An optimized and validated 384-well plate assay to test platelet function in a high-throughput screening format

Area of Science:

• Pharmacology
• Biotechnology
• Drug Discovery

Background:

• Cardiovascular disease treatments, including antiplatelet therapies, carry a high risk of hemorrhage.
• Existing platelet function assays are unsuitable for high-throughput screening (HTS) due to cost, complexity, or lack of validation.

Purpose of the Study:

• To develop and validate a simple, low-cost, HTS-compatible platelet function assay for antiplatelet drug discovery.
• To adapt platelet function measurement methods for a 384-well plate format.

Main Methods:

• Assessed agonist-induced platelet activity using absorbance, luminescence, and automated bright-field microscopy in a 384-well plate format.
• Validated assay performance using Z'-factor analysis for robustness.
• Performed drug screening using compounds identified via structure-based virtual screening.

Main Results:

• Automated bright-field imaging offers a novel, validated method for quantifying platelet activity.
• Absorbance and bright-field imaging assays achieved acceptable Z'-factor scores without replicates under specific conditions.
• Luminescence assays required triplicates to reach acceptable Z'-factor scores.

Conclusions:

• The presented assay is optimized and validated for HTS, serving as a valuable tool for antiplatelet drug discovery.
• Automated bright-field microscopy provides a robust and efficient method for platelet function assessment in HTS.
• The assay facilitates the development of safer and more effective antiplatelet therapies.