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  3. Ultra-high throughput-based screening for the discovery of antiplatelet drugs affecting receptor dependent calcium signaling dynamics

Ultra-high throughput-based screening for the discovery of antiplatelet drugs affecting receptor dependent calcium signaling dynamics

  • Sci Rep. 2024 Mar 14;14(1):6229. doi: 10.1038/s41598-024-56799-4.
Delia I Fernández 1 2,  Sara Troitiño # 2,  Vladimír Sobota # 3 4,  Bibian M E Tullemans 1 5,  Jinmi Zou 1 5,  Helma van den Hurk 6,  Ángel García 2,  Saman Honarnejad 6,  Marijke J E Kuijpers 7 8,  Johan W M Heemskerk 9 10
Affiliations

Affiliations

  • 1 The Department of Biochemistry, CARIM, Maastricht University, 6229 ER, Maastricht, The Netherlands.
  • 2 Platelet Proteomics Group, CiMUS, Universidade de Santiago de Compostela, 15782, Santiago de Compostela, Spain.
  • 3 IHU-LIRYC, Electrophysiology and Heart Modeling Institute, Fondation Bordeaux Université, 33604, Bordeaux, France.
  • 4 Institut de Mathématiques de Bordeaux, UMR5251, University of Bordeaux, 33 405, Talence, France.
  • 5 Synapse Research Institute, Kon. Emmaplein 7, 6217 KD, Maastricht, The Netherlands.
  • 6 Pivot Park Screening Centre, 5349 AB, Oss, The Netherlands.
  • 7 The Department of Biochemistry, CARIM, Maastricht University, 6229 ER, Maastricht, The Netherlands. marijke.kuijpers@maastrichtuniversity.nl.
  • 8 Thrombosis Expertise Centre, Heart and Vascular Centre, Maastricht University Medical Centre+, 6229 HX, Maastricht, The Netherlands. marijke.kuijpers@maastrichtuniversity.nl.
  • 9 The Department of Biochemistry, CARIM, Maastricht University, 6229 ER, Maastricht, The Netherlands. jwmheem722@outlook.com.
  • 10 Synapse Research Institute, Kon. Emmaplein 7, 6217 KD, Maastricht, The Netherlands. jwmheem722@outlook.com.
  • # Contributed equally.
PMID: 38486006 DOI: 10.1038/s41598-024-56799-4
Abstract

Distinct platelet activation patterns are elicited by the tyrosine kinase-linked collagen receptor Glycoprotein VI (GPVI) and the G-protein coupled protease-activated receptors (PAR1/4) for Thrombin. This is reflected in the different platelet CA2+ responses induced by the GPVI agonist collagen-related peptide (CRP) and the PAR1/4 agonist Thrombin. Using a 96 well-plate assay with human Calcium-6-loaded platelets and a panel of 22 pharmacological inhibitors, we assessed the cytosolic CA2+ signaling domains of these receptors and developed an automated CA2+ curve algorithm. The algorithm was used to evaluate an ultra-high throughput (UHT) based screening of 16,635 chemically diverse small molecules with orally active physicochemical properties for effects on platelets stimulated with CRP or Thrombin. Stringent agonist-specific selection criteria resulted in the identification of 151 drug-like molecules, of which three hit compounds were further characterized. The dibenzyl formamide derivative ANO61 selectively modulated thrombin-induced CA2+ responses, whereas the aromatic sulfonyl imidazole AF299 and the phenothiazine ethopropazine affected CRP-induced responses. Platelet functional assays confirmed selectivity of these hits. Ethopropazine retained its inhibitory potential in the presence of plasma, and suppressed collagen-dependent thrombus buildup at arterial shear rate. In conclusion, targeting of platelet CA2+ signaling dynamics in a screening campaign has the potential of identifying novel platelet-inhibiting molecules.

Keywords

Collagen; Cytosolic calcium; Glycoprotein VI; Prestwick library; Thrombin; Thrombosis.

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