Characterizing Modulators of Protease-Activated Receptors with a Calcium Mobilization Assay Using a Plate Reader

Our lab is working to understand how the modulation of protease activated receptors can inhibit inflammation. We have developed a new class of small molecules called parmodulins that modulate the activity of PAR1. The goal is to discover parmodulins with improved properties for potential use as drug candidates, and also to understand their mode of action.

In preclinical studies with our collaborators, we've determined that parmodulins may be particularly promising for the treatment of disorders driven by thromboinflammation, including kidney, liver, and cardiovascular disease. Their mode of action may also enable the development of compounds with a better safety profile than prior PAR1 targeting drug candidates. The receptor PAR1 is important for activating platelets, but it's signaling in endothelial cells is also important and relevant in numerous pathologies.

Therefore, we wanted to develop a cheap and convenient protocol for testing the activity of parmodulins and cultured endothelial cells. We're here to share an updated protocol measuring calcium mobilization that uses a standard plate reader, and does not require an automated liquid handler. It's suitable for screening PAR1 ligands in endothelial cells, but in theory, it can be used to measure the activity of any compounds affecting calcium mobilization.