AID	Name	Type	Comment
1663	MLPCN Platelet Activation -Dense Granule Release	screening	Primary HTS
1678	Broad Institute MLPCN Platelet Activation	summary	Project Summary
2518	Fluorescence Cell-Based Dose Response to Identify Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated P-Selectin Induction on Platelets.	confirmatory	Assay describing the ability of compounds to inhibit SFLLRN-induced P-selectin expression on platelets

Keywords: Platelet, cAMP, PGE1, competitive ELISA

Assay Overview:
Cell-based assay for measurement of platelet cAMP levels. Washed platelets obtained from individual donors were treated with a selection of compounds that were chosen based on activity in an SFLLRN-induced P-selectin expression assay (AID 2518). Prostaglandin E1 (PGE1) at 1 uM was used as a positive control to elevate platelet cAMP levels. Compounds were tested at 10uM. Platelets were lysed following compound or control incubation, and lysates were analyzed with a cAMP competitive ELISA kit (Thermo Scientific). Values were converted to cAMP concentration (pmol/mL) based on a cAMP standard curve measured in parallel.

Expected Outcome:
This assay was performed as a counterscreen to select against compounds that elevate cAMP levels, since many available platelet activation inhibitors are capable of elevating platelet cAMP levels. Levels of cAMP induced by treatment with compounds were compared to high levels induced by treatment with positive control PGE1. Compounds that raised cAMP levels to less than 50% of the PGE1 control were considered inactive and selected for additional assays to determine specificity in ability to inhibit platelet activation.

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

1. Platelet samples (100 ul) were incubated with 1 uM PGE1 (positive control), or 10 uM compound, or 0.3% DMSO (neutral control).

2. After a 5-minute incubation, platelets were lysed in 0.1M HCL/0.5% Triton X-100 buffer.

3. The platelet lysates were evaluated using a commercially available competitive ELISA to quantitate cAMP (Thermo Scientific; Catalog Number EMSCAMPL).

4. A dose curve of known concentrations of cAMP standard was prepared and measured in parallel using the ELISA. cAMP levels in samples were quantitated in pmol/mL by fitting optical density readings to the standard curve.

5. cAMP levels in duplicate samples from platelets exposed to compounds were compared to cAMP levels from platelets exposed to PGE1.

Data Analysis:
The neutral control was DMSO only, with no compound, in duplicate.
The positive control was PGE1 at 1 uM, in duplicate.
Compounds were tested at 10 uM, in duplicate.

PGE1-induced cAMP was calculated as:
Mean pmol/mL with PGE1 - Mean pmol/mL with DMSO

Compound-induced cAMP was calculated as:
Mean pmol/mL with compound - Mean pmol/mL with DMSO

Percent of PGE1-induced cAMP (Percent Activity) was calculated as:
100 * (Compound-induced cAMP at 10uM / PGE1-induced cAMP)


PUBCHEM_ACTIVITY_SCORE
The Percent Activity across two replicates. Negative values were adjusted to be zero. Values greater than 100 were adjusted to be 100.

PUBCHEM_ACTIVITY_OUTCOME
Activity_Outcome = 1 (inactive)
PUBCHEM_ACTIVITY_SCORE <= 50

Activity_Outcome = 2 (active)
PUBCHEM_ACTIVITY_SCORE > 50

Grant Number: 1R03DA026209-01