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BioAssay: AID 493068

SFLLRN-induced P-Selectin Platelet Surface Expression Measured in Cell-Based System Using Flow Cytometry - 2016-03_Inhibitor_Dose_DryPowder_Activity_Set3

Keywords: Platelet, activation, P-selectin, SFLLRN, PAR1, thrombin receptor, alpha-granule, secretion ..more
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 Tested Compounds
 Tested Compounds
All(5)
 
 
Active(5)
 
 
 Tested Substances
 Tested Substances
All(5)
 
 
Active(5)
 
 
 Related BioAssays
 Related BioAssays
AID: 493068
Data Source: Broad Institute (2016-03_Inhibitor_Dose_DryPowder_Activity_Set3)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-01-24

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 5
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
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
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
PRESENCE OF CONTROLS: Neutral controls (NC) were included with every run.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (HVC) was determined and the active concentration limit (AC_limit) was set to equal (10)(HVC).
NORMALIZATION:
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))
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION(AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
b) AC > AC_limit, or
c) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was not valid due to poor fit quality.
PUBCHEM_ACTIVITY_SCORE:
PUBCHEM_ACTIVITY_SCORE = 0 when PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive)
PUBCHEM_ACTIVITY_SCORE = (-10)[Log(AC)], where AC is in molar, when PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
PUBCHEM_ACTIVITY_SCORE = 100 when the curve fit is constant and showing full inhibition at all tested concentrations.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_Qualifier'>','=', or '<'String
2AC50_uM*The concentration at which activity reaches 50% of the maximumFloatμM
3Log_AC50_MThe log of the molar AC50Float
4Hill_SlopeThe slope at AC50Float
5S0The fitted activity value at zero concentrationFloat%
6SinfThe fitted activity value at infinite concentrationFloat%
7Num_PointsThe number of data points used to generate the plotInteger
8Max_ActivityThe maximum activity value observed, based on mean of replicates per concentrationFloat%
9Max_Activity_Conc_uMThe concentration at which the maximum activity is observedFloatμM
10Activity_at_0.156uM (0.156μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity_at_0.78uM (0.78μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_12.5uM (12.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_25uM (25μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_3.125uM (3.125μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_50uM (50μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_6.25uM (6.25μM**)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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