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
BioActive Compounds: 17
Keywords: Platelet, activation, granule, secretion, arterial thrombosis, PAR1, SFLLRN, thrombin receptor
Broad Institute MLPCN Platelet Activation Project
Project ID: 2016
Robert Flaumenhaft, Beth Israel Deaconess Medical Center, email@example.com
John Thomas, NHLBI, ThomasJ@nhlbi.nih.gov
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: 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.
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.
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).
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.
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 = 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.
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)