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BioAssay: AID 2656

Radioactive Cell-Based Screen to Identify Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated Dense Granule Release by Platelets

Assay Overview: Cell-based assay for measuring specific dense granule release induced by SFLLRN-mediated platelet activation. Compounds showing no activity (inhibition) in a thrombin receptor-activating peptide SFLLRN-induced P-selectin expression assay (AID 2518) were tested for ability to inhibit release of serotonin, a specific component of platelet dense granules, induced by activation with more ..
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 Tested Compounds
 Tested Compounds
All(7)
 
 
Active(1)
 
 
Inactive(6)
 
 
 Tested Substances
 Tested Substances
All(7)
 
 
Active(1)
 
 
Inactive(6)
 
 
 Related BioAssays
 Related BioAssays
AID: 2656
Data Source: Broad Institute (2016-04_INHIBITORS_SINGLE-POINT_MLPCN-CHERRYPICK)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2010-03-24
Hold-until Date: 2010-09-30
Modify Date: 2010-09-30

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compound: 1
Related Experiments
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AIDNameTypeProbeComment
1663MLPCN Platelet Activation -Dense Granule ReleaseScreening depositor-specified cross reference: Primary HTS
1678Broad Institute MLPCN Platelet ActivationSummary3 depositor-specified cross reference: Project Summary
2518Fluorescence Cell-Based Dose Response to Identify Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated P-Selectin Induction on Platelets.Confirmatory depositor-specified cross reference: Assay describing the ability of compounds to inhibit SFLLRN-induced P-selectin expression on platele
2761Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of the membrane-associated serine protease Rv3671c in M.tuberculosisScreening depositor-specified cross reference
1889Luminescence Cell-Based Dose Confirmation HTS to Identify Inhibitors of Platelet Dense Granule ReleaseConfirmatory same project related to Summary assay
1891Luminescence Biochemical Dose Response HTS to Identify Inhibitors of LuciferaseConfirmatory same project related to Summary assay
2398Luminescence Cell-Based Dose Response Followup to Identify Inhibitors of Platelet Dense Granule ReleaseConfirmatory same project related to Summary assay
2509Fluorescence Cell-Based Screen to Identify Inhibitors of Phorbol Myristate Acetate-Mediated P-Selectin Induction on PlateletsScreening same project related to Summary assay
2511Fluorescence Cell-Based Dose Response to Confirm Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated P-Selectin Induction on PlateletsConfirmatory same project related to Summary assay
2519Luminescence Cell-Based Dose Response Followup to Identify Inhibitors of Platelet Dense Granule Release.Confirmatory same project related to Summary assay
2522Fluorescence Cell-Based Screen to Identify Inhibitors of Calcium Ionophore-Mediated P-Selectin Induction on PlateletsScreening same project related to Summary assay
2527Fluorescence Cell-Based Screen to Confirm Inhibitors of Calcium Ionophore-Mediated P-Selectin Induction on PlateletsScreening same project related to Summary assay
2529Fluorescence Cell-Based Screen to Confirm Inhibitors of Phorbol Myristate Acetate-Mediated P-Selectin Induction on PlateletsScreening same project related to Summary assay
2645ELISA Cell-Based Screen to Identify Inducers of cAMP in PlateletsScreening same project related to Summary assay
2646ELISA Cell-Based Screen to Identify an Inducer of cAMP in PlateletsScreening same project related to Summary assay
2655Fluorescence Cell-Based Screen to Identify an Inhibitor of Thrombin Receptor-Activating Peptide SFLLRN-Mediated Actin Polymerization in PlateletsScreening same project related to Summary assay
2657Fluorescence Cell-Based Screen to Identify Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated Actin Polymerization in PlateletsScreening same project related to Summary assay
493031SFLLRN-induced P-Selectin Platelet Surface Expression Measured in Cell-Based System Using Flow Cytometry - 2016-03_Inhibitor_SinglePoint_DryPowder_ActivityOther same project related to Summary assay
493068SFLLRN-induced P-Selectin Platelet Surface Expression Measured in Cell-Based System Using Flow Cytometry - 2016-03_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
493100Platelet granule secretion Measured in Cell-Based System Using Plate Reader - 2016-01_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
Description:
Keywords: Platelets, dense granule secretion, serotonin, SFLLRN, PAR1

Assay Overview: Cell-based assay for measuring specific dense granule release induced by SFLLRN-mediated platelet activation. Compounds showing no activity (inhibition) in a thrombin receptor-activating peptide SFLLRN-induced P-selectin expression assay (AID 2518) were tested for ability to inhibit release of serotonin, a specific component of platelet dense granules, induced by activation with the PAR1-activating peptide SFLLRN. Washed platelets were incubated with 14C-serotonin resulting in incorporation into dense granules. Compounds were added to platelet samples at 10 uM. Platelets were activated with SFLLRN and supernatants were collected for analysis with a scintillation counter to measure released 14C-serotonin.

Expected Outcome: A moderate decrease in the level of 14C-serotonin in supernatants of compound treated samples compared to untreated samples would identify specific inhibitors of dense granule release.

Primary Collaborators(and laboratory where 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. 4.5 mL platelet rich plasma (PRP) (approx 3x10^8 platelets/mL) was incubated in the presence of 14C-serotonin (0.225 uCi ) for 45 minutes at 37oC.
2. 15% acid-citrate-dextrose (ACD)+ 150nM prostaglandin E1 (PGE1) was added to PRP and spun at 1000g for 10 minutes to maintain the resting state of platelets while removing unincorporated 14C-serotonin.
3. Platelets were re-suspended in HEPES-Tyrodes buffer to a platelet concentration of 2x10^8 platelets/mL.
4. Compounds were added to resuspended platelets at 10uM and incubated for 15 minutes.
5. Platelets were subsequently stimulated with 5 uM SFLLRN peptide and 5 uM imipramine (to prevent 14C-serotonin reuptake) for 15 minutes.
6. Platelets were separated from medium by centrifugation at 8000 x g.
7. Supernatants were collected and counted using a beta liquid scintillation counter. Disintegrations per minute (DPM) were collected for 50 ul of platelets.
Comment
Data Analysis:
The neutral control was DMSO only, with no compound.
Compounds were tested at 10 uM in duplicate.
Percent inhibition of 14C-serotonin secretion was calculated based on the assumption that complete (100%) inhibition is attainable, as:
100 - (100 * (DPM at 10uM/ Mean DPM DMSO))
A positive control compound, Cilostazol (PubChem CID 2754), was tested at 100 uM. Mean DPM for DMSO was 3245 and Mean DPM for Cilostazol was 211, resulting in 93% inhibition of 14C-serotonin secretion.
PUBCHEM_ACTIVITY_SCORE
The Mean of Percent Inhibition across two replicates.
PUBCHEM_ACTIVITY_OUTCOME
Activity_Outcome = 1 (inactive)
PUBCHEM_ACTIVITY_SCORE <= 50
Activity_Outcome = 2 (active)
PUBCHEM_ACTIVITY_SCORE > 50
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1REPLICATE_A_ACTIVITY_SCOREThe calculated activity score for the replicate AFloat%
2REPLICATE_B_ACTIVITY_SCOREThe calculated activity score for the replicate BFloat%
Additional Information
Grant Number: 1R03DA026209-01

Data Table (Concise)
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