Receptor Protease-activated Receptor-1 Research Articles

Mechanisms and Therapeutic Targets of Staphylococcus Aureus - Induced Itch in Atopic Dermatitis

Atopic dermatitis is a chronic skin disorder marked by inflammation, erythema, and pruritus, often beginning in childhood and characterized by exacerbations and remissions. Its etiology involves genetic, immunological, and environmental factors. Staphylococcus aureus, a common pathogen, exacerbates atopic dermatitis by producing toxins and enzymes such as the serine protease V8, which activates the protease-activated receptor 1 (PAR1) on sensory neurons, inducing itch. This mechanism highlights potential therapeutic targets for alleviating pruritus. Research into the physiological pathways of itch, including the role of PAR1 and other protease-activated receptors, reveals promising strategies for treatment. PAR1 antagonists could be repurposed to treat chronic itch, providing new therapeutic avenues. PAR1 and other protease-activated receptors, is crucial in developing new treatment strategies. Anti-pruritic therapies targeting these pathways, such as PAR1 antagonists, show promise in mitigating itch symptoms. Moreover, existing drugs that inhibit PAR1 could be repurposed for treating chronic itch, providing a new avenue for relief in patients with atopic dermatitis. Additionally, the evolutionary role of itch induced by microorganisms suggests pathogens may exploit neural reflexes to enhance their spread. Advancements in understanding the mechanisms behind S. aureus-induced itch and the physiological pathways involved offer promising new directions for therapeutic intervention. Advancements in understanding the mechanisms of Staphylococcal aureus-induced itch and associated physiological pathways offer promising directions for therapeutic intervention, potentially improving management and treatment outcomes for patients with atopic dermatitis and other pruritic conditions.

PAR2-dependent phosphorylation of TRPV4 at the trigeminal nerve terminals contributes to tongue cancer pain

ObjectiveThis study aimed to clarify the interactions between the tongue and primary afferent fibers in tongue cancer pain. MethodsA pharmacological analysis was conducted to evaluate mechanical hypersensitivity of the tongues of rats with squamous cell carcinoma (SCC). Changes in trigeminal ganglion (TG) neurons projecting to the tongue were analyzed using immunohistochemistry and western blotting. ResultsSCC inoculation of the tongue caused persistent mechanical sensitization and tumor formation. Trypsin expression was significantly upregulated in cancer lesions. Continuous trypsin inhibition or protease-activated receptor 2 (PAR2) antagonism in the tongue significantly inhibited SCC-induced mechanical sensitization. No changes were observed in PAR2 and transient receptor potential vanilloid 4 (TRPV4) levels in the TG or the number of PAR2-and TRPV4-expressing TG neurons after SCC inoculation. In contrast, the relative amount of phosphorylated TRPV4 in the TG was significantly increased after SCC inoculation and abrogated by PAR2 antagonism in the tongue. TRPV4 antagonism in the tongue significantly ameliorated the mechanical sensitization caused by SCC inoculation. ConclusionsOur findings indicate that tumor-derived trypsin sensitizes primary afferent fibers by PAR2 stimulation and subsequent TRPV4 phosphorylation, resulting in severe tongue pain.

Abstract 4961: Novel biased PAR2 inhibitors with best-in-class properties reduce resistance to both chemotherapy and immunotherapy in oncology models

Abstract A large-scale meta-analysis has recently identified Protease-activated receptor 2 (PAR2) gene expression to be significantly associated with resistance to immune checkpoint blockade (ICB) in cancer patients and preclinical models. PAR2 and its ligands (proteases) are indeed upregulated in different cancer types and are expressed by various cells in the tumor microenvironment. In cancer cells, the PAR2 receptor controls cell migration, proliferation, survival, and expression of inflammatory cytokines. In immune cells, it influences the infiltration and phenotype of macrophages and T cells. Therefore, PAR2 represents a promising therapeutic target in oncology and immuno-oncology. A novel series of potent and selective PAR2 inhibitors has been developed at Domain Therapeutics. In vitro experiments demonstrated unique properties of our PAR2 small molecule antagonists when compared to those of competitors. The antagonists inhibit pathogenic signaling pathways (i.e. Gz, G13, Gq, G14 and G15 protein activation as well as intracellular calcium) but not βarrestin2 recruitment, potentially reducing the risks of drug resistance. Furthermore, they maintain high potency and insurmountability in conditions that mimic the tumor microenvironment (high concentration of proteases and acidic pH). Finally, their pharmacokinetic properties are compatible with a once-a-day oral administration and demonstrate no signs of in vivo toxicity except at high doses (>500 mg/kg). Proof-of-concept experiments showed that PAR2 antagonists prevented PAR2-mediated resistance to Gefitinib in vitro and increased the potency of anti-PD1 therapy in vivo in pre-clinical syngeneic mouse models. Immunohistochemistry analyses from cancer patient biopsies confirmed that high expression levels of PAR2 in cancer and stromal cells within the tumor microenvironment significantly associates with the patient overall survival. In conclusion, new potent and selective negative allosteric modulators of PAR2 have been developed, they demonstrate strong potency by alleviating the resistance to both chemo- and immunotherapy in cancer models. These findings confirm the high value of PAR2 as a therapeutic target and demonstrates the relevance of small molecule inhibitors targeting this receptor to treat cancer. Citation Format: Thibaut Brugat, Francesco Bergami, Baptiste Rugeri, Aurélie Janvier, Edith Steinberg, Luc Baron, Mandy Recolet, Xavier Wirth, Meriem Semache, Antoine Mousson, Camille Dietsch, Quentin Ruet, Orphée Blanchard, Célia Jacoberger-Foissac, Isabelle Cousineau, Maleck Kadiri, Anne-Laure Blayo, Christel Franchet, Stanislas Mayer, John Stagg, Nathalie Lenne, Stephan Schann. Novel biased PAR2 inhibitors with best-in-class properties reduce resistance to both chemotherapy and immunotherapy in oncology models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4961.

Thrombin receptor protease-activated receptor 1 (PAR1) related cytotoxic CD8+ T cell activity is associated with atrial myopathy and pro-inflammatory immune response in early atrial fibrillation

Abstract Background and aims Cytotoxic CD8+ T cells mediate myocardial damage in patients with virus induced myocarditis, plaque erosion during acute coronary syndrome, and promote adverse post-ischemic cardiac remodeling. There is emerging evidence to support a link between inflammation and atrial fibrillation (AF). Protease-activated receptor 1 (PAR1) is the thrombin receptor expressed on platelets, but it is also expressed in endothelial cells, vascular smooth muscle cells, cardiac fibroblasts, and CD8+ T cells. The role of CD8+ T cells and PAR1 in atrial myopathy and early AF have not been studied. Methods In 80 non-anticoagulated patients with a first documented episode of AF, and 20 control subjects without AF but with a comparable cardiovascular risk profile (e.g. SCORE2, CHA2DS2-VASc, p>0.05), we studied PAR1-related cytotoxic T cell activity in the first hours (up to 24 h) of AF clinical manifestation. The Apolipoprotein E–knock out (ApoEko) mouse is a commonly used model of metabolic syndrome that develops atrial myopathy (structural correlate of AF). ApoEko male mice (Apoetm1Unc with a C57BL/6 genetic background) were fed for six weeks with a standard chew, western-type diet (high fat and high sucrose = WD) ± the specific PAR1 inhibitor vorapaxar. Results High levels of circulating, activated CD8+ T cells (CD8+CD57+ T cells with pro-inflammatory and high cytotoxic properties) were present in patients with early AF (flow cytometry). Stimulation of cytotoxic T cells, that expressed the thrombin receptor PAR1, that were isolated from patients with new onset of AF, released pro-inflammatory cytokines (e.g. IFN-γ, TNF-α). Enhanced cytotoxic activity of CD8+ T cells was suggested by elevated plasma levels of effector molecules (e.g. granulysin and granzyme). In ApoEko mice fed a WD, atrial myopathy as the structural correlate for AF, was paralleled by increased expression of effector molecules that were associated with CD8+ T cell activity (like perforin, granzyme A, granzyme B, IFN-γ). Cytotoxic T cell activity disrupted the release of the potective atrial natriuretic peptide, ANP, and increased signaling pathways associated with atrial fibrosis (e.g. TGF-β, cTGF, TGF-β receptor). The specific PAR1 inhibitor, vorapaxar, reduced inflammatory T cell activity, transcription of fibrotic mediators, and reconstituted ANP secretion in atria of ApoEko mice. Conclusions Targeting the FXa/FIIa-PAR1-CD8+ axis might be a promising approach to reduce atrial fibrosis and inflammation. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): BIH Charité Clinician Scientist Program

Protease-Activated Receptor 1 in Human Carotid Atheroma Is Significantly Related to Iron Metabolism, Plaque Vulnerability, and the Patient's Age.

(1) Background: Protease-activated receptor 1 (PAR1) has regulatory functions in inflammation, atherogenesis, and atherothrombosis. Chronic iron administration accelerates arterial thrombosis. Intraplaque hemorrhage and hemoglobin catabolism by macrophages are associated with dysregulated iron metabolism and atherosclerotic lesion instability. However, it remains unknown whether expressions of PAR1 in human atherosclerotic lesions are related to plaque severity, accumulation of macrophages, and iron-related proteins. We investigated the expression of PAR1 and its relation to the expression of ferritin and transferrin receptors in human carotid atherosclerotic plaques and then explored potential connections between their expressions, plaque development, and classical risk factors. (2) Methods: Carotid samples from 39 patients (25 males and 14 females) were immunostained with PAR1, macrophages, ferritin, and transferrin receptor. Double immunocytochemistry of PAR1 and ferritin was performed on THP-1 macrophages exposed to iron. (3) Results: PAR1 expression significantly increases with the patient’s age and the progression of human atherosclerotic plaques. Expressions of PAR1 are significantly correlated with the accumulation of CD68-positive macrophages, ferritin, and transferrin receptor 1 (TfR1), and inversely correlated with levels of high-density lipoprotein. In vitro, PAR1 is significantly increased in macrophages exposed to iron, and the expression of PAR1 is colocalized with ferritin expression. (4) Conclusions: PAR1 is significantly related to the progression of human atherosclerotic lesions and the patient’s age. PAR1 is also associated with macrophage infiltration and accumulation of iron metabolic proteins in human atherosclerotic lesions. Cellular iron-mediated induction of PAR1 and its colocalization with ferritin in macrophages may further indicate an important role of cellular iron in atherothrombosis.

Role of Chitinase-3-like Protein 1 in Cardioprotection and Angiogenesis by Post-Infarction Exercise Training.

Chitinase-3-like protein 1 (CHI3L1) is a myokine involving tissue remodeling and inflammatory processes. CHI3L1 and its receptor protease-activated receptor 2 (PAR2) are induced by exercise in skeletal muscles. However, it remains unknown if CHI3L1/PAR2 signaling also mediates exercise-induced cardioprotection after myocardial infarction. Twenty-four adult male rats were divided into three groups (n = 8/group), receiving: (1) a sham operation; (2) permanent ligation of left anterior descending coronary artery; and (3) post-MI exercise training with one-week adaptive treadmill exercise for seven days followed by four weeks of aerobic exercise. Left ventricular systolic and end-diastolic pressure indices were measured and cardiac fibrosis, and angiogenesis were examined. Furthermore, HUVEC cells were treated in vitro with AMPK agonist—AICAR (a putative pharmacological memetic of exercise), recombinant human CHI3L1, PAR2 receptor blocker (AZ3451), and PI3K inhibitor (LY294002), respectively. We found that post-MI exercise significantly upregulated CHI3L1, PAR2, pPI3K/PI3K, pAKT/AKT, pERK/ERK, improved cardiac function, and diminished fibrosis. AICAR increased HUVEC tubules formation and upregulated CHI3L1 and PAR2 and these changes were attenuated by PAR2 blocker. In conclusion, post-MI exercise training can effectively activate CHI3L1/PAR2 signaling, which led to the improved myocardial function and enhanced cardiac angiogenesis in the infarcted heart.

Modulation of Neuro-Inflammatory Signals in Microglia by Plasma Prekallikrein and Neuronal Cell Debris.

Microglia, the resident phagocytes of the central nervous system and one of the key modulators of the innate immune system, have been shown to play a major role in brain insults. Upon activation in response to neuroinflammation, microglia promote the release of inflammatory mediators as well as promote phagocytosis. Plasma prekallikrein (PKall) has been recently implicated as a mediator of neuroinflammation; nevertheless, its role in mediating microglial activation has not been investigated yet. In the current study, we evaluate the mechanisms through which PKall contributes to microglial activation and release of inflammatory cytokines assessing PKall-related receptors and their dynamics. Murine N9-microglial cells were exposed to PKall (2.5 ng/ml), lipopolysaccharide (100 ng/ml), bradykinin (BK, 0.1 μM), and neuronal cell debris (16.5 μg protein/ml). Gene expression of bradykinin 2 receptor (B2KR), protease-activated receptor 2 (PAR-2), along with cytokines and fibrotic mediators were studied. Bioinformatic analysis was conducted to correlate altered protein changes with microglial activation. To assess receptor dynamics, HOE-140 (1 μM) and GB-83 (2 μM) were used to antagonize the B2KR and PAR-2 receptors, respectively. Also, the role of autophagy in modulating microglial response was evaluated. Data from our work indicate that PKall, LPS, BK, and neuronal cell debris resulted in the activation of microglia and enhanced expression/secretion of inflammatory mediators. Elevated increase in inflammatory mediators was attenuated in the presence of HOE-140 and GB-83, implicating the engagement of these receptors in the activation process coupled with an increase in the expression of B2KR and PAR-2. Finally, the inhibition of autophagy significantly enhanced the release of the cytokine IL-6 which were validated via bioinformatics analysis demonstrating the role of PKall in systematic and brain inflammatory processes. Taken together, we demonstrated that PKall can modulate microglial activation via the engagement of PAR-2 and B2KR where PKall acts as a neuromodulator of inflammatory processes.

Development of a DHA-Losartan hybrid as a potent inhibitor of multiple pathway-induced platelet aggregation

Despite the scientific progression in the prevention and treatment of cardiovascular diseases (CVDs) they remain the leading cause of mortality and disability worldwide. The classic treatment involves the simultaneous dosing of two antiplatelet drugs, aspirin and clopidogrel/prasugrel. However, besides drug resistance, severe side effects have been also manifested including acute bleeding and toxicity. Thus, new therapeutic agents with enhanced efficacy and diminished side effects are of importance. Towards this end, omega-3 (ω-3) fatty acids have demonstrated potent efficacy against CVDs through inhibiting platelet aggregation that bears a pivotal role in atherothrombosis. Another factor that displays a critical role in the pathogenesis of cardiovascular diseases is the renin-angiotensin system (RAS), and especially the AT1R blocker losartan that has been reported to exert antiplatelet activity mediated by this receptor. Along these lines, we envisaged developing a molecular hybrid consisted of docosahexaenoic acid (ω-3 fatty acid) and losartan, that could exert a notable antiplatelet effect against CVDs. The design and synthesis of the new DHA-losartan hybrid, designated DHA-L, bestowed with the additive properties of the parent compounds, is reported. In silico studies were first exploited to validate the potential of DHA-L to retain losartan’s ability to bind AT1R. The antiplatelet activity of DHA-L was evaluated against in vitro platelet aggregation induced by several platelet agonists. Notably, the hybrid illustrated a pleiotropic antiplatelet profile inhibiting platelet aggregation through multiple platelet activation pathways including P2Y12, PAR-1 (Protease-Activated Receptor-1), PAF (Platelet Activating Factor), COX-1 (cyclooxygenase-1) and collagen receptors. The stability of DHA-L in human plasma and in a wide range of pH values was also evaluated over time using an HPLC protocol. The hybridization approach described herein could pave the way for the development of novel potent multitargeted therapeutics with enhanced antiplatelet profile. Communicated by Ramaswamy H. Sarma

Effects of Heparin and Bivalirudin on Thrombin-Induced Platelet Activation: Differential Modulation of PAR Signaling Drives Divergent Prothrombotic Responses.

Heparin and bivalirudin are widely used as anticoagulants in the setting of acute thrombosis. In this study, we investigated how these drugs affect the ability of thrombin to generate a prothrombotic platelet response via activation of the protease-activated receptors (PARs) 1 and 4. We examined the effects of heparin/antithrombin and bivalirudin on PAR1- and PAR4-mediated intracellular calcium mobilization, aggregation, α-granule release, and procoagulant membrane exposure in platelets exposed to thrombin concentrations likely to be encountered in the thrombus microenvironment during thrombosis. At physiological antithrombin levels, heparin treatment resulted in complete and sustained inhibition of thrombin-induced PAR4-mediated platelet activation, but transient PAR1 signaling was sufficient to elicit significant α-granule release and platelet aggregation. In contrast, bivalirudin treatment resulted in rapid and profound inhibition of signaling from both PAR receptors, followed by a delayed phase of PAR4-mediated platelet activation, resulting in a robust prothrombotic response. Combination treatment with bivalirudin and subtherapeutic concentrations of heparin completely inhibited the residual platelet activation observed with single drug treatment at all time-points. Our results show that heparin and bivalirudin have different and complementary inhibitory effects on the activation of PAR1 and PAR4 by thrombin.

β-arrestin–dependent PI(4,5)P2 synthesis boosts GPCR endocytosis

β-arrestins regulate many cellular functions including intracellular signaling and desensitization of G protein-coupled receptors (GPCRs). Previous studies show that β-arrestin signaling and receptor endocytosis are modulated by the plasma membrane phosphoinositide lipid phosphatidylinositol-(4, 5)-bisphosphate (PI(4,5)P2). We found that β-arrestin also helped promote synthesis of PI(4,5)P2 and up-regulated GPCR endocytosis. We studied these questions with the Gq-coupled protease-activated receptor 2 (PAR2), which activates phospholipase C, desensitizes quickly, and undergoes extensive endocytosis. Phosphoinositides were monitored and controlled in live cells using lipid-specific fluorescent probes and genetic tools. Applying PAR2 agonist initiated depletion of PI(4,5)P2, which then recovered during rapid receptor desensitization, giving way to endocytosis. This endocytosis could be reduced by various manipulations that depleted phosphoinositides again right after phosphoinositide recovery: PI(4)P, a precusor of PI(4,5)P2, could be depleted at either the Golgi or the plasma membrane (PM) using a recruitable lipid 4-phosphatase enzyme and PI(4,5)P2 could be depleted at the PM using a recruitable 5-phosphatase. Endocytosis required the phosphoinositides. Knock-down of β-arrestin revealed that endogenous β-arrestin normally doubles the rate of PIP5-kinase (PIP5K) after PAR2 desensitization, boosting PI(4,5)P2-dependent formation of clathrin-coated pits (CCPs) at the PM. Desensitized PAR2 receptors were swiftly immobilized when they encountered CCPs, showing a dwell time of ∼90 s, 100 times longer than for unactivated receptors. PAR2/β-arrestin complexes eventually accumulated around the edges or across the surface of CCPs promoting transient binding of PIP5K-Iγ. Taken together, β-arrestins can coordinate potentiation of PIP5K activity at CCPs to induce local PI(4,5)P2 generation that promotes recruitment of PI(4,5)P2-dependent endocytic machinery.

Calcium Increase and Substance P Release Induced by the Neurotoxin Brevetoxin-1 in Sensory Neurons: Involvement of PAR2 Activation through Both Cathepsin S and Canonical Signaling.

Red tides involving Karenia brevis expose humans to brevetoxins (PbTxs). Oral exposition triggers neurotoxic shellfish poisoning, whereas inhalation induces a respiratory syndrome and sensory disturbances. No curative treatment is available and the pathophysiology is not fully elucidated. Protease-activated receptor 2 (PAR2), cathepsin S (Cat-S) and substance P (SP) release are crucial mediators of the sensory effects of ciguatoxins (CTXs) which are PbTx analogs. This work explored the role of PAR2 and Cat-S in PbTx-1-induced sensory effects and deciphered the signaling pathway involved. We performed calcium imaging, PAR2 immunolocalization and SP release experiments in monocultured sensory neurons or co-cultured with keratinocytes treated with PbTx-1 or P-CTX-2. We demonstrated that PbTx-1-induced calcium increase and SP release involved Cat-S, PAR2 and transient receptor potential vanilloid 4 (TRPV4). The PbTx-1-induced signaling pathway included protein kinase A (PKA) and TRPV4, which are compatible with the PAR2 biased signaling induced by Cat-S. Internalization of PAR2 and protein kinase C (PKC), inositol triphosphate receptor and TRPV4 activation evoked by PbTx-1 are compatible with the PAR2 canonical signaling. Our results suggest that PbTx-1-induced sensory disturbances involve the PAR2-TRPV4 pathway. We identified PAR2, Cat-S, PKA, and PKC that are involved in TRPV4 sensitization induced by PbTx-1 in sensory neurons.

The transient receptor potential vanilloid 4 (TRPV4) ion channel mediates protease activated receptor 1 (PAR1)-induced vascular hyperpermeability

AbstractEndothelial barrier disruption is a hallmark of tissue injury, edema, and inflammation. Vascular endothelial cells express the G protein-coupled receptor (GPCR) protease acctivated receptor 1 (PAR1) and the ion channel transient receptor potential vanilloid 4 (TRPV4), and these signaling proteins are known to respond to inflammatory conditions and promote edema through remodeling of cell–cell junctions and modulation of endothelial barriers. It has previously been established that signaling initiated by the related protease activated receptor 2 (PAR2) is enhanced by TRPV4 in sensory neurons and that this functional interaction plays a critical role in the development of neurogenic inflammation and nociception. Here, we investigated the PAR1–TRPV4 axis, to determine if TRPV4 plays a similar role in the control of edema mediated by thrombin-induced signaling. Using Evans Blue permeation and retention as an indication of increased vascular permeability in vivo, we showed that TRPV4 contributes to PAR1-induced vascular hyperpermeability in the airways and upper gastrointestinal tract of mice. TRPV4 contributes to sustained PAR1-induced Ca2+ signaling in recombinant cell systems and to PAR1-dependent endothelial junction remodeling in vitro. This study supports the role of GPCR–TRP channel functional interactions in inflammatory-associated changes to vascular function and indicates that TRPV4 is a signaling effector for multiple PAR family members.The thrombin receptor PAR1 and mechanosensitve ion channel TRPV4 are known to modulate endothelial barriers and contribute to edema, but functional interactions between these targets are not well understood. Utilizing cell signalling studies and in vivo models, the authors have demonstrated that PAR1 activation leads to functional upregulation of endothelial TRPV4 activity, to promote vascular leakage in airways and the gastrointestinal system.

Abstract TP468: Differential Effect of Ischemia and Thrombin Mediated Toxicity on Neurovascular Unit

Objectives: Brain is a complex structure during ischemia along with neurons; components of vascular unit astrocytes, pericytes and endothelial cells play a major role in hypoxic and blood brain barrier leakage mediated injury. Thrombin acts via protease activated receptor-1 (PAR-1) and leads to neuronal survival or death, PAR-1 knockout cells survive ischemia. We hypothesize that different elements of the Brain vascular unit play different roles in responding to injury. The present study aimed to establish a correlation between astrocytes, pericytes and endothelial cells vulnerability via OGD model of ischemia and Thrombin mediated toxicity. Methods: Using highly reproducible oxygen glucose deprivation (OGD) model Primary cultured cells were exposed to OGD for various durations and Thombin doses to determine the optimum time point for 80 % cell death or cell viability measured using LDH release assay MTT assay. LD50 dose of OGD and Thrombin was used to determine the effect of various drugs mediating PAR-1 receptor pathway Results: The viability assay using MTT showed that the three elements of vascular unit respond differentially to longer durations of ischemia, with pericytes being the most vulnerable, followed by endothelial cells, followed by astrocytes. Our thrombin dose effect shows that Pericytes and endothelial respond to thrombin similarly whereas, astrocytes on lower concentration proliferate, but don’t survive the higher concentrations. Conclusion: An exciting, novel finding in our data is that different elements of the vascular unit—astrocytes, pericytes and endothelial cells—all exhibit effect of ischemia and reperfusion at different time scales. Up until recently, studies of neuroprotection addressed the brain as a homogenous unit, with the tacit assumption that all elements responded similarly. Our data on astrocytes, endothelial cells and pericytes—allows more focused investigation.

Mechanisms of β-Arrestin-Dependent PI(4,5)P2 Synthesis for GPCR Endocytosis

β-arrestins play pivotal roles in cellular functions such as insulin secretion, regulation of protein kinases and phosphatases, and desensitization of G protein-coupled receptors (GPCRs). However, how β-arrestin controls PI(4,5)P2 synthesis for internalization of GPCRs remains unclear. Activation of Gq-coupled protease-activated receptor2 (PAR2) depletes PI(4)P and PI(4,5)P2 at the plasma membrane (PM), but only transiently due to rapid desensitization of the receptor by phosphorylation and β-arrestin binding. Pharmacological assays revealed that the PI(4)P and PI(4,5)P2 pools are dynamically coupled during PAR2 desensitization. Acute depletion of PI(4)P at either Golgi or PM using the recruitable lipid 4-phosphatase enzyme Sac1, also resulted in significant depletion of PM PI(4,5)P2, suggesting that Golgi and PM PI(4)P pools interplay to refill PI(4,5)P2 at the PM. Internalization of PAR2 was also significantly blocked by this kind of depletion of PI(4)P and PI(4,5)P2. To estimate recovery rate of PI(4,5)P2 at the PM during PAR2 desensitization, cells were depolarized to activate voltage-sensitive lipid 5-phosphatase and monitored with the PI(4,5)P2 probe PH-PLCδ1. Endogenous β-arrestin augments the rate of PIP5-kinase up to 2-fold, inducing hyperactivation of PI(4,5)P2-dependent clathrin-coated pit (CCP) formation at the PM. We also confirmed that β-arrestin has physical interaction with PIP5-kinase, causes recruitment of PIP5-kinase into β-arrestin-receptor clusters. In our single-molecule tracking data, when desensitized PAR2 receptors encountered CCPs, the receptors were rapidly immobilized. Binding lifetime with pits was increased by more than 100-fold compared to that for unactivated receptors (< 0.6 s). Super-resolution imaging showed that after receptor activation, β-arrestin formed donut shape or engulfed vesicular structures at the PM, supporting accumulation of β-arrestin at the edge or inside of CCPs. Taken together, β-arrestin-receptor complex substantially increased formation of CCPs via PI(4,5)P2 synthesis from PI(4)P pools enhanced by PIP5-kinase and stable interaction with CCPs leading to more efficient internalization.

Systemic thrombin inhibition ameliorates seizures in a mouse model of pilocarpine-induced status epilepticus.

Status epilepticus (SE) is a life-threatening condition characterized by ongoing seizure activity which can lead to severe brain damage and death if not treated properly. Recent work suggests that alterations in blood-brain barrier (BBB) function and subsequent cortical exposure to coagulation factors may initiate, promote, and/or sustain SE. This suggestion is based on the observation that the serine protease thrombin, which plays a fundamental role in the blood coagulation cascade, increases neural excitability through the activation of protease-activated receptor 1 (PAR1). However, it remains unclear whether systemic inhibition of thrombin asserts "anti-epileptic" effects in vivo. We here used the pilocarpine model of SE in adult 3-month-old male mice to address the question whether intraperitoneal injection of the thrombin inhibitor α-NAPAP (0.75 mg/kg) counters SE. Indeed, pharmacological inhibition of thrombin ameliorates the behavioral outcome of pilocarpine-induced SE. Similar results are obtained when the thrombin receptor PAR1 is pharmacologically blocked using intraperitoneal injection of SCH79797 (25 μg/kg) prior to SE induction. Consistent with these results, an increase in thrombin immunofluorescence is detected in the hippocampus of pilocarpine-treated animals. Moreover, increased hippocampal serine protease activity is detected 90 min after SE induction, which is not observed in animals treated with α-NAPAP prior to SE induction. Together, these results corroborate and extend recent studies suggesting that novel oral anticoagulants which target thrombin (and PAR1) may assert anti-epileptic effects in vivo. KEY MESSAGES: Systemic thrombin/PAR1-inhibition ameliorates anticoagulants behavioral seizures. Status epilepticus increases thrombin levels in the hippocampus. Increased serine protease activity in the hippocampus after status epileptic. Anti-epileptic potential of clinically used anticoagulants must be evaluated.

Integrin Alphav-beta3 on Podocytes Orchestrates Coagulation Protease Signaling through Protease-Activated Receptors

IntroductionCoagulation protease signaling via protease-activated receptors (PARs) is essential for maintenance of cellular homeostasis. Perturbed or aberrant activation of protease-dependent signaling via PARs propagates inflammation and pathological responses in disease models such as sepsis, neurological diseases and metabolic diseases including atherosclerosis, obesity and diabetic nephropathy (dNP). Disruption of protease-activated protein C (aPC) signaling in renal epithelial cells, i.e. podocytes, compromises adaptive endoplasmic reticulum (ER) signaling, promoting maladaptive ER-stress and ultimately dysfunction of the glomerular filtration barrier and dNP. While these results demonstrate that aPC-dependent ER-reprogramming is an essential component of its cytoprotective effect, currently the co-receptors that are involved in mediating aPC's cytoprotective effects in podocytes remain unclear. In endothelial cells, endothelial protein C receptor (EPCR) facilitates the binding and alternative proteolytic cleavage of PAR1 propagating biased anti-inflammatory and cytoprotective effects. Intriguingly, unlike in endothelial cells, podocytes do not express EPCR and the cytoprotective effects of aPC requires PAR3/PAR2 heterodimer (human podocytes) and PAR3/PAR1 heterodimer (mouse podocytes). Podocytes express integrins β1 and β3, which could potentially mediate biased protease signaling.MethodsTo characterize the receptor-dependent mechanism through which aPC-mediates its cytoprotective effects in podocytes, we generated transgenic mice that expresses constitutively higher levels of wild type-human aPC (APChigh mice) and integrin binding deficient mutant (RGE-APChigh mice). Additionally, the receptor-dependent mechanisms in dNP were evaluated in mouse models with podocyte specific genetic deletion of the integrin β3. After 26 weeks of persistent hyperglycaemia (streptozotocin-induced diabetes), markers of dNP were determined. In wild type diabetic mice, integrin β3 antagonist Cyclo-RGDfv and PAR1 antagonist (SCH79797) was administered to pharmacologically inhibit integrin and PAR1 signaling, respectively.ResultsIn vitro data show that integrin αvβ3 in podocytes controls protease-dependent spatial and temporal regulation of PAR signaling by aPC. This protease binds to integrin αvβ3 and temporally regulates PAR signaling. Abolishing aPC-αvβ3 interaction or knockdown of αvβ3 integrin perturbs endosomal trafficking of PARs resulting in aberrant cell-disruptive signaling. Likewise, in vivo overexpression of integrin binding deficient aPC-mutant or podocyte-specific genetic deletion of integrin β3 abrogates aPC-dependent cytoprotective effects in a mouse model of dNP. Akin to wild type aPC, pharmacological inhibition of aberrant PAR1 signaling protects against dNP. Corroborating our results in murine dNP, analysis of human dNP samples revealed increased expression and activation of αvβ3 associated with altered PAR receptor localization specifically within the podocytes.ConclusionOur results show that aPC-αvβ3 interaction on podocytes modulates endosomal trafficking of PAR receptors and that loss of this interaction drives aberrant protease-dependent signaling and pathological podocyte migration. Complementary in vivo studies in newly developed integrin binding deficient aPC mutant mice demonstrate that integrin αvβ3 ligation is essential for nephroprotective aPC signaling. These results identify integrin αvβ3 as a rheostat that provides temporal and spatial regulation of PAR signaling in diabetic disease. DisclosuresNo relevant conflicts of interest to declare.

Dabigatran Reduces Liver Fibrosis in Thioacetamide-Injured Rats.

Liver fibrosis can progress to cirrhosis, hepatocellular carcinoma, or liver failure. Unfortunately, the antifibrotic agents are limited. Thrombin activates hepatic stellate cells (HSCs). Therefore, we investigated the effects of a direct thrombin inhibitor, dabigatran, on liver fibrosis. Adult male Sprague-Dawley rats were injected intraperitoneally with thioacetamide (TAA, 200mg/kg twice per week) for 8 or 12weeks to induce liver fibrosis. The injured rats were assigned an oral gavage of dabigatran etexilate (30mg/kg/day) or vehicle in the last 4weeks of TAA administration. Rats receiving an injection of normal saline and subsequent oral gavage of dabigatran etexilate or vehicle served as controls. In the 8-week TAA-injured rats, dabigatran ameliorated fibrosis, fibrin deposition, and phosphorylated ERK1/2 in liver, without altering the transcript expression of thrombin receptor protease-activated receptor-1. In vitro, dabigatran inhibited thrombin-induced HSC activation. Furthermore, dabigatran reduced intrahepatic angiogenesis and portal hypertension in TAA-injured rats. Similarly, in the 12-week TAA-injured rats, a 4-week treatment with dabigatran reduced liver fibrosis and portal hypertension. By inhibiting thrombin action, dabigatran reduced liver fibrosis and intrahepatic angiogenesis. Dabigatran may be a promising therapeutic agent for treatment of liver fibrosis.

Selective inhibition of the thrombin receptor protease-activated receptor-1 attenuates hepatic ischemia-reperfusion injury in mice

Selective inhibition of the thrombin receptor protease-activated receptor-1 attenuates hepatic ischemia-reperfusion injury in mice

The effect of vorapaxar on the interaction of CD34+ progenitor cells with platelets

Aim: Platelets interact with circulating CD34+ progenitor cells inducing their differentiation into endothelial progenitor cells (EPCs) and further promoting EPC maturation into endothelial cells, a beneficial interaction for endothelial regeneration. Protease-activated receptor-1 (PAR-1) mainly mediates thrombin-induced platelet activation. The aim of this study was to investigate the effect of vorapaxar, a potent and specific PAR-1 antagonist, on the differentiation of CD34+ cells into EPCs induced by activated, through PAR-1 receptor, platelets, as well as on the interaction of activated platelets with these cells, in vitro.

Protease Activated Receptor‐2 Mediates Giardia‐Induced Disruptions of the Intestinal Mucus Barrier

BackgroundGiardia duodenalis has been associated with disruptions of the mucus layers of the small and large intestines, in part via Giardia's cysteine proteases. Disruption of the intestinal mucus barrier has been implicated in a variety of intestinal disorders, but mechanisms currently remain unclear.HypothesisGoblet cell function and mucus disruptions are modulated by activation of protease activated receptor 2 (PAR2), which is highly expressed through the gastrointestinal tract and is known to regulate mucus production in the airway and stomach. Giardia's cysteine proteases cleave and activate PAR2 on intestinal goblet cells.MethodsMUC2 mucin and PAR gene expression were assessed in the human colonic epithelial cell line LS174T using quantitative PCR (qPCR). LS174T were pre‐treated with the PAR2 antagonist p2Pal‐218S pepducin, and incubated with Giardia trophozoites or the PAR1 and PAR2 agonist peptides TFLLRN and 2fLIGRLO, respectively. Ability of Giardia to cleave PAR1 and PAR2 at the N‐terminal domain was determined using CHO cells transformed with N‐luciferase tagged PAR1 or PAR2. Following incubation with Giardia, culture supernatant was collected, reacted with luciferin, and luminescence was measured. Wild‐type and PAR2 deficient mice were infected with Giardia trophozoites. Thickness of the mucus layer was measured by fluorescently staining mucus with fluorescein‐coupled WGA, and expression of Muc2 and Muc5ac genes were determined in the colon and jejunum via qPCR.ResultsLS174T cells express functional and responsive PAR2, but little or no PAR1. Treatment of cells with 2fLIGRLO PAR2 agonist, but not TFLLRN PAR1 agonist, increased MUC2 gene expression. Incubation of cells with Giardia trophozoites increased MUC2 mRNA production, and this increase was abolished by pre‐treatment of cells with a PAR2 antagonist. Giardia also caused decreased PAR2 expression in LS174T. Giardia NF and Giardia GSM are both able to cleave PAR1 and PAR2 at the N terminal domain. Cleavage by NF was more efficient than GSM, and PAR1 was cleaved more extensively than PAR2 by both strains. Giardia infection increased Muc2 and Muc5ac expression in the colon and increased Muc5ac expression in the jejunum of wild‐type mice compared to uninfected controls. In PAR2−/− mice, Giardia infection reduced expression of Muc5ac in the jejunum, and had no impact on either gene in the colon. Giardia infection caused thinning of the mucus layer in wild‐type mice. In contrast, infection induced thickening of the mucus layer in PAR2−/− mice.ConclusionsUsing a model of Giardia infection, the findings demonstrate that PAR2 plays a significant role in mucin gene regulation and function in mice and in a human colonic goblet cell line, and that Giardia is capable of cleaving both PAR1 and PAR2 receptors with strain‐specific efficiencies.Support or Funding InformationCrohn's Colitis Canada (CCC), NSERCThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.