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BioAssay: AID 2518

Fluorescence Cell-Based Dose Response to Identify Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated P-Selectin Induction on Platelets.

Keywords: Platelet, activation, P-selectin, SFLLRN, PAR1, thrombin receptor, alpha-granule, secretion ..more
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 Tested Compounds
 Tested Compounds
All(28)
 
 
Active(22)
 
 
Inactive(6)
 
 
 Tested Substances
 Tested Substances
All(28)
 
 
Active(22)
 
 
Inactive(6)
 
 
 Related BioAssays
 Related BioAssays
AID: 2518
Data Source: Broad Institute (2016-03_INHIBITORS_DOSE-TITRATION_MLPCN-CHERRYPICK)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2010-03-12
Hold-until Date: 2010-09-30
Modify Date: 2010-09-30

Data Table ( Complete ):           Active    All
BioActive Compounds: 22
Depositor Specified Assays
AIDNameTypeProbeComment
1663MLPCN Platelet Activation -Dense Granule Releasescreening Primary HTS
1678Broad Institute MLPCN Platelet Activationsummary3 Project Summary
1889Luminescence Cell-Based Dose Confirmation HTS to Identify Inhibitors of Platelet Dense Granule Releaseconfirmatory retest at dose
1891Luminescence Biochemical Dose Response HTS to Identify Inhibitors of Luciferaseconfirmatory counterscreen
2645ELISA Cell-Based Screen to Identify Inducers of cAMP in Plateletsscreening
2646ELISA Cell-Based Screen to Identify an Inducer of cAMP in Plateletsscreening
2656Radioactive Cell-Based Screen to Identify Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated Dense Granule Release by Plateletsscreening
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 cherry picked compounds chosen based on activity in a retest at dose (AID 1889), a counterscreen for non-specific inhibitors to luciferase (AID 1891), not having known platelet activation inhibitory activity, and having potentially interesting chemical structure. Compounds were tested at 100uM, 30uM, 1uM, and 0.3uM. 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 in a dose-dependent manner 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.

Primary Collaborators(and laboratory where this 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. Platelet samples (10 ul) were tested at different concentrations of compound (100uM, 30uM, 1uM, 0.3uM, and 0uM for cherry-picks and 30uM, 10uM, 1uM, 0.3uM, and 0uM for powders).

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.
Four compound concentrations were tested: 100uM, 30uM, 1uM, 0.3uM.

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))

EC50 was determined to be the lowest concentration that supported >= 50% of Maximum Activity.

PUBCHEM_ACTIVITY_SCORE
The percent inhibition value at the tested concentration 1uM.

PUBCHEM_ACTIVITY_OUTCOME
Activity_Outcome = 1 (inactive)
EC50 >= 30uM

Activity_Outcome = 2 (active)
EC50 < 30uM
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50 Qualifier'>', '=', or '<'String
2EC50*the concentration whereupon perceived activity reaches 50% of the maximumFloatμM
3Activity at 100uM (100μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
4Activity at 30uM (30μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
5Activity at 1uM (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
6Activity at 0.3uM (0.3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
7Activity at 0uM (0μM**)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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