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BioAssay: AID 2639

Platelet Aggregation Secondary Assay for Inhibitors of Platelet Integrin alphallb-beta3

The alphaIIbbeta3 receptor plays a vital role in hemostasis and thrombosis, where receptor deficiency causes the hemorrhagic disorder, Glanzmann thrombasthenia, and uncontrolled receptor activation causes thrombosis and blood vessel occlusion. Small molecule inhibitors of alphaIIbbeta3, tirofiban and eptifibatide, have undesirable side effects thought to arise from conformational changes in more ..
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 Tested Compounds
 Tested Compounds
All(32)
 
 
Active(18)
 
 
Inactive(14)
 
 
 Tested Substances
 Tested Substances
All(32)
 
 
Active(18)
 
 
Inactive(14)
 
 
AID: 2639
Data Source: NCGC (RUC737)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-03-22
Hold-until Date: 2010-09-22
Modify Date: 2010-09-23

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 18
Related Experiments
AIDNameTypeComment
2663Inhibitors of Platelet Integrin alphallb-beta3: SummarySummarydepositor-specified cross reference
2628Selectivity Counterscreen Cell Adhesion Assay for Inhibitors of Platelet Integrin alphallb-beta3Confirmatorysame project related to Summary assay
2634Platelet Adhesion Secondary Assay for Inhibitors of Platelet Integrin alphallb-beta3Confirmatorysame project related to Summary assay
Description:
NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]

MLPCN Grant: 1 R03 MH083257-01
Assay Provider: Barry Coller, Rockefeller University

NCGC Assay Overview:

The alphaIIbbeta3 receptor plays a vital role in hemostasis and thrombosis, where receptor deficiency causes the hemorrhagic disorder, Glanzmann thrombasthenia, and uncontrolled receptor activation causes thrombosis and blood vessel occlusion. Small molecule inhibitors of alphaIIbbeta3, tirofiban and eptifibatide, have undesirable side effects thought to arise from conformational changes in alphaIIbbeta3. These changes transiently activate the receptor to cause spontaneous platelet aggregation and thrombus formation. The goal of this project is to identify new alphaIIbbeta3 antagonists that do not activate the receptor or initiate conformational changes that expose novel epitopes. This secondary assay measures platelet aggregation (Blue et al., 2008). Platelets are induced to aggregate and aggregation is tracked by the change in absorbance over time. Inhibitors of alphaIIbbeta3 block platelet aggregation and decrease the rate of change in absorbance.
Protocol
NCGC Assay Protocol Summary:

Untreated 96-well polystyrene plates (Nunc, Rochester, NY) were blocked for at least 1 hour with HBMT (138 mM NaCl, 12 mM NaHCO3, 10 mM HEPES, 2.7 mM KCl, 0.4 mM NaH2PO4, 0.1% glucose, 0.35% BSA, pH 7.4). Compounds (33 uL in HBMT) and citrated platelet-rich plasma (67 uL) were added to the wells. After plates equilibrated at 37 C in the plate reader (SpectraMax; Molecular Devices, Sunnyvale, CA) for 10 minutes, aggregation was initiated by adding ADP (5 uM final concentration; Chrono-Log Corp, Havertown, PA). Absorbance (Abs) was measured at 563 nm every 10 seconds with 3 seconds of shaking between determinations for 8 minutes.

Reference

Blue R, Murcia M, Karan C, Jirouskova M, and Coller BS. Application of high-throughput screening to identify a novel alphaIIb-specific small- molecule inhibitor of alphaIIbbeta3-mediated platelet interaction with fibrinogen. Blood. 2008, 111(3):1248-56
Comment
Compound Ranking:

Active compounds were assigned a score between 30 and 100 based on compound potency. Inactive compounds were assigned a score of 0 and inconclusive compounds were assigned a score of 25.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ActivityIndicates type of activity observed: active or inactive.String
2Potency*The concentration of sample yielding half-maximal inhibition.FloatμM
3Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration.
Additional Information
Grant Number: 1 R03 MH083257-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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