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

Protease-activated receptors (PARs)^1,2,3 are a subfamily of class A G protein-coupled receptors (GPCRs) that are expressed in a variety of cell types, including platelets and endothelial cells^4,5,6,7. Unlike the majority of GPCRs, PARs have a unique intramolecular mode of activation. Most GPCRs are activated by soluble ligands interacting with a distinct binding pocket, but PARs are activated by the proteolytic cleavage of the N-terminus, which results in a new tethered ligand that can interact with the extracellular loop 2 domain on the surface of a cell^6,8,9. This interaction activates the receptor and can initiate several signaling pathways, promoting effects such as inflammation and platelet activation^4,10,11,12. Different proteases can activate PARs through cleavage at unique sites on the N-terminus, revealing different tethered ligands (TL) that stabilize receptor conformations, which initiate different signaling pathways^9,13,14,15. For example, in the most well-studied member of the subfamily, PAR1, cleavage by thrombin is used to support numerous biological processes, including platelet activation and leukocyte recruitment to the endothelium, but can lead to deleterious effects when the receptor is overexpressed or overactivated^{4,16,17,18,19,20,21}. Conversely, cleavage by activated protein C (aPC) can promote anti-inflammatory effects and maintenance of endothelial barriers^{15,22,23,24,25,26,27,28,29}. PARs can also be activated by peptide analogs of the TLs in an intermolecular fashion^13,30,31. These peptides are routinely used to measure PAR inhibition (modulation) in place of PAR-targeting proteases, and they are used in this protocol.

Numerous disorders are associated with pathological PAR1 signaling, including sepsis^22,32, cardiovascular disease^{33,34,35,36,37,38}, kidney disease^39,40,41,42, sickle cell disease⁴³, fibrosis⁴⁴, osteoporosis and osteoarthritis^45,46, neurodegeneration^{47,48,49,50,51}, and cancer^{52,53,54,55,56,57,58,59}. Antagonists of PAR1 have been studied since the 1990s as antiplatelet agents for cardiovascular disease, and the growing list of diseases associated with the receptor necessitates the identification of novel ligands for use as biological probes (tool compounds) or as potential therapeutics. Currently, there is only one FDA-approved PAR1 antagonist, vorapaxar, which is used to treat coronary artery disease in high-risk patients^34,36,37,60. An alternative PAR1 antagonist, the pepducin PZ-128, completed a successful phase II study to prevent thrombosis in cardiac catheterization patients³⁸. The Dockendorff group has focused on the medicinal chemistry and pharmacology of a separate class of small molecules, PAR1 ligands known as parmodulins^61,62. Unlike reported PAR1 antagonists such as vorapaxar, parmodulins are allosteric, biased modulators of PAR1 that selectively block the G_αq pathway while promoting cytoprotective effects similar to aPC. Unlike potent orthosteric PAR1 antagonists such as vorapaxar, published parmodulins are also reversible^63,64,65.

Initially, parmodulins were identified by Flaumenhaft and coworkers for their ability to inhibit P-selectin expression or granule secretion in platelets^61,66. However, an alternative method was required to study the effects of parmodulins on endothelial cells. One common method to monitor GPCR-related signaling is to measure intracellular Ca²⁺ mobilization, an important secondary messenger that can be measured using a suitable intracellular calcium-binding dye^67,68. Substantial evidence has been provided showing that calcium mobilization induced by PAR1 is through the activation of G_αq^69,70. Once activated by its tethered ligand (or a suitable exogenous ligand), PAR1 undergoes a conformational change which causes guanosine diphosphate (GDP) bound to the G_αq subunit to be replaced by guanosine triphosphate (GTP)⁶⁸. The G_αq subunit then activates phospholipase Cβ (PLC-β), which catalyzes the hydrolysis of phosphatidylinositol 4,5 bisphosphate (PIP2), forming 1,4,5-inositol triphosphate (IP₃) and diacylglycerol (DAG). Finally, IP₃ binds to IP₃-sensitive Ca²⁺ channels in the membrane of the endoplasmic reticulum, allowing Ca²⁺ to be released into the cytoplasm, where it can bind to Ca²⁺-dependent fluorescent dyes, such as Fluo-4, that are added to the cells⁷¹. This process occurs within seconds and can increase the concentration of Ca²⁺ 100-fold, leading to a drastic change in the amount of calcium-bound dye and a robust fluorescence signal.

In 2018, the Dockendorff group disclosed a medium-throughput Ca²⁺ mobilization assay that could be used to identify antagonists of the G_αq pathway of PAR1⁷². The assay used EA.hy926⁷³, a hybrid human endothelial cell line, which can be used for multiple passages without a noticeable change in PAR1 expression, and is established for in vitro measurements of cytoprotective effects. 

The original protocol used EA.hy926 cells in 96-well plates and loaded with Fluo-4/AM dye, which was chosen due to its intense fluorescence at 488 nm and high cell permeability. Once the dye was loaded into the cells, lengthy washing steps were performed with an automated 8-channel liquid handler (faster methods of liquid handling, such as a 96-channel washer, were inaccessible). The reproducibility of this assay was superior to that without the careful, automated robotic media changes. Antagonists were then incubated with the cells, PAR1 was activated through the sequential addition of a selective agonist (16 wells at a time), and changes in fluorescence resulting from calcium mobilization and dye binding were measured to determine activity.

While this protocol allows for the measurement of PAR1-mediated calcium mobilization, it is limited by the time required to assay each 96-well plate. Long experiment times are problematic not only because the number of compounds that can be screened each day is limited, but also because dye efflux occurs over time, narrowing the assay window by increasing the basal fluorescence. One contributor to the long experiment time is the use of an 8-channel liquid handler for plate washing, which adds over 30 min to each experiment. The required tips also became difficult to obtain due to supply chain problems. Here an updated protocol for the PAR-mediated calcium mobilization assay that does not require a liquid handler, and therefore can be run in higher throughput, is reported. This protocol should also be suitable for measuring signaling with other GPCRs that lead to intracellular calcium mobilization. This updated plate reader protocol is ideal for academic and small industrial labs that do not have the resources for expensive cell imaging instruments but have a need to rapidly screen numerous compounds. For an example of a calcium mobilization assay using a plate imager, see Caers et al.⁷⁴.