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BioAssay: AID 1889

Luminescence Cell-Based Dose Confirmation HTS to Identify Inhibitors of Platelet Dense Granule Release

Keywords: Platelet, activation, granule, secretion, arterial thrombosis, PAR1, SFLLRN, thrombin receptor ..more
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 Tested Compounds
 Tested Compounds
All(1584)
 
 
Active(651)
 
 
Inactive(933)
 
 
 Tested Substances
 Tested Substances
All(1584)
 
 
Active(651)
 
 
Inactive(933)
 
 
 Related BioAssays
 Related BioAssays
AID: 1889
Data Source: Broad Institute (2016-01_INHIBITORS_DOSE-TITRATION_MLPCN-CHERRYPICK)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-07-27

Data Table ( Complete ):           Active    All
BioActive Compounds: 651
Depositor Specified Assays
AIDNameTypeComment
1663MLPCN Platelet Activation -Dense Granule ReleasescreeningPrimary HTS
1678Broad Institute MLPCN Platelet ActivationsummarySummary of Project
2398Luminescence Cell-Based Dose Response Followup to Identify Inhibitors of Platelet Dense Granule Releaseconfirmatory
2518Fluorescence Cell-Based Dose Response to Identify Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated P-Selectin Induction on Platelets.confirmatory
Description:
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.
Protocol
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.
Comment
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
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50 Qualifier'>', '=', or '<'String
2EC50*the concentration whereupon activity reaches 50% of the maximumFloatμM
3Donor 1 EC50 Qualifier'>', '=', or '<'String
4Donor 1 EC50the concentration whereupon activity reaches 50% of the maximumFloatμM
5Donor 1 EC50 Stderrthe standard error for the calculated EC50 valueFloatμM
6Donor 1 S0the fitted activity level at zero concentrationFloat%
7Donor 1 SInfthe fitted activity level at infinite concentrationFloat%
8Donor 1 Hill Slopethe slope at EC50Float
9Donor 1 # of Points Fitthe number of data points included in the plotInteger
10Donor 1 Max Activitythe maximum activity value observedFloat%
11Donor 1 Activity at 0.09uM (0.09μM**)The average measured activity of all accepted replicates at the specified concentration for Donor 1Float%
12Donor 1 Activity at 0.18uM (0.18μM**)The average measured activity of all accepted replicates at the specified concentration for Donor 1Float%
13Donor 1 Activity at 0.35uM (0.35μM**)The average measured activity of all accepted replicates at the specified concentration for Donor 1Float%
14Donor 1 Activity at 0.70uM (0.7μM**)The average measured activity of all accepted replicates at the specified concentration for Donor 1Float%
15Donor 1 Activity at 1.40uM (1.4μM**)The average measured activity of all accepted replicates at the specified concentration for Donor 1Float%
16Donor 1 Activity at 2.81uM (2.81μM**)The average measured activity of all accepted replicates at the specified concentration for Donor 1Float%
17Donor 1 Activity at 5.61uM (5.61μM**)The average measured activity of all accepted replicates at the specified concentration for Donor 1Float%
18Donor 1 Activity at 11.22uM (11.22μM**)The average measured activity of all accepted replicates at the specified concentration for Donor 1Float%
19Donor 2 EC50 Qualifier'>', '=', or '<'String
20Donor 2 EC50the concentration whereupon activity reaches 50% of the maximumFloatμM
21Donor 2 EC50 Stderrthe standard error for the calculated EC50 valueFloatμM
22Donor 2 S0the fitted activity level at zero concentrationFloat%
23Donor 2 SInfthe fitted activity level at infinite concentrationFloat%
24Donor 2 Hill Slopethe slope at EC50Float
25Donor 2 # of Points Fitthe number of data points included in the plotInteger
26Donor 2 Max Activitythe maximum activity value observedFloat%
27Donor 2 Activity at 0.09uM (0.09μM**)The average measured activity of all accepted replicates at the specified concentration for Donor 2Float%
28Donor 2 Activity at 0.18uM (0.18μM**)The average measured activity of all accepted replicates at the specified concentration for Donor 2Float%
29Donor 2 Activity at 0.35uM (0.35μM**)The average measured activity of all accepted replicates at the specified concentration for Donor 2Float%
30Donor 2 Activity at 0.70uM (0.7μM**)The average measured activity of all accepted replicates at the specified concentration for Donor 2Float%
31Donor 2 Activity at 1.40uM (1.4μM**)The average measured activity of all accepted replicates at the specified concentration for Donor 2Float%
32Donor 2 Activity at 2.81uM (2.81μM**)The average measured activity of all accepted replicates at the specified concentration for Donor 2Float%
33Donor 2 Activity at 5.61uM (5.61μM**)The average measured activity of all accepted replicates at the specified concentration for Donor 2Float%
34Donor 2 Activity at 11.22uM (11.22μM**)The average measured activity of all accepted replicates at the specified concentration for Donor 2Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R03DA026209-01

Data Table (Concise)
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