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Characterization of Protease-Activated Receptor (PAR) Ligands: Parmodulins Are Reversible Allosteric Inhibitors of PAR1-Driven Calcium Mobilization in Endothelial Cells

preprint
submitted on 21.02.2018 and posted on 21.02.2018 by Disha M. Gandhi, Mark W. Majewski, Ricardo Rosas, Jr., Kaitlin Kentala, Trevor J. Foster, Eric Greve, Chris Dockendorff
We report a detailed protocol for an intracellular calcium mobilization assay with adherent endothelial cells in multiwell plates that was used to study a number of different PAR1 and PAR2 ligands, including an alkynylated version of the PAR1 antagonist RWJ-58259 that is suitable for the preparation of tagged or conjugate compounds. Using the cell line EA.hy926, it was necessary to perform media exchanges with automated liquid handling equipment in order to obtain optimal and reproducible antagonist concentration-response curves. The assay was used to confirm that vorapaxar acts as an irreversible antagonist of PAR1 in endothelium, and parmodulin 2 (ML161) and the related parmodulin RR-90 were found to inhibit PAR1 reversibly, in a manner consistent with negative allosteric modulation. Detailed synthetic protocols are also provided for several known and novel PAR ligands.

Funding

NIH R15HL127636

History

Email Address of Submitting Author

christopher.dockendorff@mu.edu

Institution

Marquette University

Country

USA

ORCID For Submitting Author

0000-0002-4092-5636

Declaration of Conflict of Interest

C.D. is co-inventor of a patent describing compounds in this manuscript (WO 2012/040636; US 9,422,262).

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