Inhibition Of Protease-activated Receptor-1 Research Articles

PAR1 regulates sepsis-induced vascular endothelial barrier dysfunction by mediating ERM phosphorylation via the RhoA/ROCK signaling pathway

Sepsis begins with vascular endothelial barrier breakdown and causes widespread organ failure. Protease-activated receptor 1 (PAR1) is an important target for modulating vascular endothelial permeability; however, little research has been undertaken in sepsis, and its putative molecular mechanism remains unknown. The vascular endothelial permeability was examined by detecting FITC-dextran flux. F-actin was examined by immunofluorescence (IF). PAR1, ERM phosphorylation, and RhoA/ROCK signaling pathway expression in lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs) line were examined by IF and Western blot. To develop the sepsis model, cecal ligation and puncture (CLP) were conducted. The PAR1 inhibitor SCH79797 was utilized to inhibit PAR1 expression in vivo. Vascular permeability in main organs weres measured by Evans blue dye extravasation. The pathological changes in main organs were examined by HE staining. The expression of PAR1, ERM phosphorylation, and the RhoA/ROCK signaling pathway was examined using IF, immunohistochemical and WB in CLP mice. In vitro, in response to LPS stimulation of HUVECs, PAR1 mediated the phosphorylation of ERM, promoted F-actin rearrangement, and increased endothelial hyperpermeability, all of which were prevented by inhibiting PAR1 or RhoA. Additionally, inhibiting PAR1 expression reduced RhoA and ROCK expression. In vivo, we showed that inhibiting PAR1 expression will reduce ezrin/radixin/moesin (ERM) phosphorylation to relieve vascular endothelial barrier dysfunction and thereby ameliorate multiorgan dysfunction syndrome (MODS) in CLP-induced septic mice. This study revealed that PAR1-mediated phosphorylation of ERM induced endothelial barrier dysfunction, which in turn led to MODS in sepsis, and that the RhoA/ROCK signaling pathway underlay these effects.

Tubulovascular protection from protease-activated receptor-1 depletion during AKI-to-CKD transition.

Thromboembolic events are prevalent in chronic kidney disease (CKD) patients due to increased thrombin generation leading to a hypercoagulable state. We previously demonstrated that inhibition of protease-activated receptor-1 (PAR-1) by vorapaxar reduces kidney fibrosis. We used an animal model of unilateral ischemia-reperfusion injury-induced CKD to explore the tubulovascular crosstalk mechanisms of PAR-1 in acute kidney injury (AKI)-to-CKD transition. During the early phase of AKI, PAR-1-deficient mice exhibited reduced kidney inflammation, vascular injury, and preserved endothelial integrity and capillary permeability. During the transition phase to CKD, PAR-1 deficiency preserved kidney function and diminished tubulointerstitial fibrosis via downregulated transforming growth factor-β/Smad signaling. Maladaptive repair in the microvasculature after AKI further exacerbated focal hypoxia with capillary rarefaction, which was rescued by stabilization of hypoxia-inducible factor and increased tubular vascular endothelial growth factor A in PAR-1-deficient mice. Chronic inflammation was also prevented with reduced kidney infiltration by both M1- and M2-polarized macrophages. In thrombin-induced human dermal microvascular endothelial cells (HDMECs), PAR-1 mediated vascular injury through activation of NF-κB and ERK MAPK pathways. Gene silencing of PAR-1 exerted microvascular protection via a tubulovascular crosstalk mechanism during hypoxia in HDMECs. Finally, pharmacologic blockade of PAR-1 with vorapaxar improved kidney morphology, promoted vascular regenerative capacity, and reduced inflammation and fibrosis depending on the time of initiation. Our findings elucidate a detrimental role of PAR-1 in vascular dysfunction and profibrotic responses upon tissue injury during AKI-to-CKD transition and provide an attractive therapeutic strategy for post-injury repair in AKI.

Single-Cell Analysis Reveals Distinct Immune and Smooth Muscle Cell Populations that Contribute to Chronic Thromboembolic Pulmonary Hypertension.

Rationale: Chronic thromboembolic pulmonary hypertension (CTEPH) is a sequela of acute pulmonary embolism (PE) in which the PE remodels into a chronic scar in the pulmonary arteries. This results in vascular obstruction, pulmonary microvasculopathy, and pulmonary hypertension. Objectives: Our current understanding of CTEPH pathobiology is primarily derived from cell-based studies limited by the use of specific cell markers or phenotypic modulation in cell culture. Therefore, our main objective was to identify the multiple cell types that constitute CTEPH thrombusy and to study their dysfunction. Methods: Here we used single-cell RNA sequencing of tissue removed at the time of pulmonary endarterectomy surgery from five patients to identify the multiple cell types. Using invitro assays, we analyzed differences in phenotype between CTEPH thrombus and healthy pulmonary vascular cells. We studied potential therapeutic targets in cells isolated from CTEPH thrombus. Measurements and Main Results: Single-cell RNA sequencing identified multiple cell types, including macrophages, T cells, and smooth muscle cells (SMCs), that constitute CTEPH thrombus. Notably, multiple macrophage subclusters were identified but broadly split into two categories, with the larger group characterized by an upregulation of inflammatory signaling predicted to promote pulmonary vascular remodeling. CD4+ and CD8+ T cells were identified and likely contribute to chronic inflammation in CTEPH. SMCs were a heterogeneous population, with a cluster of myofibroblasts that express markers of fibrosis and are predicted to arise from other SMC clusters based on pseudotime analysis. Additionally, cultured endothelial, smooth muscle, and myofibroblast cells isolated from CTEPH fibrothrombotic material have distinct phenotypes from control cells with regard to angiogenic potential and rates of proliferation and apoptosis. Last, our analysis identified PAR1 (protease-activated receptor 1) as a potential therapeutic target that links thrombosis to chronic PE in CTEPH, with PAR1 inhibition decreasing SMC and myofibroblast proliferation and migration. Conclusions: These findings suggest a model for CTEPH similar to atherosclerosis, with chronic inflammation promoted by macrophages and T cells driving vascular remodeling through SMC modulation, and suggest new approaches for pharmacologically targeting this disease.

The Effect of Protease-Activated Receptor-1 (PAR-1) Inhibition on Endothelial-Related Biomarkers in Patients with Coronary Artery Disease.

Vorapaxar has been shown to reduce cardiovascular mortality in post-myocardial infarction (MI) patients. Pharmacodynamic biomarker research related to protease-activated receptor-1 (PAR-1) inhibition with vorapaxar in humans has short follow-up (FU) duration and is mainly focused on platelets rather than endothelial cells. This article assesses systemic changes in endothelial-related biomarkers during vorapaxar treatment compared with placebo at 30 days' FU and beyond, in patients with coronary heart disease. Local substudy patients in Norway were included consecutively from two randomized controlled trials; post-MI subjects from TRA2P-TIMI 50 and non-ST-segment elevation MI (NSTEMI) patients from TRACER. Aliquots of citrated blood were stored at -80°C. Angiopoietin-2, angiopoietin-like 4, vascular endothelial growth factor, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin, von Willebrand factor, thrombomodulin, and plasminogen activator inhibitor-1 and -2 were measured at 1-month FU and at study completion (median 2.3 years for pooled patients). A total of 265 consecutive patients (age median 62.0, males 83%) were included. Biomarkers were available at both FUs in 221 subjects. In the total population, angiopoietin-2 increased in patients on vorapaxar as compared with placebo at 1-month FU (p = 0.034). Angiopoietin-like 4 increased (p = 0.028) and plasminogen activator inhibitor-2 decreased (p = 0.025) in favor of vorapaxar at final FU. In post-MI subjects, a short-term increase in E-selectin favoring vorapaxar was observed, p = 0.029. Also, a short-term increase in von Willebrand factor (p = 0.032) favoring vorapaxar was noted in NSTEMI patients. Significant endothelial biomarker changes during PAR-1 inhibition were observed in post-MI and NSTEMI patients.

Protease Activated Receptor-1 (PAR-1) Inhibition By Parmodulin 2, but Not Vorapaxar, Protects Sickle Cell Mice from Heme-Induced Acute Chest Syndrome

Background: Acute chest syndrome (ACS) is a leading cause of ICU admission and mortality in sickle cell patients, characterized by hypoxemia, fever, and significant chest pain. The main cause for ACS in SCD is hemolytic anemia, which elevates circulating heme levels. A model of ACS identified that excess heme induces acute lung injury (ALI) dependent on TLR4 and endothelial p-selectin in sickle cell mice. Despite increased understanding of the etiology of ACS, current treatments are either supportive or not targeted. We have shown that thrombin-dependent protease activated receptor-1 (PAR-1) signaling contributes to heme-induced vascular stasis via upregulation of endothelial p-selectin. Thrombin/PAR-1 signaling is cytotoxic and pro-inflammatory, whereas activation of PAR-1 by activated protein C (APC) is cytoprotective. We hypothesize that PAR-1 signaling contributes to the pathogenesis of ACS. Objective: To investigate the role of PAR-1 in a mouse model of heme-induced ACS using two PAR-1 inhibitors: vorapaxar or parmodulin-2 (PM2). Methods: Townes wild type (AA) and sickle (SS) mice (3-4 months) were used in three experiments. 1) AA females (n=4), SS males (n=6) and SS females (n=11) received 140 µmol/kg heme via retro-orbital injection. 2) SS females received vehicle (n=6) or Vorapaxar (105 µmolar/kg BW, oral gavage) (n=7) daily for 5 days, then treated with heme (140 µmol/kg). 3) SS females received vehicle (n=5) or PM2 (10 mg/kg, intraperitoneal, n=5) 30 minutes prior to heme (140µmol/kg). In all experiments, mice were monitored for signs of ALI (endpoint criteria: severe discomfort with disability) and lungs were collected for histopathology at endpoint or 120 mins after heme infusion. Results: Heme infusion caused 11/11 of SS females to reach endpoint (median survival = 18 min, p=0.0006 vs AA and SS males), whereas only 2/6 of SS males and 0/4 AA mice reached endpoint. Correlation analysis for RBCs and endpoint time showed that SS females with RBC counts less than 7 x 1012/L were vulnerable to heme-induced ACS; yet even males with significant anemia did not reach endpoint. The lung histology showed alveolar thickening, edema and hemorrhage in heme-treated SS females. Hence, only female SS mice were used for the remaining studies. Interestingly, vorapaxar did not protect SS mice from heme-induced ACS (5/7 mice, median survival 17 min) compared to vehicle treatment (4/6 mice, median survival 16 min). In contrast, PM2 treatment significantly protected the SS mice from heme induced ACS with 0/5 reaching endpoint, compared to 4/5 of vehicle -treated SS mice reaching endpoint (p=0.013), and prevented lung damage observed by histology. Conclusions: We found that female mice are more susceptible to heme-induced ACS than males. Vorapaxar, an irreversible antagonist of PAR-1 which blocks all PAR-1 signaling pathways, did not protect SS mice against heme-induced ACS. In contrast, PM2 completely alleviated heme-induced lung injury and death in SS mice. This could be attributed to the fact that PM2 allosterically blocks thrombin/PAR-1 signaling while preserving cytoprotective signaling similar to that mediated by APC. Targeting PAR-1 by preserving (or inducing) beneficial signaling might be effective in preventing the development of ACS.

Abstract 10315: The Effect of Protease-Activated Receptor-1 Inhibition With Vorapaxar in Post-mi and Nstemi Patients on Selected Biomarkers Reflecting Endothelial Function

Introduction: Biomarker research related to protease-activated receptor-1 (PAR-1) inhibition with vorapaxar in humans has essentially focused on platelets. Follow-up (FU) time has been short, whereas in the current study, focus has been on endothelial function during both short- and long-term PAR-1 inhibition. Aim: To assess short- and long-term effects of vorapaxar (V) as compared to Placebo (PL) on the following biomarkers: Angiopoietin-2 (ANGPT2), Angiopoietin-like 4 (ANGPTL4), VEGF, ICAM-1, VCAM-1, E-Selectin (ESEL), von Willebrand Factor (VWF), Thrombomodulin (TM), PAI-1 and PAI-2. Methods: In an independent collective subproject performed in Norway, post-MI patients at steady state were recruited from the “Trial to Assess the Effects of Vorapaxar in Preventing Heart Attack and Stroke in Patients with Atherosclerosis” (TRA2ᵒP-TIMI 50) and NSTEMI patients were recruited from the “Thrombin Receptor Antagonist for Clinical Event Reduction in acute coronary syndrome” (TRACER) trial. Biomarkers were measured in duplicate by enzyme immunoassays (EIA) in citrated plasma at one month follow-up and at study completion (median 2.3 years) for subjects recruited from both trials. Results: Biomarkers were measured in 265 consecutive patients [age median 62.0 years (Q1-Q3: 55.0-68.0 years), males 83%] with at least one change from baseline value. Among these, biomarkers were available at both short- and long-term follow-up in 221 subjects. ANGPT2 increased significantly in V as compared to PL at 1-month follow-up in the total population (p=0.034), and in males in both post-MI (p=0.031) and NSTEMI subjects (p=0.012), respectively. ANGPTL4 increased (p=0.028) and PAI-2 decreased (p=0.025) significantly in the total population in favor of V at final FU. In the total post-MI subgroup and among males of that group, a short-term significant increase in ESEL in favor of V was observed, p=0.029 and p=0.018, respectively. Also, a transient significant increase in VWF (p=0.032) in favor of V was seen at one month in NSTEMI patients. Conclusions: Significant changes suggesting potential harmful effects in some biomarkers were observed during 1-month and long-term PAR-1 inhibition as compared to placebo in post-MI and acute coronary syndrome patients.

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

Pleiotropic Effects of the Protease-Activated Receptor 1 (PAR1) Inhibitor, Vorapaxar, on Atherosclerosis and Vascular Inflammation

Background: Protease-activated receptor 1 (PAR1) and toll-like receptors (TLRs) are inflammatory mediators contributing to atherogenesis and atherothrombosis. Vorapaxar, which selectively antagonizes PAR1-signaling, is an approved, add-on antiplatelet therapy for secondary prevention. The non-hemostatic, platelet-independent, pleiotropic effects of vorapaxar have not yet been studied. Methods and Results: Cellular targets of PAR1 signaling in the vasculature were identified in three patient cohorts with atherosclerotic disease. Evaluation of plasma biomarkers (n = 190) and gene expression in endomyocardial biopsies (EMBs) (n = 12) revealed that PAR1 expression correlated with endothelial activation and vascular inflammation. PAR1 colocalized with TLR2/4 in human carotid plaques and was associated with TLR2/4 gene transcription in EMBs. In addition, vorapaxar reduced atherosclerotic lesion size in apolipoprotein E–knock out (ApoEko) mice. This reduction was associated with reduced expression of vascular adhesion molecules and TLR2/4 presence, both in isolated murine endothelial cells and the aorta. Thrombin-induced uptake of oxLDL was augmented by additional TLR2/4 stimulation and abrogated by vorapaxar. Plaque-infiltrating pro-inflammatory cells were reduced in vorapaxar-treated ApoEko mice. A shift toward M2 macrophages paralleled a decreased transcription of pro-inflammatory cytokines and chemokines. Conclusions: PAR1 inhibition with vorapaxar may be effective in reducing residual thrombo-inflammatory event risk in patients with atherosclerosis independent of its effect on platelets.

Lack of miRNA-17 family mediates high glucose-induced PAR-1 upregulation in glomerular mesangial cells.

Upregulation of thrombin receptor protease-activated receptor 1 (PAR-1) is verified to contribute to chronic kidney diseases, including diabetic nephropathy; however, the mechanisms are still unclear. In this study, we investigated the effect of PAR-1 on high glucose-induced proliferation of human glomerular mesangial cells (HMCs), and explored the mechanism of PAR-1 upregulation from alteration of microRNAs. We found that high glucose stimulated proliferation of the mesangial cells whereas PAR-1 inhibition with vorapaxar attenuated the cell proliferation. Moreover, high glucose upregulated PAR-1 in mRNA level and protein expression while did not affect the enzymatic activity of thrombin in HMCs after 48h culture. Then high glucose induced PAR-1 elevation was likely due to the alteration of the transcription or post-transcriptional processing. It was found that miR-17 family members including miR-17-5p, -20a-5p, and -93-5p were significantly decreased among the eight detected microRNAs only in high glucose-cultured HMCs, but miR-129-5p, miR-181a-5p, and miR-181b-5p were markedly downregulated in both high glucose-cultured HMCs and equivalent osmotic press control compared with normal glucose culture. So miR-20a was selected to confirm the role of miR-17 family on PAR-1 upregulation, finding that miR-20a-5p overexpression reversed the upregulation of PAR-1 in mRNA and protein levels induced by high glucose in HMCs. In summary, our finding indicated that PAR-1 upregulation mediated proliferation of glomerular mesangial cells induced by high glucose, and deficiency of miR-17 family resulted in PAR-1 upregulation.

Hirudin, a thrombin inhibitor, attenuates TGF-β-induced fibrosis in renal proximal tubular epithelial cells by inhibition of protease-activated receptor 1 expression via S1P/S1PR2/S1PR3 signaling.

Renal interstitial fibrosis (RIF) is the final common outcome of numerous chronic kidney diseases, contributing to end-stage renal disease. Hirudin, a thrombin inhibitor, has attracted increased attention as a potential treatment approach for renal fibrosis. The present study aimed to investigate the molecular mechanism underlying the effect of hirudin on fibrosis in renal proximal tubular epithelial cells. An in vivo mouse RIF model established using unilateral ureteral obstruction (UUO) and an in vitro of RIF using the renal tubular epithelial cell line HK-2 treated with TGF-β were used. Expressions of sphingosine-1-phosphate (S1P) receptors (S1PR)1-4 and protease-activated receptor 1 (PAR1) were measured by reverse transcription-quantitative PCR and western blotting in mice with UUO and TGF-β induced HK-2 cells. Western blotting was used to detect the expression of N-cadherin, Slug, E-cadherin, Collagen IV, fibronectin, MMP9 and monocyte chemoattractant protein-1. Immunofluorescence staining was conducted to measure α-SMA level expression. The results demonstrated that the expression levels of S1PR1, S1PR2, S1PR3, S1PR4 and PAR1 were upregulated in both TGF-β-induced HK-2 cells and renal tissues from mice with unilateral ureteral ligation. Notably, hirudin inhibited TGF-β-induced PAR1, S1PR2 and S1PR3 upregulation in both HK-2 cells and renal tissues. Additionally, the inhibition of S1PR2 and S1PR3 resulted in PAR1 downregulation. Furthermore, treatment with S1P and PAR1 agonists abolished the effect of hirudin on the expression of EMT, fibrosis-related proteins and monocyte chemoattractant protein 1. In conclusion, hirudin attenuated TGF-β-induced fibrosis in proximal renal tubular epithelial HK-2 cells by inhibiting PAR1 expression via the S1P/S1PR2/S1PR3 signaling pathway. Therefore, hirudin may be considered as a promising therapeutic agent for RIF.

Deficiency of MMP1a (Matrix Metalloprotease 1a) Collagenase Suppresses Development of Atherosclerosis in Mice: Translational Implications for Human Coronary Artery Disease.

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Inhibition of protease-activated receptor 1 (PAR1) ameliorates cognitive performance and synaptic plasticity impairments in animal model of Alzheimer's diseases.

Alzheimer's disease (AD) is a progressive brain disorder accompanied with synaptic failures and decline in cognitive and learning processes. Protease-activated receptor 1 (PAR1) is the major thrombin receptor in the brain that is implicated in synaptic plasticity and memory formation. In the current study, we hypothesized that inhibition of PAR1 would theoretically prevent amyloid beta (Aβ) accumulation in the brain and then contribute to reduce risk of AD. The aim of the present study was to evaluate the effect of PAR1 inhibition by using SCH (as an inhibitor of PAR1) on spatial learning, memory, and synaptic plasticity in the CA1 region of the hippocampus in rat model of Alzheimer's disease. For the induction of Alzheimer's disease, amyloid beta (Aβ) 1-42 was injected in the CA1 region of the hippocampus. The rats were divided into four groups: group I (surgical sham); group II rat mode of Alzheimer's disease (AD); group III (SCH) (25 μg/kg) intraperitoneally (i.p.), and group IV (AD + SCH). After 14 days of protocol, the rats in group III received SCH and 30 min after injection behavioral and electrophysiological tests were performed. Learning and memory ability was assessed by Morris water maze and novel object recognition tests. Extracellular evoked field excitatory postsynaptic potentials (fEPSP) were recorded in the stratum radiatum of the CA1 area. Our results showed that AD rats showed impairments in learning and memory, and long-term potentiation (LTP) was not induced in these rats. However, injection of SCH overcame the AD-induced impairment in LTP generation in the CA1 area of the hippocampus and improved learning and memory impairment.

Protease-Activated Receptor 1 Contributes to Microcirculation Failure and Tubular Damage in Renal Ischemia-Reperfusion Injury in Mice.

Ischemia-reperfusion- (IR-) induced kidney injury is difficult to avoid during renal transplantation and robot-assisted partial nephrectomy. Renal IR injury is characterized by tubular damage, microcirculation failure, and inflammation, which coordinately augment renal injury; however, no specific treatment is available for these conditions. Protease-activated receptor-1 (PAR-1) and its ligand, thrombin, are involved in coagulation and were shown to be associated with epithelial cell injury. Here, we hypothesized that PAR-1 exaggerated renal IR-induced tubular cell damage and microcirculation failure and that pharmacological inhibition of PAR-1 by Q94 could prevent these injuries. Renal warm IR increased the expression of PAR-1 in the renal tubules. Q94 attenuated renal IR-induced changes and histopathological damage. Microcirculation failure analyzed by congestion in the histopathology and blood cell flow examined by intravital multiphoton microscopy were suppressed by Q94 treatment. Q94 also dramatically increased tubular cell proliferation despite the lower renal damage. Thrombin suppressed cell proliferation and induced apoptosis in the tubules; these effects were prevented by Q94 treatment. Taken together, PAR-1 was associated with renal IR injury. Inhibition of PAR-1 ameliorated injury possibly by improving renal microcirculation and tubular cell survival/proliferation.

The thrombin receptor links brain derived neurotrophic factor to neuron cholesterol production, resiliency and repair after spinal cord injury

Despite concerted efforts to identify CNS regeneration strategies, an incomplete understanding of how the needed molecular machinery is regulated limits progress. Here we use models of lateral compression and FEJOTA clip contusion-compression spinal cord injury (SCI) to identify the thrombin receptor (Protease Activated Receptor 1 (PAR1)) as an integral facet of this machine with roles in regulating neurite growth through a growth factor- and cholesterol-dependent mechanism. Functional recovery and signs of neural repair, including expression of cholesterol biosynthesis machinery and markers of axonal and synaptic integrity, were all increased after SCI in PAR1 knockout female mice, while PTEN was decreased. Notably, PAR1 differentially regulated HMGCS1, a gene encoding a rate-limiting enzyme in cholesterol production, across the neuronal and astroglial compartments of the intact versus injured spinal cord. Pharmacologic inhibition of cortical neuron PAR1 using vorapaxar in vitro also decreased PTEN and promoted neurite outgrowth in a cholesterol dependent manner, including that driven by suboptimal brain derived neurotrophic factor (BDNF). Pharmacologic inhibition of PAR1 also augmented BDNF-driven HMGCS1 and cholesterol production by murine cortical neurons and by human SH-SY5Y and iPSC-derived neurons. The link between PAR1, cholesterol and BDNF was further highlighted by demonstrating that the deleterious effects of PAR1 over-activation are overcome by supplementing cultures with BDNF, cholesterol or by blocking an inhibitor of adenylate cyclase, Gαi. These findings document PAR1-linked neurotrophic coupling mechanisms that regulate neuronal cholesterol metabolism as an important component of the machinery regulating CNS repair and point to new strategies to enhance neural resiliency after injury.

Increased Mucosal Thrombin is Associated with Crohn's Disease and Causes Inflammatory Damage through Protease-activated Receptors Activation.

Background and AimsThrombin levels in the colon of Crohn’s disease patients have recently been found to be elevated 100-fold compared with healthy controls. Our aim was to determine whether and how dysregulated thrombin activity could contribute to local tissue malfunctions associated with Crohn’s disease.MethodsThrombin activity was studied in tissues from Crohn’s disease patients and healthy controls. Intracolonic administration of thrombin to wild-type or protease-activated receptor-deficient mice was used to assess the effects and mechanisms of local thrombin upregulation. Colitis was induced in rats and mice by the intracolonic administration of trinitrobenzene sulphonic acid.ResultsActive forms of thrombin were increased in Crohn’s disease patient tissues. Elevated thrombin expression and activity were associated with intestinal epithelial cells. Increased thrombin activity and expression were also a feature of experimental colitis in rats. Colonic exposure to doses of active thrombin comparable to what is found in inflammatory bowel disease tissues caused mucosal damage and tissue dysfunctions in mice, through a mechanism involving both protease-activated receptors -1 and -4. Intracolonic administration of the thrombin inhibitor dabigatran, as well as inhibition of protease-activated receptor-1, prevented trinitrobenzene sulphonic acid-induced colitis in rodent models.ConclusionsOur data demonstrated that increased local thrombin activity, as it occurs in the colon of patients with inflammatory bowel disease, causes mucosal damage and inflammation. Colonic thrombin and protease-activated receptor-1 appear as possible mechanisms involved in mucosal damage and loss of function and therefore represent potential therapeutic targets for treating inflammatory bowel disease.

Vorapaxar-modified polysulfone membrane with high hemocompatibility inhibits thrombosis

Hemodialysis therapy is intended for patients suffering from renal insufficiency, pancreatitis, and other serious diseases. Platelets are an important active ingredient in the thrombosis induced by hemodialysis membranes. So far, there are few studies of hemodialysis membranes focusing on the effects of protease-activated receptor 1 (PAR1) activation on the platelet membrane. Among various antithrombotic agents, vorapaxar is a novel PAR1 inhibitor with high efficacy. In this study, we constructed a vorapaxar-modified polysulfone (VMPSf) membrane using immersion-precipitation phase transformation methods and characterized the microstructure in terms of hydrophilicity and mechanical properties. The water contact angle of the VMPSf membrane was 22.45% lower than that of the PSf membrane. A focused determination of platelet morphology was obtained using scanning electron microscopy. Meanwhile, we evaluated the effects of a VMPSf membrane on platelet adhesion. We observed that the VMPSf membrane could reduce the number of adhered platelets without altering their spherical or elliptical shape. The PAR1 levels in VMPSf membranes were 7.4 MFI lower than those in PSf membranes, suggesting that this modified membrane can effectively inhibit platelet activation. Activated partial thromboplastin time (APTT, 5.3 s extension) and thrombin time (TT, 2.1 s extension) reflect good anticoagulant properties. Recalcification time (80.6 s extension) and fibrinogen adsorption (9.9 μg/cm2 reduction) were related to antithrombotic properties. To determine the biosafety of VMPSf membranes, we investigated antianaphylactic and anti-inflammatory properties in vitro and acute toxicity in vivo, it was obvious that C3a and C5a had decreased to 9.6 and 0.8 ng/mL, respectively. The results indicated that the VMPSf membrane has potential for clinical application.

Testing the effect of PAR1 inhibitors on Plasmodium falciparum-induced loss of endothelial cell barrier function.

Background: Sequestration and cytoadherence of Plasmodium falciparum-infected erythrocytes (IE) to microvascular endothelium alters endothelial barrier function and plays a role in the pathogenesis of severe malaria. Binding of IE is mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1) and the PfEMP1 variants that binds to endothelial protein C receptor (EPCR) have, in particular, been associated with the dysregulation of the coagulation/inflammation pathways in endothelial cells. This has prompted speculation about the role of protease-activated receptor-1 (PAR1) activation and signalling in causing endothelial activation and loss of barrier function in cerebral malaria. Methods: We used a co-culture of primary human brain microvascular endothelial cells (HBMEC) with P. falciparum material, recombinant PfEMP1 or lysates from IE, and measured barrier function by trans endothelial electrical resistance (TEER). A selection of PAR1 inhibitors was tested for their ability to reverse the P. falciparum and thrombin induced decrease in barrier function. Results: An initial screen in the presence of recombinant PfEMP1 identified a few inhibitors that were able to reduce the rapid thrombin-induced barrier disruption even when activated protein C (aPC) was unable to do so. However, PAR1 inhibitors did not rescue the barrier dysfunction after co-culture with IE lysate. Conclusions: The selected PAR1 inhibitors were able to reverse the disruption of barrier function by thrombin but did not reverse the IE lysate induced disruption of barrier function, implicating a different PAR1-independent mechanism. These findings have implications for the design of adjunct therapies to reduce brain swelling in cerebral malaria.

Twist-mediated PAR1 induction is required for breast cancer progression and metastasis by inhibiting Hippo pathway

Breast cancer is considered to be the most prevalent cancer in women worldwide, and metastasis is the primary cause of death. Protease-activated receptor 1 (PAR1) is a GPCR family member involved in the invasive and metastatic processes of cancer cells. However, the functions and underlying mechanisms of PAR1 in breast cancer remain unclear. In this study, we found that PAR1 is highly expressed in high invasive breast cancer cells, and predicts poor prognosis in ER-negative and high-grade breast cancer patients. Mechanistically, Twist transcriptionally induces PAR1 expression, leading to inhibition of Hippo pathway and activation of YAP/TAZ; Inhibition of PAR1 suppresses YAP/TAZ-induced epithelial-mesenchymal transition (EMT), invasion, migration, cancer stem cell (CSC)-like properties, tumor growth and metastasis of breast cancer cells in vitro and in vivo. These findings suggest that PAR1 acts as a direct transcriptionally target of Twist, can promote EMT, tumorigenicity and metastasis by controlling the Hippo pathway; this may lead to a potential therapeutic target for treating invasive breast cancer.

Testing the effect of PAR1 inhibitors on Plasmodium falciparum-induced loss of endothelial cell barrier function

Background: Sequestration and cytoadherence of Plasmodium falciparum-infected erythrocytes (IE) to microvascular endothelium alters endothelial barrier function and plays a role in the pathogenesis of severe malaria. Binding of IE is mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1) and the PfEMP1 variants that binds to endothelial protein C receptor (EPCR) have, in particular, been associated with the dysregulation of the coagulation/inflammation pathways in endothelial cells. This has prompted speculation about the role of protease-activated receptor-1 (PAR1) activation and signalling in causing endothelial activation and loss of barrier function in cerebral malaria. Methods: We used a co-culture of primary human brain microvascular endothelial cells (HBMEC) with P. falciparum material, recombinant PfEMP1 or lysates from IE, and measured barrier function by trans endothelial electrical resistance (TEER). A selection of PAR1 inhibitors was tested for their ability to reverse the P. falciparum and thrombin induced decrease in barrier function. Results: An initial screen in the presence of recombinant PfEMP1 identified a few inhibitors that were able to reduce the rapid thrombin-induced barrier disruption even when activated protein C (aPC) was unable to do so. However, PAR1 inhibitors did not rescue the barrier dysfunction after co-culture with IE lysate. Conclusions: The selected PAR1 inhibitors were able to reverse the disruption of barrier function by thrombin but did not reverse the IE lysate induced disruption of barrier function, implicating a different PAR1-independent mechanism. These findings have implications for the design of adjunct therapies to reduce brain swelling in cerebral malaria.

Testing the effect of PAR1 inhibitors on Plasmodium falciparum-induced loss of endothelial cell barrier function

Background: Sequestration and cytoadherence of Plasmodium falciparum-infected erythrocytes (IE) to microvascular endothelium alters endothelial barrier function and plays a role in the pathogenesis of severe malaria. Binding of IE is mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1) and the PfEMP1 variants that binds to endothelial protein C receptor (EPCR) have, in particular, been associated with the dysregulation of the coagulation/inflammation pathways in endothelial cells. This has prompted speculation about the role of protease-activated receptor-1 (PAR1) activation and signalling in causing endothelial activation and loss of barrier function in cerebral malaria. Methods: We used a co-culture of primary human brain microvascular endothelial cells (HBMEC) with P. falciparum material, recombinant PfEMP1 or lysates from IE, and measured barrier function by trans endothelial electrical resistance (TEER). A selection of PAR1 inhibitors was tested for their ability to reverse the P. falciparum and thrombin induced decrease in barrier function. Results: An initial screen in the presence of recombinant PfEMP1 identified a few inhibitors that were able to reduce the rapid thrombin-induced barrier disruption even when activated protein C (aPC) was unable to do so. However, PAR1 inhibitors did not rescue the barrier dysfunction after co-culture with IE lysate. Conclusions: The selected PAR1 inhibitors were able to reverse the disruption of barrier function by thrombin but did not reverse the IE lysate induced disruption of barrier function, implicating a different PAR1-independent mechanism. These findings have implications for the design of adjunct therapies to reduce brain swelling in cerebral malaria.