Keywords: Platelet, activation, P-selectin, SFLLRN, PAR1, thrombin receptor, alpha-granule, secretion ..more
Related BioAssays
Related BioAssays
Target
BioActive Compounds: 11
Depositor Specified Assays
| AID | Name | Type | Comment |
|---|---|---|---|
| 1678 | Broad Institute MLPCN Platelet Activation | summary | Summary assay |
Description:
Keywords: Platelet, activation, P-selectin, SFLLRN, PAR1, thrombin receptor, alpha-granule, secretion
Assay Overview:
Cell-based assay for inhibition of SFLLRN-induced P-selectin surface expression. Washed platelets obtained from individual donors were treated with a selection of compounds either at dose or at a single point of 10uM. Following compound addition, platelets were subsequently stimulated with 5 mM SFLLRN. 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 gate for the defined probe paths. A moderate decrease in the amount of P-selectin expressed on the surface of platelets will identify compounds that inhibit pathways that regulate granule secretion or G-protein coupled receptors (GPCR) expressed on the surface of platelets. Lack of substantial inhibition of surface expression of P-selectin is indicative of compounds that might selectively effect dense granule secretion. Additional secondary testing will further delineate the specificity of the probes.
Assay Overview:
Cell-based assay for inhibition of SFLLRN-induced P-selectin surface expression. Washed platelets obtained from individual donors were treated with a selection of compounds either at dose or at a single point of 10uM. Following compound addition, platelets were subsequently stimulated with 5 mM SFLLRN. 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 gate for the defined probe paths. A moderate decrease in the amount of P-selectin expressed on the surface of platelets will identify compounds that inhibit pathways that regulate granule secretion or G-protein coupled receptors (GPCR) expressed on the surface of platelets. Lack of substantial inhibition of surface expression of P-selectin is indicative of compounds that might selectively effect dense granule secretion. Additional secondary testing will further delineate the specificity of the probes.
Protocol
Protocol:
1. Platelet samples (10 ul) were treated with compounds at dose or at a single point of 10uM.
2. 20 minutes following compound addition, platelets were stimulated with 5 uM thrombin-receptor-derived hexapeptide SFLLRN from 100 uM stock.
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 fluorescence values were collected for each sample.
1. Platelet samples (10 ul) were treated with compounds at dose or at a single point of 10uM.
2. 20 minutes following compound addition, platelets were stimulated with 5 uM thrombin-receptor-derived hexapeptide SFLLRN from 100 uM stock.
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 fluorescence values were collected for each sample.
Comment
Data Analysis:
The neutral control was: DMSO only, with no compound.
Compounds were tested at 10uM
Percent inhibition of induced P-selectin expression was calculated based on the assumption that complete (100% ) inhibition is attainable, as:
100 - (100 * (Mean Fluorescence at dose/ Mean Fluorescence at DMSO))
PUBCHEM_ACTIVITY_SCORE
The percent inhibition value at 10uM.
#PUBCHEM_ACTIVITY_OUTCOME
Activity_Outcome = 1 (inactive)
PUBCHEM_ACTIVITY_SCORE<50
Activity_Outcome = 2 (active)
PUBCHEM_ACTIVITY_SCORE>=50
The neutral control was: DMSO only, with no compound.
Compounds were tested at 10uM
Percent inhibition of induced P-selectin expression was calculated based on the assumption that complete (100% ) inhibition is attainable, as:
100 - (100 * (Mean Fluorescence at dose/ Mean Fluorescence at DMSO))
PUBCHEM_ACTIVITY_SCORE
The percent inhibition value at 10uM.
#PUBCHEM_ACTIVITY_OUTCOME
Activity_Outcome = 1 (inactive)
PUBCHEM_ACTIVITY_SCORE<50
Activity_Outcome = 2 (active)
PUBCHEM_ACTIVITY_SCORE>=50
Result Definitions
| TID | Name | Description | Histogram | Type | Unit | |
|---|---|---|---|---|---|---|
| Outcome | The BioAssay activity outcome | Outcome | ||||
| Score | The BioAssay activity ranking score |  | Integer | |||
| 1 | REPRODUCIBILITY_COSINE_TRANSFORM | A measure of how well the activity reproduced across the two samples. Computed as the absolute value of the cosine between the 'replicate vector' (ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility. NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was Invalid# | | Float | ||
| 2 | REPLICATE_A_ACTIVITY_SCORE | The calculated percent activity for the indicated sample | | Float | % | |
| 3 | REPLICATE_B_ACTIVITY_SCORE | #The calculated percent activity for the indicated sample | | Float | % |
Additional Information
Grant Number: 1R03DA026209-01
Data Table (Concise)
Classification
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