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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 ):           Active    All
Target
BioActive Compounds: 11
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
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
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)
Classification
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