chen B, Trejo J. Signal transduction by protease-activated receptors. Br J Pharmacol. 2010;160:191–203.PubMedPubMedCentralCrossRefGoogle Scholar
  206. 206.
    Jacob C, Cottrell GS, Gehringer D, Schmidlin F, Grady EF, Bunnett NW. c-Cbl mediates ubiquitination, degradation, and down-regulation of human protease-activated receptor 2. J Biol Chem. 2005;280:16076–87.PubMedCrossRefPubMedCentralGoogle Scholar
  207. 207.
    Dery O, Thoma MS, Wong H, Grady EF, Bunnett NW. Trafficking of proteinase-activated receptor-2 and beta-arrestin-1 tagged with green fluorescent protein. Beta-Arrestin-dependent endocytosis of a proteinase receptor. J Biol Chem. 1999;274:18524–35.CrossRefGoogle Scholar
  208. 208.
    Ricks TK, Trejo J. Phosphorylation of protease-activated receptor-2 differentially regulates desensitization and internalization. J Biol Chem. 2009;284:34444–57.PubMedPubMedCentralCrossRefGoogle Scholar
  209. 209.
    Jimenez-Vargas NN, Pattison LA, Zhao P, Lieu T, Latorre R, Jensen DD, Castro J, Aurelio L, Le GT, Flynn B, et al. Protease-activated receptor-2 in endosomes signals persistent pain of irritable bowel syndrome. Proc Natl Acad Sci U S A. 2018;115:E7438–47.PubMedPubMedCentralCrossRefGoogle Scholar
  210. 210.
    Steinhoff M, Buddenkotte J, Shpacovitch V, Rattenholl A, Moormann C, Vergnolle N, Luger TA, Hollenberg MD. Proteinase-activated receptors: transducers of proteinase-mediated signaling in inflammation and immune response. Endocr Rev. 2005;26:1–43.PubMedCrossRefPubMedCentralGoogle Scholar
  211. 211.
    Smith TH, Coronel LJ, Li JG, Dores MR, Nieman MT, Trejo J. Protease-activated receptor-4 signaling and trafficking is regulated by the Clathrin adaptor protein complex-2 independent of beta-Arrestins. J Biol Chem. 2016;291:18453–64.PubMedPubMedCentralCrossRefGoogle Scholar
  212. 212.
    Shapiro MJ, Weiss EJ, Faruqi TR, Coughlin SR. Protease-activated receptors 1 and 4 are shut off with distinct kinetics after activation by thrombin. J Biol Chem. 2000;275:25216–21.PubMedCrossRefPubMedCentralGoogle Scholar
  213. 213.
    Smith TH, Li JG, Dores MR, Trejo J. Protease-activated receptor-4 and purinergic receptor P2Y12 dimerize, co-internalize, and activate Akt signaling via endosomal recruitment of beta-arrestin. J Biol Chem. 2017;292:13867–78.PubMedPubMedCentralCrossRefGoogle Scholar
  214. 214.
    Kahn ML, Nakanishi-Matsui M, Shapiro MJ, Ishihara H, Coughlin SR. Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin. J Clin Invest. 1999;103:879–87.PubMedPubMedCentralCrossRefGoogle Scholar
  215. 215.
    Andrade-Gordon P, Derian CK, Maryanoff BE, Zhang HC, Addo MF, Cheung W, Damiano BP, D'Andrea MR, Darrow AL, de Garavilla L, et al. Administration of a potent antagonist of protease-activated receptor-1 (PAR-1) attenuates vascular restenosis following balloon angioplasty in rats. J Pharmacol Exp Ther. 2001;298:34–42.PubMedPubMedCentralGoogle Scholar
  216. 216.
    Bae JS, Rezaie AR. Thrombin inhibits nuclear factor kappaB and RhoA pathways in cytokine-stimulated vascular endothelial cells when EPCR is occupied by protein C. Thromb Haemost. 2009;101:513–20.PubMedPubMedCentralCrossRefGoogle Scholar
  217. 217.
    Buddenkotte J, Stroh C, Engels IH, Moormann C, Shpacovitch VM, Seeliger S, Vergnolle N, Vestweber D, Luger TA, Schulze-Osthoff K, Steinhoff M. Agonists of proteinase-activated receptor-2 stimulate upregulation of intercellular cell adhesion molecule-1 in primary human keratinocytes via activation of NF-kappa B. J Invest Dermatol. 2005;124:38–45.PubMedCrossRefPubMedCentralGoogle Scholar
  218. 218.
    Howells GL, Macey MG, Chinni C, Hou L, Fox MT, Harriott P, Stone SR. Proteinase-activated receptor-2: expression by human neutrophils. J Cell Sci. 1997;110(Pt 7):881–7.PubMedPubMedCentralGoogle Scholar
  219. 219.
    Borbiev T, Birukova A, Liu F, Nurmukhambetova S, Gerthoffer WT, Garcia JG, Verin AD. p38 MAP kinase-dependent regulation of endothelial cell permeability. Am J Physiol Lung Cell Mol Physiol. 2004;287:L911–8.PubMedCrossRefPubMedCentralGoogle Scholar
  220. 220.
    Schuepbach RA, Feistritzer C, Fernandez JA, Griffin JH, Riewald M. Protection of vascular barrier integrity by activated protein C in murine models depends on protease-activated receptor-1. Thromb Haemost. 2009;101:724–33.PubMedPubMedCentralCrossRefGoogle Scholar
  221. 221.
    Birukova AA, Alekseeva E, Mikaelyan A, Birukov KG. HGF attenuates thrombin-induced endothelial permeability by Tiam1-mediated activation of the Rac pathway and by Tiam1/Rac-dependent inhibition of the rho pathway. FASEB J. 2007;21:2776–86.PubMedCrossRefPubMedCentralGoogle Scholar
  222. 222.
    Grimsey NJ, Aguilar B, Smith TH, Le P, Soohoo AL, Puthenveedu MA, Nizet V, Trejo J. Ubiquitin plays an atypical role in GPCR-induced p38 MAP kinase activation on endosomes. J Cell Biol. 2015;210:1117–31.PubMedPubMedCentralCrossRefGoogle Scholar
  223. 223.
    Hamilton JR, Frauman AG, Cocks TM. Increased expression of protease-activated receptor-2 (PAR2) and PAR4 in human coronary artery by inflammatory stimuli unveils endothelium-dependent relaxations to PAR2 and PAR4 agonists. Circ Res. 2001;89:92–8.PubMedCrossRefPubMedCentralGoogle Scholar
  224. 224.
    Saifeddine M, al-Ani B, Cheng CH, Wang L, Hollenberg MD. Rat proteinase-activated receptor-2 (PAR-2): cDNA sequence and activity of receptor-derived peptides in gastric and vascular tissue. Br J Pharmacol. 1996;118:521–30.PubMedPubMedCentralCrossRefGoogle Scholar
  225. 225.
    Moffatt JD, Cocks TM. Endothelium-dependent and -independent responses to protease-activated receptor-2 (PAR-2) activation in mouse isolated renal arteries. Br J Pharmacol. 1998;125:591–4.PubMedPubMedCentralCrossRefGoogle Scholar
  226. 226.
    Sobey CG, Moffatt JD, Cocks TM. Evidence for selective effects of chronic hypertension on cerebral artery vasodilatation to protease-activated receptor-2 activation. Stroke. 1999;30:1933–40 discussion 1941.PubMedCrossRefPubMedCentralGoogle Scholar
  227. 227.
    McGuire JJ, Hollenberg MD, Andrade-Gordon P, Triggle CR. Multiple mechanisms of vascular smooth muscle relaxation by the activation of proteinase-activated receptor 2 in mouse mesenteric arterioles. Br J Pharmacol. 2002;135:155–69.PubMedPubMedCentralCrossRefGoogle Scholar
  228. 228.
    Villari A, Giurdanella G, Bucolo C, Drago F, Salomone S. Apixaban enhances vasodilatation mediated by protease-activated receptor 2 in isolated rat arteries. Front Pharmacol. 2017;8:480.PubMedPubMedCentralCrossRefGoogle Scholar
  229. 229.
    Roy SS, Saifeddine M, Loutzenhiser R, Triggle CR, Hollenberg MD. Dual endothelium-dependent vascular activities of proteinase-activated receptor-2-activating peptides: evidence for receptor heterogeneity. Br J Pharmacol. 1998;123:1434–40.PubMedPubMedCentralCrossRefGoogle Scholar
  230. 230.
    Arisato T, Sarker KP, Kawahara K, Nakata M, Hashiguchi T, Osame M, Kitajima I, Maruyama I. The agonist of the protease-activated receptor-1 (PAR) but not PAR3 mimics thrombin-induced vascular endothelial growth factor release in human smooth muscle cells. Cell Mol Life Sci. 2003;60:1716–24.PubMedCrossRefPubMedCentralGoogle Scholar
  231. 231.
    Seymour ML, Binion DG, Compton SJ, Hollenberg MD, MacNaughton WK. Expression of proteinase-activated receptor 2 on human primary gastrointestinal myofibroblasts and stimulation of prostaglandin synthesis. Can J Physiol Pharmacol. 2005;83:605–16.PubMedCrossRefPubMedCentralGoogle Scholar
  232. 232.
    Iablokov V, Hirota CL, Peplowski MA, Ramachandran R, Mihara K, Hollenberg MD, MacNaughton WK. Proteinase-activated receptor 2 (PAR2) decreases apoptosis in colonic epithelial cells. J Biol Chem. 2014;289:34366–77.PubMedPubMedCentralCrossRefGoogle Scholar
  233. 233.
    Rolland-Fourcade C, Denadai-Souza A, Cirillo C, Lopez C, Jaramillo JO, Desormeaux C, Cenac N, Motta JP, Larauche M, Tache Y, et al. Epithelial expression and function of trypsin-3 in irritable bowel syndrome. Gut. 2017;66:1767–78.PubMedPubMedCentralCrossRefGoogle Scholar
  234. 234.
    Vergnolle N, Macnaughton WK, Al-Ani B, Saifeddine M, Wallace JL, Hollenberg MD. Proteinase-activated receptor 2 (PAR2)-activating peptides: identification of a receptor distinct from PAR2 that regulates intestinal transport. Proc Natl Acad Sci U S A. 1998;95:7766–71.PubMedPubMedCentralCrossRefGoogle Scholar
  235. 235.
    Buresi MC, Schleihauf E, Vergnolle N, Buret A, Wallace JL, Hollenberg MD, MacNaughton WK. Protease-activated receptor-1 stimulates Ca(2+)-dependent Cl(-) secretion in human intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol. 2001;281:G323–32.PubMedCrossRefPubMedCentralGoogle Scholar
  236. 236.
    Chin AC, Vergnolle N, MacNaughton WK, Wallace JL, Hollenberg MD, Buret AG. Proteinase-activated receptor 1 activation induces epithelial apoptosis and increases intestinal permeability. Proc Natl Acad Sci U S A. 2003;100:11104–9.PubMedPubMedCentralCrossRefGoogle Scholar
  237. 237.
    Idell S, Gonzalez K, Bradford H, Macarthur CK, Fein AM, Maunder RJ, Garcia JGN, Griffith DE, Weiland J, Martin TR, et al. Procoagulant activity in Bronchoalveolar lavage in the adult respiratory-distress syndrome - contribution of tissue factor associated with factor-vii. Am Rev Respir Dis. 1987;136:1466–74.PubMedCrossRefPubMedCentralGoogle Scholar
  238. 238.
    Jarjour NN, Calhoun WJ, Schwartz LB, Busse WW. Elevated bronchoalveolar lavage fluid histamine levels in allergic asthmatics are associated with increased airway obstruction. Am Rev Respir Dis. 1991;144:83–7.PubMedCrossRefPubMedCentralGoogle Scholar
  239. 239.
    Clark JM, Abraham WM, Fishman CE, Forteza R, Ahmed A, Cortes A, Warne RL, Moore WR, Tanaka RD. Tryptase inhibitors block allergen-induced airway and inflammatory responses in allergic sheep. Am J Respir Crit Care Med. 1995;152:2076–83.PubMedCrossRefPubMedCentralGoogle Scholar
  240. 240.
    Rice KD, Tanaka RD, Katz BA, Numerof RP, Moore WR. Inhibitors of tryptase for the treatment of mast cell-mediated diseases. Curr Pharm Des. 1998;4:381–96.PubMedPubMedCentralGoogle Scholar
  241. 241.
    Hauck RW, Schulz C, Schomig A, Hoffman RK, Panettieri RA Jr. Alpha-thrombin stimulates contraction of human bronchial rings by activation of protease-activated receptors. Am J Phys. 1999;277:L22–9.Google Scholar
  242. 242.
    Cocks TM, Fong B, Chow JM, Anderson GP, Frauman AG, Goldie RG, Henry PJ, Carr MJ, Hamilton JR, Moffatt JD. A protective role for protease-activated receptors in the airways. Nature. 1999;398:156–60.PubMedCrossRefPubMedCentralGoogle Scholar
  243. 243.
    Cocks TM, Moffatt JD. Protease-activated receptor-2 (PAR2) in the airways. Pulm Pharmacol Ther. 2001;14:183–91.PubMedCrossRefPubMedCentralGoogle Scholar
  244. 244.
    Schmidlin F, Amadesi S, Vidil R, Trevisani M, Martinet N, Caughey G, Tognetto M, Cavallesco G, Mapp C, Geppetti P, Bunnett NW. Expression and function of proteinase-activated receptor 2 in human bronchial smooth muscle. Am J Respir Crit Care Med. 2001;164:1276–81.PubMedCrossRefPubMedCentralGoogle Scholar
  245. 245.
    Schmidlin F, Amadesi S, Dabbagh K, Lewis DE, Knott P, Bunnett NW, Gater PR, Geppetti P, Bertrand C, Stevens ME. Protease-activated receptor 2 mediates eosinophil infiltration and hyperreactivity in allergic inflammation of the airway. J Immunol. 2002;169:5315–21.PubMedCrossRefPubMedCentralGoogle Scholar
  246. 246.
    Knight DA, Lim S, Scaffidi AK, Roche N, Chung KF, Stewart GA, Thompson PJ. Protease-activated receptors in human airways: upregulation of PAR-2 in respiratory epithelium from patients with asthma. J Allergy Clin Immunol. 2001;108:797–803.PubMedCrossRefPubMedCentralGoogle Scholar
  247. 247.
    Miotto D, Hollenberg MD, Bunnett NW, Papi A, Braccioni F, Boschetto P, Rea F, Zuin A, Geppetti P, Saetta M, et al. Expression of protease activated receptor-2 (PAR-2) in central airways of smokers and non-smokers. Thorax. 2002;57:146–51.PubMedPubMedCentralCrossRefGoogle Scholar
  248. 248.
    Ricciardolo FL, Steinhoff M, Amadesi S, Guerrini R, Tognetto M, Trevisani M, Creminon C, Bertrand C, Bunnett NW, Fabbri LM, et al. Presence and bronchomotor activity of protease-activated receptor-2 in Guinea pig airways. Am J Respir Crit Care Med. 2000;161:1672–80.PubMedCrossRefPubMedCentralGoogle Scholar
  249. 249.
    Carr MJ, Schechter NM, Undem BJ. Trypsin-induced, neurokinin-mediated contraction of Guinea pig bronchus. Am J Respir Crit Care Med. 2000;162:1662–7.PubMedCrossRefPubMedCentralGoogle Scholar
  250. 250.
    Lin C, von der Thusen J, Daalhuisen J, ten Brink M, Crestani B, van der Poll T, Borensztajn K, Spek CA. Protease-activated receptor (PAR)-2 is required for PAR-1 signalling in pulmonary fibrosis. J Cell Mol Med. 2015;19:1346–56.PubMedPubMedCentralCrossRefGoogle Scholar
  251. 251.
    Cork MJ, Danby SG, Vasilopoulos Y, Hadgraft J, Lane ME, Moustafa M, Guy RH, Macgowan AL, Tazi-Ahnini R, Ward SJ. Epidermal barrier dysfunction in atopic dermatitis. J Invest Dermatol. 2009;129:1892–908.PubMedCrossRefPubMedCentralGoogle Scholar
  252. 252.
    Derian CK, Eckardt AJ, Andrade-Gordon P. Differential regulation of human keratinocyte growth and differentiation by a novel family of protease-activated receptors. Cell Growth Differ. 1997;8:743–9.PubMedPubMedCentralGoogle Scholar
  253. 253.
    D'Andrea MR, Derian CK, Santulli RJ, Andrade-Gordon P. Differential expression of protease-activated receptors-1 and -2 in stromal fibroblasts of normal, benign, and malignant human tissues. Am J Pathol. 2001;158:2031–41.PubMedPubMedCentralCrossRefGoogle Scholar
  254. 254.
    Wakita H, Furukawa F, Takigawa M. Thrombin and trypsin induce granulocyte-macrophage colony-stimulating factor and interleukin-6 gene expression in cultured normal human keratinocytes. Proc Assoc Am Physicians. 1997;109:190–207.PubMedPubMedCentralGoogle Scholar
  255. 255.
    Kanke T, Macfarlane SR, Seatter MJ, Davenport E, Paul A, McKenzie RC, Plevin R. Proteinase-activated receptor-2-mediated activation of stress-activated protein kinases and inhibitory kappa B kinases in NCTC 2544 keratinocytes. J Biol Chem. 2001;276:31657–66.PubMedCrossRefPubMedCentralGoogle Scholar
  256. 256.
    Kawagoe J, Takizawa T, Matsumoto J, Tamiya M, Meek SE, Smith AJ, Hunter GD, Plevin R, Saito N, Kanke T, et al. Effect of protease-activated receptor-2 deficiency on allergic dermatitis in the mouse ear. Jpn J Pharmacol. 2002;88:77–84.PubMedCrossRefPubMedCentralGoogle Scholar
  257. 257.
    Lee SE, Jeong SK, Lee SH. Protease and protease-activated receptor-2 signaling in the pathogenesis of atopic dermatitis. Yonsei Med J. 2010;51:808–22.PubMedPubMedCentralCrossRefGoogle Scholar
  258. 258.
    Steinhoff M, Neisius U, Ikoma A, Fartasch M, Heyer G, Skov PS, Luger TA, Schmelz M. Proteinase-activated receptor-2 mediates itch: a novel pathway for pruritus in human skin. J Neurosci. 2003;23:6176–80.PubMedCrossRefPubMedCentralGoogle Scholar
  259. 259.
    Calabresi E, Petrelli F, Bonifacio AF, Puxeddu I, Alunno A. One year in review 2018: pathogenesis of rheumatoid arthritis. Clin Exp Rheumatol. 2018;36:175–84.PubMedPubMedCentralGoogle Scholar
  260. 260.
    Kelso EB, Lockhart JC, Hembrough T, Dunning L, Plevin R, Hollenberg MD, Sommerhoff CP, McLean JS, Ferrell WR. Therapeutic promise of proteinase-activated receptor-2 antagonism in joint inflammation. J Pharmacol Exp Ther. 2006;316:1017–24.PubMedCrossRefPubMedCentralGoogle Scholar
  261. 261.
    Ferrell WR, Lockhart JC, Kelso EB, Dunning L, Plevin R, Meek SE, Smith AJ, Hunter GD, McLean JS, McGarry F, et al. Essential role for proteinase-activated receptor-2 in arthritis. J Clin Invest. 2003;111:35–41.PubMedPubMedCentralCrossRefGoogle Scholar
  262. 262.
    McCulloch K, McGrath S, Huesa C, Dunning L, Litherland G, Crilly A, Hultin L, Ferrell WR, Lockhart JC, Goodyear CS. Rheumatic disease: protease-activated Receptor-2 in synovial joint pathobiology. Front Endocrinol (Lausanne). 2018;9:257.CrossRefGoogle Scholar
  263. 263.
    Crilly A, Palmer H, Nickdel MB, Dunning L, Lockhart JC, Plevin R, McInnes IB, Ferrell WR. Immunomodulatory role of proteinase-activated receptor-2. Ann Rheum Dis. 2012;71:1559–66.PubMedPubMedCentralCrossRefGoogle Scholar
  264. 264.
    Andrade-Gordon P, Maryanoff BE, Derian CK, Zhang HC, Addo MF, Darrow AL, Eckardt AJ, Hoekstra WJ, McComsey DF, Oksenberg D, et al. Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor. Proc Natl Acad Sci U S A. 1999;96:12257–62.PubMedPubMedCentralCrossRefGoogle Scholar
  265. 265.
    Brass LF, Vassallo RR Jr, Belmonte E, Ahuja M, Cichowski K, Hoxie JA. Structure and function of the human platelet thrombin receptor. Studies using monoclonal antibodies directed against a defined domain within the receptor N terminus. J Biol Chem. 1992;267:13795–8.PubMedPubMedCentralGoogle Scholar
  266. 266.
    Chackalamannil S, Wang Y, Greenlee WJ, Hu Z, Xia Y, Ahn HS, Boykow G, Hsieh Y, Palamanda J, Agans-Fantuzzi J, et al. Discovery of a novel, orally active himbacine-based thrombin receptor antagonist (SCH 530348) with potent antiplatelet activity. J Med Chem. 2008;51:3061–4.PubMedCrossRefPubMedCentralGoogle Scholar
  267. 267.
    Kogushi M, Matsuoka T, Kawata T, Kuramochi H, Kawaguchi S, Murakami K, Hiyoshi H, Suzuki S, Kawahara T, Kajiwara A, Hishinuma I. The novel and orally active thrombin receptor antagonist E5555 (Atopaxar) inhibits arterial thrombosis without affecting bleeding time in guinea pigs. Eur J Pharmacol. 2011;657:131–7.PubMedCrossRefPubMedCentralGoogle Scholar
  268. 268.
    Covic L, Gresser AL, Talavera J, Swift S, Kuliopulos A. Activation and inhibition of G protein-coupled receptors by cell-penetrating membrane-tethered peptides. Proc Natl Acad Sci U S A. 2002;99:643–8.PubMedPubMedCentralCrossRefGoogle Scholar
  269. 269.
    Aisiku O, Peters CG, De Ceunynck K, Ghosh CC, Dilks JR, Fustolo-Gunnink SF, Huang M, Dockendorff C, Parikh SM, Flaumenhaft R. Parmodulins inhibit thrombus formation without inducing endothelial injury caused by vorapaxar. Blood. 2015;125:1976–85.PubMedPubMedCentralCrossRefGoogle Scholar
  270. 270.
    Becker RC, Moliterno DJ, Jennings LK, Pieper KS, Pei J, Niederman A, Ziada KM, Berman G, Strony J, Joseph D, et al. Safety and tolerability of SCH 530348 in patients undergoing non-urgent percutaneous coronary intervention: a randomised, double-blind, placebo-controlled phase II study. Lancet. 2009;373:919–28.PubMedCrossRefPubMedCentralGoogle Scholar
  271. 271.
    Morrow DA, Braunwald E, Bonaca MP, Ameriso SF, Dalby AJ, Fish MP, Fox KA, Lipka LJ, Liu X, Nicolau JC, et al. Vorapaxar in the secondary prevention of atherothrombotic events. N Engl J Med. 2012;366:1404–13.PubMedCrossRefPubMedCentralGoogle Scholar
  272. 272.
    Tricoci P, Huang Z, Held C, Moliterno DJ, Armstrong PW, Van de Werf F, White HD, Aylward PE, Wallentin L, Chen E, et al. Thrombin-receptor antagonist vorapaxar in acute coronary syndromes. N Engl J Med. 2012;366:20–33.PubMedCrossRefPubMedCentralGoogle Scholar
  273. 273.
    Goto S, Ogawa H, Takeuchi M, Flather MD, Bhatt DL, Investigators JL. Double-blind, placebo-controlled phase II studies of the protease-activated receptor 1 antagonist E5555 (atopaxar) in Japanese patients with acute coronary syndrome or high-risk coronary artery disease. Eur Heart J. 2010;31:2601–13.PubMedPubMedCentralCrossRefGoogle Scholar
  274. 274.
    Wiviott SD, Flather MD, O'Donoghue ML, Goto S, Fitzgerald DJ, Cura F, Aylward P, Guetta V, Dudek D, Contant CF, et al. Randomized trial of atopaxar in the treatment of patients with coronary artery disease: the lessons from antagonizing the cellular effect of thrombin-coronary artery disease trial. Circulation. 2011;123:1854–63.PubMedCrossRefPubMedCentralGoogle Scholar
  275. 275.
    O'Donoghue ML, Bhatt DL, Wiviott SD, Goodman SG, Fitzgerald DJ, Angiolillo DJ, Goto S, Montalescot G, Zeymer U, Aylward PE, et al. Safety and tolerability of atopaxar in the treatment of patients with acute coronary syndromes: the lessons from antagonizing the cellular effects of thrombin-acute coronary syndromes trial. Circulation. 2011;123:1843–53.PubMedCrossRefPubMedCentralGoogle Scholar
  276. 276.
    Gurbel PA, Bliden KP, Turner SE, Tantry US, Gesheff MG, Barr TP, Covic L, Kuliopulos A. Cell-penetrating Pepducin therapy targeting PAR1 in subjects with coronary artery disease. Arterioscler Thromb Vasc Biol. 2016;36:189–97.PubMedPubMedCentralCrossRefGoogle Scholar
  277. 277.
    Wilson SJ, Ismat FA, Wang Z, Cerra M, Narayan H, Raftis J, Gray TJ, Connell S, Garonzik S, Ma X, et al. PAR4 (protease-activated receptor 4) antagonism with BMS-986120 inhibits human ex vivo Thrombus formation. Arterioscler Thromb Vasc Biol. 2018;38:448–56.PubMedPubMedCentralCrossRefGoogle Scholar
  278. 278.
    Rasmussen UB, Gachet C, Schlesinger Y, Hanau D, Ohlmann P, Van Obberghen-Schilling E, Pouyssegur J, Cazenave JP, Pavirani A. A peptide ligand of the human thrombin receptor antagonizes alpha- thrombin and partially activates platelets. J Biol Chem. 1993;268:14322–8.PubMedPubMedCentralGoogle Scholar
  279. 279.
    Seiler SM, Peluso M, Tuttle JG, Pryor K, Klimas C, Matsueda GR, Bernatowicz MS. Thrombin receptor activation by thrombin and receptor-derived peptides in platelet and CHRF-288 cell membranes: receptor-stimulated GTPase and evaluation of agonists and partial agonists. Mol Pharmacol. 1996;49:190–7.PubMedPubMedCentralGoogle Scholar
  280. 280.
    Cisowski J, O'Callaghan K, Kuliopulos A, Yang J, Nguyen N, Deng Q, Yang E, Fogel M, Tressel S, Foley C, et al. Targeting protease-activated receptor-1 with cell-penetrating pepducins in lung cancer. Am J Pathol. 2011;179:513–23.PubMedPubMedCentralCrossRefGoogle Scholar
  281. 281.
    Cheung WM, D'Andrea MR, Andrade-Gordon P, Damiano BP. Altered vascular injury responses in mice deficient in protease-activated receptor-1. Arterioscler Thromb Vasc Biol. 1999;19:3014–24.PubMedCrossRefPubMedCentralGoogle Scholar
  282. 282.
    Young SE, Duvernay MT, Schulte ML, Lindsley CW, Hamm HE. Synthesis of indole derived protease-activated receptor 4 antagonists and characterization in human platelets. PLoS One. 2013;8:e65528.PubMedPubMedCentralCrossRefGoogle Scholar
  283. 283.
    Zhang HC, Derian CK, Andrade-Gordon P, Hoekstra WJ, McComsey DF, White KB, Poulter BL, Addo MF, Cheung WM, Damiano BP, et al. Discovery and optimization of a novel series of thrombin receptor (par-1) antagonists: potent, selective peptide mimetics based on indole and indazole templates. J Med Chem. 2001;44:1021–4.PubMedCrossRefPubMedCentralGoogle Scholar
  284. 284.
    Xu QL, Guo XH, Liu JX, Chen B, Liu ZF, Su L. Blockage of protease-activated receptor 1 ameliorates heat-stress induced intestinal high permeability and bacterial translocation. Cell Biol Int. 2015;39:411–7.PubMedCrossRefPubMedCentralGoogle Scholar
  285. 285.
    Nawata S, Murakami A, Hirabayashi K, Sakaguchi Y, Ogata H, Suminami Y, Numa F, Nakamura K, Kato H. Identification of squamous cell carcinoma antigen-2 in tumor tissue by two-dimensional electrophoresis [in process citation]. Electrophoresis. 1999;20:614–7.PubMedCrossRefPubMedCentralGoogle Scholar
  286. 286.
    Kato Y, Kita Y, Nishio M, Hirasawa Y, Ito K, Yamanaka T, Motoyama Y, Seki J. In vitro antiplatelet profile of FR171113, a novel non-peptide thrombin receptor antagonist. Eur J Pharmacol. 1999;384:197–202.PubMedCrossRefPubMedCentralGoogle Scholar
  287. 287.
    Tanaka M, Arai H, Liu N, Nogaki F, Nomura K, Kasuno K, Oida E, Kita T, Ono T. Role of coagulation factor Xa and protease-activated receptor 2 in human mesangial cell proliferation. Kidney Int. 2005;67:2123–33.PubMedCrossRefPubMedCentralGoogle Scholar
  288. 288.
    Kogushi M, Matsuoka T, Kobayashi H, Sato N, Suzuki S, Kawahara T, Kajiwara A, Hishinuma I. Biological characterization of ER129614-06, a novel, non-peptide protease-activated receptor-1 (PAR-1) antagonist. Atheroscler Suppl. 2003;4:245–6.CrossRefGoogle Scholar
  289. 289.
    Kawahara T, Suzuki S, Kogushi M, Matsuoka T, Kobayashi H, Kajiwara A, Hishinuma I. Discovery and optimization of potent orally active small molecular thrombin receptor PAR-1 antagonists. Abstr Pap Am Chem Soc. 2003;225:U215.Google Scholar
  290. 290.
    Perez M, Lamothe M, Maraval C, Mirabel E, Loubat C, Planty B, Horn C, Michaux J, Marrot S, Letienne R, et al. Discovery of novel protease activated receptors 1 antagonists with potent antithrombotic activity in vivo. J Med Chem. 2009;52:5826–36.PubMedCrossRefPubMedCentralGoogle Scholar
  291. 291.
    Monjotin N, Gillespie J, Farrie M, Le Grand B, Junquero D, Vergnolle N. F16357, a novel protease-activated receptor 1 antagonist, improves urodynamic parameters in a rat model of interstitial cystitis. Br J Pharmacol. 2016;173:2224–36.PubMedPubMedCentralCrossRefGoogle Scholar
  292. 292.
    Ahn HS, Foster C, Boykow G, Stamford A, Manna M, Graziano M. Inhibition of cellular action of thrombin by N3-cyclopropyl-7-[[4-(1-methylethyl)phenyl]methyl]-7H-pyrrolo[3, 2-f]quinazoline-1,3-diamine (SCH 79797), a nonpeptide thrombin receptor antagonist. Biochem Pharmacol. 2000;60:1425–34.PubMedCrossRefPubMedCentralGoogle Scholar
  293. 293.
    Gupta N, Liu R, Shin S, Sinha R, Pogliano J, Pogliano K, Griffin JH, Nizet V, Corriden R. SCH79797 improves outcomes in experimental bacterial pneumonia by boosting neutrophil killing and direct antibiotic activity. J Antimicrob Chemother. 2018;73:1586–94.PubMedCrossRefPubMedCentralGoogle Scholar
  294. 294.
    Kogushi M, Kobayashi H, Matsuoka T, Suzuki S, Kawahara T, Kajiwara A, Hishinuma L. Anti-thrombotic and bleeding time effects of E5555, an orally active protease-activated receptor-1 antagonist, in Guinea pigs. Circulation. 2003;108:280.Google Scholar
  295. 295.
    Asteriti S, Daniele S, Porchia F, Dell'anno MT, Fazzini A, Pugliesi I, Trincavelli ML, Taliani S, Martini C, Mazzoni MR, Gilchrist A. Modulation of PAR(1) signaling by benzimidazole compounds. Br J Pharmacol. 2012;167(1):80–94.PubMedPubMedCentralCrossRefGoogle Scholar
  296. 296.
    Deng X, Mercer PF, Scotton CJ, Gilchrist A, Chambers RC. Thrombin induces fibroblast CCL2/JE production and release via coupling of PAR1 to Galphaq and cooperation between ERK1/2 and rho kinase signaling pathways. Mol Biol Cell. 2008;19:2520–33.PubMedPubMedCentralCrossRefGoogle Scholar
  297. 297.
    Tressel SL, Kaneider NC, Kasuda S, Foley C, Koukos G, Austin K, Agarwal A, Covic L, Opal SM, Kuliopulos A. A matrix metalloprotease-PAR1 system regulates vascular integrity, systemic inflammation and death in sepsis. EMBO Mol Med. 2011;3:370–84.PubMedPubMedCentralCrossRefGoogle Scholar
  298. 298.
    Lin C, Duitman J, Daalhuisen J, Ten Brink M, von der Thusen J, van der Poll T, Borensztajn K, Spek CA. Targeting protease activated receptor-1 with P1pal-12 limits bleomycin-induced pulmonary fibrosis. Thorax. 2014;69:152–60.PubMedCrossRefPubMedCentralGoogle Scholar
  299. 299.
    Dockendorff C, Aisiku O, Verplank L, Dilks JR, Smith DA, Gunnink SF, Dowal L, Negri J, Palmer M, Macpherson L, et al. Discovery of 1,3-diaminobenzenes as selective inhibitors of platelet activation at the PAR1 receptor. ACS Med Chem Lett. 2012;3:232–7.PubMedPubMedCentralCrossRefGoogle Scholar
  300. 300.
    De Ceunynck K, Peters CG, Jain A, Higgins SJ, Aisiku O, Fitch-Tewfik JL, Chaudhry SA, Dockendorff C, Parikh SM, Ingber DE, Flaumenhaft R. PAR1 agonists stimulate APC-like endothelial cytoprotection and confer resistance to thromboinflammatory injury. Proc Natl Acad Sci U S A. 2018;115:E982–91.PubMedPubMedCentralCrossRefGoogle Scholar
  301. 301.
    Zhong W, Chen S, Zhang Q, Xiao T, Qin Y, Gu J, Sun B, Liu Y, Jing X, Hu X, et al. Doxycycline directly targets PAR1 to suppress tumor progression. Oncotarget. 2017;8:16829–42.PubMedPubMedCentralGoogle Scholar
  302. 302.
    Zhong W, Chen S, Qin Y, Zhang H, Wang H, Meng J, Huai L, Zhang Q, Yin T, Lei Y, et al. Doxycycline inhibits breast cancer EMT and metastasis through PAR-1/NF-kappaB/miR-17/E-cadherin pathway. Oncotarget. 2017;8:104855–66.PubMedPubMedCentralGoogle Scholar
  303. 303.
    Bendeck MP, Conte M, Zhang M, Nili N, Strauss BH, Farwell SM. Doxycycline modulates smooth muscle cell growth, migration, and matrix remodeling after arterial injury. Am J Pathol. 2002;160:1089–95.PubMedPubMedCentralCrossRefGoogle Scholar
  304. 304.
    Wei H, Wei Y, Tian F, Niu T, Yi G. Blocking proteinase-activated receptor 2 alleviated neuropathic pain evoked by spinal cord injury. Physiol Res. 2016;65:145–53.PubMedPubMedCentralGoogle Scholar
  305. 305.
    Al-Ani B, Saifeddine M, Wijesuriya SJ, Hollenberg MD. Modified proteinase-activated receptor-1 and -2 derived peptides inhibit proteinase-activated receptor-2 activation by trypsin. J Pharmacol Exp Ther. 2002;300:702–8.PubMedCrossRefPubMedCentralGoogle Scholar
  306. 306.
    Kanke T, Kabeya M, Kubo S, Kondo S, Yasuoka K, Tagashira J, Ishiwata H, Saka M, Furuyama T, Nishiyama T, et al. Novel antagonists for proteinase-activated receptor 2: inhibition of cellular and vascular responses in vitro and in vivo. Br J Pharmacol. 2009;158:361–71.PubMedPubMedCentralCrossRefGoogle Scholar
  307. 307.
    Goh FG, Ng PY, Nilsson M, Kanke T, Plevin R. Dual effect of the novel peptide antagonist K-14585 on proteinase-activated receptor-2-mediated signalling. Br J Pharmacol. 2009;158:1695–704.PubMedPubMedCentralCrossRefGoogle Scholar
  308. 308.
    Boitano S, Hoffman J, Flynn AN, Asiedu MN, Tillu DV, Zhang Z, Sherwood CL, Rivas CM, DeFea KA, Vagner J, Price TJ. The novel PAR2 ligand C391 blocks multiple PAR2 signalling pathways in vitro and in vivo. Br J Pharmacol. 2015;172:4535–45.PubMedPubMedCentralCrossRefGoogle Scholar
  309. 309.
    Suen JY, Barry GD, Lohman RJ, Halili MA, Cotterell AJ, Le GT, Fairlie DP. Modulating human proteinase activated receptor 2 with a novel antagonist (GB88) and agonist (GB110). Br J Pharmacol. 2012;165:1413–23.PubMedPubMedCentralCrossRefGoogle Scholar
  310. 310.
    Gardell LR, Ma JN, Seitzberg JG, Knapp AE, Schiffer HH, Tabatabaei A, Davis CN, Owens M, Clemons B, Wong KK, et al. Identification and characterization of novel small-molecule protease-activated receptor 2 agonists. J Pharmacol Exp Ther. 2008;327:799–808.PubMedCrossRefPubMedCentralGoogle Scholar
  311. 311.
    Chanakira A, Westmark PR, Ong IM, Sheehan JP. Tissue factor-factor VIIa complex triggers protease activated receptor 2-dependent growth factor release and migration in ovarian cancer. Gynecol Oncol. 2017;145:167–75.PubMedPubMedCentralCrossRefGoogle Scholar
  312. 312.
    Sun Q, Wang Y, Zhang J, Lu J. ENMD-1068 inhibits liver fibrosis through attenuation of TGF-beta1/Smad2/3 signaling in mice. Sci Rep. 2017;7:5498.PubMedPubMedCentralCrossRefGoogle Scholar
  313. 313.
    Barry GD, Suen JY, Le GT, Cotterell A, Reid RC, Fairlie DP. Novel agonists and antagonists for human protease activated receptor 2. J Med Chem. 2010;53:7428–40.PubMedCrossRefPubMedCentralGoogle Scholar
  314. 314.
    Lieu T, Savage E, Zhao P, Edgington-Mitchell L, Barlow N, Bron R, Poole DP, McLean P, Lohman RJ, Fairlie DP, Bunnett NW. Antagonism of the proinflammatory and pronociceptive actions of canonical and biased agonists of protease-activated receptor-2. Br J Pharmacol. 2016;173:2752–65.PubMedPubMedCentralCrossRefGoogle Scholar
  315. 315.
    Cheng RKY, Fiez-Vandal C, Schlenker O, Edman K, Aggeler B, Brown DG, Brown GA, Cooke RM, Dumelin CE, Dore AS, et al. Structural insight into allosteric modulation of protease-activated receptor 2. Nature. 2017;545:112–5.PubMedCrossRefPubMedCentralGoogle Scholar
  316. 316.
    Sevigny LM, Austin KM, Zhang P, Kasuda S, Koukos G, Sharifi S, Covic L, Kuliopulos A. Protease-activated receptor-2 modulates protease-activated receptor-1-driven neointimal hyperplasia. Arterioscler Thromb Vasc Biol. 2011;31:e100–6.PubMedPubMedCentralCrossRefGoogle Scholar
  317. 317.
    Michael ES, Kuliopulos A, Covic L, Steer ML, Perides G. Pharmacological inhibition of PAR2 with the pepducin P2pal-18S protects mice against acute experimental biliary pancreatitis. Am J Physiol Gastrointest Liver Physiol. 2013;304:G516–26.PubMedCrossRefPubMedCentralGoogle Scholar
  318. 318.
    Ishikawa C, Tsuda T, Konishi H, Nakagawa N, Yamanishi K. Tetracyclines modulate protease-activated receptor 2-mediated proinflammatory reactions in epidermal keratinocytes. Antimicrob Agents Chemother. 2009;53:1760–5.PubMedPubMedCentralCrossRefGoogle Scholar
  319. 319.
    Liu XJ, Mu ZL, Zhao Y, Zhang JZ. Topical tetracycline improves MC903-induced atopic dermatitis in mice through inhibition of inflammatory cytokines and Thymic stromal Lymphopoietin expression. Chin Med J. 2016;129:1483–90.PubMedPubMedCentralCrossRefGoogle Scholar
  320. 320.
    Castro ML, Franco GC, Branco-de-Almeida LS, Anbinder AL, Cogo-Muller K, Cortelli SC, Duarte S, Saxena D, Rosalen PL. Downregulation of proteinase-activated Receptor-2, Interleukin-17, and other Proinflammatory genes by subantimicrobial doxycycline dose in a rat periodontitis model. J Periodontol. 2016;87:203–10.PubMedCrossRefPubMedCentralGoogle Scholar
  321. 321.
    Hollenberg MD, Saifeddine M. Proteinase-activated receptor 4 (PAR4): activation and inhibition of rat platelet aggregation by PAR4-derived peptides. Can J Physiol Pharmacol. 2001;79:439–42.PubMedCrossRefPubMedCentralGoogle Scholar
  322. 322.
    Kim HY, Goo JH, Joo YA, Lee HY, Lee SM, Oh CT, Ahn SM, Kim NH, Hwang JS. Impact on inflammation and recovery of skin barrier by nordihydroguaiaretic acid as a protease-activated receptor 2 antagonist. Biomol Ther (Seoul). 2012;20:463–9.CrossRefGoogle Scholar
  323. 323.
    Yang J, Wu J, Kowalska MA, Dalvi A, Prevost N, O'Brien PJ, Manning D, Poncz M, Lucki I, Blendy JA, Brass LF. Loss of signaling through the G protein, Gz, results in abnormal platelet activation and altered responses to psychoactive drugs. Proc Natl Acad Sci U S A. 2000;97:9984–9.PubMedPubMedCentralCrossRefGoogle Scholar
  324. 324.
    Wu CC, Huang SW, Hwang TL, Kuo SC, Lee FY, Teng CM. YD-3, a novel inhibitor of protease-induced platelet activation. Br J Pharmacol. 2000;130:1289–96.PubMedPubMedCentralCrossRefGoogle Scholar
  325. 325.
    Wu CC, Hwang TL, Liao CH, Kuo SC, Lee FY, Lee CY, Teng CM. Selective inhibition of protease-activated receptor 4-dependent platelet activation by YD-3. Thromb Haemost. 2002;87:1026–33.PubMedCrossRefPubMedCentralGoogle Scholar
  326. 326.
    Young SE, Duvernay MT, Schulte ML, Nance KD, Melancon BJ, Engers J, Wood MR, Hamm HE, Lindsley CW. A novel and selective PAR4 antagonist: ML354. In: Probe reports from the NIH molecular libraries program. Bethesda: National Center for Biotechnology Information; 2015.Google Scholar
  327. 327.
    Wen W, Young SE, Duvernay MT, Schulte ML, Nance KD, Melancon BJ, Engers J, Locuson CW 2nd, Wood MR, Daniels JS, et al. Substituted indoles as selective protease activated receptor 4 (PAR-4) antagonists: discovery and SAR of ML354. Bioorg Med Chem Lett. 2014;24:4708–13.PubMedPubMedCentralCrossRefGoogle Scholar
  328. 328.
    Peek GJ, Elbourne D, Mugford M, Tiruvoipati R, Wilson A, Allen E, Clemens F, Firmin R, Hardy P, Hibbert C, et al. Randomised controlled trial and parallel economic evaluation of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure (CESAR). Health Technol Assess. 2010;14:1–46.PubMedCrossRefPubMedCentralGoogle Scholar
  329. 329.
    Wong PC, Seiffert D, Bird JE, Watson CA, Bostwick JS, Giancarli M, Allegretto N, Hua J, Harden D, Guay J, et al. Blockade of protease-activated receptor-4 (PAR4) provides robust antithrombotic activity with low bleeding. Sci Transl Med. 2017;9.  https://doi.org/10.1126/scitranslmed.aaf5294.
  330. 330.
    Hollenberg MD, Saifeddine M, Sandhu S, Houle S, Vergnolle N. Proteinase-activated receptor-4: evaluation of tethered ligand-derived peptides as probes for receptor function and as inflammatory agonists in vivo. Br J Pharmacol. 2004;143:443–54.PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© The Author(s). 2019

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  1. 1.Institute of Intensive Care Medicine, University Hospital Zurich, University of ZurichZurichSwitzerland
  2. 2.Surgical Research DivisionUniversity Hospital Zurich, University of ZurichZurichSwitzerland

Personalised recommendations