Proteinase-activated receptors (PARs) as targets for antiplatelet therapy.

Since the identification of the proteinase-activated receptor (PAR) family as mediators of serine protease activity in the 1990s, there has been tremendous progress in the elucidation of their pathophysiological roles. The development of drugs that target PARs has been the focus of many laboratories for the potential treatment of thrombosis, cancer and other inflammatory diseases. Understanding the mechanisms of PAR activation and G protein signalling pathways evoked in response to the growing list of endogenous proteases has yielded great insight into receptor regulation at the molecular level. This has led to the development of new selective modulators of PAR activity, particularly PAR1. The mixed success of targeting PARs has been best exemplified in the context of inhibiting PAR1 as a new antiplatelet therapy. The development of the competitive PAR1 antagonist, vorapaxar (Zontivity), has clearly shown the value in targeting PAR1 in acute coronary syndrome (ACS); however the severity of associated bleeding with this drug has limited its use in the clinic. Due to the efficacy of thrombin acting via PAR1, strategies to selectively inhibit specific PAR1-mediated G protein signalling pathways or to target the second thrombin platelet receptor, PAR4, are being devised. The rationale behind these alternative approaches is to bias downstream thrombin activity via PARs to allow for inhibition of pro-thrombotic pathways but maintain other pathways that may preserve haemostatic balance and improve bleeding profiles for widespread clinical use. This review summarizes the structural determinants that regulate PARs and the modulators of PAR activity developed to date.

Vorapaxar and diplopia: Possible off-target PAR-receptor mismodulation.

Vorapaxar, a novel antiplatelet thrombin PAR-1 inhibitor, has been evaluated in the successful TRA2P trial and the failed TRACER trial. The drug is currently approved for post myocardial infarction and peripheral artery disease indications with concomitant use of clopidogrel and/or aspirin. The FDA ruled that the vorapaxar safety profile is acceptable. However, both trials revealed excess diplopia (double vision) usually reversible after vorapaxar. The diplopia risk appears to be small (about 1 extra case per 1,000 treated subjects), but real. Overall, there were 10 placebo and 34 vorapaxar diplopia cases (p=0.018) consistent for TRACER (2 vs 13 cases; p=0.010) and for TRA2P (8 vs 21 cases; p=0.018). Hence, we review the FDA-confirmed evidence and discuss potential causes and implications of such a surprising adverse association, which may be related to off-target PAR receptor mismodulation in the eye.

Proteinase activated-receptors-associated signaling in the control of gastric cancer.

Gastric cancer (GC) is the fourth most common cancer in the world and the second cause of cancer-related death. Gastric carcinogenesis is a multifactorial process, in which environmental and genetic factors interact to activate multiple intracellular signals thus leading to uncontrolled growth and survival of GC cells. One such a pathway is regulated by proteinase activated-receptors (PARs), seven transmembrane-spanning domain G protein-coupled receptors, which comprise four receptors (i.e., PAR-1, PAR-2, PAR-3, and PAR-4) activated by various proteases. Both PAR-1 and PAR-2 are over-expressed on GC cells and their activation triggers and/or amplifies intracellular pathways, which sustain gastric carcinogenesis. There is also evidence that expression of either PAR-1 or PAR-2 correlates with depth of wall invasion and metastatic dissemination and inversely with the overall survival of patients. Consistently, data emerging from experimental models of GC suggest that both these receptors can be important targets for therapeutic interventions in GC patients. In contrast, PAR-4 levels are down-regulated in GC and correlate inversely with the aggressiveness of GC, thus suggesting a negative role of this receptor in the control of GC. In this article we review the available data on the expression and role of PARs in GC and discuss whether manipulation of PAR-driven signals may be useful for interfering with GC cell behavior.

Luminal trypsin induces enteric nerve-mediated anion secretion in the mouse cecum.

Proteases play a diverse role in health and disease. An excessive concentration of proteases has been found in the feces of patients with inflammatory bowel disease or irritable bowel syndrome and been implicated in the pathogenesis of such disorders. This study examined the effect of the serine protease, trypsin, on intestinal epithelial anion secretion when added to the luminal side. A mucosal-submucosal sheet of the mouse cecum was mounted in Ussing chambers, and the short-circuit current (I sc) was measured. Trypsin added to the mucosal (luminal) side increased I sc with an ED50 value of approximately 10 µM. This I sc increase was suppressed by removing Cl(-) from the bathing solution. The I sc increase induced by 10-100 µM trypsin was substantially suppressed by tetrodotoxin, and partially inhibited by a neurokinin-1 receptor antagonist, but not by a muscarinic or nicotinic ACh-receptor antagonist. The trypsin-induced I sc increase was also significantly inhibited by a 5-hydroxytryptamine-3 receptor (5-HT3) antagonist and substantially suppressed by the simultaneous addition of both 5-HT3 and 5-HT4 receptor antagonists. We conclude that luminal trypsin activates the enteric reflex to induce anion secretion, 5-HT and substance P playing important mediating roles in this secreto-motor reflex. Luminal proteases may contribute to the cause of diarrhea occurring with some intestinal disorders.

Coagulation, protease-activated receptors, and viral myocarditis.

The coagulation protease cascade plays an essential role in hemostasis. In addition, a clot contributes to host defense by limiting the spread of pathogens. Coagulation proteases induce intracellular signaling by cleavage of cell surface receptors called protease-activated receptors (PARs). These receptors allow cells to sense changes in the extracellular environment, such as infection. Viruses activate the coagulation cascade by inducing tissue factor expression and by disrupting the endothelium. Virus infection of the heart can cause myocarditis, cardiac remodeling, and heart failure. A recent study using a mouse model have shown that tissue factor, thrombin, and PAR-1 signaling all positively regulate the innate immune during viral myocarditis. In contrast, PAR-2 signaling was found to inhibit interferon-ß expression and the innate immune response. These observations suggest that anticoagulants may impair the innate immune response to viral infection and that inhibition of PAR-2 may be a new strategy to reduce viral myocarditis.

Biological activity of Helicobacter pylori components in mammalian cells: is it independent of proteinase-activated receptors?

To clarify the relationship between Helicobacter pylori (H. pylori), a risk factor for gastritis, peptic ulcer and gastric cancer, and proteinase-activated receptors (PARs) that contribute to inflammatory responses, we determined and characterized the biological activity of H. pylori components in the mammalian cells that express PARs. The activity of H. pylori extracts was assessed in distinct cell lines with high expression of PAR1 (RGM1 cells), PAR2 (A549 cells), or PAR2 and PAR4 (HCT-15 cells). A PAR1-activating peptide (AP), but not H. pylori extracts, caused prostaglandin E2 (PGE2) release in RGM1 cells. On the other hand, H. pylori extracts produced release of PGE2 and interleukin-8 (IL-8) in A549 and HCT-15 cells, respectively, as a PAR2-AP did. The activity of H. pylori extracts in A549 cells was not affected by a proteinase inhibitor or exposure to boiling, but abolished by inhibitors of lipopolysaccharide (LPS), IRAK-1/4 or NF-κB. The activity of H. pylori extracts in HCT-15 cells was partially suppressed by boiling or the proteinase inhibitor. In rat platelets that express PAR4 and PAR3, like a PAR4-AP, H. pylori extracts induced aggregation when assessed in platelet rich plasma, an effect unaffected by the proteinase inhibitor, but did not cause aggregation of washed rat platelets that responded to the PAR4-AP or thrombin. The present study thus shows the biological activities of H. pylori extracts in A549 and HCT-15 cells or rat platelets, and suggests that they are not mediated by any PAR-activating proteinases, but may involve the other pathogenic factors including LPS.

Signal transduction by protease-activated receptors.

The family of G protein-coupled receptors (GPCRs) constitutes the largest class of signalling receptors in the human genome, controlling vast physiological responses and are the target of many drugs. After activation, GPCRs are rapidly desensitized by phosphorylation and beta-arrestin binding. Most classic GPCRs are internalized through a clathrin, dynamin and beta-arrestin-dependent pathway and then recycled back to the cell surface or sorted to lysosomes for degradation. Given the vast number and diversity of GPCRs, different mechanisms are likely to exist to precisely regulate the magnitude, duration and spatial aspects of receptor signalling. The G protein-coupled protease-activated receptors (PARs) provide elegant examples of GPCRs that are regulated by distinct desensitization and endocytic sorting mechanisms, processes that are critically important for the spatial and temporal fidelity of PAR signalling. PARs are irreversibly activated through proteolytic cleavage and transmit cellular responses to extracellular proteases. Activated PAR(1) internalizes through a clathrin- and dynamin-dependent pathway independent of beta-arrestins. Interestingly, PAR(1) is basally ubiquitinated and deubiquitinated after activation and traffics from endosomes to lysosomes independent of ubiquitination. In contrast, beta-arrestins mediate activated PAR(2) internalization and function as scaffolds that promote signalling from endocytic vesicles. Moreover, activated PAR(2) is modified with ubiquitin, which facilitates lysosomal degradation. Activated PARs also adopt distinct active conformations that signal to diverse effectors and are likely regulated by different mechanisms. Thus, the identification of the molecular machinery important for PAR signal regulation will enable the development of new strategies to manipulate receptor signalling and will provide novel targets for the development of drugs.

Protease-activated receptors as drug targets in inflammation and pain.

Proteases have been shown to signal to cells through the activation of a novel class of receptors coupled to G proteins: the protease-activated receptors (PARs). Those receptors are expressed in a wide range of cells, which ultimately are all involved in mechanisms of inflammation and pain. Numerous studies have considered the role of PARs in cells, organ systems or in vivo, highlighting the fact that PAR activation results in signs of inflammation. A growing body of evidences discussed here suggests that these receptors, and the proteases that activate them, interfere with inflammation and pain processes. Whether a role for PARs has been clearly defined in inflammatory and pain pathologies is discussed in this review. Further, the pros and cons for considering PARs as targets for the development of therapeutic options for the treatment of inflammation and pain are discussed.

Protease-activated receptors in cardiovascular health and diseases.

The platelet, once thought to be solely involved in clot formation, is now known to be a key mediator in various other processes such as inflammation, thrombosis, and atherosclerosis. Therefore, antiplatelet agents have become paramount in the prevention and management of various cardiovascular diseases. However, the currently most widely used antiplatelet drugs, aspirin and clopidogrel, have been shown to reduce the risk of serious vascular events only by approximately one quarter. Similarly, oral glycoprotein IIb/IIIa antagonists have been associated with excess mortality, thus restricting the use of parental glycoprotein IIb/IIIa antagonists to the treatment of acute clinical conditions. Thus, for the prevention of cardiovascular diseases, there is still a clinical need for antiplatelet drugs with higher antithrombotic efficacy but with safety profiles that allow for a preventive long-term administration. Thrombin signaling through the protease-activated receptors (PARs) has been shown to influence a wide range of physiologic and pathologic responses in cardiovascular systems. Thus, interference with PARs appears to be a promising strategy to develop new antiplatelet agents with higher efficacy. This review focuses on the cardiovascular actions of PARs that play a role in normal cardiovascular physiology and that are likely to contribute to cardiovascular diseases.

Coagulation factor Xa signaling: the link between coagulation and inflammatory bowel disease?

Inflammatory bowel disease (IBD) is characterized by activation of the coagulation cascade and it has long been suspected that coagulation is an essential component of this still largely idiopathic group of diseases. The realization that coagulation factors are not only passive mediators in the propagation of coagulation, but also actively engage different cell types by activating proteinase-activated receptors (PARs) has provided mechanistic insight into how coagulation cascade propagation might participate in the progression of IBD. The emergence of PAR(2) as a key player in the progression of IBD has focused attention on its agonist, coagulation factor Xa (FXa). Recent findings on FXa and PAR(2) link the coagulation cascade to the progression of IBD. Here, we propose that FXa-induced PAR(2) activation has an important role in orchestrating intestinal inflammatory and fibroproliferative responses, and that PAR(2) might provide a novel therapeutic target for the management of IBD.

Should we target allergen protease activity to decrease the burden of allergic airway inflammation?

The prevalence of allergy and asthma has been increasing steadily in the developed world. Despite the growing health problems associated with these conditions few new therapeutic options are becoming available. Relatively recent developments have identified a role for proteases in promoting allergic sensitisation and allergic airway inflammation. It is also known that most major allergens have associated protease activity that is closely related to their efficacy to induce sensitization and inflammation. Here we review the evidence for the significance of protease activity in allergic airway disease and offer suggestions as to where the field could progress.

The periodontal pathogen Porphyromonas gingivalis sensitises human blood platelets to epinephrine.

Recent studies indicate connections between periodontitis and atherothrombosis, and the periodontal pathogen Porphyromonas gingivalis has been found within atherosclerotic lesions. P. gingivalis-derived proteases, designated gingipains activate human platelets, probably through a "thrombin-like" activity on protease-activated receptors (PARs). However, the potential interplay between P. gingivalis and other physiological platelet activators has not been investigated. The aim of this study was to elucidate consequences and mechanisms in the interaction between P. gingivalis and the stress hormone epinephrine. By measuring changes in light transmission through platelet suspensions, we found that P. gingivalis provoked aggregation, whereas epinephrine alone never had any effect. Intriguingly, pre-treatment of platelets with a low, sub-threshold number of P. gingivalis (i.e. a density that did not directly provoke platelet aggregation) resulted in a marked aggregation response when epinephrine was added. This synergistic action was not inhibited by the cyclooxygenas inhibitor aspirin. Furthermore, fura-2-measurements revealed that epinephrine caused an intracellular Ca(2+) mobilization in P. gingivalis pre-treated platelets, whereas epinephrine alone had no effect. Inhibition of the arg-specific gingipains, but not the lys-specific gingipains, abolished the aggregation and the Ca(2+) response provoked by epinephrine. Similar results were achieved by separate blockage of platelet alpha(2)-adrenergic receptors and PARs. In conclusion, the present study shows that a sub-threshold number of P. gingivalis sensitizes platelets to epinephrine. We suggest that P. gingivalis-derived arg-specific gingipains activates a small number of PARs on the surface of the platelets. This leads to an unexpected Ca(2+) mobilization and a marked aggregation response when epinephrine subsequently binds to the alpha(2)-adrenergic receptor. The present results are consistent with a direct connection between periodontitis and stress, and describe a novel mechanism that may contribute to pathological platelet activation.

Proinflammatory cytokine production by cultured neonatal rat microglia after exposure to blood products.

Periventricular germinal matrix hemorrhage is a devastating complication of preterm birth. Inflammation appears to play a role in brain damage after premature birth and hypoxia. The effects of rat blood plasma and serum on cytokine expression by cultured rat microglial cells were investigated. We analyzed mRNA expression levels of tumor necrosis factor (TNF)-alpha, interleukin-6 and protease activated receptor-1 and -4 by quantitative RT-PCR. Protein expression for TNFalpha was done using immunocytochemistry and ELISPOT assays. Plasma and serum had dose dependent toxic effects on microglia as measured by lactate dehydrogenase release assay and activated caspase-3 immunocytochemistry. High concentrations of plasma enhanced TNFalpha mRNA expression and protein production, while high concentrations of serum enhanced IL-6 mRNA expression. This study suggests that soluble components of blood might be differentially responsible for up regulating production of the cytokines TNFalpha and IL-6 by microglia from immature rodent brain.

Proteinases and signalling: pathophysiological and therapeutic implications via PARs and more.

Proteinases like thrombin, trypsin and tissue kallikreins are now known to regulate cell signaling by cleaving and activating a novel family of G-protein-coupled proteinase-activated receptors (PARs 1-4) via exposure of a tethered receptor-triggering ligand. On their own, short synthetic PAR-selective PAR-activating peptides (PAR-APs) mimicking the tethered ligand sequences can activate PARs 1, 2 and 4 and cause physiological responses both in vitro and in vivo. Using the PAR-APs as sentinel probes in vivo, it has been found that PAR activation can affect the vascular, renal, respiratory, gastrointestinal, musculoskeletal and nervous systems (both central and peripheral nervous system) and can promote cancer metastasis and invasion. In general, responses triggered by PARs 1, 2 and 4 are in keeping with an innate immune inflammatory response, ranging from vasodilatation to intestinal inflammation, increased cytokine production and increased or decreased nociception. Further, PARs have been implicated in a number of disease states, including cancer and inflammation of the cardiovascular, respiratory, musculoskeletal, gastrointestinal and nervous systems. In addition to activating PARs, proteinases can cause hormone-like effects by other signalling mechanisms, like growth factor receptor activation, that may be as important as the activation of PARs. We, therefore, propose that the PARs themselves, their activating serine proteinases and their associated signalling pathways can be considered as attractive targets for therapeutic drug development. Thus, proteinases in general must now be considered as 'hormone-like' messengers that can signal either via PARs or other mechanisms.

Modulation of visceral nociceptive pathways.

Increased sensitivity of visceral nociceptive pathways contributes to symptoms in an array of clinical gastrointestinal conditions, however, the search for a consistently effective pharmacological agent to treat these conditions remain elusive. Modulation of visceral nociceptive pathways can occur at peripheral, spinal and supra-spinal sites and a dizzying array of potential drug targets exists. Till date, only tricyclic anti-depressants (TCAs) such as amitriptyline and, more recently, selective serotonin reuptake inhibitors (SSRIs) such as citalopram have demonstrated convincing visceral anti-nociceptive properties and clinical benefit in a limited population of patients with visceral hypersensitivity. Unfortunately, there is an incomplete understanding of the receptors and/or primary site of action at which these compounds exert their effects and significant side effects are often encountered. There is a continuing and concerted effort underway to develop target-specific visceral analgesic/anti-hyperalgesic compounds and the aim of this article is to provide a concise update on the most recent advances in this area.

Protease activated receptors: clinical relevance to hemostasis and inflammation.

The protease-activated receptors (PARs) are a unique family of vascular receptors that confer on cells an ability to sense, and respond to, local changes in the proteolytic environment. They are activated by serine proteases of the blood coagulation cascade, notably thrombin, and are linked to thrombotic and inflammatory effector pathways. In surgery with cardiopulmonary bypass (CPB), thrombin is generated in large quantities in the extracorporeal circuit and can exert systemic effects by way of platelet and endothelial PAR1. Aprotinin (Trasylol), a serine protease inhibitor used in cardiac surgery, preserves platelet function, and attenuates the inflammatory response by protecting the PAR 1 receptor on platelets and endothelium.

Do heterotrimeric G proteins redistribute upon G protein-coupled receptor stimulation in platelets?

Previous studies have proposed that stimulation of G protein-coupled receptors can cause a redistribution of G proteins to other receptors. The redistribution would cause a greater functional sensitivity of unsensitized 'secondary' receptors toward their agonists. Using platelets as a model system, we utilized a proximal signaling event, intracellular calcium mobilization, to determine if agonist stimulation of particular Gq-coupled receptors would result in increased sensitivity for stimulation of other Gq-coupled receptors. Platelets express three Gq-coupled receptors for thrombin, thromboxane A2, and ADP with different potencies. Varying concentrations of a primary agonist (PAR-1 agonist SFLLRN, or the TXA2 agonist U46619) was followed by a constant submaximal concentration of a secondary agonist (U46619, or the P2Y1 agonist ADP). We observed that initial stimulation by SFLLRN was followed by a decrease in the extent of secondary U46619 or ADP-mediated calcium mobilization in comparison to control responses (i.e. without primary stimulation). To extend these studies we examined calcium mobilization in platelets from mice that were either wild-type or homozygous null for the PAR-4 or P2Y1 receptors, hypothesizing that the loss of PAR-4 or P2Y1 receptors would cause redistribution of its Galphaq proteins to other receptors, and elicit a greater response when stimulated with other agonists than in platelets from a wild-type mouse. However, our results showed almost identical levels of peak calcium between wild-type or PAR-4 null mice when stimulated with either ADP or U46619. Similar results were obtained for the P2Y1 null mice stimulated with AYPGKF or U46619. We conclude that stimulation of one Gq coupled receptor does not result in redistribution of Gq to other Gq-coupled receptors.

Proteinase-activated receptors, targets for kallikrein signaling.

Serine proteinases like thrombin can signal to cells by the cleavage/activation of proteinase-activated receptors (PARs). Although thrombin is a recognized physiological activator of PAR(1) and PAR(4), the endogenous enzymes responsible for activating PAR(2) in settings other than the gastrointestinal system, where trypsin can activate PAR(2), are unknown. We tested the hypothesis that the human tissue kallikrein (hK) family of proteinases regulates PAR signaling by using the following: 1) a high pressure liquid chromatography (HPLC)-mass spectral analysis of the cleavage products yielded upon incubation of hK5, -6, and -14 with synthetic PAR N-terminal peptide sequences representing the cleavage/activation motifs of PAR(1), PAR(2), and PAR(4); 2) PAR-dependent calcium signaling responses in cells expressing PAR(1), PAR(2), and PAR(4) and in human platelets; 3) a vascular ring vasorelaxation assay; and 4) a PAR(4)-dependent rat and human platelet aggregation assay. We found that hK5, -6, and -14 all yielded PAR peptide cleavage sequences consistent with either receptor activation or inactivation/disarming. Furthermore, hK14 was able to activate PAR(1), PAR(2), and PAR(4) and to disarm/inhibit PAR(1). Although hK5 and -6 were also able to activate PAR(2), they failed to cause PAR(4)-dependent aggregation of rat and human platelets, although hK14 did. Furthermore, the relative potencies and maximum effects of hK14 and -6 to activate PAR(2)-mediated calcium signaling differed. Our data indicate that in physiological settings, hKs may represent important endogenous regulators of the PARs and that different hKs can have differential actions on PAR(1), PAR(2), and PAR(4).

Proteinase-activated receptors (PARs): crossroads between innate immunity and coagulation.

Coagulation cascade and innate immunity are intimately linked in their endeavor to organize the body's response to injury. Protease-activated receptors (PARs) are important mediators of inflammatory response that can be activated by proteases of the coagulation cascade. Their recent discovery has shed new light on the crosstalk between coagulation and innate immunity. Recent studies have investigated the physiological relevance of PARs in the context of immunity and vascular injury, suggesting that these receptors could be used as therapeutic targets for the treatment of pathologies related to innate immunity, endothelial functions and coagulation processes.