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Proteinase-Activated Receptors

The search for a functional thrombin receptor, using expression cloning methods, led to the discovery of a G protein-coupled receptor that mediates the actions of thrombin on platelets and endothelial cells. The thrombin receptor, in contrast with other G protein-coupled receptors, lacks a circulating ligand. Rather, its unique mechanism of activation involves the proteolytic unmasking of an N-terminal receptor-triggering sequence buried in the "pro-receptor". The "revealed" N-terminal sequence acts as a "tethered" or anchored receptor-stimulating ligand. Remarkably, the Proteinase Activated Receptor for thrombin (termed, PAR1) can be activated in the absence of thrombin by relatively short peptides based on the sequence of the proteolytically revealed N-terminal domain (so-called Thrombin Receptor-Activating Polypeptides, or TRAPs). The TRAPs (termed, PAR1-Activating Peptides, or PAR1APs) have been used as surrogate activators of PAR1 to evaluate the potential actions of thrombin in tissues wherein the effects of the proteinase itself might be difficult to interpret. The TRAPs have also served as a basis for the development of receptor-selective PAR1-targeted agonists and antagonists.

Results with the originally designed PAR1APs, including peptide structure-activity studies, interspecies studies of platelet aggregation and the development of PAR1-knockout mice, clearly pointed to the existence of other members of the PAR-family. Thus, the serendipitous discovery of a second G protein-coupled proteinase-activated receptor (PAR2), that could be stimulated preferentially by trypsin in comparison with thrombin, was not entirely unexpected. Like PAR1, PAR2 is activated by a proteolytically-revealed tethered-ligand mechanism. Also like PAR1, PAR2 can be activated by short peptides (e.g. SLIGRL... from rat PAR2) based on the N-terminal trypsin-revealed “tethered-ligand” sequence. Whereas the PAR2AP, SLIGRL-NH2 cannot activate PAR1, it was quickly realized that the originally described PAR1APs (or TRAPs) could activate both PAR2 and PAR1; and that peptide structure-activity studies were required to design PAR1APs that could selectively activate PAR1, without activating PAR2. Because of the cross-reactivity of PAR1-targeted ligands with PAR2, receptor-selective antagonists and binding probes have proved difficult to synthesize. For instance, the peptide PAR1 antagonists that can block thrombin-mediated platelet activation have been shown to be agonists for PAR2. Nonetheless, non-peptide PAR1 antagonists (RWJ 56110; SCH 79797) have now been described that can block both thrombin- and peptide-mediated receptor activation.

The discovery of PAR2 did not, however, explain the activity of thrombin in murine platelets that lack PAR1. The absence of PAR1 in murine platelets prompted a continued search for more thrombin receptors, resulting in the discovery of two more family members, PAR3 and PAR4, each of which has a unique thrombin-revealed tethered ligand. Unexpectedly, PAR3 does not appear to signal itself, but rather acts as a cofactor for the activation of PAR4. Synthetic peptides modeled on the thrombin-revealed sequence of PAR3 (e.g. TFRGAP...) do not activate PAR3, but are now known to activate both PAR1 and PAR2. The PAR4 tethered ligands (murine-GYPGKF; human-GYPGQV) fail to activate other PARs, but are of low potencies (active in the 100-400 µM range). The more potent PAR4AP, AYPGKF-NH2 (active in the 10 to 50 µM range), is more useful for physiological studies of PAR4 function. In keeping with trans-cinnamoyl-subsituted PAR1-derived peptides that are PAR1 antagonists, the peptide, trans-cinnamoyl-YPGKF-NH2, is a selective PAR4 antagonist that can be of use in studies done in vitro. Because of its relatively low potency, this trans-cinnamoyl derivative cannot be used in vivo, in contrast with the 'pepducin' PAR4 antagonist, N-palmitoyl-SGRRYGHALR-NH2 (P4pal-10).

In summary, four proteinase-activated receptors that are regulated by a proteolytically-revealed tethered ligand mechanism are known. In addition to the recognized roles for PARs 1, 3 and 4 in regulating platelet and endothelial cell function, data point to prominent roles for the PARs in physiological processes ranging from inflammation and pain sensation to the regulation of the vascular, pulmonary and gastrointestinal systems.

 

The Table below contains accepted modulators and additional information. For a list of additional products, see the "Similar Products" section below.

 

Currently Accepted Name PAR1 PAR2
Alternate Names Thrombin receptor
PAR-1
PAR-2
Structural Information 425 aa (human) 397 aa (human)
Activating Proteinases Thrombin (T7513 (b), T1063 (h)) > Trypsin (T1426) >> Plasmin (P1867)
Cysteine proteinase (RgpB) produced by Porphyromonas gingivalis
Factor Xa (F9302 (b))
Trypsin (T1426)a
Tryptase (T7063)
Trypsin-2
Trypsin IV
Factor Xa TF
Factor VIIa
Matriptase/MT-serine protease 1
Cysteine proteinase (RgpB) produced by Porphyromonas gingivalis
Dust mite proteinases Der p3 and Der p9
Subtype Selective Agonists TFRIFDb
TFLLR-NH2 (T7830)
SLIGRL-NH2 (S9317)
2-furoyl-LIGLRO-NH2br>
Putative Antagonists BMS 200261d (B4188)
Mercaptopropionyl-Phe-Cha-Arg-Lys-Pro-Lys-Pro-Asn-Asp-Lys-NH2e
N-palmitoyl-RCLSSSAVANRS (Pepducin P1pal 12)
RWJ56110
SCH 79797
Not Known
Signal Transduction Mechanisms Gq/11 (increase IP3/DAG)
Gi (cAMP modulation)
G12/13 (actin rearrangement)
Gq/11 (increase IP3/DAG)
Gi (cAMP modulation)
Tissue Expression Platelets
Vasculature (edothelium and smooth muscle)
G.I. tract (neurons epithelia and smooth muscle)
CNS (neurons and astrocytes)
Lung
Kidney
Liver
Leukocytes
Heart
Vasculature (endothelium)
GI tract (neurons, epithelia)
CNS (neurons and astrocytes)
Lung
Kidney
Liver
Leukocytes
Heart
Physiological Function Platelet aggregation and secretion
Vasoregulation
Gastric motility
Inflammation
Nociception
Neuronal regulation
Vasoregulation
Gastric motility
Inflammation
Nociception
Neuronal regulation
Disease Relevance Coronary thrombosis
Inflammatory bowel disease
Cancer
Arthritis
Inflammatory bowel disease
Cancer
Infectious colitis

 

 

Currently Accepted Name PAR3 PAR4
Alternate Names Thrombin receptor
PAR-3
Thrombin receptor
PAR-4
Structural Information 374 aa (human) 385 aa (human)
Activating Proteinases Thrombin (T7513 (b), T1063 (h)) >> Trypsin (T1426) > Factor Xa (F9302 (b))
Thrombin (T7513 (b), T1063 (h)) ~ Trypsin (T1426)
Trypsin IV Cathepsin G (C4428)
Factor VIIa
Factor Xa (F9302 (b))
Subtype Selective Agonists Thrombin cleaves, but does not activate
GYPGKF-NH2c
GYPGQV-NH2
AYPGKF-NH2 (A3227)
Putative Antagonists Not Known trans-cinnamoyl-YPGKF-NH2
N-palmitoyl-SGRRYGHALR-NH2 (Pepducin P4pal-10)
Signal Transduction Mechanisms Not Known
Gq/11 (increase IP3/DAG)
Tissue Expression Platelets, vasculature (endothelium), liver, leukocytes
Platelets, vasculature (endothelium), GI tract (epithelia), leukocytes, lung, heart
Physiological Function Not Known
Platelet aggregation and secretion
Inflammation
Disease Relevance Not Known
Coronary thrombosis
Inflammatory bowel disease
Cancer

 

Footnotes

a) Thrombin is inactive.

b) TFRIFD is the Xenopus thrombin receptor tethered-ligand domain (the human PAR1 tethered-ligand domain sequence SFLLRN activates both PAR1 and PAR2 receptors).

c) AYPGKF is 10-fold more potent than the natural human (GYPGQV) or murine (GYPGKF) tethered ligand sequences, which are also PAR4 selective.

d) May also act at PAR2.

e) Antagonizes PAR1; acts as an agonist at PAR2.

 

Abbreviation

BMS 200261: Transcinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-NH2 RWJ 56110: N-[(1S)-3-Amino-1-[[(phenylmethyl)amino]carbonyl]propyl]-a-[[[[1-[(2,6-dichlorphenyl) methyl]-3-(1-pyrrolidinylmethyl]-1H-indol-6-yl]amino]carbonyl]amino]-3,-difluoro-benzenepropanamide SCH 79797: N3-cyclopropyl-7-[[4-(1-methylethyl)phenyl]methyl]-7H-pyrrolo[3, 2-f]quinazoline-1,3-diamine

TF: Tissue factor
b: bovine
h: human

 

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References