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BioAssay: AID 652255

Discovery of Novel Antagonists of Protease activated receptor 4: PAR 1 Selectivity CRC Assay

Protease activated receptor-4 (PAR4) is one of the thrombin receptors on human platelets and is a potential target for the management of thrombotic disorders. PAR1 antagonism was efficacious for the prevention of cardiovascular events in two phase III clinical trials, though patients also had an increased risk of intracranial hemmorage. PAR4 is the low affinity thrombin receptor and thus inhibition of PAR4 may also prove to be efficacious for the prevention of cardiovascular events without the increased risk of bleeding. ..more
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 Tested Compounds
 Tested Compounds
All(2)
 
 
Probe(1)
 
 
Active(1)
 
 
Inactive(1)
 
 
 Tested Substances
 Tested Substances
All(2)
 
 
Probe(1)
 
 
Active(1)
 
 
Inactive(1)
 
 
AID: 652255
Data Source: Vanderbilt Specialized Chemistry Center (Protease activated receptor 4 (PAR4) Antagonist GPIIbIIIa In..)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2013-04-02
Hold-until Date: 2014-04-01
Modify Date: 2014-04-01

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compound: Chemical Probe: 1    Active: 1
Related Experiments
AIDNameTypeComment
652261ML354 Discovery of Novel Antagonists of Protease activated receptor 4Summarydepositor-specified cross reference
652250Discovery of Novel Antagonists of Protease activated receptor 4: Single PointConfirmatorysame project related to Summary assay
652253Discovery of Novel Antagonists of Protease activated receptor 4: CRC AssayConfirmatorysame project related to Summary assay
686926ML354 Eurofin Panel Assay for PAR4 Antagonists Inhibitor (Probe Compound)Othersame project related to Summary assay
Description:
Assay Provider: Heidi Hamm
Assay Provider Affiliation: Vanderbilt University

Protease activated receptor-4 (PAR4) is one of the thrombin receptors on human platelets and is a potential target for the management of thrombotic disorders. PAR1 antagonism was efficacious for the prevention of cardiovascular events in two phase III clinical trials, though patients also had an increased risk of intracranial hemmorage. PAR4 is the low affinity thrombin receptor and thus inhibition of PAR4 may also prove to be efficacious for the prevention of cardiovascular events without the increased risk of bleeding.

There are no probes available to study the role of PAR4 in thrombosis and hemostasis in vivo. The current PAR4 antagonist YD-3 was first described as an antagonist in 2001 and no further characterization or optimization has been conducted. YD-3 has several drawbacks including, a lengthy synthetic scheme, and high lipophilicity making it unsuitable for chemical modification and having limited in vivo use. The field's understanding of the role of PAR4 in physiology is limited, because of the lack of a sufficient tool compound to study PAR4. An optimized compound will allow us to determine basic pharmacological information about PAR4 including receptor density, possible patient-to-patient variability, and mechanism of action for PAR4 antagonists and agonists, data which currently cannot be collected.
Protocol
60 muL of washed platelets at a concentration of 0.15x108 platelets/mL were added to polystyrene tubes. Anti-CD62P antibody antibody was diluted (to the manufacturers suggested concentrations) in Tyrode's buffer containing 0.1% BSA. 40 muL of diluted antibody was added to the platelets and allowed to bind for 5 minutes. Platelets were pre-treated with indicated concentrations of antagonist or DMSO control for 5 minutes followed by addition of PAR1-AP or PAR4-AP for 10 minutes. Platelet activity was quenched by the addition ice cold 1.5% paraformaldehyde followed by dilution in 1X phosphate buffered saline. The final DMSO concentration was 0.5%. Platelets were stored up to 18 hours at 4 oC before flow cytometric analysis. Analysis was carried out on a BD FACS Canto II (Franklin Lakes, NJ). Fluorescent intensity was determined for 100,000 events within the platelet gate. Data was collected and analyzed via FACS DiVa software.
These compounds had IC50s less than 1 micromolar for PAR4 in the flow cytometry assay, and therefore, 'Outcome' was assigned as 'Active'. Compound (VU0099704) SID XXXXXXXXX was more than 100-fold selective for PAR4 versus and thus assigned a 'Score' of '100'.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1Average_IC50_uM*Calculated Avg IC50 for the 3 replicatesFloatμM
2SD_IC50_uMCalculated Stand. Dev of IC50 for the 3 replicatesFloatμM
3SEM_IC50_uMCalculated SEM for IC50 for the 3 replicatesFloatμM
4Average_%_PAR1-AP_Max_InhibitionCalculated Average for PAR4-AP Max InhibitionFloat%
5SD_Average_%_PAR1-AP_Max_InhibitionCalculated Std. Deviation for PAR4-AP Max InhibitionFloat%
6SEM_Average_%_PAR1-AP_Max_InhibitionCalculated S.E.M. for the PAR4-AP Max InhibitionFloat%
7CategoryString
8Value at_0.0001_uM_1 (0.0001μM**)See ProtocolFloat%
9Value at_0.000316_uM_1 (0.000316μM**)See ProtocolFloat%
10Value at_0.001_uM_1 (0.001μM**)See ProtocolFloat%
11Value at_0.00316_uM_1 (0.00316μM**)See ProtocolFloat%
12Value at_0.01_uM_1 (0.01μM**)See ProtocolFloat%
13Value at_0.0316_uM_1 (0.0316μM**)See ProtocolFloat%
14Value at_0.1_uM_1 (0.1μM**)See ProtocolFloat%
15Value at_0.316_uM_1 (0.316μM**)See ProtocolFloat%
16Value at_1_uM_1 (1μM**)See ProtocolFloat%
17Value at_3_uM_1 (3μM**)See ProtocolFloat%
18Value at_10_uM_1 (10μM**)See ProtocolFloat%
19IC50_uM_1IC50 Replicate 1FloatμM
20%_PAR1-AP_Max_1See ProtocolFloat%
21Value at_0.0001_uM_2 (0.0001μM**)See ProtocolFloat%
22Value at_0.000316_uM_2 (0.000316μM**)See ProtocolFloat%
23Value at_0.001_uM_2 (0.001μM**)See ProtocolFloat%
24Value at_0.00316_uM_2 (0.00316μM**)See ProtocolFloat%
25Value at_0.01_uM_2 (0.01μM**)See ProtocolFloat%
26Value at_0.0316_uM_2 (0.0316μM**)See ProtocolFloat%
27Value at_0.1_uM_2 (0.1μM**)See ProtocolFloat%
28Value at_0.316_uM_2 (0.316μM**)See ProtocolFloat%
29Value at_1_uM_2 (1μM**)See ProtocolFloat%
30Value at_3_uM_2 (3μM**)See ProtocolFloat%
31Value at_10_uM_2 (10μM**)See ProtocolFloat%
32IC50_uM_2IC50 Replicate2FloatμM
33%_PAR1-AP_Max_2PERCENTFloat%
34Value at_0.0001_uM_3 (0.0001μM**)See ProtocolFloat%
35Value at_0.000316_uM_3 (0.000316μM**)See ProtocolFloat%
36Value at_0.001_uM_3 (0.001μM**)See ProtocolFloat%
37Value at_0.00316_uM_3 (0.00316μM**)See ProtocolFloat%
38Value at_0.01_uM_3 (0.01μM**)See ProtocolFloat%
39Value at_0.0316_uM_3 (0.0316μM**)See ProtocolFloat%
40Value at_0.1_uM_3 (0.1μM**)See ProtocolFloat%
41Value at_0.316_uM_3 (0.316μM**)See ProtocolFloat%
42Value at_1_uM_3 (1μM**)See ProtocolFloat%
43Value at_3_uM_3 (3μM**)See ProtocolFloat%
44Value at_10_uM_3 (10μM**)See ProtocolFloat%
45IC50_uM_3IC50 of replicate3FloatμM
46%_PAR1-AP_Max_3See ProtocolFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 5P50HL081009-03

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