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BioAssay: AID 493100

Platelet granule secretion Measured in Cell-Based System Using Plate Reader - 2016-01_Inhibitor_Dose_DryPowder_Activity

Keywords: Platelet, activation, granule, secretion, arterial thrombosis, PAR1, SFLLRN, thrombin receptor ..more
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 Tested Compounds
 Tested Compounds
All(49)
 
 
Active(17)
 
 
Inactive(27)
 
 
Inconclusive(5)
 
 
 Tested Substances
 Tested Substances
All(49)
 
 
Active(17)
 
 
Inactive(27)
 
 
Inconclusive(5)
 
 
 Related BioAssays
 Related BioAssays
AID: 493100
Data Source: Broad Institute (2016-01_Inhibitor_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-01-27
Hold-until Date: 2011-02-05
Modify Date: 2011-02-07

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 17
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
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
Description:
Keywords: Platelet, activation, granule, secretion, arterial thrombosis, PAR1, SFLLRN, thrombin receptor

Broad Institute MLPCN Platelet Activation Project

Project ID: 2016

Primary Collaborators:

Robert Flaumenhaft, Beth Israel Deaconess Medical Center, rflaumen@bidmc.harvard.edu

John Thomas, NHLBI, ThomasJ@nhlbi.nih.gov

Project Overview:

The goal of this project is to identify small molecules that inhibit the activation of platelets by measuring the secretion of ATP-rich dense granules. The aim is to identify inhibitory compounds that act on the diverse mechanisms responsible for platelet activation. Probes demonstrating the specific ability to inhibit platelet activation will be further evaluated as potential antithrombic agents. Those potential probes will fall into the 3 attribute classes in order of priority:
1. Small molecule probes that are dense granule specific (Class 1)
2. Small molecule probes that are granule specific (Class 2)
3. Inhibitors of G-protein coupled receptors (GPCRs) (Class 3)


Assay Overview:

Assay: Dense granule release of platelet-rich plasma (PRP). Expired units of PRP obtained from various blood centers were plated in 384-well white assay plates (Aurora, 00030721) on average of 15,600,000 platelets/well in 20ul. PRP was exposed to 7.5uM compounds from the MLPCN collection or various doses of the positive control, Cilostazol (Sigma #C0737, Lot# 042K4704, BRD-K67017579-001-04-2 ) for 30 minutes prior to addition of the thrombin receptor (Par1) activator SFLLRN (5uM, Bachem, H-8365) and detection reagent CellTiter-Glo (Promega, G755) using a modified protocol, for measurement of ATP released from the dense granules. PBS is used in place of CellTiter-Glo Buffer thus preventing platelet lysis and a high background of ATP. Luminescence measurements are taken 15 minutes after reagent addition.


Outcome: A decrease in the luminescent signal will identify compounds that either inhibit the release of dense granules from the platelets, or inhibit the luciferase enzyme. The results are reported as % inhibition. Specificity for platelet inhibition will be determined in secondary assays.
Protocol
Protocol:

1) Plasma bag(s) from a single donor were emptied into a sterile bottle in a hood. If multiple bags exist from a single donor, they were combined as to increase batch volume. Multiple donors' samples could not be pooled due to possible immunological reactions, therefore multiple batches were run daily with each being prepared singly. All information provided on each unit used was recorded (source, donor number, blood type, etc.).

2) Samples were taken to count the number of platelets/ml and checked for activation activity by addition of SFLLRN and CellTiter-Glo.
3) A liquid handler in a hood was used to dispense 20ul of plasma per well. While filling, platelets were kept in homogeneous suspension by gentle agitation.
4) Assay plates were loaded into racks for placement into an automated incubator. The incubator is docked to an enclosed automated screening system (HighRes Biosolutions). Compound plates have their foil seals removed off-line and placed on a plate hotel on the deck of the screening system.
5) CellTiter-Glo/SFLLRN was prepared for each day to a volume to accommodate the number of assay plates estimated for the day.
6) Screening was initiated on the screening system. 100nl of compounds were pinned into assay plates.
7) Assay plates were returned to the incubator for a 30 min. incubation.
8) At the completion of incubation, plates were moved to a liquid handler for addition of 10ul CellTiter-Glo/SFLLRN solution per well, with a final well concentration of 0.083X CellTiter-Glo and 5uM SFLLRN.
9) Plates were moved to a plate hotel on deck for a 15 min. incubation.
10) At completion of incubation, plates were moved to an Envision 2104 Multilabel Reader (Perkin Elmer) for luminescence detection. The ultra sensitive detection was used, with the 1536 aperture in place, to decrease bleed-through from adjacent wells. Read time is 0.1s/well.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.


EXPECTED OUTCOME: Active compounds result in decreasing readout signal.


The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:

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:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.


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.
The columns labeled 'Log_AC50' are equal to (-1)(log10(AC)), often referred to as pAC50.


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.

DATA AGGREGATION:
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:

1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.

2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations (the SEM was also calculated);
b. If the final PUBCHEM_ACTIVITY_OUTCOME != 2, AC was left empty.

3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.
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
4PUBCHEM_ACTIVITY_OUTCOME_TEST1The Activity Summary for every Substance has two parts, the outcome and the score. The outcome for each Substance is reported as an integer value in this column and must be one of three different values:
1 - Substance is considered inactive
2 - Substance is considered active
3 - Substance activity outcome is inconclusive
5 - Substance is considered probe
Integer
5PUBCHEM_ACTIVITY_SCORE_TEST1The Activity Summary for every Substance has two parts, the outcome and the score. The score for this Substance is reported in this column and must be an integer value, where larger values are more active and smaller values are less active. Please make sure your scores are on a linear scale because that's how they will be interpreted. We encourage depositors to consider using the range 0-100, although values larger and smaller are allowed. The score values are used to allow PubChem users to partition, sort, and profile Assay Data results within and between biological assays.Integer
6AC50_Qualifier_TEST1'>','=', or '<'String
7PUBCHEM_ASSAYDATA_COMMENT_TEST1String
8AC50_uM_TEST1The concentration at which activity reaches 50% of the maximumFloatμM
9Log_AC50_M_TEST1The log of the molar AC50Float
10S0_TEST1The fitted activity value at zero concentrationFloat%
11Sinf_TEST1The fitted activity value at infinite concentrationFloat%
12Hill_Slope_TEST1The slope at AC50Float
13Num_Points_TEST1The number of data points used to generate the plotInteger
14Max_Activity_TEST1The maximum activity value observed, based on mean of replicates per concentrationFloat%
15Max_Activity_Conc_uM_TEST1
The concentration at which the maximum activity is observed
FloatμM
16Activity_at_0.005uM_TEST1 (0.005μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_0.015uM_TEST1 (0.015μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.046uM_TEST1 (0.046μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_0.135uM_TEST1 (0.135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_0.42uM_TEST1 (0.42μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_1.2uM_TEST1 (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_3.8uM_TEST1 (3.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_11uM_TEST1 (11μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_35uM_TEST1 (35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25PUBCHEM_ACTIVITY_OUTCOME_TEST2The Activity Summary for every Substance has two parts, the outcome and the score. The outcome for each Substance is reported as an integer value in this column and must be one of three different values:
1 - Substance is considered inactive
2 - Substance is considered active
3 - Substance activity outcome is inconclusive
5 - Substance is considered probe
Integer
26PUBCHEM_ACTIVITY_SCORE_TEST2The Activity Summary for every Substance has two parts, the outcome and the score. The score for this Substance is reported in this column and must be an integer value, where larger values are more active and smaller values are less active. Please make sure your scores are on a linear scale because that's how they will be interpreted. We encourage depositors to consider using the range 0-100, although values larger and smaller are allowed. The score values are used to allow PubChem users to partition, sort, and profile Assay Data results within and between biological assays.Integer
27PUBCHEM_ASSAYDATA_COMMENT_TEST2String
28AC50_Qualifier_TEST2'>','=', or '<'String
29AC50_uM_TEST2The concentration at which activity reaches 50% of the maximumFloatμM
30Log_AC50_M_TEST2The log of the molar AC50Float
31Hill_Slope_TEST2The slope at AC50Float
32S0_TEST2The fitted activity value at zero concentrationFloat%
33Sinf_TEST2The fitted activity value at infinite concentrationFloat%
34Num_Points_TEST2The number of data points used to generate the plotInteger
35Max_Activity_TEST2The maximum activity value observed, based on mean of replicates per concentrationFloat%
36Max_Activity_Conc_uM_TEST2The concentration at which the maximum activity is observedFloatμM
37Activity_at_0.005uM_TEST2 (0.005μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
38Activity_at_0.015uM_TEST2 (0.015μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
39Activity_at_0.046uM_TEST2 (0.046μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
40Activity_at_0.135uM_TEST2 (0.135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
41Activity_at_0.42uM_TEST2 (0.42μM**)
The average measured activity of all accepted replicates at the specified concentration
Float%
42Activity_at_1.2uM_TEST2 (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
43Activity_at_3.8uM_TEST2 (3.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
44Activity_at_11uM_TEST2 (11μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
45Activity_at_35uM_TEST2 (35μM**)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)
Data Table ( Complete ):     View Active Data    View All Data
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