Luminescence Cell-Based Dose Response Followup to Identify Inhibitors of Platelet Dense Granule Release.
Keywords: Platelet, activation, granule, secretion, arterial thrombosis, PAR1, SFLLRN, thrombin receptor ..more
BioActive Compounds: 8
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 a subset of the positive compounds from AID 1889, which were selected for high inhibitory activity in the assay, no known platelet inhibitory properties, and interesting chemical properties. Compounds were plated at dose, with final testing concentrations ranging from 33.35uM to 0.005um. 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 50uM or 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. All powders were tested on at least two donor samples of PRP.
Expected Outcome: A decrease in the luminescent signal will identify compounds that inhibit the release of dense granules from the platelets. Compounds that retest positively will be those that show inhibition of signal in a dose-dependent manner in multiple donor PRP samples. Normalization and curve fitting of the retest data was performed using the Genedata Screener applications. Specific mode of action in platelets will be further delineated in a suite of secondary assays.
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 30C, 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 22C, 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
The positive control was: Cilostazol, PubChem CID 2754
The neutral control was: DMSO only, with no compound
Neutral control (NC) wells and positive control (PC) wells were included on every plate.
Active compounds result in decreased signal.
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate NC wells was set to a normalized activity value of 0.
The median raw signal of the intraplate PC wells was set to a normalized activity value of -100.
Experimental wells were scaled to this range, giving an activity score as percent change in signal relative to the intraplate controls.
No plate pattern correction algorithm was applied.
IC50 values were calculated using the curve fitting strategies in Genedata
Screener Condoseo (7.0.3). IC50 values were extrapolated up to 1 log
over the highest tested concentration.
Assignment of PUBCHEM_ACTIVITY_SCORE:
Inactive compounds = 0
Active compounds = -10*Log(IC50)
Assignment of PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive)
IC50 > 1 log over the highest tested concentration
Activity_Outcome = 2 (active)
IC50 <= 1 log over the highest tested concentration
Activity_Outcome = 3 (inconclusive)
Curve fitting strategy resulted in a constant fit with activity >30% but <70%
The fit was not valid due to poor fit quality.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)