Abstract/Summary

Antiplatelet drugs targeting G-protein coupled receptors (GPCR), 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 rises to differentiate GPVI and GPCR effects on platelets. In 96-, 384- or 1536-well formats, Calcium-6-loaded human platelets displayed a slow-prolonged or fast-transient [Ca2+]i rise when stimulated with the GPVI agonist collagen-related peptide (CRP), or with thrombin and other GPCR agonists, respectively. Semi-automated curve fitting revealed five early and late 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 provide proof-of-principle of distinct receptor-type dependent Ca2+ signaling curves platelets, which allow identification of new inhibitors in a UHT way.