We thank Irene Hernandez, Trudy Holyst, Dr. Hartmut Weiler (Versiti Blood Research Institute), and Dr. Leggy Arnold (University of Wisconsin-Milwaukee) for providing space and indirect support of this project, and Dr. John McCorvy (Medical College of Wisconsin) for pertinent advice. We thank the National Heart, Lung, and Blood Institute (R15HL127636), the U.S. Dept. of Defense (W81XWH22101), and the National Science Foundation (2223225) for grant support.

All media exchanges/additions made in steps 1 and 2 of the following protocol are performed in a sterile hood. Unless otherwise noted, all plasticware used in the sterile hood must be purchased sterilized and sealed or sterilized appropriately via autoclave.
1. Initiation of EA.hy926 cell line
<ol>
	<li>Acquire EA.hy926 cells.</li>
	<li>Store vial(s) of cells in the vapor phase of a liquid nitrogen tank.</li>
	<li>Prepare DMEM complete medium by first warming DMEM, FBS, and ...

Studying Protease-Activated Receptor Ligands in Endothelial Cells

While the previously reported protocol72 was generally reliable and allowed us to identify a new lead parmodulin, NRD-21,62 a more efficient protocol was desired. The assay was further compromised during the supply shortage caused by the COVID-19 pandemic. Acquiring tips for the automated liquid handler became difficult, and attempting to wash, sterilize, and reuse the tips produced CRCs with significant variance. This facilitated an urgent series of experiments designed to...

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 cells4,5,6,7. 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 cell6,8,9. This interaction activates the receptor and can initiate several signaling pathways, promoting effects such as inflammation and platelet activation4,10,11,12. 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 pathways9,13,14,15. 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 overactivated4,16,17,18,19,20,21. Conversely, cleavage by activated protein C (aPC) can promote anti-inflammatory effects and maintenance of endothelial barriers15,22,23,24,25,26,27,28,29. PARs can also be activated by peptide analogs of the TLs in an intermolecular fashion13,30,31. 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 sepsis22,32, cardiovascular disease33,34,35,36,37,38, kidney disease39,40,41,42, sickle cell disease43, fibrosis44, osteoporosis and osteoarthritis45,46, neurodegeneration47,48,49,50,51, and cancer52,53,54,55,56,57,58,59. 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 patients34,36,37,60. 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 parmodulins61,62. Unlike reported PAR1 antagonists such as vorapaxar, parmodulins are allosteric, biased modulators of PAR1 that selectively block the G&#945;q pathway while promoting cytoprotective effects similar to aPC. Unlike potent orthosteric PAR1 antagonists such as vorapaxar, published parmodulins are also reversible63,64,65.
Initially, parmodulins were identified by Flaumenhaft and coworkers for&#160;their ability to inhibit P-selectin expression or granule secretion in platelets61,66. 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 dye67,68. Substantial evidence has been provided showing that calcium mobilization induced by PAR1 is through the activation of G&#945;q69,70. 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&#945;q subunit to be replaced by guanosine triphosphate (GTP)68. The G&#945;q subunit then activates phospholipase C&#946; (PLC-&#946;), 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.&#160;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&#945;q pathway of PAR172. The assay used&#160;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.&#160;
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.&#160;This updated plate reader protocol&#160;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell Culture Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adherent EA.hy926 cells</td>
			<td>ATCC</td>
			<td>CRL-2922</td>
			<td></td>
		</tr>
		<tr>
			<td>CellStripper cell dissociation reagent</td>
			<td>Corning</td>
			<td>25-056-CI</td>
			<td>Trypsin can optionally be used, but should definitely be avoided with PAR2 assays.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) w/phenol red</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Avantor</td>
			<td>97068-091</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from porcine skin</td>
			<td>MilliporeSigma</td>
			<td>G2500</td>
			<td>Use to make an aqueous 0.4% (w/v) solution with deionized water. Autoclave before use to sterilize.</td>
		</tr>
		<tr>
			<td>Pen/Strep (100X)</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue (0.4% w/v)</td>
			<td>Corning</td>
			<td>25-900-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Mobilization Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>Thermo</td>
			<td>172571000</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Avantor</td>
			<td>97061-420</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Thermo</td>
			<td>42352-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Thermo</td>
			<td>J66650-AD</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-4/AM</td>
			<td>Invitrogen</td>
			<td>F14201</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution (Ca/Mg/phenol-red free)</td>
			<td>Corning</td>
			<td>21-022-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>MilliporeSigma</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127 (Poloxamer 407)</td>
			<td>Spectrum Chemical</td>
			<td>P1166</td>
			<td></td>
		</tr>
		<tr>
			<td>Probenecid</td>
			<td>TCI America</td>
			<td>P1975</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>VWR International</td>
			<td>BDH9292</td>
			<td></td>
		</tr>
		<tr>
			<td>TFLLRN-NH2 (TFA salt)</td>
			<td></td>
			<td></td>
			<td>Prepared by Trudy Holyst at the Versiti Blood Research Institute</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well culture-treated, black-walled, clear bottom assay plate</td>
			<td>Corning</td>
			<td>3603</td>
			<td>with transparent lids</td>
		</tr>
		<tr>
			<td>Centrifuge tube, 15 mL</td>
			<td>Avantor</td>
			<td>89039-664</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube, 50 mL</td>
			<td>Avantor</td>
			<td>89039-656</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture flask, T-75</td>
			<td>Corning</td>
			<td>353136</td>
			<td>tissue culture treated</td>
		</tr>
		<tr>
			<td>Disposable reagent reservoir, 50 mL</td>
			<td>Corning</td>
			<td>RES-V-50-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Enspire plate reader</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>Discontinued</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube, 1.5 mL</td>
			<td>Avantor</td>
			<td>20170-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette, 9&#34;</td>
			<td>Fisher</td>
			<td>13-678-6B</td>
			<td>must be sterilized</td>
		</tr>
		<tr>
			<td>PCR tube strip with separate flat cap strips</td>
			<td>Avantor</td>
			<td>76318-802</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips, 20 &#181;L</td>
			<td>Biotix</td>
			<td>63300042</td>
			<td>sterile, filtered tips</td>
		</tr>
		<tr>
			<td>Pipette tips, 200 &#181;L</td>
			<td>Biotix</td>
			<td>63300044</td>
			<td>sterile, filtered tips</td>
		</tr>
		<tr>
			<td>Pipette tips, 1250 &#181;L</td>
			<td>Biotix</td>
			<td>63300047</td>
			<td>sterile, filtered tips</td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad</td>
			<td></td>
			<td>volume 6 used</td>
		</tr>
		<tr>
			<td>Serological pipette, 5 mL</td>
			<td>Tradewinds Direct</td>
			<td>&#160;07-5005</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 10 mL</td>
			<td>Tradewinds Direct</td>
			<td>&#160;07-5010</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 25 mL</td>
			<td>Tradewinds Direct</td>
			<td>&#160;07-5025</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterizing modulators of protease-activated receptors with a calcium mobilization assay using a plate reader

Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding dyes and imaging instruments that can measure fluorescent emissions from up to 1,536 wells simultaneously. However, these instruments are much more expensive and can be challenging to maintain relative to widely available plate readers that scan wells individually. Described here is an optimized plate reader assay for use with an endothelial cell line (EA.hy926) to measure the protease-activated receptor (PAR)-driven activation of G&#945;q signaling and subsequent calcium mobilization using the calcium-binding dye Fluo-4. This assay has been used to characterize a range of PAR ligands, including the allosteric PAR1-targeting anti-inflammatory "parmodulin" ligands identified in the Dockendorff lab. This protocol obviates the need for an automated liquid handler and permits the medium-throughput screening of PAR ligands in 96-well plates and should be applicable to the study of other receptors that initiate calcium mobilization.

Author Spotlight: Developing Parmodulins to Target Protease-Activated Receptors for Inflammation Control

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.

The purpose of this assay is generally to produce concentration-response curves (CRCs) for three to four new parmodulins. On each assay plate, an additional CRC for a known compound, such as NRD-21, is often generated that acts as a quality check for the experiment due to its known IC50. To generate CRCs, a plate map such as the one depicted in Table 1 should be planned. If single-point concentration-responses are desired instead, compounds at 10 &#181;M final concentrations (or other preferre...

An improved protocol for a calcium mobilization assay with endothelial cells, used to identify ligands of protease-activated receptors (PARs), has been developed. The new protocol reduces total assay time by 90-120 min and yields reproducible concentration-response curves.

Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding ...