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BioAssay: AID 2655

Fluorescence Cell-Based Screen to Identify an Inhibitor of Thrombin Receptor-Activating Peptide SFLLRN-Mediated Actin Polymerization in Platelets

Cell-based assay for measurement of actin polymerization in response to SFLLRN-induced platelet activation. Platelets were obtained from individual donors, gel-filtered, and treated with DMSO (neutral control) or compounds (10 uM) that did not induce elevation of cAMP in platelets as measured in a cAMP assay. Following incubation with compounds, platelets were either treated or not treated more ..
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 Tested Compounds
 Tested Compounds
All(2)
 
 
Active(2)
 
 
 Tested Substances
 Tested Substances
All(2)
 
 
Active(2)
 
 
 Related BioAssays
 Related BioAssays
AID: 2655
Data Source: Broad Institute (2016-06_INHIBITORS_SINGLE-POINT_MLPCN-DRYPOWDER)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2010-03-24
Hold-until Date: 2010-09-30
Modify Date: 2010-09-30

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 2
Related Experiments
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AIDNameTypeProbeComment
1663MLPCN Platelet Activation -Dense Granule ReleaseScreening depositor-specified cross reference: Primary HTS
1678Broad Institute MLPCN Platelet ActivationSummary3 depositor-specified cross reference: Project Summary
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
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
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, SFLLRN, PAR1, actin, phalloidin

Assay Overview:
Cell-based assay for measurement of actin polymerization in response to SFLLRN-induced platelet activation. Platelets were obtained from individual donors, gel-filtered, and treated with DMSO (neutral control) or compounds (10 uM) that did not induce elevation of cAMP in platelets as measured in a cAMP assay. Following incubation with compounds, platelets were either treated or not treated with the thrombin receptor peptide agonist, SFLLRN. Both populations of platelets were fixed, and then incubated in the presence of FITC-phalloidin and Triton X-100. Samples were analyzed by flow cytometry to determine levels of actin polymerization upon activation. Actin filament formation results in higher fluorescent measurements. Geometric mean values were collected for each sample.

Expected Outcome:
This assay further delineates the specificity of the probes. Compounds that demonstrated <50% inhibition of FITC-phalloidin binding upon stimulation with SFLLRN were considered Granule Specific Probes (Class ii probes), as defined in the probe definition. Compounds that demonstrated >50% inhibition were further evaluated in additional assays for their potential as Class iii probes, G-coupled receptor specific probes.

Primary Collaborators(and laboratory where 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. Gel-filtered platelet samples (50 ul) were incubated in the presence of 0.3% DMSO (neutral control) or 10 uM compounds for 20 minutes at 37oC.
2. Platelet samples were then incubated in the presence or absence of 5uM SFLLRN for 15 minutes at 37oC.
3. 37% Formaldehyde was added to each sample at 10% of the sample volume and incubated at 30oC for 45 minutes.
4. 10uM FITC-Phalloidin + 0.1% Triton X-100 was added to each sample and incubated at 30oC for 1 hour.
5. Samples were added to 500uL of BD Biosciences FACSFlow buffer (Catalog Number 342003) and then analyzed by flow cytometry. Geometric mean fluorescence was measured to quantitate FITC-phalloidin binding.
Comment
Data Analysis:
The resting (background) control was DMSO only, with no SFFLRN stimulation, in duplicate.
The neutral control was DMSO only, with SFFLRN stimulation, in duplicate.
Compounds were tested at 10 uM, with SFFLRN stimulation, in duplicate.
Mean Corrected Fluorescence DMSO was calculated as:
Mean Fluorescence DMSO with SFFLRN stimulation - Mean Fluorescence DMSO with no stimulation
Mean Corrected Fluorescence at 10 uM was calculated as:
Mean Fluorescence at 10 uM with SFFLRN stimulation - Mean Fluorescence DMSO with no stimulation
Mean Percent Inhibition of SFFLRN-induced phalloidin binding was calculated based on the assumption that complete (100% ) inhibition is attainable, as:
100 - (100 * (Mean Corrected Fluorescence at 10 uM / Mean Corrected Fluorescence DMSO))
PUBCHEM_ACTIVITY_SCORE
The Mean Percent Inhibition value at the tested concentration 10 uM.
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
1Percent InhibitionMean Percent Inhibition of SFFLRN-induced phalloidin binding Float%
Additional Information
Grant Number: 1R03DA026209-01

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