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BioAssay: AID 2511

Fluorescence Cell-Based Dose Response to Confirm Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated P-Selectin Induction on Platelets

Keywords: Platelet, activation, P-selectin, SFLLRN, PAR1, thrombin receptor, alpha-granule, secretion ..more
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 Tested Compounds
 Tested Compounds
All(8)
 
 
Active(8)
 
 
 Tested Substances
 Tested Substances
All(8)
 
 
Active(8)
 
 
 Related BioAssays
 Related BioAssays
AID: 2511
Data Source: Broad Institute (2016-03_INHIBITORS_DOSE-TITRATION_MLPCN-DRYPOWDER)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2010-03-11
Hold-until Date: 2010-09-30
Modify Date: 2010-09-30

Data Table ( Complete ):           Active    All
BioActive Compounds: 8
Depositor Specified Assays
AIDNameTypeComment
1663MLPCN Platelet Activation -Dense Granule ReleasescreeningPrimary HTS
1678Broad Institute MLPCN Platelet ActivationsummaryProject Summary
Description:
Keywords: Platelet, activation, P-selectin, SFLLRN, PAR1, thrombin receptor, alpha-granule, secretion

Assay Overview:
Cell-based assay for inhibition of SFLLRN-induced P-selectin surface expression. Washed platelets obtained from individual donors were treated with a selection of compounds resourced as powders found to be active in secondary assays. Platelet samples were tested at 10uM, 3uM, 1uM, 0.3uM, and 0uM concentrations. Following compound addition, platelets were subsequently stimulated with 5 mM SFLLRN. After a 15-minute incubation, phycoerythrin-labeled anti-P-selectin antibody (BD Biosciences) was added for a 20-minute incubation. The samples were analyzed by flow cytometry to determine P-selectin expression on the surface of the platelets as a response to activation. Geometric mean values were collected for each sample.

Expected Outcome:
This assay serves as a gate for the defined probe paths. A moderate decrease in the amount of P-selectin expressed on the surface of platelets in a dose-dependent manner will identify compounds that inhibit pathways that regulate granule secretion or G-protein coupled receptors (GPCR) expressed on the surface of platelets. Lack of substantial inhibition of surface expression of P-selectin is indicative of compounds that might selectively effect dense granule secretion. Additional secondary testing will further delineate the specificity of the probes.

Primary Collaborators(and laboratory where this assay was performed):
Robert Flaumenhaft,Beth Israel Deaconess Medical Center,rflaumen@bidmc.harvard.edu,617-754-1204
John Thomas,NHLBI,ThomasJ@nhlbi.nih.gov
Protocol
1. Platelet samples (10 ul) were tested at different concentrations of compound (100uM, 30uM, 1uM, 0.3uM, and 0uM for cherry-picks and 30uM, 10uM, 1uM, 0.3uM, and 0uM for powders).

2. 20 minutes following compound addition, platelets were stimulated with 5 uM thrombin-receptor-derived hexapeptide SFLLRN from 100 uM stock.

3. After a 15-minute incubation, phycoerythrin-labeled anti-P-selectin antibody (5 ul; BD Biosciences) was added. The samples were agitated gently.

4. After a 20-minute incubation, 500 ul of FACS buffer (BD Biosciences) was added to each of the samples.

5. Samples were then analyzed by flow cytometry to determine P-selectin expression on the surface of the platelets as a response to activation.

6. Geometric mean fluorescence values were collected for each sample.
Comment
Data Analysis:

The neutral control was: DMSO only, with no compound.
Four compound concentrations were tested: 10uM, 3uM, 1uM, 0.3uM.

Percent inhibition of induced P-selectin expression was calculated based on the assumption that complete (100% ) inhibition is attainable, as:
100 - (100 * (Mean Fluorescence at dose/ Mean Fluorescence at DMSO))
The average of percent inhibition across two replicates is reported for each compound concentration in each run.

EC50 was determined to be the lowest concentration that supported >= 50% of Maximum Activity.

PUBCHEM_ACTIVITY_SCORE
The average percent inhibition value at the tested concentration 1uM.

PUBCHEM_ACTIVITY_OUTCOME
Activity_Outcome = 1 (inactive)
EC50 >= 3uM

Activity_Outcome = 2 (active)
EC50 < 3uM

Several compounds were retested at least once. These retests were analyzed as independent tests; therefore each test generated a separate set of curve data, including PUBCHEM_ACTIVITY_SCORE, PUBCHEM_ACTIVITY_OUTCOME, and EC50.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50 Qualifier'>', '=', or '<'String
2EC50*the concentration whereupon perceived activity reaches 50% of the maximumFloatμM
3Activity at 10uM (10μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
4Activity at 3uM (3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
5Activity at 1uM (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
6Activity at 0.3uM (0.3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
7Activity at 0uM (0μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
8outcome_Run2The activity outcome for Run2Integer
9score_Run2The activity ranking score for Run2Integer
10EC50 Qualifier_Run2'>', '=', or '<'String
11EC50_Run2the concentration whereupon activity reaches 50% of the maximumFloatμM
12Activity at 10uM_Run2 (10μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity at 3uM_Run2 (3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity at 1uM_Run2 (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity at 0.3uM_Run2 (0.3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity at 0uM_Run2 (0μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

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

Data Table (Concise)
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