Protease-activated Receptor Research Articles

Human Pulmonary Neuroendocrine Cells Respond to House Dust Mite Extract With PAR-1 Dependent Release of CGRP.

Pulmonary neuroendocrine cells (PNEC) are rare airway epithelial cells that have recently gained attention as potential amplifiers of allergic asthma. However, studying PNEC function in humans has been challenging due to a lack of cell isolation methods, and little is known about human PNEC function in response to asthma-relevant stimuli. Here we developed and characterized an invitro human PNEC model and investigated the neuroendocrine response to extracts of the common aeroallergen house dust mite (HDM). PNEC-enriched cultures were generated from human induced pluripotent stem cells (iPNEC) and primary bronchial epithelial cells (ePNEC). Characterized PNEC cultures were exposed to HDM extract, a volatile chemical odorant (Bergamot oil), or the bacterial membrane component, lipopolysaccharide (LPS), and neuroendocrine gene expression and neuropeptide release determined. Both iPNEC and ePNEC models demonstrated similar baseline neuroendocrine characteristics and a stimuli-specific modulation of gene expression. Most notably, exposure to HDM but not Bergamot oil or LPS, leads to dose-dependent induction of the CGRP encoding gene, CALCB, and corresponding release of the neuropeptide. HDM-induced CALCB expression and CGRP release could be inhibited by a protease-activated receptor 1 (PAR1) antagonist or protease inhibitors and was mimicked by a PAR1 agonist. We have characterized a novel model of PNEC-enriched human airway epithelium and utilized this model to demonstrate a previously unrecognized role for human PNEC in mediating a direct neuroendocrine response to aeroallergen exposure.

Gastrodin attenuates diabetic cardiomyopathy characterized by myocardial fibrosis by inhibiting the KLK8-PAR1 signaling axis

BackgroundDiabetic cardiomyopathy (DCM), characterized by myocardial fibrosis, is a major cause of mortality and morbidity in diabetic patients; the inhibition of cardiac fibrosis is a fundamental strategy for treating DCM. Gastrodin (GAS), a compound extracted from Gastrodia elata protects against DCM, but the molecular mechanism underlying its antifibrotic effect has not been elucidated.MethodsIn vivo, the effects of GAS were investigated using C57BL/6 mice with DCM, which was induced by administering a high-sugar, high-fat (HSF) diet and streptozotocin (STZ). We assessed the cardiac function in these mice and detected histopathological changes in their hearts and the degree of cardiac fibrosis. In vitro, neonatal rat cardiac fibroblasts (CFs) were transformed into myofibroblasts by exposing them to high glucose combined with high palmitic acid (HG-PA), and CFs were induced by pEX-1 (pGCMV/MCS/EGFP/Neo) plasmid-mediated overexpression of KLK8, which contains the rat KLK8 gene. The KLK8 siRNA was knocked down to study the effects of GAS on CF differentiation, collagen synthesis, and cell migration by specific mechanisms of action of GAS.ResultsGAS attenuated pathological changes in the hearts of DCM mice, rescued impaired cardiac function, and attenuated cardiac fibrosis. Additionally, the results of molecular docking analysis showed that GAS binds to kinin-releasing enzyme-related peptidase 8 (KLK8) to inhibit the increase in protease-activated receptor-1 (PAR-1), thus attenuating myocardial fibrosis. Specifically, GAS attenuated the transformation of neonatal rat CFs to myofibroblasts exposed to HG-PA. Overexpressing KLK8 promoted CF differentiation, collagen synthesis, and cell migration, and KLK8 siRNA attenuated HG-PA-induced CF differentiation, collagen synthesis, and cell migration. Further studies revealed that a PAR-1 antagonist, but not a PAR-2 antagonist, could attenuate CF differentiation, collagen synthesis, and cell migration. Additionally, GAS inhibited KLK8 upregulation and PAR1 activation, thus blocking the differentiation, collagen synthesis, and cell migration of HG-PA-exposed CFs and triggering TGF-β1/Smad3 signaling.ConclusionGAS alleviated pathological changes in the hearts of DCM model mice induced by an HSF diet combined with STZ. KLK8 mediated HG-PA-induced differentiation, collagen synthesis, and the migration of CFs. GAS attenuated the differentiation, collagen synthesis, and migration of CFs by inhibiting the KLK8-PAR1 signaling axis, a process in which TGF-β1 and Smad3 are involved.Graphical

House dust mites stimulate thymic stromal lymphopoietin production in human bronchial epithelial cells and promote airway remodeling through activation of PAR2 and ERK signaling pathway.

House dust mites (HDM) are common aeroallergens linked to airway inflammation and remodeling in asthma. Protease-activated receptor 2 (PAR2) and thymic stromal lymphopoietin (TSLP) may mediate these immune responses. However, how the epithelium influences fibroblasts toward airway remodeling remains unclear. We hypothesize that HDM stimulates human bronchial epithelial cells (HBECs) to produce TSLP via PAR2 activation, driving fibroblasts toward remodeling processes. HBECs were treated with HDM, with or without the PAR2 antagonist FSLLRY-NH2 (FSL), and TSLP expression was measured by qPCR and ELISA. Phosphorylation of MAPKs was assessed by western blotting. Human lung fibroblasts (HLFs) were exposed to recombinant TSLP or conditioned medium (CM) from HDM-stimulated HBECs, with or without anti-TSLP antibodies. Fibroblast proliferation and collagen production were assessed as remodeling markers. HDM increased ERK phosphorylation (not p38 or JNK) and TSLP expression at mRNA and protein levels. FSL preincubation significantly reduced ERK phosphorylation and TSLP production: HDM-stimulated CM induced fibroblast proliferation and collagen production, effects suppressed by anti-TSLP or FSL. Direct treatment with recombinant TSLP also promoted fibroblast proliferation and collagen synthesis. These findings suggest that HDM promotes HBEC-to-HLF paracrine interactions via PAR2-ERK-TSLP axis, participating in airway remodeling. PAR2 antagonists may represent potential therapeutic targets for HDM-induced remodeling processes.

The Role of Platelets in Atherosclerosis: A Historical Review.

Atherosclerosis is a chronic, multifactorial inflammatory disorder of large and medium-size arteries, which is the leading cause of cardiovascular mortality and morbidity worldwide. Although platelets in cardiovascular disease have mainly been studied for their crucial role in the thrombotic event triggered by atherosclerotic plaque rupture, over the last two decades it has become clear that platelets participate also in the development of atherosclerosis, owing to their ability to interact with the damaged arterial wall and with leukocytes. Platelets participate in all phases of atherogenesis, from the initial functional damage to endothelial cells to plaque unstabilization. Platelets deposit at atherosclerosis predilection sites before the appearance of manifest lesions to the endothelium and contribute to induce endothelial dysfunction, thus supporting leukocyte adhesion to the vessel wall. In particular, platelets release matrix metalloproteinases, which interact with protease-activated receptor 1 on endothelial cells triggering adhesion molecule expression. Moreover, P-selectin and glycoprotein Ibα expressed on the surface of vessel wall-adhering platelets bind PSGL-1 and β2 integrins on leukocytes, favoring their arrest and transendothelial migration. Platelet-leukocyte interactions promote the formation of radical oxygen species which are strongly involved in the lipid peroxidation associated with atherosclerosis. Platelets themselves actively migrate through the endothelium toward the plaque core where they release chemokines that modify the microenvironment by modulating the function of other inflammatory cells, such as macrophages. While current antiplatelet agents seem unable to prevent the contribution of platelets to atherogenesis, the inhibition of platelet secretion, of the release of MMPs, and of some specific pathways of platelet adhesion to the vessel wall may represent promising future strategies for the prevention of atheroprogression.

IMMU-41. GBM PLATELET HYPERACTIVITY DRIVES THE IMMUNOSUPPRESSIVE TME IN A SEX-DEPENDENT MANNER

Abstract Cancer-related thrombosis is the second leading cause of death for cancer patients, including those with glioblastoma (GBM), the most common primary malignant brain tumor. Patients with GBM have a 30% risk of venous thromboembolism. Moreover, sex biases are well established in GBM, with men 1.6-fold more likely to develop GBM and exhibiting a poorer prognosis than females. GBM is extremely difficult to treat due to its highly immunosuppressive tumor microenvironment (TME), often infiltrated with suppressive myeloid populations and a reduction in T-cells intratumorally and systemically. Our group has recently demonstrated these sex-biases in survival are due to differences in the immune cell populations making up the TME. Despite these findings, there is limited information as to how a hyper-thrombotic state contribute to the immunosuppressive TME. Additionally, the role that sex biases play in these mechanisms has not been explored. Our previous work demonstrated that thrombin, a major platelet activator through PAR1 and PAR4 receptor signaling, is secreted from cancer stem cells into the tumor microenvironment. We now show that GBM patient have elevated levels of the thrombin-antithrombin complex and have increased PAR4 receptor mediated platelet activation. Similarly, we show that tumor bearing mice have elevated PAR4 receptor mediated platelet activation relative to non-tumor bearing mice. We further demonstrate pharmacologically targeting the thrombin-PAR4 signaling axis in murine GBM models prolongs survival in females but has not affect in males by preventing female platelet hyperactivity and subsequently increasing the number and function of tumor infiltrating CD8+ T-cells. These findings demonstrate how activated platelets interact and regulate immune cell populations in GBM. These results suggest that the hyper-thrombotic state seen in many GBM patients simultaneously contributes to the immunosuppressive TME. In addition, these results identify therapeutic strategies to leverage the platelet hyperactivity seen in GBM by hyperactive thrombin-PAR4 for sex-dependent therapeutic purposes.

L-DOPA Promotes Functional Proliferation Through GPR143, Specific L-DOPA Receptor of Astrocytes.

l-3,4-Dihydroxyphenylalanine (levodopa and L-DOPA in this text), alongside dopamine, boasts high biocompatibility, prompting industrial demand for its use as a coating material. Indeed, the effectiveness of L-DOPA is steadily rising as it serves as an oral therapeutic agent for neurodegenerative brain diseases, particularly Parkinson's disease (PD). However, the effects of L-DOPA on the growth and function of astrocytes, the main glial cells, and the most numerous glial cells in the brain, are unknown. Here, we investigated whether L-DOPA is possible as a coating material on cover glass and polystyrene for rat primary astrocytes. The coating state of L-DOPA on the cover glass and polystyrene was characterized by X-ray photoelectron spectroscopy (XPS) and static water contact angle (WCA). Interestingly, L-DOPA coated on the cover glass promoted the proliferation of astrocytes but not neurons. Furthermore, L-DOPA coated on the cover glass, as opposed to polystyrene, facilitated the proliferation of the astrocytes. The astrocytes grown on L-DOPA-coated cover glasses exhibited functional receptor-activated Ca2+ transients through the activation of protease-activated receptor subtype 1 (PAR-1), recognized as an astrocytic functional marker. However, cover glass coated with 0, 500, 1000, 2000, and 4000 μg/mL L-DOPA maintained astrocyte viability, while supplementation with 500 and 1000 μM L-DOPA significantly decreased astrocyte viability. This suggests that treatments with free 500 and 1000 μM L-DOPA significantly reduced the number of astrocytes. Both Pimozide, an inhibitor of G protein-coupled receptor 143 (GPR143), also known as Ocular albinism type 1 (OA1), and CCG2046, an inhibitor of regulator of G protein signaling 4 (RGS4), reduced the viability of astrocytes on cover glass coated with L-DOPA compared to astrocytes on cover glass coated with poly-d-lysine (PDL). This suggests that L-DOPA promotes astrocyte proliferation through activation of the GPR143 signaling pathway. These findings imply that L-DOPA proliferates functional astrocytes through the activation of GPR143. These results are the first report that L-DOPA coating cover glass proliferates rat primary astrocytes with the activation of GPR143. The discovery that levodopa enhances cell adhesion can significantly influence research in multiple ways. It provides insights into cell behavior, disease mechanisms, and potential therapeutic applications in tissue engineering and regenerative medicine. Additionally, it offers opportunities to explore novel approaches for improving cell-based therapies and tissue regeneration. Overall, this finding opens up new avenues for research, with broad implications across various scientific fields.

Rivaroxaban as a protector of Oxidative Stress-induced Vascular Endothelial Glycocalyx Damage via The IQGAP1/PAR1-2/PI3K/Akt Pathway.

The vascular endothelial glycocalyx, crucial for blood vessel integrity and homeostasis, is vulnerable to oxidative stress, leading to endothelial dysfunction, which strongly correlates with cardiovascular disease (CVD). This study investigates the protective effects of rivaroxaban, a FXa inhibitor, on the glycocalyx under oxidative stress condition. We examined the impact of rivaroxaban on human umbilical vein endothelial cells (HUVECs) exposed to acute and chronic H₂O₂-induced oxidative stress. Rivaroxaban dose-dependently suppressed syndecan-1, a key component of the glycocalyx, shedding from cell surface, and enhanced protease-activated receptor (PAR)1-PAR2/ phosphatidylinositol-3-kinase (PI3K)-dependent cell viability after acute induction of H2O2. This protective effect was linked to the translocation of IQGAP1, a scaffold protein that modulates the actin cytoskeleton, to the perinucleus from the cell membrane. Under chronic H2O2 treatments, rivaroxaban improves cell viability accompanied by an increase in hyaluronidase activities, aiding the turnover and remodeling of hyaluronic acid within the glycocalyx. We identify that rivaroxaban protects against oxidative stress-induced endothelial glycocalyx damage and cell viability through IQGAP1/PAR1-2/PI3K/Akt pathway, offering a potential to be a therapeutic target for CVD prevention.

Endothelial NMMHC IIA dissociation from PAR1 activates the CREB3/ARF4 signaling in thrombin-mediated intracerebral hemorrhage

IntroductionThere is an urgent need for cerebroprotective interventions to improve the suboptimal outcomes with intracerebral hemorrhage (ICH). Despite the important role of nonmuscle myosin heavy chain IIA (NMMHC IIA) in the blood–brain barrier (BBB), its function in ICH remains unclear. ObjectivesThe objective of this study is to explore how NMMHC IIA functions in ICH and to evaluate the effectiveness of targeting NMMHC IIA as a treatment for ICH. MethodsWe firstly examined the protein expression of NMMHC IIA in clinical patients and animal models with ICH. The function of NNMMHC IIA was then corroborated by using overexpress or knockdown NMMHC IIA specifically in ECs mice and pBMECs. In addition, we explored protein interacts with NMMHC IIA and signaling pathways after ICH by LC-MS/MS and transcriptomics analysis with an emphasis on the function of PAR1 and the CREB3/ARF4 signaling pathway, and validated them in three kind of animal models. To support the clinical translation of our results, we targeted NMMHC IIA to bicalutamide selected from a library of marketed drugs and examined to validate its ameliorative effect on ICH. ResultsWe observed an upregulation of endothelial NMMHC IIA in the brain following the onset of ICH in both patients and mice, while inhibited NMMHC ⅡA improved ICH induced by thrombin, warfarin or tissue plasminogen activator (tPA) after ischemic stroke. Mechanistically, the head domain of NMMHC IIA interacted with protease-activated receptor 1 (PAR1) at the 380–430 aa region and subsequently dissociated and activated the CREB3/ARF4 signaling pathway. We found that bicalutamide and blebbistatin could bind to NMMHC IIA and effectively protect mice from thrombin-mediated ICH. ConclusionThe findings indicated that NMMHC IIA dissociated from PAR1 and activated CREB3/ARF4 pathway, which aggravated BBB damage induced by thrombin. This suggested that NMMHC IIA was a novel potential therapeutic target for BBB-related diseases.

Fibrosis signaling in endometrial cells and endometriosis development

In endometriosis, the tissues similar to the endometrial tissue attaches outside the uterine cavity, causing inflammation and fibrosis. The retrograde menstruation theory is the most plausible mechanism, though the detailed pathogenesis remains unclear. Our observations suggest that endometriosis-like lesions occur more often at sites of ovarian excision causing bleeding in mouse models. Additionally, prostaglandin E2 (PGE2) and thrombin, a protease-activated receptor (PAR) agonist in menstrual blood exacerbate inflammation in these lesions. Focusing on the hypoxic conditions of menstrual blood, we investigated the effects of PGE2/thrombin on inflammation and fibrosis using primary cultured endometrial stromal cells (ESCs) and glandular epithelial cells (EECs) under low oxygen conditions. Chemokine CXCL12 secreted by endometrial stromal cells under hypoxia acts on CXCR4 receptors on glandular epithelial cells, inducing epithelial-mesenchymal transition (EMT), suggesting a possible role in endometriosis progression. RNA-seq analysis of PGE2/thrombin effects on endometrial stromal cells revealed activation of the transforming growth factor (TGF)-β pathway, particularly increased production and secretion of activin A, a member of the TGFβ family. Activin A, via increased connective tissue growth factor (CTGF) expression, promotes differentiation of endometrial stromal cells from fibroblast-like to myofibroblast transdifferentiation (FMT) of ESCs. In conclusion, targeting the CXCL12/CXCR4 and activin A/CTGF signaling pathways holds promise for improving fibrosis in endometriosis lesions.

Divergent modulation of activated protein C pleiotropic functions by antibodies that differ by a single amino acid

AbstractActivated protein C (APC) is a pleiotropic plasma protease with diverse functions derived from its anticoagulant, anti-inflammatory, and cytoprotective activities. The selective uncoupling and/or modulation of these APC activities by antibodies may have therapeutic benefit in diseases such as traumatic bleeding, hemophilia, sepsis, and ischemia. TPP-26870 is an antibody that targets a nonactive site of APC for the selective modulation of APC activities. To optimize the potency of TPP-26870, variants with single amino acid mutation in the complementarity-determining regions (CDRs) were screened, and 21 variants with improved KD were identified. Interestingly, the affinity maturation of TPP-26870 did not merely generate a panel of variants with higher potency in functional assays. Functional data demonstrated that the pleiotropic functions of APC were very sensitive to epitope-CDR interactions. Single amino acid mutations within the CDRs of TPP-26870 were sufficient to elicit divergent antagonistic and agonistic effects on the various APC functional activities. These include prolonged in vitro APC plasma half-life, increased inhibition of anticoagulant activity, and agonistic enhancement of histone H3 cleavage, while having less impact on protease-activated receptor 1 cleavage, compared with TPP-26870. This study illustrates that APC is highly sensitive to non–active site targeting that can lead to unpredictable changes in its activity profile of this pleiotropic enzyme. Furthermore, this study demonstrates the ability to modify APC functions to advance the potential development of APC-targeted antibodies as therapeutics for the treatment of diseases including trauma bleeding, hemophilia, ischemia, and sepsis.

1-Piperidine Propionic Acid Protects from Septic Shock Through Protease Receptor 2 Inhibition.

Sepsis is a complex disorder caused by a dysregulated host response to infection, with high levels of morbidity and mortality. Treatment aimed to modulate immune response and maintain vascular function is still one of the major clinical challenges. This study was designed to test the effect of the small molecule 1-Piperidine Propionic Acid (1-PPA) as molecular targeted agent to block protease-activated receptor 2 (PAR2), one of the major modulators of inflammatory response in LPS-induced experimental endotoxemia. In the THP-1 cell line, LPS-induced cytokine expression was inhibited by 1-PPA in a dose-dependent manner. In LPS-injected mice, treatment with 1-PPA was effective in reducing mortality and sepsis-related symptoms and improved cardiac function parameters. After 6 h from LPS injection, a significant decrease in IL-6, IL-1β, and IL-10 was observed in the lung tissue of 1-PPA-treated mice, compared to controls. In these mice, a significant decrease in vasoactive molecules, especially kininogen-1, was also observed, mainly in the liver. Histopathological analysis confirmed typical features of sepsis in different organs and these findings were markedly reduced in mice treated with 1-PPA. These data demonstrate the effectiveness of 1-PPA in protecting the whole organism from sepsis-induced damage.

PAR1 Is a Candidate Target for the Treatment of Peritoneal Dissemination in Gastric Cancer.

Peritoneal dissemination (PD) is a frequent cause of death in gastric cancer (GC), and there is evidence of an association between protease-activated receptor-1 (PAR1) and the development of PD. This study hypothesized that PD in GC might be influenced by PAR1. The cytotoxic effect of paclitaxel (PTX) on PAR1-transfected MKN45 (MKN45/PAR1) cells was analyzed using the MTT assay, and IC50 values were determined. In female athymic nude mice, MKN45/PAR1 cells were suspended in 0.05 ml phosphate-buffered saline (PBS) medium and inoculated into the stomach mid-wall. In each group, intraperitoneal injections of PBS, PTX, SCH79797 (PAR1-antagonist), or PTX plus SCH79797 were administered on days 8, 15, and 22 following tumor inoculation. At 56 days after tumor inoculation, mice were examined for both abdominal tumor nodule status and size and weight of the tumors. The IC50 of PTX for MKN45/PAR1 cells was 0.0697 μM and that of SCH79797 was 0.0145 μM. Mean survival of the MKN45/PAR1 mice in the PBS group was 28.75 days, whereas survival times for the mice treated with SCH79797, PTX, or a combination of PTX and SCH79797 were 31.2, 49.2, and 48.5 days, respectively. Tumor weight was smaller in the group receiving PTX and SCH79797 intraperitoneally compared with that in the PBS group (1,086±127.2 mg vs. 33.2±19.9 mg; p<0.001). The PAR1 antagonist was found to inhibit PD in a PAR1-expressing GC cell line. PAR1 may serve as a promising therapeutic target for managing PD in gastric cancer, as it plays a crucial role in its progression.

Protease-activated receptor 2 (PAR2) inhibition reduces complement-associated cardiac remodelling in obesity-related heart failure

Abstract Background and Aims Obesity is considered a major risk factor for the development of heart failure (HF) due to lipid accumulation, oxidative stress and inflammation which results in cardiac remodeling. Protease activated receptor 2 (PAR2) has been linked to various cardiovascular and inflammatory diseases. The role of PAR2 has not yet been studied in obesity-related HF. Methods In the experimental mouse study, we used 1-year-old ApoE knockout (ApoE-/-) and ApoE PAR2 double knockout (ApoE-/-PAR2-/-) mice with a C57BL/6J background on a high fat (HF) diet (n=5/ group). Cell culture experiments were performed with human primary cardiac fibroblasts and HepG2 hepatocytes. Patients (n=50) included in the clinical study had a BMI &amp;gt; 25kg/m², symptoms of heart failure (HF), coronary angiography (CA) that excluded coronary artery disease (CAD) and endomyocardial biopsy (EMB) that confirmed the absence of active myocarditis or primary cardiac amyloidosis. Patients were divided into PAR2low (n=22) and PAR2high (n=22) based on the median PAR2 expression measured via qPCR in the EMB of the cohort, and six patients served as healthy controls. Results Evaluation of endomyocardial biopsies (EMB) from overweight/ obese non-ischemic cardiomyopathy (NICM) patients with heart failure (HF) (n=44) revealed that a reduced cardiac PAR2 expression is associated with less cardiac C3 (p≤0.054), C4 (p≤0.041) and C5 gene expression (p≤0.045), as well as plasma levels of complement C3 (1.1g/l± 0.7 vs 2.1g/l±0.2; p≤0.016) and C5b-9 (303.4ng/ml± 215.7 vs 992.7ng/ml± 756.2; p≤0.0002) and as a result, better systolic function i.e. LVEF (46.1%±17.1 vs 36.8%±15.2; p≤0.046) and LV GLS (-14.9%± 5.7 vs -9.5%± 4.6; p≤0.009), less cardiac remodeling and damage. Correspondingly, aged ApoE-/-PAR2-/-mice on a Western diet showed less hepatic and cardiac complement expression (i.e. C1q, C3, C4, C5, C9), cardiac C3 deposition (p≤0.047) and C5b-9 plasma levels (p≤0.049), as well as less cardiac necrosis (p≤0.046) and lower BNP (p≤0.016) than ApoE-/-. PAR2 overexpression in human cardiac fibroblasts (HCFs) independently enhanced and PAR2 antagonism reduced interferon γ (IFNγ)-mediated STAT1-dependent complement C3, C4 and C5 expression. Conclusions Protease-activated receptor 2 regulates complement-mediated cardiac damage and remodeling in HF. The PAR2-IFNγ/STAT1/C3-C4-C5 complement-axis might be a promising prognostic and therapeutic approach to identify and treat high-risk cases of HF in overweight/obese patients.

Elderly patients are hyperresponsive to potent P2Y12 inhibitors

Abstract Background Aging has recently been associated with increased basal platelet activation and adenosine diphosphate (ADP) hyperreactivity on the one hand, but decreased platelet response to thrombin receptor stimulation on the other hand in individuals without antiplatelet therapy. In the current study, we investigated platelet response to agonist stimulation in elderly patients (70 years or older) on dual antiplatelet therapy with potent P2Y12 inhibitors. Methods Platelet aggregation in response to arachidonic acid (AA), ADP, collagen, the protease-activated receptor (PAR)-1 agonist SFLLRN and the PAR-4 agonist AYPGKF was assessed by multiple electrode aggregometry in 79 prasugrel- and 77 ticagrelor-treated patients 3 days after acute percutaneous coronary intervention. Results In the overall study population (n=156), patients aged ≥70 years (n=33) had lower platelet aggregation in response to AA, ADP and SFLLRN than younger patients (all p&amp;lt;0.05). In prasugrel-treated patients (n=79), those aged ≥70 years (n=13) showed significantly lower platelet aggregation in response to all agonists compared to younger patients (all p&amp;lt;0.05). In contrast, in ticagrelor-treated patients (n=77), those aged ≥70 years (n=20) only had significantly lower ADP-inducible platelet aggregation than younger patients (p=0.03), whereas platelet aggregation in response to AA, collagen, SFLLRN and AYPGKF was similar between elderly and younger patients on ticagrelor (all p&amp;gt;0.05). Among patients aged ≥70 years, prasugrel-treated patients showed significantly lower platelet aggregation in response to AA, collagen and AYPGKF than those receiving ticagrelor (all p&amp;lt;0.05). Conclusion Patients aged ≥70 years on potent P2Y12 inhibitors exhibit increased inhibition of ADP-inducible platelet aggregation. In addition, elderly patients on prasugrel show a significantly lower response to AA, collagen, SFLLRN and AYPGKF than younger patients.Platelet aggregation prasugrel patientsPlatelet aggregation ticagrelor patients

From bench to bedside and back to bench: investigating the role of platelet heterogeneity in coronary artery disease

Abstract Background/Introduction Platelets exhibit considerable heterogeneity in RNA content, size, and thrombogenicity. Our preliminary investigation of the transcriptome of the RNA-rich reticulated platelets(RPs) in healthy donors revealed an enrichment of prothrombotic transcripts compared to mature platelets(MPs). Recently, we validated the prognostic role of elevated RPs at clinical level in a modern cohort of ACS patients treated with potent P2Y12 inhibitors. However, the pathophysiology of RP hyperreactivity remains unclear, particularly its correlation with adverse events and cardiovascular death. Purpose To study the biology of RPs in patients with chronic coronary syndrome(CCS) and to elucidate their role in disease progression. Methods RPs were isolated from peripheral blood of CCS patients(N=20) and compared with MPs. Cell viability and reactivity of RPs were quantified by FACS. Deep transcriptomic profiling was performed using post-sorting bulk RNA sequencing of RPs and MPs and analyzed with ad-hoc bioinformatic pipelines. Proteomic profiling using mass cytometry(CyTOF) was used to validate the transcriptomic findings with single cell resolution. Results FACS analysis confirmed RPs hyperreactivity showing increased P-Selectin expression upon activation with TRAP, ADP and Collagen(Fig. 1A). Total RNA-sequencing detected 2213 differentially regulated genes with an enrichment in RPs of key functional transcript such as, the von Willebrand Factor VWF(p=3.06*10-33), the thromboxane receptor TBXA2R(p=2.07*10-29) and the collagen receptor GP6(p=1.29*10-38, Fig. 1B-C). Gene ontology and gene set enrichment analyses detected an upregulation of relevant categories as "Regulation of platelet activation" and "Platelet aggregation"(Fig. 1D-F). In addition, we detected a novel alternative splicing event on the collagen receptor transcript GP6 upregulated in RPs(Fig. 1G). We detected 33 differentially regulated miRNAs, including miR-409, miR-134 and miR-432, which have been already linked to prothrombotic phenotypes(Fig. 1H). Additionally, we detected an enrichment of circular RNAs in RPs compared to MPs with several circular RNA upregulated in RPs, that have not been described before(Fig. 1I-K). Mass cytometry detected a significant upregulation in RPs of relevant proteins including the collagen receptor GPVI(p=8.98*10-12, Fig.2) and the thrombin receptor PAR1(p=5.21*10-11). Validation assays in an independent cohort detected an upregulation of the PI3K/AKT pathway in RPs which is downstream of the receptor PAR1 and GP6. Conclusion This study represents the first comprehensive multiomic characterization of RPs in CCS patients, providing insights into their prothrombotic phenotype and into their correlation with cardiovascular events. These findings suggest potential avenues for tailored therapeutic interventions targeting the identified upregulated pathways in patients with elevated RPs, warranting further investigation at the clinical level.

Modulation of persistent bladder pain in mice: The role of macrophage migration inhibitory factor, high mobility group box-1, and downstream signaling pathways.

Repeated intravesical activation of protease-activated receptor-4 (PAR4) serves as a model of persistent bladder hyperalgesia (BHA) in mice, which lasts several days after the final stimulus. Spinal macrophage migration inhibitory factor (MIF) and high mobility group box 1 (HMGB1) are critical mediators in the persistence of BHA. We aimed to identify effective systemic treatments for persistent BHA using antagonists or transgenic deletions. Persistent BHA was induced through transurethral instillations of a PAR4-activating peptide (PAR4-AP; 100 μM, 1 h; scrambled peptide, control) under anesthesia, administered on Days 0, 2, and 4. Lower abdominal hypersensitivity was measured on Days 0-4 and 7-9. Systemic injections from Days 2-8 included ISO-1 (a MIF antagonist), ethyl pyruvate (an inhibitor of HMGB1 release), phosphate-buffered saline, or 10% DMSO (vehicle control) in C57BL/6 mice. To examine the role of HMGB1 receptors, Toll-like receptor-4 (TLR4)-null mice or systemic treatment with FPS-ZM1 (receptor for advanced glycation end product [RAGE] antagonist) were used. In addition, TIR-domain-containing adaptor-inducing interferon-β (TRIF)-null mice were tested to assess the involvement of TLR4 signaling pathways. Micturition volume and frequency were assessed on Day 9, and the bladder was histopathologically examined to assess inflammation and edema. MIF antagonism significantly reversed persistent BHA, whereas HMGB1 antagonism led to a partial reduction of persistent BHA. TLR4 deficiency or systemic administration of FPS-ZM1 significantly mitigated persistent BHA, while TRIF-deficient mice experienced a faster onset of BHA. Only MIF or HMGB1 inhibition resulted in increased micturition volume. The histopathological examination revealed no changes in inflammation or edema. MIF and HMGB1, acting through TLR4 and RAGE, mediated persistent BHA, while TRIF might modulate its onset. Further exploration of downstream TLR4 signaling may uncover novel therapeutic targets for treating persistent bladder pain.

Protease activated receptor 2 deficiency retards progression of abdominal aortic aneurysms by modulating phenotypic transformation of vascular smooth muscle cells via ERK signaling

Abdominal aortic aneurysm (AAA) is characterized by localized structural deterioration of the aortic wall, leading to progressive dilatation and rupture. Protease activated receptor 2 (PAR2) dependent signaling has been implicated in the pathophysiology of atherosclerosis through the regulation of smooth muscle cell function. However, its role in AAA remains unclear. This study investigates the function and potential mechanism of PAR2 in AAA progression. Angiotensin II (Ang II) and β-aminopropionitrile (BAPN) were administered to wild type (WT) mice to induce AAA. Increased PAR2 expression was observed in the aneurysmal tissues of these mice and in Ang II-treated vascular smooth muscle cells (VSMCs). We demonstrated that PAR2 deficiency markedly inhibited aorta dilatation and vascular remodeling in the AAA model relative to WT mice. Immunohistochemical staining showed significant upregulation of contractile markers and a reduction in synthetic markers in PAR2 knockout mice. Consistent with in vivo results, PAR2 knockdown diminished the effects of Ang II on VSMCs phenotypic switching, resulting in reduced proliferation and migration. Conversely, a PAR2 agonist (SLIGRL) induced the opposite effect, which was partially mitigated by pretreatment with an extracellular signal-regulated kinase (ERK) inhibitor (PD98059). This study suggests that PAR2 deficiency restrains aortic expansion and mitigates adverse vascular remodeling in AAA models, mediated in part by the ERK signaling pathway, indicating that PAR2 could be a potential therapeutic target for mitigating AAA development or progression.

Discovery of quinazoline-benzothiazole derivatives as novel potent protease-activated receptor 4 antagonists with improved pharmacokinetics and low bleeding liability

Protease-activated receptor 4 (PAR4) plays a critical role in the development of pathological thrombosis, and targeting PAR4 is considered a promising strategy for improving antiplatelet therapies. Here, we reported the design of a series of quinazoline-benzothiazole-based PAR4 antagonists using a scaffold-hopping strategy. Systematic structure-activity relationship exploration leads to the discovery of compounds 20f and 20g, which displayed optimal activity (h. PAR4-AP PRP IC50 = 6.39 nM and 3.45 nM, respectively) on human platelets and high selectivity for PAR4. Both of them also showed excellent metabolic stability in human liver microsomes (compound 20f, T1/2 = 249.83 min, compound 20g, T1/2 = 282.60 min) and favourable PK profiles in rats (compound 20f, T1/2 = 5.16 h, F = 50.5 %, compound 20g, T1/2 = 7.05 h, F = 27.3 %). More importantly, neither compound prolonged the bleeding time in the mouse tail-cutting model (10 mg/kg, p.o.). These results suggest that these compounds have great potential for use in antiplatelet therapies.

Protease activated receptor 2 as a novel druggable target for the treatment of metabolic dysfunction-associated fatty liver disease and cancer.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is spreading worldwide, largely due to unhealthy lifestyles that contribute to the rise in diabetes, metabolic syndrome, and obesity. In this situation, the progression of injury to metabolic steatohepatitis can evolve to cirrhosis and, eventually, to hepatocellular carcinoma (HCC). It is well known that serine protease enzymes with different functions in cellular homeostasis act as signaling molecules that regulate liver inflammation by activating the protease-activated receptors (PARs) family members, expressed on the cellular plasma membrane. Among them, PAR2 plays a central role in the activation of signaling pathways in response to changes in the extracellular microenvironment. Experimental data have provided evidence that PAR2 is involved not only in inflammatory response but also in insulin resistance, lipid metabolism, and cancer. The major aims of this narrative review are addressed to assess PAR2 involvement in inflammation, metabolism, and liver disease progression and to explore possible therapeutic strategies, based on PAR2 inhibition, in order to prevent its biological effects in the context of MAFLD and cancer.

N-terminomics profiling of naïve and inflamed murine colon reveals proteolytic signatures of legumain.

Legumain is a cysteine protease broadly associated with inflammation. It has been reported to cleave and activate protease-activated receptor 2 to provoke pain associated with oral cancer. Outside of gastric and colon cancer, little has been reported on the roles of legumain within the gastrointestinal tract. Using a legumain-selective activity-based probe, LE28, we report that legumain is activated within colonocytes and macrophages of the murine colon, and that it is upregulated in models of acute experimental colitis. We demonstrated that loss of legumain activity in colonocytes, either through pharmacological inhibition or gene deletion, had no impact on epithelial permeability in vitro. Moreover, legumain inhibition or deletion had no obvious impacts on symptoms or histological features associated with dextran sulfate sodium-induced colitis, suggesting its proteolytic activity is dispensable for colitis initiation. To gain insight into potential functions of legumain within the colon, we performed field asymmetric waveform ion mobility spectrometry-facilitated quantitative proteomics and N-terminomics analyses on naïve and inflamed colon tissue from wild-type and legumain-deficient mice. We identified 16 altered cleavage sites with an asparaginyl endopeptidase signature that may be direct substrates of legumain and a further 16 cleavage sites that may be indirectly mediated by legumain. We also analyzed changes in protein abundance and proteolytic events broadly associated with colitis in the gut, which permitted comparison to recent analyses on mucosal biopsies from patients with inflammatory bowel disease. Collectively, these results shed light on potential functions of legumain and highlight its potential roles in the transition from inflammation to colorectal cancer.