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BioAssay: AID 652261

ML354 Discovery of Novel Antagonists of Protease activated receptor 4

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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AID: 652261
Data Source: Vanderbilt Specialized Chemistry Center (ML 354: Protease activated receptor 4 (PAR4) Antagonist Disc..)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2013-04-02
Modify Date: 2013-04-16
Target
Related Experiments
AIDNameTypeComment
652250Discovery of Novel Antagonists of Protease activated receptor 4: Single PointConfirmatorydepositor-specified cross reference: Single Point
652253Discovery of Novel Antagonists of Protease activated receptor 4: CRC AssayConfirmatorydepositor-specified cross reference: CRC Assay
652255Discovery of Novel Antagonists of Protease activated receptor 4: PAR 1 Selectivity CRC AssayConfirmatorydepositor-specified cross reference: PAR 1 Selectivity CRC Assay
686926ML354 Eurofin Panel Assay for PAR4 Antagonists Inhibitor (Probe Compound)Otherdepositor-specified cross reference: Eurofins PanLabs Panel 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'. Selectiivity studies were carried against PAR 1 receptor.
Additional Information
Grant Number: 5P50HL081009-03

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