The activation fragment of PAR2 is elevated in serum from patients with rheumatoid arthritis and reduced in response to anti-IL6R treatment.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are serious and painful diseases. Protease-activated receptor 2 (PAR2) is involved in the pathology of both OA and RA including roles in synovial hyperplasia, cartilage destruction, osteophyogenesis and pain. PAR2 is activated via cleavage of its N-terminus by serine proteases. In this study a competitive ELISA assay was developed targeting the 36-amino acid peptide that is cleaved and released after PAR2 activation (PRO-PAR2). Technical assay parameters including antibody specificity, intra- and inter-assay variation (CV%), linearity, accuracy, analyte stability and interference were evaluated. PRO-PAR2 release was confirmed after in vitro cleavage of PAR2 recombinant protein and treatment of human synovial explants with matriptase. Serum levels of 22 healthy individuals, 23 OA patients and 15 RA patients as well as a subset of RA patients treated with tocilizumab were evaluated. The PRO-PAR2 antibody was specific for the neo-epitope and intra-inter assay CV% were 6.4% and 5.8% respectively. In vitro cleavage and matriptase treated explants showed increased PRO-PAR2 levels compared to controls. In serum, PRO-PAR2 levels were increased in RA patients and decreased in RA patients treated with tocilizumab. In conclusion, PRO-PAR2 may be a potential biomarker for monitoring RA disease and pharmacodynamics of treatment.

Peripheral blood intermediate monocyte protease-activated receptor-2 expression increases during asthma exacerbations and after inhalation allergen challenge.

BACKGROUND: Myeloid cells, especially dendritic cells and macrophages, play important roles in asthma pathophysiology. Monocytes (Mo) and macrophages express protease-activated receptor-2 (PAR-2), a proinflammatory serine protease receptor implicated in the pathophysiology of allergic airway inflammation. We have revealed that patients with severe asthma and those with a history of frequent asthma exacerbations exhibit increased PAR-2 expression on peripheral blood monocytes. OBJECTIVE: To determine PAR-2 expression on peripheral blood intermediate monocytes (IMMo) in subjects with increased airway inflammation, either as a result of an asthma exacerbation or after an inhalation allergen challenge. METHODS: A total of 16 adults who presented to the emergency department with asthma exacerbations were recruited after giving an informed consent. After 2 weeks, 10 patients returned for follow-up. A total of 11 patients with mild asthma treated only with as-needed bronchodilators were recruited and underwent inhalation allergen challenge after providing an informed consent. Immune cell profiling was performed by whole blood flow cytometry in both groups of patients. RESULTS: PAR-2 expression in peripheral blood IMMo increased in patients with an asthma exacerbation compared with those with stable disease, but this expression decreased after treatment of the asthma exacerbation. Subjects with mild asthma had an increase in percentages of IMMo expressing PAR-2 after an allergen challenge. Patients who presented to the emergency department had lower dendritic cell and dendritic cell subset numbers in peripheral blood during exacerbation compared with after treatment. CONCLUSION: Increased PAR-2 expression on Mo during periods of increased airway inflammation may initiate a positive feedback loop leading to systemic inflammatory changes.

Upregulation of Protease-Activated Receptor 2 Promotes Proliferation and Migration of Human Vascular Smooth Muscle Cells (VSMCs).

BACKGROUND Protease-Activated Receptor 2 (PAR2), a G-protein-coupled receptor, has been proved to be enhanced in human coronary atherosclerosis lesions. We aimed to investigate whether PAR2 actively participates in the atherosclerosis process. MATERIAL AND METHODS PAR2 expression was assessed in blood samples by RT-qPCR from healthy controls and patients with atherosclerosis. Human vascular smooth muscle cells (VSMCs) were treated with oxidative low-density lipoprotein (ox-LDL). After PAR2 overexpression by transfection, cell proliferation was determined by CCK-8, and cell migration was evaluated by Transwell assay. The protein expressions associated with cell growth and migration were measured by Western blot. The distribution of alpha-SMA in VSMCs was evaluated by immunofluorescence. RESULTS Expression of PAR2 was higher in patients with atherosclerosis compared with normal controls. PAR2 mRNA and protein expression was increased in ox-LDL-treated VSMCs compared with control cells. Induced overexpression of PAR2 in VSMCs led to a reduction in alpha-SMA expression compared to controls. In addition, PAR2 overexpression caused increased migration compared to normal controls, and upregulated MMP9 and MMP14 expression. PAR-2 overexpression promoted cell proliferation compared to control cells, and increased expression levels of CDK2, and CyclinE1, but reduced levels of p27. We preliminary explored the potential mechanism of PAR2, and results showed that overexpression of PAR2 increased expression levels of VEGFA and Angiopoietin 2 compared to controls. Moreover, overexpression of PAR2 enhanced production of tissue factor and IL-8 compared to normal controls. CONCLUSIONS PAR2 promotes cell proliferation and disrupts the quiescent condition of VSMCs, which may be a potential therapeutic target for atherosclerosis.

Tissue factor at the crossroad of coagulation and cell signaling.

The tissue factor (TF) pathway plays a central role in hemostasis and thrombo-inflammatory diseases. Although structure-function relationships of the TF initiation complex are elucidated, new facets of the dynamic regulation of TF's activities in cells continue to emerge. Cellular pathways that render TF non-coagulant participate in signaling of distinct TF complexes with associated proteases through the protease-activated receptor (PAR) family of G protein-coupled receptors. Additional co-receptors, including the endothelial protein C receptor (EPCR) and integrins, confer signaling specificity by directing subcellular localization and trafficking. We here review how TF is switched between its role in coagulation and cell signaling through thiol-disulfide exchange reactions in the context of physiologically relevant lipid microdomains. Inflammatory mediators, including reactive oxygen species, activators of the inflammasome, and the complement cascade play pivotal roles in TF procoagulant activation on monocytes, macrophages and endothelial cells. We furthermore discuss how TF, intracellular ligands, co-receptors and associated proteases are integrated in PAR-dependent cell signaling pathways controlling innate immunity, cancer and metabolic inflammation. Knowledge of the precise interactions of TF in coagulation and cell signaling is important for understanding effects of new anticoagulants beyond thrombosis and identification of new applications of these drugs for potential additional therapeutic benefits.

Thrombin is a selective inducer of heparanase release from platelets and granulocytes via protease-activated receptor-1.

Heparanase, known to be involved in angiogenesis and metastasis, was shown to form a complex with tissue factor (TF) and to enhance the generation of factor Xa. Platelets and granulocytes contain abundant amounts of heparanase that may enhance the coagulation system upon discharge. It was the aim of this study to identify the inducer and pathway of heparanase release from these cells. Platelets and granulocytes were purified from pooled normal plasma and were incubated with ATP, ADP, epinephrine, collagen, ristocetin, arachidonic acid, serotonin, LPS and thrombin. Heparanase levels were assessed by ELISA, heparanase procoagulant activity assay and western blot analysis. The effects of selective protease-activated receptor (PAR)-1 and 2 inhibitors and PAR-1 and 4 activators were studied. An in-house synthesised inhibitory peptide to heparanase was used to evaluate platelet heparanase involvement in activation of the coagulation system. Heparanase was released from platelets only by thrombin induction while other inducers exerted no such effect. The heparanase level in a platelet was found to be 40 % higher than in a granulocyte. Heparanase released from platelets or granulocytes increased factor Xa generation by three-fold. PAR-1 activation via ERK intracellular pathway was found to induce heparanase release. In conclusion, heparanase is selectively released from platelets and granulocytes by thrombin interacting with PAR-1. Heparanase derived from platelets and granulocytes is involved in activation of the extrinsic coagulation pathway. The present study implies on a potential anticoagulant effect, in addition to anti-platelet effect, of the new clinically studied PAR-1 inhibitors.

Increased Protease-Activated Receptor-2 (PAR-2) Expression on CD14++CD16+ Peripheral Blood Monocytes of Patients with Severe Asthma.

BACKGROUND: Protease-Activated Receptor-2 (PAR-2), a G protein coupled receptor activated by serine proteases, is widely expressed in humans and is involved in inflammation. PAR-2 activation in the airways plays an important role in the development of allergic airway inflammation. PAR-2 expression is known to be upregulated in the epithelium of asthmatic subjects, but its expression on immune and inflammatory cells in patients with asthma has not been studied. METHODS: We recruited 12 severe and 24 mild/moderate asthmatics from the University of Alberta Hospital Asthma Clinics and collected baseline demographic information, medication use and parameters of asthma severity. PAR-2 expression on blood inflammatory cells was analyzed by flow cytometry. RESULTS: Subjects with severe asthma had higher PAR-2 expression on CD14++CD16+ monocytes (intermediate monocytes) and also higher percentage of CD14++CD16+PAR-2+ monocytes (intermediate monocytes expressing PAR-2) in blood compared to subjects with mild/moderate asthma. Receiver operating characteristics (ROC) curve analysis showed that the percent of CD14++CD16+PAR-2+ in peripheral blood was able to discriminate between patients with severe and those with mild/moderate asthma with high sensitivity and specificity. In addition, among the whole populations, subjects with a history of asthma exacerbations over the last year had higher percent of CD14++CD16+ PAR-2+ cells in peripheral blood compared to subjects without exacerbations. CONCLUSIONS: PAR-2 expression is increased on CD14++CD16+ monocytes in the peripheral blood of subjects with severe asthma and may be a biomarker of asthma severity. Our data suggest that PAR-2 -mediated activation of CD14++CD16+ monocytes may play a role in the pathogenesis of severe asthma.

Effects of clopidogrel and aspirin in combination versus aspirin alone on platelet activation and major receptor expression in diabetic patients: the PLavix Use for Treatment Of Diabetes (PLUTO-Diabetes) trial.

BACKGROUND: Clopidogrel is widely used in diabetic patients after vascular events; however, the ability of this thienopyridine to yield additional antiplatelet protection on top of aspirin has never been explored in a controlled study with comprehensive assessment of platelet activity. The objective of this study was to compare the antiplatelet profiles of clopidogrel + aspirin in combination (C + ASA) versus aspirin alone (ASA) in patients with type 2 diabetes mellitus. METHODS: Seventy patients with documented diabetes already treated with antecedent aspirin were randomly assigned to receive C + ASA or ASA in the PLUTO-Diabetes trial. Platelet studies included adenosine diphosphate-, collagen-, and arachidonic acid-induced aggregometry; PFA-100 (Dade-Behring, Miami, FL) and Ultegra (Accumetrics, San Diego, CA) analyzers; and expression of 6 major receptors by flow cytometry at baseline and at day 30 after randomization. RESULTS: There were no differences in the baseline clinical and platelet characteristics between the C + ASA and ASA groups, or subsequent significant changes in platelet biomarkers in the ASA group, except for diminished collagen-induced aggregation (P = .02). In contrast, when compared with the ASA group, therapy with C + ASA resulted in significant inhibition of platelet activity assessed by adenosine diphosphate aggregation (P = .0001); closure time prolongation (P = .0003) and reduction of platelet activation units with Ultegra (P = .0001); and expression of platelet/endothelial cell adhesion molecule 1 (P = .002), glycoprotein IIb/IIIa antigen (P = .0002), and activity (P = .0001). CONCLUSION: Treatment with C + ASA for 1 month provides significantly greater inhibition of platelet activity than ASA alone in diabetic patients in this small randomized trial. However, despite dual antiplatelet regimen, diabetic patients exhibit high residual activity of some platelet biomarkers, including unaffected protease-activated receptor 1 receptor expression.