Assay Overview: Cell-based assay for inhibition of PMA (phorbol myristate acetate)-induced P-selectin surface expression. Washed platelets obtained from individual donors were treated with compounds found to show less than 50% inhibition in an SFLLRN-induced FITC phalloidin assay. Platelet samples were treated with 30 uM compound. Following compound addition, platelets were stimulated with 5 more ..
Tested Compounds:
Depositor Specified Assays
Description:
Keywords: Platelet, activation, P-selectin, PMA, Protein kinase C
Assay Overview: Cell-based assay for inhibition of PMA (phorbol myristate acetate)-induced P-selectin surface expression. Washed platelets obtained from individual donors were treated with compounds found to show less than 50% inhibition in an SFLLRN-induced FITC phalloidin assay. Platelet samples were treated with 30 uM compound. Following compound addition, platelets were stimulated with 5 nM PMA. After a 15-minute incubation, phycoerythrin-labeled anti-P-selectin antibody (BD Biosciences) was added for a 20-minute incubation. The samples were analyzed by flow cytometry to determine P-selectin expression on the surface of the platelets as a response to activation. Geometric mean values were collected for each sample.
Expected Outcome: This assay serves as a specificity assay, along with an analogous assay using Ca2+-ionophore-induced activation of platelets, to determine selectivity towards G-protein coupled receptors (GPCR). Compounds that do not inhibit surface expression of P-selectin from PMA-induced and Ca2+-ionophore-induced activation are potential GPCR-selective inhibitors.
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
Assay Overview: Cell-based assay for inhibition of PMA (phorbol myristate acetate)-induced P-selectin surface expression. Washed platelets obtained from individual donors were treated with compounds found to show less than 50% inhibition in an SFLLRN-induced FITC phalloidin assay. Platelet samples were treated with 30 uM compound. Following compound addition, platelets were stimulated with 5 nM PMA. After a 15-minute incubation, phycoerythrin-labeled anti-P-selectin antibody (BD Biosciences) was added for a 20-minute incubation. The samples were analyzed by flow cytometry to determine P-selectin expression on the surface of the platelets as a response to activation. Geometric mean values were collected for each sample.
Expected Outcome: This assay serves as a specificity assay, along with an analogous assay using Ca2+-ionophore-induced activation of platelets, to determine selectivity towards G-protein coupled receptors (GPCR). Compounds that do not inhibit surface expression of P-selectin from PMA-induced and Ca2+-ionophore-induced activation are potential GPCR-selective inhibitors.
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
Protocol
1. Platelet samples (10 ul) were incubated with 30 uM compound.
2. 20 minutes following compound addition, platelets were stimulated with 5 nM PMA.
3. After a 15-minute incubation, phycoerythrin-labeled anti-P-selectin antibody (5 ul; BD Biosciences) was added. The samples were agitated gently.
4. After a 20-minute incubation, 500 ul of FACS buffer (BD Biosciences) was added to each of the samples.
5. Samples were then analyzed by flow cytometry to determine P-selectin expression on the surface of the platelets as a response to activation.
6. Geometric mean values were collected for each sample.
2. 20 minutes following compound addition, platelets were stimulated with 5 nM PMA.
3. After a 15-minute incubation, phycoerythrin-labeled anti-P-selectin antibody (5 ul; BD Biosciences) was added. The samples were agitated gently.
4. After a 20-minute incubation, 500 ul of FACS buffer (BD Biosciences) was added to each of the samples.
5. Samples were then analyzed by flow cytometry to determine P-selectin expression on the surface of the platelets as a response to activation.
6. Geometric mean values were collected for each sample.
Comment
Data analysis:
The neutral control was: DMSO only, with no compound.
Compounds were tested at 30uM.
Percent inhibition of induced P-Selectin expression was calculated as:
100 - (100 * (Mean Fluorescence at 30uM/ Mean Fluorescence at DMSO))
The percent inhibition is reported for two replicates in each run.
PUBCHEM_ACTIVITY_SCORE
The average Percent Inhibition value at the tested concentration 30uM, calculated across all runs.
PUBCHEM_ACTIVITY_OUTCOME
Activity_Outcome = 1 (inactive)
score <= 50 in all runs
Activity_Outcome = 2 (active)
score > 50 in all runs
Activity_Outcome = 3 (inconclusive)
score > 50 in one or more runs and score <= 50 in one or more runs
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 (average percent inhibition across replicates) and an outcome (2 (active) for score > 50).
The neutral control was: DMSO only, with no compound.
Compounds were tested at 30uM.
Percent inhibition of induced P-Selectin expression was calculated as:
100 - (100 * (Mean Fluorescence at 30uM/ Mean Fluorescence at DMSO))
The percent inhibition is reported for two replicates in each run.
PUBCHEM_ACTIVITY_SCORE
The average Percent Inhibition value at the tested concentration 30uM, calculated across all runs.
PUBCHEM_ACTIVITY_OUTCOME
Activity_Outcome = 1 (inactive)
score <= 50 in all runs
Activity_Outcome = 2 (active)
score > 50 in all runs
Activity_Outcome = 3 (inconclusive)
score > 50 in one or more runs and score <= 50 in one or more runs
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 (average percent inhibition across replicates) and an outcome (2 (active) for score > 50).
Result Definitions
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| TID | Name | Description | Histogram | Type | Unit | |
|---|---|---|---|---|---|---|
| Outcome | The BioAssay activity outcome | Outcome | ||||
| Score | The BioAssay activity ranking score |  | Integer | |||
| 1 | outcome_Run1 | The activity outcome for Run1 | | Integer | ||
| 2 | score_Run1 | The activity ranking score for Run1 | | Integer | ||
| 3 | REPLICATE_A_ACTIVITY_SCORE_Run1 | The calculated activity score for the sample A, a NULL here denotes data was not used or was not produced | | Float | % | |
| 4 | REPLICATE_B_ACTIVITY_SCORE_Run1 | The calculated activity score for the sample A, a NULL here denotes data was not used or was not produced | | Float | % | |
| 5 | outcome_Run2 | The activity outcome for Run2 | | Integer | ||
| 6 | score_Run2 | The activity ranking score for Run2 | | Integer | ||
| 7 | REPLICATE_A_ACTIVITY_SCORE_Run2 | The calculated activity score for the sample A, a NULL here denotes data was not used or was not produced | | Float | % | |
| 8 | REPLICATE_B_ACTIVITY_SCORE_Run2 | The calculated activity score for the sample A, a NULL here denotes data was not used or was not produced | | Float | % |
Additional Information
Grant Number: 1R03DA026209-01
Data Table (Concise)
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