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
2761	Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of the membrane-associated serine protease Rv3671c in M.tuberculosis	screening

Keywords: Platelets, dense granule secretion, serotonin, SFLLRN, PAR1

Assay Overview: Cell-based assay for measuring specific dense granule release induced by SFLLRN-mediated platelet activation. Compounds showing no activity (inhibition) in a thrombin receptor-activating peptide SFLLRN-induced P-selectin expression assay (AID 2518) were tested for ability to inhibit release of serotonin, a specific component of platelet dense granules, induced by activation with the PAR1-activating peptide SFLLRN. Washed platelets were incubated with 14C-serotonin resulting in incorporation into dense granules. Compounds were added to platelet samples at 10 uM. Platelets were activated with SFLLRN and supernatants were collected for analysis with a scintillation counter to measure released 14C-serotonin.

Expected Outcome: A moderate decrease in the level of 14C-serotonin in supernatants of compound treated samples compared to untreated samples would identify specific inhibitors of dense granule release.

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. 4.5 mL platelet rich plasma (PRP) (approx 3x10^8 platelets/mL) was incubated in the presence of 14C-serotonin (0.225 uCi ) for 45 minutes at 37oC.

2. 15% acid-citrate-dextrose (ACD)+ 150nM prostaglandin E1 (PGE1) was added to PRP and spun at 1000g for 10 minutes to maintain the resting state of platelets while removing unincorporated 14C-serotonin.

3. Platelets were re-suspended in HEPES-Tyrodes buffer to a platelet concentration of 2x10^8 platelets/mL.

4. Compounds were added to resuspended platelets at 10uM and incubated for 15 minutes.

5. Platelets were subsequently stimulated with 5 uM SFLLRN peptide and 5 uM imipramine (to prevent 14C-serotonin reuptake) for 15 minutes.

6. Platelets were separated from medium by centrifugation at 8000 x g.

7. Supernatants were collected and counted using a beta liquid scintillation counter. Disintegrations per minute (DPM) were collected for 50 ul of platelets.

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

Percent inhibition of 14C-serotonin secretion was calculated based on the assumption that complete (100%) inhibition is attainable, as:
100 - (100 * (DPM at 10uM/ Mean DPM DMSO))

A positive control compound, Cilostazol (PubChem CID 2754), was tested at 100 uM. Mean DPM for DMSO was 3245 and Mean DPM for Cilostazol was 211, resulting in 93% inhibition of 14C-serotonin secretion.


PUBCHEM_ACTIVITY_SCORE
The Mean of Percent Inhibition across two replicates.

PUBCHEM_ACTIVITY_OUTCOME
Activity_Outcome = 1 (inactive)
PUBCHEM_ACTIVITY_SCORE <= 50

Activity_Outcome = 2 (active)
PUBCHEM_ACTIVITY_SCORE > 50

Grant Number: 1R03DA026209-01