Therapeutic Intervention Targeting Mucosal Thrombin Or Protease‐Activated‐Receptor 1 Are Protective Against Colitis
Authors: Motta Jean-Paul , Palese Simone , Sagnat David , Rousset Perrine , Guiraud Laura , Edir Anissa , Seguy Carine , Alric Laurent , Bonnet Delphine , Bournet Barbara , Buscail Louis , Gilletta Cyrielle , Barocelli Elisabetta , Grand Sylvie Le , Grand Bruno Le , Deraison Celine , Vergnolle Nathalie 
Published: April 2020

Background

Current therapies for Inflammatory Bowel Disease (IBD) are unsatisfactory for proper tissue healing. Serine proteases belong to locally produced host factors that can fuel inflammatory processes in tissue from IBD patients, in part through activation of Protease‐Activated Receptors (PAR). We have recently discovered that intestinal epithelium was able to produce active thrombin, suggesting that mucosa itself could be an important source of high thrombin in IBD.

Objectives

We first aimed to determine whether mucosal thrombin was upregulated in animal models of colitis and in tissues from IBD patients. We then determined whether local thrombin upregulation could contribute to local tissue malfunctions. Finally, we evaluated therapeutic feasibility of local delivering of either direct thrombin inhibitors or PAR antagonist in animal models of colitis.

Methods

Colonic tissue samples were obtained from diagnosed IBD patients undergoing colonoscopy at the Toulouse Hospital. Colitis was induced by administering trinitrobenzene sulfonic acid (TNBS) in the colon of Wistar rats or C57Bl6 mice. Human tissue collection and animal procedures received ethical approval from local ethic committees. Thrombin (100 U/ml, 10 days), direct thrombin inhibitor (dabigatran, 1 μg/kg, 4 days) and PAR1 antagonist (Vorapaxar, 2.5 mg/kg, 7 days) were administered in the colon of healthy or TNBS animals under light anesthesia. At time of the sacrifice, colonic tissues were harvested and disease severity was assessed. Thrombin expression was detected using PCR, western blot and immunofluorescence. Thrombin activity was quantified in tissue supernatants using specific enzymatic assays.

Results

We confirmed an increased thrombin protein expression in human mucosal tissue by immunofluorescence and western blots. We found that some, but not all, forms of active thrombin were upregulated, particularly in tissues from Crohn’s disease patients. As observed in human, we found that increased thrombin mRNA expression and activity is also a feature of colitis in animal models of colitis. We demonstrated in vivo that colonic exposure to high dose of active thrombin can cause mucosal damage and tissue dysfunctions. Specific inhibition of thrombin activity, and PAR1 antagonists prevent some intestinal damage in TNBS colitis.

Conclusions

In this study, using both animal models and human IBD tissues, we showed that upregulation of mucosal thrombin alone can lead to inflammatory insults. We propose that targeting downstream events from high thrombin activity, rather than inhibiting thrombin directly, might be a better option for IBD because mucosal thrombin at low dose plays an important role on maintaining tissue homeostasis. Considering these promising preclinical results on PAR1 antagonist, future clinical studies in IBD patients could therefore be rapidly envisioned, particularly in patients with the strongest upregulation of thrombin activity.