Radioactive Cell-Based Screen to Identify Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated Dense Granule Release by Platelets
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 more ..
BioActive Compound: 1
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, firstname.lastname@example.org, 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.
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.
The Mean of Percent Inhibition across two replicates.
Activity_Outcome = 1 (inactive)
PUBCHEM_ACTIVITY_SCORE <= 50
Activity_Outcome = 2 (active)
PUBCHEM_ACTIVITY_SCORE > 50
Data Table (Concise)