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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 ):           Active    All
BioActive Compounds: 5
Depositor Specified Assays
AIDNameTypeComment
1678Broad Institute MLPCN Platelet ActivationsummarySummary 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.
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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