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BioAssay: AID 493031

SFLLRN-induced P-Selectin Platelet Surface Expression Measured in Cell-Based System Using Flow Cytometry - 2016-03_Inhibitor_SinglePoint_DryPowder_Activity

Keywords: Platelet, activation, P-selectin, SFLLRN, PAR1, thrombin receptor, alpha-granule, secretion ..more
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 Tested Compounds
 Tested Compounds
All(21)
 
 
Active(11)
 
 
Inactive(10)
 
 
 Tested Substances
 Tested Substances
All(21)
 
 
Active(11)
 
 
Inactive(10)
 
 
AID: 493031
Data Source: Broad Institute (2016-03_Inhibitor_SinglePoint_DryPowder_Activity)
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-01-14
Hold-until Date: 2011-02-05
Modify Date: 2011-02-07

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 11
Related Experiments
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AIDNameTypeProbeComment
1678Broad Institute MLPCN Platelet ActivationSummary3 depositor-specified cross reference: Summary assay
1663MLPCN Platelet Activation -Dense Granule ReleaseScreening same project related to Summary assay
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
2518Fluorescence Cell-Based Dose Response to Identify Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated P-Selectin Induction on Platelets.Confirmatory 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
2656Radioactive Cell-Based Screen to Identify Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated Dense Granule Release by 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
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: 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 either at dose or at a single point of 10uM. 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 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.
Protocol
Protocol:
1. Platelet samples (10 ul) were treated with compounds at dose or at a single point of 10uM.
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.
Compounds were tested at 10uM
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))
PUBCHEM_ACTIVITY_SCORE
The percent inhibition value at 10uM.
#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
1REPRODUCIBILITY_COSINE_TRANSFORMA measure of how well the activity reproduced across the two samples. Computed as the absolute value of the cosine between the 'replicate vector' (ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility. NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was Invalid# Float
2REPLICATE_A_ACTIVITY_SCOREThe calculated percent activity for the indicated sampleFloat%
3REPLICATE_B_ACTIVITY_SCORE#The calculated percent activity for the indicated sampleFloat%
Additional Information
Grant Number: 1R03DA026209-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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