Ultra-high-throughput Ca2+ assay in platelets to distinguish ITAM-linked and G-protein-coupled receptor activation

iScience

卷:25

Alicia Veninga¹  Jan van Groningen²  Joanne L. Dunster³  Isabella Provenzale⁴  Marijke J.E. Kuijpers⁵  Delia I. Fernández⁵  Bibian M.E. Tullemans⁵  Hilaire Y.F. Cheung⁵  Helma van den Hurk⁵  Johan W.M. Heemskerk⁶  Saman Honarnejad⁷ 

[1] ISASLeibniz-Institut fur Analytische Wissenschaften-ISAS-e.V., 44227 Dortmund, Germany;

[2] Institute for Cardiovascular and Metabolic Research, University of Reading, RG6 6AX Reading, UK;

[3] Institute of Cardiovascular Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK;

[4] Platelet Proteomics Group, Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain;

[5] Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, 6229 ER Maastricht, The Netherlands;

[6] Pivot Park Screening Centre, 5349 AB Oss, the Netherlands;

关键词: Cell biology;    Functional aspects of cell biology;    Methodology in biological sciences;   

DOI  :  

来源: DOAJ

【 摘 要 】

Summary: Antiplatelet drugs targeting G-protein-coupled receptors (GPCRs), used for the secondary prevention of arterial thrombosis, coincide with an increased bleeding risk. Targeting ITAM-linked receptors, such as the collagen receptor glycoprotein VI (GPVI), is expected to provide a better antithrombotic-hemostatic profile. Here, we developed and characterized an ultra-high-throughput (UHT) method based on intracellular [Ca2+]i increases to differentiate GPVI and GPCR effects on platelets. In 96-, 384-, or 1,536-well formats, Calcium-6-loaded human platelets displayed a slow-prolonged or fast-transient [Ca2+]i increase when stimulated with the GPVI agonist collagen-related peptide or with thrombin and other GPCR agonists, respectively. Semi-automated curve fitting revealed five parameters describing the Ca2+ responses. Verification of the UHT assay was done with a robustness compound library and clinically relevant platelet inhibitors. Taken together, these results present proof of principle of distinct receptor-type-dependent Ca2+ signaling curves in platelets, which allow identification of new inhibitors in a UHT way.

【 授权许可】

Unknown