AID	Name	Type	Comment
1663	MLPCN Platelet Activation -Dense Granule Release	screening	Primary HTS
1678	Broad Institute MLPCN Platelet Activation	summary	Project Summary

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

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.

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, calculated across all runs.

PUBCHEM_ACTIVITY_OUTCOME
Activity_Outcome = 1 (inactive)
PUBCHEM_ACTIVITY_SCORE <= 50

Activity_Outcome = 2 (active)
PUBCHEM_ACTIVITY_SCORE > 50

Some compounds were retested at least once. These retests were analyzed as independent tests; therefore each test generated a separate set of replicate data, including a score (mean percent inhibition across replicates) and an outcome (2 (active) for score > 50).

Grant Number: 1R03DA026209-01