PAR1 Activation, via LMBR1/BMP Axis, Promotes the Osteogenesis of Periodontal Ligament Stem Cells (PDLSCs) and Alleviates the Inhibitory Effect of Sodium Butyrate on PDLSCs Osteogenesis.

BACKGROUND: Periodontitis is the leading cause of tooth loss and can exacerbate various systemic inflammatory conditions. Periodontal ligament stem cells (PDLSCs) stand out as prominent and favorable candidates for promoting periodontal tissue regeneration. This study aimed to investigate whether the protease-activated receptor type 1 (PAR1) can mitigate the sodium butyrate (NaB)-induced PDLSCs osteogenesis inhibition and unravel the underlying mechanism. METHODS: Public datasets from the Gene Expression Omnibus (GEO) were utilized to analyze differentially expressed genes (DEGs) in periodontitis and subsequent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. PDLSCs were cultured normally in control medium (CM) as the negative control or in osteogenic medium (OM) to induce osteogenesis. PAR1 was either activated or suppressed using a selective agonist or antagonist (OM+agonist and OM+antagonist). The evaluation of PDLSCs osteogenesis was based on the levels of osteogenesis-related markers, including runt-related transcription factor 2 (RUNX2), osterix (OSX), osteocalcin (OCN), and osteopontin (OPN), alkaline phosphatase (ALP) activity, and calcium concentration. Additionally, cell proliferation and osteogenic differentiation were measured through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Alizarin Red Staining. To determine the PAR1 targeting the limb development membrane protein 1 (LMBR1)/bone morphogenetic protein (BMP) pathway, LMBR1 was upregulated through cell transfection and BMP2 was inhibited using the selective inhibitor Noggin protein. Finally, NaB was introduced into PDLSCs to investigate the effect on NaB-induced inhibition of PDLSCs osteogenesis. RESULTS: PAR1, RUNX2, OSX, OCN, OPN, proliferation, ALP activity, calcium concentration, osteogenic differentiation, BMP2, and BMP4 exhibited significant increases in PDLSCs cultured in OM (p < 0.01). These parameters were further elevated by PAR1 agonist and conversely reduced by PAR1 antagonist (p < 0.01). Conversely, LMBR1 was decreased in PDLSCs cultured in OM (p < 0.001), with further reduction induced by PAR1 agonist and a reverse increase observed with PAR1 antagonist (p < 0.001). OE-LMBR1 transfection successfully elevated LMBR1 levels, subsequently inhibiting BMP2 and BMP4 (p < 0.001). Meanwhile, the Noggin protein effectively suppressed BMP2 and BMP4 (p < 0.001). All observed osteogenesis-related changes were reversed by the increased LMBR1 or inhibition of the BMP pathway (p < 0.001). Furthermore, NaB suppressed osteogenesis-related changes in OM-cultured PDLSCs (p < 0.001), and these effects were entirely reversed by PAR1 agonist (p < 0.001). Conversely, the increased LMBR1 or inhibited BMP pathway disrupted the osteogenesis reversion induced by PAR1 agonist (p < 0.001). CONCLUSION: The activation of PAR1, through suppressing LMBR1 signaling and activating BMP pathway, demonstrates the ability to enhance the osteogenesis of PDLSCs and mitigate the inhibitory effects on PDLSCs osteogenesis caused by NaB.

Therapeutic Effect of Proteinase-Activated Receptor-1 Antagonist on Colitis-Associated Carcinogenesis.

BACKGROUND & AIMS: Inflammatory bowel disease is associated with carcinogenesis, which limits the prognosis of the patients. The local expression of proteinases and proteinase-activated receptor 1 (PAR1) increases in inflammatory bowel disease. The present study investigated the therapeutic effects of PAR1 antagonism on colitis-associated carcinogenesis. METHODS: A colitis-associated carcinogenesis model was prepared in mice by treatment with azoxymethane (AOM) and dextran sulfate sodium (DSS). PAR1 antagonist E5555 was administered in long- and short-term protocol, starting on the day of AOM injection and 1 week after completing AOM/DSS treatment, respectively. The fecal samples were collected for metagenome analysis of gut microbiota. The intestinal myofibroblasts of the Crohn's disease patients were used to elucidate underlying cellular mechanisms. Caco-2 cells were used to investigate a possible source of PAR1 agonist proteinases. RESULTS: AOM/DSS model showed weight loss, diarrhea, tumor development, inflammation, fibrosis, and increased production of inflammatory cytokines. The ß-diversity, but not α-diversity, of microbiota significantly differed between AOM/DSS and control mice. E5555 alleviated these pathological changes and altered the microbiota ß-diversity in AOM/DSS mice. The thrombin expression was up-regulated in tumor and non-tumor areas, whereas PAR1 mRNA expression was higher in tumor areas compared with non-tumor areas. E5555 inhibited thrombin-triggered elevation of cytosolic Ca2+ concentration and ERK1/2 phosphorylation, as well as IL6-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation in intestinal myofibroblasts. Caco-2 cell-conditioned medium contained immunoreactive thrombin, which cleaved the recombinant protein containing the extracellular domain of PAR1 at the thrombin cleavage site. CONCLUSIONS: PAR1 antagonism is proposed to be a novel therapeutic strategy for treatment of inflammatory bowel disease and its associated carcinogenesis.

Recombinant hirudin suppresses angiogenesis of diffuse large B-cell lymphoma through regulation of the PAR-1-VEGF.

Hirudin is one of the specific inhibitors of thrombin, which has been confirmed to have strong bioactivities, including inhibiting tumors. However, the function and mechanism of hirudin and protease-activated receptor 1 (PAR-1) in diffuse large B-cell lymphoma (DLBCL) have not been clear. Detecting the expression PAR-1 in DLBCL tissues and cells by RT-qPCR and IHC. Transfected sh-NC, sh-PAR-1, or pcDNA3.1-PAR-1 in DLBCL cells or processed DLBCL cells through added thrombin, Vorapaxar, Recombinant hirudin (RH), or Na2S2O4 and co-culture with EA.hy926. And built DLBCL mice observed tumor growth. Detecting the expression of related genes by RT-qPCR, Western blot, IHC, and immunofluorescence, measured the cellular hypoxia with Hypoxyprobe-1 Kit, and estimated the cell inflammatory factors, proliferation, migration, invasion, and apoptosis by ELISA, CCK-8, flow cytometry, wound-healing and Transwell. Co-immunoprecipitation and pull-down measurement were used to verify the relationship. PAR-1 was highly expressed in DLBCL tissues and cells, especially in SUDHL2. Na2S2O4 induced SUDHL2 hypoxia, and PAR-1 did not influence thrombin-activated hypoxia. PAR-1 could promote SUDHL2 proliferation, migration, and invasion, and it was unrelated to cellular hypoxia. PAR-1 promoted proliferation, migration, and angiogenesis of EA.hy926 or SUDHL2 through up-regulation vascular endothelial growth factor (VEGF). RH inhibited tumor growth, cell proliferation, and migration, promoted apoptosis of DLBCL, and inhibited angiogenesis by down-regulating PAR-1-VEGF. RH inhibits proliferation, migration, and angiogenesis of DLBCL cells by down-regulating PAR-1-VEGF.

PARIN5, a Novel Thrombin Receptor Antagonist Modulates a Streptozotocin Mice Model for Diabetic Encephalopathy.

Diabetic encephalopathy (DE) is an inflammation-associated diabetes mellitus (DM) complication. Inflammation and coagulation are linked and are both potentially modulated by inhibiting the thrombin cellular protease-activated receptor 1 (PAR1). Our aim was to study whether coagulation pathway modulation affects DE. Diabetic C57BL/6 mice were treated with PARIN5, a novel PAR1 modulator. Behavioral changes in the open field and novel object recognition tests, serum neurofilament (NfL) levels and thrombin activity in central and peripheral nervous system tissue (CNS and PNS, respectively), brain mRNA expression of tumor necrosis factor α (TNF-α), Factor X (FX), prothrombin, and PAR1 were assessed. Subtle behavioral changes were detected in diabetic mice. These were accompanied by an increase in serum NfL, an increase in central and peripheral neural tissue thrombin activity, and TNF-α, FX, and prothrombin brain intrinsic mRNA expression. Systemic treatment with PARIN5 prevented the appearance of behavioral changes, normalized serum NfL and prevented the increase in peripheral but not central thrombin activity. PARIN5 treatment prevented the elevation of both TNF-α and FX but significantly elevated prothrombin expression. PARIN5 treatment prevents behavioral and neural damage in the DE model, suggesting it for future clinical research.

A KLK4 proteinase substrate capture approach to antagonize PAR1.

Proteinase-activated receptor-1 (PAR1), triggered by thrombin and other serine proteinases such as tissue kallikrein-4 (KLK4), is a key driver of inflammation, tumor invasiveness and tumor metastasis. The PAR1 transmembrane G-protein-coupled receptor therefore represents an attractive target for therapeutic inhibitors. We thus used a computational design to develop a new PAR1 antagonist, namely, a catalytically inactive human KLK4 that acts as a proteinase substrate-capture reagent, preventing receptor cleavage (and hence activation) by binding to and occluding the extracellular R41-S42 canonical PAR1 proteolytic activation site. On the basis of in silico site-saturation mutagenesis, we then generated KLK4S207A,L185D, a first-of-a-kind 'decoy' PAR1 inhibitor, by mutating the S207A and L185D residues in wild-type KLK4, which strongly binds to PAR1. KLK4S207A,L185D markedly inhibited PAR1 cleavage, and PAR1-mediated MAPK/ERK activation as well as the migration and invasiveness of melanoma cells. This 'substrate-capturing' KLK4 variant, engineered to bind to PAR1, illustrates proof of principle for the utility of a KLK4 'proteinase substrate capture' approach to regulate proteinase-mediated PAR1 signaling.

Coagulation Signaling through PAR1 as a Therapeutic Target in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal disease with a 5-year survival rate of less than 10% following diagnosis. The aggressive and invasive properties of pancreatic cancer tumors coupled with poor diagnostic options contribute to the high mortality rate since most patients present with late-stage disease. Accordingly, PDAC is linked to the highest rate of cancer-associated venous thromboembolic disease of all solid tumor malignancies. However, in addition to promoting clot formation, recent studies suggest that the coagulation system in PDAC mediates a reciprocal relationship, whereby coagulation proteases and receptors promote PDAC tumor progression and dissemination. Here, upregulation of tissue factor (TF) by tumor cells can drive local generation of the central coagulation protease thrombin that promotes cell signaling activity through protease-activated receptors (PARs) expressed by both tumor cells and multiple stromal cell subsets. Moreover, the TF-thrombin-PAR1 signaling axis appears to be a major mechanism of cancer progression in general and PDAC in particular. Here, we summarize the current literature regarding the role of PAR1 in PDAC and review possibilities for pharmacologically targeting PAR1 as a PDAC therapeutic approach.

The PAR-1 antagonist vorapaxar ameliorates kidney injury and tubulointerstitial fibrosis.

Protease-activated receptor (PAR)-1 has emerged as a key profibrotic player in various organs including kidney. PAR-1 activation leads to deposition of extracellular matrix (ECM) proteins in the tubulointerstitium and induction of epithelial-mesenchymal transition (EMT) during renal fibrosis. We tested the anti-fibrotic potential of vorapaxar, a clinically approved PAR-1 antagonist for cardiovascular protection, in an experimental kidney fibrosis model of unilateral ureteral obstruction (UUO) and an AKI-to-chronic kidney disease (CKD) transition model of unilateral ischemia-reperfusion injury (UIRI), and dissected the underlying renoprotective mechanisms using rat tubular epithelial cells. PAR-1 is activated mostly in the renal tubules in both the UUO and UIRI models of renal fibrosis. Vorapaxar significantly reduced kidney injury and ameliorated morphologic changes in both models. Amelioration of kidney fibrosis was evident from down-regulation of fibronectin (Fn), collagen and α-smooth muscle actin (αSMA) in the injured kidney. Mechanistically, inhibition of PAR-1 inhibited MAPK ERK1/2 and transforming growth factor-ß (TGF-ß)-mediated Smad signaling, and suppressed oxidative stress, overexpression of pro-inflammatory cytokines and macrophage infiltration into the kidney. These beneficial effects were recapitulated in cultured tubular epithelial cells in which vorapaxar ameliorated thrombin- and hypoxia-induced TGF-ß expression and ECM accumulation. In addition, vorapaxar mitigated capillary loss and the expression of adhesion molecules on the vascular endothelium during AKI-to-CKD transition. The PAR-1 antagonist vorapaxar protects against kidney fibrosis during UUO and UIRI. Its efficacy in human CKD in addition to CV protection warrants further investigation.

Ankaferd hemostat (ABS) as a potential mucosal topical agent for the management of COVID-19 syndrome based on its PAR-1 inhibitory effect and oestrogen content.

COVID-19 due to the SARS-CoV-2 infection is a multi-systemic immune syndrome affecting mainly the lungs, oropharyngeal region, and other vascular endothelial beds. There are tremendous ongoing efforts for the aim of developing drugs against the COVID-19 syndrome-associated inflammation. However, currently no specific medicine is present for the absolute pharmacological cure of COVID-19 mucositis. The re-purposing/re-positioning of already existing drugs is a very important strategy for the management of ongoing pandemy since the development of a new drug needs decades. Apart from altering angiotensin signaling pathways, novel drug candidates for re-purposing comprise medications shall target COVID-19 pathobiology, including pharmaceutical formulations that antagonize proteinase-activated receptors (PARs), mainly PAR-1. Activation of the PAR-1, mediators and hormones impact on the hemostasis, endothelial activation, alveolar epithelial cells and mucosal inflammatory responses which are the essentials of the COVID-19 pathophysiology. In this context, Ankaferd hemostat (Ankaferd Blood Stopper, ABS) which is an already approved hemostatic agent affecting via vital erythroid aggregation and fibrinogen gamma could be a potential topical remedy for the mucosal management of COVID-19. ABS is a clinically safe and effective topical hemostatic agent of plant origin capable of exerting pleiotropic effects on the endothelial cells, angiogenesis, cell proliferation and vascular dynamics. ABS had been approved as a topically applied hemostatic agent for the management of post-surgical/dental bleedings and healing of infected inflammatory mucosal wounds. The anti-inflammatory and proteinase-activated receptor axis properties of ABS with a considerable amount of oestrogenic hormone presence highlight this unique topical hemostatic drug regarding the clinical re-positioning for COVID-19-associated mucositis. Topical ABS as a biological response modifier may lessen SARS-CoV-2 associated microthrombosis, endothelial dysfunction, oropharyngeal inflammation and mucosal lung damage. Moreover, PAR-1 inhibition ability of ABS might be helpful for reducing the initial virus propagation and mocasal spread of COVID-19.

Blockade of PAR-1 Signaling Attenuates Cardiac Hypertrophy and Fibrosis in Renin-Overexpressing Hypertensive Mice.

Background Although PAR-1 (protease-activated receptor-1) exerts important functions in the pathophysiology of the cardiovascular system, the role of PAR-1 signaling in heart failure development remains largely unknown. We tested the hypothesis that PAR-1 signaling inhibition has protective effects on the progression of cardiac remodeling induced by chronic renin-angiotensin system activation using renin-overexpressing hypertensive (Ren-Tg) mice. Methods and Results We treated 12- to 16-week-old male wild-type (WT) mice and Ren-Tg mice with continuous subcutaneous infusion of the PAR-1 antagonist SCH79797 or vehicle for 4 weeks. The thicknesses of interventricular septum and the left ventricular posterior wall were greater in Ren-Tg mice than in WT mice, and SCH79797 treatment significantly decreased these thicknesses in Ren-Tg mice. The cardiac fibrosis area and monocyte/macrophage deposition were greater in Ren-Tg mice than in WT mice, and both conditions were attenuated by SCH79797 treatment. Cardiac mRNA expression levels of PAR-1, TNF-α (tumor necrosis factor-α), TGF-ß1 (transforming growth factor-ß1), and COL3A1 (collagen type 3 α1 chain) and the ratio of ß-myosin heavy chain (ß-MHC) to α-MHC were all greater in Ren-Tg mice than in WT mice; SCH79797 treatment attenuated these increases in Ren-Tg mice. Prothrombin fragment 1+2 concentration and factor Xa in plasma were greater in Ren-Tg mice than in WT mice, and both conditions were unaffected by SCH79797 treatment. In isolated cardiac fibroblasts, both thrombin and factor Xa enhanced ERK1/2 (extracellular signal-regulated kinase 1/2) phosphorylation, and SCH79797 pretreatment abolished this enhancement. Furthermore, gene expression of PAR-1, TGF-ß1, and COL3A1 were enhanced by factor Xa, and all were inhibited by SCH79797. Conclusions The results indicate that PAR-1 signaling is involved in cardiac remodeling induced by renin-angiotensin system activation, which may provide a novel therapeutic target for heart failure.

pHLIP(Var7)-P1AP suppresses tumor cell proliferation in MDA-MB-231 triple-negative breast cancer by targeting protease activated receptor 1.

PURPOSE: Protease-activated receptor 1 (PAR1) is a signaling protein ubiquitously present on the surface of tumor cells, and its homologous protein fragment, PAR1-activating peptide (P1AP), can inhibit protein signal transduction of PAR1/G in tumor cells. pH (Low) insertion peptide (pHLIP) can target the acidic tumor microenvironment (TME) and can be used as an excellent carrier to deliver P1AP to tumor cells for therapeutic purposes. METHODS: PAR1 expression on the surface of MDA-MB-231 cells and human MCF10A mammary epithelial cells was observed. The binding between fluorescent-labeled pHLIP(Var7)-P1AP and MDA-MB-231 cells under different pH values was analyzed. The effect of pHLIP(Var7)-P1AP on the proliferation of MDA-MB-231 cells was analyzed under the conditions of pH 7.4 and 6.0. RESULTS: PAR1 was highly expressed on the surface of MDA-MB-231 cells. In an acidic environment (pH 6.0 and 5.0), fluorescent-labeled pHLIP(Var7)-P1AP and MDA-MB-231 cells had a high binding ability, and the binding ability increased with the decrease in pH. In an acidic environment (pH 6.0), pHLIP(Var7)-P1AP significantly inhibited MDA-MB-231 cell proliferation. With 0.5 µg, 1 µg, 2 µg, 4 µg, and 8 µg of pHLIP(Var7)-P1AP, the cell proliferation inhibition rates were 3.39%, 5.27%, 14.29%, 22.14%, and 35.69%, respectively. CONCLUSION: PAR1 was highly expressed on the surface of MDA-MB-231 cells. pHLIP(Var7)-P1AP can effectively target MDA-MB-231 cells in an acidic environment and inhibit the growth of MDA-MB-231 cells by inhibiting the signal transduction of PAR1/G protein.

A double-blind, randomized, placebo-controlled pilot trial to evaluate safety and efficacy of vorapaxar on arteriovenous fistula maturation.

BACKGROUND: Protease-activated receptor-1 antagonism by vorapaxar could facilitate arteriovenous fistula maturation but may increase bleeding risk. OBJECTIVE: The primary objective of the Vorapaxar Study for Maturation of arteriovenous fistula for Hemodialysis Access (VorapAccess) was to determine if vorapaxar improves arteriovenous fistula functional maturation in patients with end-stage renal disease. METHODS: VorapAccess was a randomized, placebo-controlled, double-blind pilot trial comparing 2.5 mg vorapaxar per day with placebo for twelve weeks starting on day two after arteriovenous fistula creation. The primary outcome was time to functional maturation defined as successful cannulation for six hemodialysis sessions within three weeks. The planned sample size was 50 participants. The study was terminated early after withdrawal of planned financial support. Given the small number of randomized patients, we performed descriptive analyses without inference testing. RESULTS: A total of 13 participants were randomly allocated study drug (six vorapaxar and seven placebo). The median age was 56 years and seven participants (54%) were female. The median (minimum-maximum) days to functional maturation were 169 (77-287) days in the vorapaxar group and 145 (48-198) days in the placebo group. Six of the 13 (46%) participants had arteriovenous fistula functional maturation within 180 days; two of six (33%) in the vorapaxar group and four of seven (57%) in the placebo group. There was one bleeding event in the placebo group. CONCLUSION: Fewer than half of participants had functional maturation within 180 days after surgery, suggesting a major need for agents or strategies that enhance arteriovenous fistula maturation.

Thrombin Preconditioning Boosts Biogenesis of Extracellular Vesicles from Mesenchymal Stem Cells and Enriches Their Cargo Contents via Protease-Activated Receptor-Mediated Signaling Pathways.

We investigated the role of protease-activated receptor (PAR)-mediated signaling pathways in the biogenesis of human umbilical cord blood-derived mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) and the enrichment of their cargo content after thrombin preconditioning. Immunoblot analyses showed that MSCs expressed two PAR subtypes: PAR-1 and PAR-3. Thrombin preconditioning significantly accelerated MSC-derived EV biogenesis more than five-fold and enriched their cargo contents by more than two-fold via activation of Rab5, early endosomal antigen (EEA)-1, and the extracellular signal regulated kinase (ERK)1/2 and AKT signaling pathways. Blockage of PAR-1 with the PAR-1-specific antagonist, SCH79797, significantly suppressed the activation of Rab5, EEA-1, and the ERK1/2 and AKT pathways and subsequently increased EV production and enriched EV cargo contents. Combined blockage of PAR-1 and PAR-3 further and significantly inhibited the activation of Rab5, EEA-1, and the ERK1/2 and AKT pathways, accelerated EV production, and enriched EV cargo contents. In summary, thrombin preconditioning boosted the biogenesis of MSC-derived EVs and enriched their cargo contents largely via PAR-1-mediated pathways and partly via PAR-1-independent, PAR-3-mediated activation of Rab5, EEA-1, and the ERK1/2 and AKT signaling pathways.

Activation of calcium signaling in human gingival fibroblasts by recombinant Porphyromonas gingivalis RgpB protein.

Arginine-specific cysteine proteinases, such as Arg-gingipain B (RgpB), mediate inflammation by activating protease-activated receptors (PARs). Arg-gingipain B is produced by Porphyromonas gingivalis, and is implicated in the causation of periodontal disease. The purpose of the present study was to observe the influence of recombinant RgpB protein (rRgpB) on PAR activation by monitoring intracellular Ca2+ ion concentration ([Ca2+]i) and inositol-1,4,5-triphosphate (IP3) levels in human gingival fibroblasts (HGFs). Our findings showed that rRgpB could cause a transient increase in [Ca2+]i. This increase in [Ca2+]i was completely suppressed by vorapaxar, a PAR-1 antagonist. Recombinant Arg-gingipain B increased the concentration of IP3, reaching a maximum at 60 s after treatment; this was completely inhibited by vorapaxar. We therefore conclude that rRgpB-induced calcium signaling in HGFs is mainly caused by PAR-1 activation. This suggests that PAR-1 activation plays a significant role in chronic inflammatory periodontal disease induced by P. gingivalis RgpB.

Unveiling novel 2-cyclopropyl-3-ethynyl-4-(4-fluorophenyl)quinolines as GPCR ligands via PI3-kinase/PAR-1 antagonism and platelet aggregation valuations; development of a new class of anticancer drugs with thrombolytic effects.

In the present study, novel 2-cyclopropyl-3-ethynyl-4-(4-fluorophenyl) quinolines (4a-l) were recognized and evaluated as G-Protein Coupled Receptor (GPCR) ligands through molecular evaluations. Thrombin mediates adhesion of mast cell, a type of cell abundantly found in connective tissue and releasing histamine and other substances during inflammatory and allergic reactions, through phosphoinositol 3-kinase pathway. With this background, as preliminary, 4a-l are resolute to be potential leads, designated from their effective phosphoinositol 3-kinase (PI3-Kinase) inhibition potentials, best-docked scores, comparative ligand efficiency, and significant structural attributes evaluated by ab initio simulations. Since thrombin is one of the main reason for various cancer invasion in association with PI3Kinase, a thrombolytic potential of the compounds also analyzed. The experimental in vitro studies confirmed the significant enhancement as PI3Kinase inhibitors and appreciable enhancement in MTT assay of breast and skin cancer cell lines. Significantly, acetophenone substituent in the quinoline scaffold could be coherent to note the significant binding affinity to all the evaluated drug targets.

Vorapaxar for HIV-associated inflammation and coagulopathy (ADVICE): a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Increased D-dimer concentrations are associated with poor cardiovascular and other clinical outcomes in people with treated HIV infection. Proteinase activated receptor-1 (PAR-1) is activated by thrombin and overexpressed by immune cells from HIV-infected people. We aimed to study the efficacy of vorapaxar, a licensed inhibitor of PAR-1, in reducing HIV-associated hypercoagulation and inflammation. METHODS: This was a multicentre, double-blind, randomised, placebo-controlled trial done in seven hospital clinics in Australia and the USA. Eligible participants were HIV-infected, aviraemic, were receiving stable antiretroviral therapy, and had D-dimer concentrations greater than 200 ng/mL. We randomly assigned participants (1:1) using computer-generated block lists of size two to receive vorapaxar (2·5 mg orally daily) or matched placebo for 12 weeks. Participants were reviewed and had a blood sample taken at weeks 1, 4, 8, and 12 during treatment, and at a final visit at week 18. The primary endpoint was treatment group difference in changes from baseline D-dimer concentrations after 8-12 weeks of treatment, and was assessed in the modified intention-to-treat population (participants who had at least one dose of study drug or one follow-up visit). This trial is registered with ClinicalTrials.gov, number NCT02394730, and is closed to new participants. FINDINGS: Between Oct 21, 2015, and July 14, 2017, 65 eligible patients were randomly assigned to the placebo group (n=31) or vorapaxar group (n=34). One patient from the vorapaxar group did not receive any study drug, and the modified intention-to-treat population was comprised of 33 patients. D-dimer concentrations after 8-12 weeks of treatment did not differ significantly between groups (difference -0·02 log10 ng/mL, 95% CI -0·10 to 0·05; p=0·56). Vorapaxar treatment was safe and well tolerated in this cohort. There were 161 adverse events (n=84 in the placebo group and n=77 in the vorapaxar group), and five protocol-defined serious adverse events that required hospital admission for more than 24 h (n=2 in the placebo group and n=3 in the vorapaxar group). One patient ceased taking vorapaxar because of an adverse event. There were 25 bleeding events, 23 of which were mild, one was moderate, and one was severe. INTERPRETATION: Vorapaxar had no effect on D-dimer concentrations in HIV-infected patients receiving stable antiretroviral therapy but at risk of poor outcomes. Alternative approaches are needed to reduce hypercoagulation, inflammation, and adverse long-term outcomes in patients with treated HIV infection. FUNDING: Australian National Health and Medical Research Council, US National Cancer Institute, National Institutes of Health.

Molecular targeting of breast and colon cancer cells by PAR1 mediated apoptosis through a novel pro-apoptotic peptide.

A novel activating peptide was designed and synthesized from V. cholerae hemagglutinine protease (HAP) mediated cleavage site of mouse PAR1. The peptide "PFISED" interacts with PAR1 in a new site which is different from its thrombin mediated conventional activation site and induced a series of new downstream signaling pathways. The peptide showed apoptosis in human and mouse breast (MCF-7 and EAC) and colon (HT29 and CT26) cancer cells where as in the same peptide concentration in normal human breast epithelial cells (MCF-10A), normal human fibroblast cells (MRC-5), normal mouse peritoneal macrophage cells and normal mouse breast and colon tissues did not show any effect. Treatment with this peptide enhanced the survival kinetics of EAC induced mice. The peptide mediated apoptosis was inhibited in presence of PAR1 inhibitor and was significantly reduced in si-PAR1 treated cells that indicate the activating peptide "PFISED" induced PAR1 mediated apoptosis of colon and breast cancer cells. This peptide induced over expression and activation of PAR1 and its downstream MAP kinase and NFκB signaling pathways. These signaling pathways enhanced the cellular ROS level to kill malignant cells. We report a novel pro-apoptotic peptide which can selectively kill malignant cells via its specific target receptor PAR1 which is over expressed in the malignant cells and can be used as a molecular target therapy for cancer treatment.

Protease-Activated Receptor 1 as Therapeutic Target in Breast, Lung, and Ovarian Cancer: Pepducin Approach.

The G-protein coupled receptors (GPCRs) belong to a large family of diverse receptors that are well recognized as pharmacological targets. However, very few of these receptors have been pursued as oncology drug targets. The Protease-activated receptor 1 (PAR1), which is a G-protein coupled receptor, has been shown to act as an oncogene and is an emerging anti-cancer drug target. In this paper, we provide an overview of PAR1's biased signaling role in metastatic cancers of the breast, lungs, and ovaries and describe the development of PAR1 inhibitors that are currently in clinical use to treat acute coronary syndromes. PAR1 inhibitor PZ-128 is in a Phase II clinical trial and is being developed to prevent ischemic and thrombotic complication of patients undergoing cardiac catheterization. PZ-128 belongs to a new class of cell-penetrating, membrane-tethered peptides named pepducins that are based on the intracellular loops of receptors targeting the receptor G-protein interface. Application of PZ-128 as an anti-metastatic and anti-angiogenic therapeutic agent in breast, lung, and ovarian cancer is being reviewed.

Designing Natural Product Hybrids Bearing Triple Antiplatelet Profile and Evaluating Their Human Plasma Stability.

Cardiovascular diseases (CVDs) are becoming major contributors to the burden of disease due to genetic and environmental factors. Despite current standard oral care, cardiovascular risk remains relatively high. A triple antiplatelet therapy with a cyclooxygenase-1 (COX-1) inhibitor, a P2Y12 receptor antagonist, and a protease-activated receptor-1 (PAR-1) antagonist has been established in the secondary prevention of atherothrombosis in patients with acute myocardial infraction and in those with peripheral artery disease. However, due to the combinatorial use of three different drugs, patients receiving this triple therapy are exposed to enhanced risk of bleeding. Conforming to polypharmacology principles, the discovery of a single compound that can simultaneously block the three platelet activation pathways (PAR-1, P2Y12, and COX-1) is of importance. Natural products have served as an inexhaustible source of bioactive compounds presenting a diverse pharmaceutical profile, including anti-inflammatory, antioxidant, anticancer, and antithrombotic activity. Indeed, principal component analysis indicated that natural products have the potential to inhibit the three aforementioned pathways, though existed reports refer to single inhibition mechanism on specific receptor(s) implicated in platelet activation. We thus set out to explore possibilities that take advantage of this potential of natural products and shape the basis to produce novel compounds that could simultaneously target PAR-1, P2Y12, and COX-1 platelet activation pathways. Polyunsaturated fatty acids (PUFAs) have multiple effects leading to improvements in blood pressure and cardiac function and arterial compliance. A promising approach to achieve the desirable goal is the bioconjugation of natural products with PUFAs. Herein, we describe the principles that should be followed to develop molecular hybrids bearing triple antiplatelet activity profile.

Inhibition of Protease-Activated Receptor (PAR1) Reduces Activation of the Endothelium, Coagulation, Fibrinolysis and Inflammation during Human Endotoxemia.

The protease-activated receptor-1 (PAR-1) is critically involved in the co-activation of coagulation and inflammatory responses. Vorapaxar is a reversible, orally active, low molecular weight, competitive antagonist of PAR-1.We investigated the effects of PAR-1 inhibition by vorapaxar on the inflammatory response, the activation of coagulation, fibrinolysis and endothelium during experimental endotoxemia. In this randomized, double blind, crossover trial, 16 healthy volunteers received a bolus infusion of 2 ng/kg lipopolysaccharide (LPS) ± placebo/vorapaxar with a washout period of 8 weeks. Vorapaxar dosing was guided by thrombin receptor-activating peptide-6-induced whole blood aggregometry. Participants received 10 mg vorapaxar or placebo as an initial dose and, depending on the aggregometry, potentially an additional 10 mg. Goal was > 80% inhibition of aggregation compared with baseline. Vorapaxar significantly reduced the LPS-induced increase in pro-thrombin fragments F1 + 2 by a median of 27% (quartiles: 11-49%), thrombin-anti-thrombin concentrations by 22% (-3 to 46%) and plasmin-anti-plasmin levels by 38% (23-53%). PAR-1 inhibition dampened peak concentrations of tumour necrosis factor -α, interleukin-6 and consequently C-reactive protein by 66% (-11-71%), 50% (15-79%) and 23% (16-38%), respectively. Vorapaxar decreased maximum von Willebrand factor levels by 29% (26-51%) and soluble E-selectin concentrations by 30% (25-38%) after LPS infusion. PAR-1 inhibition did not affect thrombomodulin, soluble P-selectin and platelet factor-4 concentrations.PAR-1 inhibition significantly reduced the activation of coagulation, fibrinolysis, the inflammatory response and endothelial activation during experimental human endotoxemia.

Thrombin induces protease-activated receptor 1 signaling and activation of human atrial fibroblasts and dabigatran prevents these effects.

BACKGROUND: Data with animal cells and models suggest that thrombin activates cardiac fibroblasts (Fib) to myofibroblasts (myoFib) via protease-activated receptor 1 (PAR1) cleavage, and in this way promotes adverse atrial remodeling and, thereby, atrial fibrillation (AF). OBJECTIVE: Here, we explored the effects of thrombin on human atrial Fib and whether they are antagonized by the clinically available direct thrombin inhibitor, dabigatran. METHODS: Fib isolated from atrial appendages of patients without AF undergoing elective cardiac surgery were evaluated for PAR expression and treated with thrombin with or without dabigatran. PAR1 cleavage, downstream signaling and myoFib markers were investigated by immunofluorescence and Western blot. Collagen synthesis, activity of matrix metalloprotease (MMP)-2 and proliferation were assessed by Picro-Sirius red staining, gelatinolytic zymography and BrdU incorporation, respectively. Fib function was studied as capability to contract a collagen gel and stimulate the chemotaxis of peripheral blood monocytes from healthy volunteers. RESULTS: Primary human atrial Fib expressed PAR1, while levels of the other PARs were very low. Thrombin triggered PAR1 cleavage and phosphorylation of ERK1/2, p38 and Akt, elicited a switch to myoFib enriched for αSMA, fibronectin and type I collagen, and induced paracrine/autocrine transforming growth factor beta-1, cyclooxygenase-2, endothelin-1 and chemokine (C-C motif) ligand 2 (CCL2); conversely, MMP-2 activity decreased. Thrombin-primed cells displayed enhanced proliferation, formed discrete collagen-containing cellular nodules, and stimulated the contraction of a collagen gel. Furthermore, their conditioned medium caused monocytes to migrate. All these effects were prevented by dabigatran. CONCLUSION: These results with human cells complete the knowledge about thrombin actions on cardiac Fib and strengthen the translational potential of the emerging paradigm that pharmacological blockade of thrombin may counteract molecular and cellular events underlying AF.