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BioAssay: AID 2529

Fluorescence Cell-Based Screen to Confirm Inhibitors of Phorbol Myristate Acetate-Mediated P-Selectin Induction on Platelets

Assay Overview: Cell-based assay for inhibition of PMA (phorbol myristate acetate)-induced P-selectin surface expression. Washed platelets obtained from individual donors were treated with compounds found to show less than 50% inhibition in an SFLLRN-induced FITC phalloidin assay. Platelet samples were treated with 30 uM compound. Following compound addition, platelets were stimulated with 5 more ..
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 Tested Compounds
 Tested Compounds
All(2)
 
 
Inactive(2)
 
 
 Tested Substances
 Tested Substances
All(2)
 
 
Inactive(2)
 
 
 Related BioAssays
 Related BioAssays
AID: 2529
Data Source: Broad Institute (2016-07_INHIBITORS_SINGLE-POINT_MLPCN-DRYPOWDER)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2010-03-12
Hold-until Date: 2010-09-30
Modify Date: 2010-09-30

Data Table ( Complete ):           All
Tested Compounds:
Depositor Specified Assays
AIDNameTypeComment
1663MLPCN Platelet Activation -Dense Granule ReleasescreeningPrimary HTS
1678Broad Institute MLPCN Platelet ActivationsummaryProject Summary
Description:
Keywords: Platelet, activation, P-selectin, PMA, Protein kinase C

Assay Overview: Cell-based assay for inhibition of PMA (phorbol myristate acetate)-induced P-selectin surface expression. Washed platelets obtained from individual donors were treated with compounds found to show less than 50% inhibition in an SFLLRN-induced FITC phalloidin assay. Platelet samples were treated with 30 uM compound. Following compound addition, platelets were stimulated with 5 nM PMA. 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 specificity assay, along with an analogous assay using Ca2+-ionophore-induced activation of platelets, to determine selectivity towards G-protein coupled receptors (GPCR). Compounds that do not inhibit surface expression of P-selectin from PMA-induced and Ca2+-ionophore-induced activation are potential GPCR-selective inhibitors.

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 incubated with 30 uM compound.

2. 20 minutes following compound addition, platelets were stimulated with 5 nM PMA.

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 values were collected for each sample.
Comment
Data analysis:
The neutral control was: DMSO only, with no compound.
Compounds were tested at 30uM.

Percent inhibition of induced P-Selectin expression was calculated as:
100 - (100 * (Mean Fluorescence at 30uM/ Mean Fluorescence at DMSO))
The percent inhibition is reported for two replicates in each run.

PUBCHEM_ACTIVITY_SCORE
The average Percent Inhibition value at the tested concentration 30uM, calculated across all runs.

PUBCHEM_ACTIVITY_OUTCOME
Activity_Outcome = 1 (inactive)
score <= 50 in all runs

Activity_Outcome = 2 (active)
score > 50 in all runs

Activity_Outcome = 3 (inconclusive)
score > 50 in one or more runs and score <= 50 in one or more runs

Compounds were retested at least once. These retests were analyzed as independent tests; therefore each test generated a separate set of replicate data, including a score (average percent inhibition across replicates) and an outcome (2 (active) for score > 50).
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1outcome_Run1The activity outcome for Run1Integer
2score_Run1The activity ranking score for Run1Integer
3REPLICATE_A_ACTIVITY_SCORE_Run1The calculated activity score for the sample A, a NULL here denotes data was not used or was not producedFloat%
4REPLICATE_B_ACTIVITY_SCORE_Run1The calculated activity score for the sample A, a NULL here denotes data was not used or was not producedFloat%
5outcome_Run2The activity outcome for Run2Integer
6score_Run2The activity ranking score for Run2Integer
7REPLICATE_A_ACTIVITY_SCORE_Run2The calculated activity score for the sample A, a NULL here denotes data was not used or was not producedFloat%
8REPLICATE_B_ACTIVITY_SCORE_Run2The calculated activity score for the sample A, a NULL here denotes data was not used or was not producedFloat%
Additional Information
Grant Number: 1R03DA026209-01

Data Table (Concise)
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