Implantation Serine Proteinase 1 Exhibits Mixed Substrate Specificity that Silences Signaling via Proteinase-Activated Receptors

Implantation S1 family serine proteinases (ISPs) are tryptases involved in embryo hatching and uterine implantation in the mouse. The two different ISP proteins (ISP1 and ISP2) have been detected in both pre- and post-implantation embryo tissue. To date, native ISP obtained from uterus and blastocyst tissues has been isolated only as an active hetero-dimer that exhibits trypsin-like substrate specificity. We hypothesised that in isolation, ISP1 might have a unique substrate specificity that could relate to its role when expressed alone in individual tissues. Thus, we isolated recombinant ISP1 expressed in Pichia pastoris and evaluated its substrate specificity. Using several chromogenic substrates and serine proteinase inhibitors, we demonstrate that ISP1 exhibits trypsin-like substrate specificity, having a preference for lysine over arginine at the P1 position. Phage display peptide mimetics revealed an expanded but mixed substrate specificity of ISP1, including chymotryptic and elastase activity. Based upon targets observed using phage display, we hypothesised that ISP1 might signal to cells by cleaving and activating proteinase-activated receptors (PARs) and therefore assessed PARs 1, 2 and 4 as potential ISP1 targets. We observed that ISP1 silenced enzyme-triggered PAR signaling by receptor-disarming. This PAR-disarming action of ISP1 may be important for embryo development and implantation

Thrombin: To PAR or Not to PAR, and the regulation of inflammation

© Springer Science+Business Media, LLC 2009. All rights reserved. Thrombin, a key final common pathway coagulation cascade proteinase, can be envisioned as one of the body\u27s main sentries, always on the lookout to be rendered active at sites of injury or other stress inducers, and always ready to generate a variety of signals that trigger the defense responses that comprise the process termed inflammation. Thrombin does this job in a clever way, using mechanisms that range from the generation of fibrin from fibrinogen, to the activation of G-protein-coupled receptors. The novel way that thrombin acts on human platelets, by cleaving and stimulating proteolytically activated receptors (PARs), has defined a new role not only for thrombin but also for proteinases in general, as hormone-like agents. Thus, thrombin can be seen as a prototype for a number of proteinases that can regulate cell function either by unmasking the receptor-activating tethered ligand sequence of PARs or by silencing PARs by removing the tethered ligand, thereby preventing activation by other proteinases such as thrombin. To play its role in inflammation, thrombin acts not only via the PARs but also by other mechanisms, such as the activation of metalloproteinases, the generation of active peptides from fibrin and by using non-catalytic mechanisms to trigger cell signalling. This chapter summarizes the several mechanisms (both PAR and non-PAR-related) that thrombin can use to regulate cell and tissue function, with a particular focus on the inflammatory response

Neutrophil elastase and proteinase-3 trigger G proteinbiased signaling through proteinase-activated receptor-1 (PAR1)

Neutrophil proteinases released at sites of inflammation can affect tissue function by either activating or disarming signal transduction mediated by proteinase-activated receptors (PARs). BecausePAR1is expressed at sites where abundant neutrophil infiltration occurs, we hypothesized that neutrophil-derived enzymes might also regulate PAR1 signaling. We report here that both neutrophil elastase and proteinase-3 cleave the human PAR1 N terminus at sites distinct from the thrombin cleavage site. This cleavage results in a disarming of thrombinactivated calcium signaling through PAR1. However, the distinct non-canonical tethered ligands unmasked by neutrophil elastase and proteinase-3, as well as synthetic peptides with sequences derived from these novel exposed tethered ligands, selectively stimulated PAR1-mediated mitogen-activated protein kinase activation. This signaling was blocked by pertussis toxin, implicating a Gαi-triggered signal pathway. We conclude that neutrophil proteinases trigger biased PAR1 signaling and we describe a novel set of tethered ligands that are distinct from the classical tethered ligand revealed by thrombin. We further demonstrate the function of this biased signaling in regulating endothelial cell barrier integrity. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc

Proteinase-activated receptors 1 and 2 and the regulation of porcine coronary artery contractility: A role for distinct tyrosine kinase pathways

Background and Purpose Because angiotensin-II-mediated porcine coronary artery (PCA) vasoconstriction is blocked by protein tyrosine kinase (PYK) inhibitors, we hypothesized that proteinase-activated receptors (PARs), known to regulate vascular tension, like angiotensin-II, would also cause PCA contractions via PYK-dependent signalling pathways. Experimental Approach Contractions of intact and endothelium-free isolated PCA rings, stimulated by PAR1/PAR2-activating peptides, angiotensin-II, PGF 2α, EGF, PDGF and KCl, were monitored with/without multiple signalling pathway inhibitors, including AG-tyrphostins AG18 (non-specific PYKs), AG1478 (EGF-receptor kinase), AG1296 (PDGF receptor kinase), PP1 (Src kinase), U0126 and PD98059 (MEK/MAPKinase kinase), indomethacin/SC-560/NS-398 (COX-1/2) and L-NAME (NOS). Key Results AG18 inhibited the contractions induced by all the agonists except KCl, whereas U0126 attenuated contractions induced by PAR1/PAR2 agonists, EGF and angiotensin-II, but not by PGF2α, the COX-produced metabolites of arachidonate and KCl. PP1 only affected the responses to PAR1/PAR2-activating peptides and angiotensin-II the EGF-kinase inhibitor, AG1478, attenuated contractions initiated by the PARs (PAR2 \u3e\u3e PAR1) and EGF itself, but not by angiotensin-II, PGF2α or KCl. COX-1/2 inhibitors blocked the contractions induced by all the agonists, except KCl and PGF2α. Conclusion and Implications PAR 1/2-mediated contractions of the PCA are dependent on Src and MAPKinase and, in part, involve EGF-receptor-kinase transactivation and the generation of a COX-derived contractile agonist. However, the PYK signalling pathways used by PARs are distinct from each other and from those triggered by angiotensin-II and EGF these signalling pathways may be therapeutic targets for managing coagulation-proteinase-induced coronary vasospasm. © 2014 The British Pharmacological Society

Derivatized 2-furoyl-LIGRLO-amide, a versatile and selective probe for proteinase-activated receptor 2: Binding and visualization

The proteinase-activated receptor-2 (PAR2)-activating peptide with an N-terminal furoyl group modification, 2-furoyl-LIGRLO-NH2 (2fLI), was derivatized via its free ornithine amino group to yield [ 3H]propionyl-2fLI and Alexa Fluor 594-2fLI that were used as receptor probes for ligand binding assays and receptor visualization both for cultured cells in vitro and for colonic epithelial cells in vivo. The binding of the radiolabeled and fluorescent PAR2 probes was shown to be present in PAR2-transfected Kirsten normal rat kidney cells, but not in vectoralone-transfected cells, and was abolished by pretreatment of cells with saturating concentrations of receptor-selective PAR2 peptide agonists such as SLIGRL-NH2 and the parent agonist 2fLI but not by reverse-sequence peptides such as 2-furoyl-OLRGIL-NH2 that cannot activate PAR2. The relative orders of potencies for a series of PAR2 peptide agonists to compete for the binding of [ 3H]propionyl-2fLI (2fLI ≫ SLIGRL-NH2 ≅ transcinnamoyl-LIGRLO-NH2 \u3e SLIGKV-NH2 \u3e SLIGKT-NH2) mirrored qualitatively their relative potencies for PAR2-mediated calcium signaling in the same cells or for vasorelaxation in a rat aorta vascular assay. In the vascular assay, the potency of Alexa Fluor 594-2fLI was the same as 2fLI. We conclude that ornithine-derivatized 2fLI peptides are conveniently synthesized PAR2 probes that will be of value for future studies of receptor binding and visualization. Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics

Kallikreins and proteinase-mediated signaling: Proteinase-activated receptors (PARs) and the pathophysiology of inflammatory diseases and cancer

Proteinases such as thrombin and trypsin can affect tissues by activating a novel family of G protein-coupled proteinase-activated receptors (PARs 1-4) by exposing a \u27tethered\u27 receptor-triggering ligand (TL). Work with synthetic TL-derived PAR peptide sequences (PAR-APs) that stimulate PARs 1, 2 and 4 has shown that PAR activation can play a role in many tissues, including the gastrointestinal tract, kidney, muscle, nerve, lung and the central and peripheral nervous systems, and can promote tumor growth and invasion. PARs may play roles in many settings, including cancer, arthritis, asthma, inflammatory bowel disease, neurodegeneration and cardiovascular disease, as well as in pathogen-induced inflammation. In addition to activating or disarming PARs, proteinases can also cause hormone-like effects via PAR-independent mechanisms, such as activation of the insulin receptor. In addition to proteinases of the coagulation cascade, recent data suggest that members of the family of kallikrein-related peptidases (KLKs) represent endogenous PAR regulators. In summary: (1) proteinases are like hormones, signaling in a paracrine and endocrine manner via PARs or other mechanisms; (2) KLKs must now be seen as potential hormone-like PAR regulators in vivo; and (3) PAR-regulating proteinases, their target PARs, and their associated signaling pathways appear to be novel therapeutic targets. © 2008 by Walter de Gruyter

Proteinase-activated receptors (PARs): Differential signalling by kallikrein-related peptidases KLK8 and KLK14

We compared signalling pathways such as calcium transients, MAPK activation, β-arrestin interactions and receptor internalization triggered by kallikrein-related peptidases (KLKs) 8 and 14 in human and rat proteinase-activated receptor (PAR)2-expressing human embryonic kidney (HEK) and Kirsten transformed rat kidney (KNRK) cells. Further, we analy sed processing by KLK8 vs. KLK14 of synthetic human and rat PAR2-derived sequences representing the cleavageactivation domain of PAR2. Our data show that like KLK14, KLK8 can unmask a PAR2 receptor-activating sequence from a peptide precursor. However, whilst KLK8, like KLK14, can signal in rat-PAR2-expressing KNRK cells, this enzyme cannot signal via human PAR2 in HEK or KNRK cells, but rather, disarms HEK PAR1. Thus, KLK8 and KLK14 can signal differentially via the PARs to affect tissue function. © 2012 by Walter de Gruyter · Berlin · Boston

Targeting a proteinase-activated receptor 4 (PAR4) carboxyl terminal motif to regulate platelet function

© 2017 by The American Society for Pharmacology and Experimental Therapeutics. Thrombin initiates human platelet aggregation by coordinately activating proteinase-activated receptors (PARs) 1 and 4. However, targeting PAR1 with an orthosteric-tethered ligand bindingsite antagonist results in bleeding, possibly owing to the important role of PAR1 activation on cells other than platelets. Because of its more restricted tissue expression profile, we have therefore turned to PAR4 as an antiplatelet target. We have identified an intracellular PAR4 C-terminal motif that regulates calcium signaling and b-arrestin interactions. By disrupting this PAR4 calcium/b-arrestin signaling process with a novel cellpenetrating peptide, we were able to inhibit both thrombintriggered platelet aggregation in vitro and clot consolidation in vivo. We suggest that targeting PAR4 represents an attractive alternative to blocking PAR1 for antiplatelet therapy in humans