AID	Name	Type	Comment
1663	MLPCN Platelet Activation -Dense Granule Release	screening	Primary HTS
1678	Broad Institute MLPCN Platelet Activation	summary	Summary of Project
2398	Luminescence Cell-Based Dose Response Followup to Identify Inhibitors of Platelet Dense Granule Release	confirmatory
2518	Fluorescence Cell-Based Dose Response to Identify Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated P-Selectin Induction on Platelets.	confirmatory

Keywords: Platelet, activation, granule, secretion, arterial thrombosis, PAR1, SFLLRN, thrombin receptor

Assay Overview: Dense granule release of platelet-rich plasma (PRP) retest at dose. Expired units of PRP obtained from a blood-distribution center were plated in 384-well white assay plates (Aurora, 00030721) on average of 15,600,000 platelets/well in 20ul. PRP was exposed to 1584 cherry picked compounds chosen based on activity of the primary screen and those with similar structure to provide some SAR data. Compounds were plated at dose, with final testing concentrations ranging from 11.25uM to 0.875um. The positive control, Cilostazol (Sigma #C0737, Lot# 042K4704, BRD-K67017579-001-04-2) was included on all plates to give a final screening concentration of 100uM. Compounds were added to PRP for 30 minutes prior to addition of the thrombin receptor (Par1) activator SFLLRN (5uM, Bachem, H-8365) and detection reagent CellTiter-Glo (Promega, G755) using a modified protocol, for measurement of ATP released from the dense granules. PBS is used in place of CellTiter-Glo Buffer thus preventing platelet lysis and a high background of ATP. Luminescence measurements are taken 15 minutes after reagent addition. The 1584 compounds were tested on two donor samples of PRP.

Expected Outcome: A decrease in the luminescent signal will identify compounds that either inhibit the release of dense granules from the platelets, or inhibit the luciferase enzyme. Compounds that retest positively will be those that show inhibition of signal in a dose-dependent manner in at least one of the donor PRP samples. Normalization and curve fitting of the retest data was performed using the Genedata Screener applications. Specificity for the inhibition of platelet dense granule release will be determined in secondary assay testing for inhibition of luciferase.

Taken from 2016-01-A01-012

1) Plasma bag(s) from a single donor were emptied into a sterile bottle (250 or 500ml, depending on volume of plasma) in a hood. If multiple bags exist from a single donor (matching barcodes), they were combined as to increase batch volume. Multiple donors' samples could not be pooled due to possible immunological reactions, therefore multiple batches were run daily with each being prepared singly. All information provided on each unit used was recorded (source, donor number, blood type, etc.).
2) Samples were taken to count the number of platelets/ml and checked for activation activity by addition of SFLLRN and CellTiter-Glo.
3) The Thermo MultiDrop Combi in a hood was prepared for dispensing 20ul of plasma per well. As many plates allowable with volume of plasma were filled from a single donor. Batch size ranged from 200ml-700ml. While filling, platelets were kept in homogeneous suspension by gentle agitation.
4) Assay plates were loaded into racks for placement into a Liconic STR240 HRIT incubator set at 30'C, 95% humidity, 5% CO2. The incubator is docked to the screening system. Compound plates have their foil seals pierced off-line. Those plates are loaded into a Liconic STR240 DRIT incubator set to 22'C, 15% humidity.
5) CellTiter-Glo/SFLLRN was prepared for each day to a volume to accommodate the number of assay plates estimated for the day. The CellTiter-Glo Substrate was previously resuspended in 100ml PBS, aliquoted and frozen. An aliquot was thawed and diluted 1:4 with PBS. SFLLRN was previously resuspended in stocks of 10mM and frozen. An aliquot was thawed and was added to the mixture at 15uM. The Combi on the screening system was prepared for run and primed with the reagent.
6) Screening was performed on an enclosed, contained screening system (HighRes Biosolutions). The run was initiated by set-up in CBIP (Broad Chemical Biology Informatics Platform) and scheduled with Cellario (HighRes Biosolutions). Staubli arms moved plates from the different instruments on the system. Compounds were pinned into assay plates using a MicroPin (High Res Biosolutions) using a 25nl head calibrated to deliver 50nl.
7) Assay plates were returned to the incubator for a 30 min. incubation.
8) At the completion of incubation, plates moved to the Combi for addition of 10ul CellTiter-Glo/SFLLRN solution per well.
9) Plates were moved to a plate hotel on deck for a 15 min. incubation.
10) At completion of incubation, plates were moved to an Envision 2104 Multilabel Reader (Perkin Elmer) for luminescence detection. The ultra sensitive detection was used, with the 1536 aperture in place, to decrease bleed-through from adjacent wells. Read time is 0.1s/well.

Dose Response Data Analysis:
32 Negative control (DMSO) and 32 positive control (Cilostazol, PubChem CID 2754) wells were included on every plate.

All wells tested were background subtracted using the median of the negative controls wells on the same plate and scaled using the median value of the positive control wells on the same plate. DMSO plates were run before and after the compound plate and Genedata's runwise additive correction was used with those as calibration plates to correct for plate effects.

EC50 values were calculated using the Smart Fit strategy of Genedata
Screener Condoseo (v6.0.1). EC50 values were extrapolated up to 1 log
over the highest tested concentration.

PUBCHEM_ACTIVITY_SCORE
Inactive compounds = 0
Active compounds = -10*Log(EC50)

PUBCHEM_ACTIVITY_OUTCOME
Activity_Outcome = 1 (inactive)
EC50 > 1 log over the highest tested concentration

Activity_Outcome = 2 (active)
EC50 <= 1 log over the highest tested concentration

Grant Number: 1R03DA026209-01