Abstract Number: OC 08.1
Meeting: ISTH 2020 Congress
Theme: Diagnostics and OMICs » Nanotechnology and Novel Biomolecules
Background: Antiplatelet drugs targeting G-protein coupled receptors (GPCR) are currently used for the secondary prevention of atherothrombotic events. A general limitation of these drugs is that they increase the bleeding risk. First clinical studies suggest that the targeting of ITAM-linked receptors such as glycoprotein VI (GPVI) provide a better antithrombotic-haemostatic profile. This urges for high-throughput assays to screen for potential inhibitory drugs of the latter receptor pathways.
Aims: To develop a high-throughput screening assay for finding new inhibitory molecules that differentiate between ITAM-linked receptor and GPCR-induced platelet activation.
Methods: Washed human platelets were loaded with Calcium-6 dye and activated with collagen-related peptide (CRP) or thrombin in 96- or 1536-well plates by automated pipetting by Flex Station-3 or FLIPR-Tetra robots, respectively. Using a panel of 25 known antiplatelet agents, each inhibiting specific signalling steps (receptors, phospholipases, protein kinases, Ca2+-ATPases), we developed an algorithm to identify drugs that maximally differentiate in the suppression of GPVI- and PAR1/4-responses. This information was used for the evaluation of a 10,000 small molecule compound screen in 1536-well plates.
Results: Both in 96- and 1536-well plates, the GPVI agonist CRP showed time-prolonged [Ca2+]i increases, whereas the PAR1/4 agonist thrombin evoked quick monophasic transient [Ca2+]i rises. The activation profile was described using three curve parameters: area under the curve, calcium increase, and slope. Most panel antagonists differed in their effects on CRP- or thrombin-induced parameters. Pathway analysis revealed separate signalling domains, (in)directly downstream of ITAM-linked or G-protein coupled receptors. The separate mode of inhibition in response to CRP or thrombin was confirmed by a screening of 10,000 publicly available small molecule compounds. Discriminative molecules, not affecting cell viability, were selected for further analysis.
Conclusions: Ultra high-throughput screening assay of agonist-induced [Ca2+]i rises has the potential to identify new antiplatelet drugs with a discriminative mode of receptor-dependent action.
To cite this abstract in AMA style:
Fernández DI, Tullemans BME, Provenzalle I, van Groningen J, Heemskerk JWM, van den Hurk H, Kuijpers MJE. Ultra High-throughput Screening Assay to Identify New Antiplatelet Drugs with Discriminative Mode of Receptor-dependent Action [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/ultra-high-throughput-screening-assay-to-identify-new-antiplatelet-drugs-with-discriminative-mode-of-receptor-dependent-action/. Accessed March 21, 2024.« Back to ISTH 2020 Congress
ISTH Congress Abstracts - https://abstracts.isth.org/abstract/ultra-high-throughput-screening-assay-to-identify-new-antiplatelet-drugs-with-discriminative-mode-of-receptor-dependent-action/