Detection of proteinase activated receptor stimulating enzymes in human arthritic knee joint fluids

Abstract

Purpose: Osteoarthritis (OA) is a major health care burden worldwide. OA can affect all joints but is manifest most often in the knees, hips, big toe hands and spine, and results in joint inflammation, pain and stiffness. There is currently no effective cure or therapeutic interventions for OA. Therefore, there is a need for further studies into mechanisms underlying disease progression and for the identification of novel therapeutic targets. A key feature of OA is the progressive degradation of the cartilage. It is well established that there is an increase in proteolytic enzyme activity that is linked to this damage. We hypothesized that in addition to degrading the extracellular matrix, increased proteolytic activity in the arthritic joint could also signal through the enzymatically activated Proteinase Activated Receptor (PAR) family of G-protein coupled receptors. Methods: To study the hypothesis, twenty human knee joint synovial fluid samples derived from OA patients undergoing realignment osteotomy were screened for PAR cleavage/activity in CHO cells stably transfected with genetically encoded PAR1 or PAR2 sensors (nluc-hPAR1-eYFP and nluc-hPAR2-eYFP). The N-terminal nano-luciferase released upon proteolytic cleavage of the receptor was measured as a luminescence signal in the presence of the substrate furimazine in a 96-well microplate reader. To determine the class/type of enzymes, the test samples were pretreated with enzyme inhibitors and the receptor cleavage was measured using the PAR sensors. In addition, fluorogenic enzyme substrates were used to identify the class of enzymes using a fluorescence microplate reader. Results: PAR activating enzymes were identified at significant levels in the OA synovial fluid samples derived from twenty patients. Through the use of class/type selective enzyme inhibitors and fluorogenic substrates it was found that serine protease class enzymes including thrombin and trypsin-like, and matrix metalloproteinases are present in the inflamed knee joint samples. Further work aimed at identifying the specific enzymes is underway using activity-based probes and mass-spectrometry analysis. Conclusions: It is known that PAR activation is pro-inflammatory and inhibition of these receptors with specific antagonists may be beneficial in treating OA. A better understanding of PAR activators and the pathological signaling pathways triggered by these receptors will guide development of novel therapies to treat joint diseases.