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

Abstract

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 [Ca 2+ ] 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 [Ca 2+ ] 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 Ca 2+ 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 Ca 2+ signaling curves in platelets, which allow identification of new inhibitors in a UHT way.

Anti‐GPVI nanobody blocks collagen‐ and atherosclerotic plaque–induced GPVI clustering, signaling, and thrombus formation

“…To investigate this further, we inhibited all integrins by removing free ions with ethylenediaminetetraacetic acid (EDTA). Thrombus formation on plaque and collagen I was not affected by 2 mM EDTA; 33,34 however, at higher concentrations (9 mM) 35 a significant decrease in thrombus formation and platelet activation markers was observed on collagen I. On plaque a slight, but not significant, reduction was noted with 9 mM EDTA (Figure 2A, Figure S2).…”

“…To investigate this further, we inhibited all integrins by removing free ions with ethylenediaminetetraacetic acid (EDTA). Thrombus formation on plaque and collagen I was not affected by 2 mM EDTA; 33,34 however, at higher concentrations (9 mM) 35…”

Roles of Focal Adhesion Kinase PTK2 and Integrin αIIbβ3 Signaling in Collagen- and GPVI-Dependent Thrombus Formation under Shear

“…For optimal, diffusion-limited mixing, the speed of agonist injection was set at 125 μL/s [ 41 ]. Calibrated, nanomolar changes in cytosolic [Ca 2+ ] i were calculated as before [ 57 , 58 ]. Measurements were performed in triplicate wells, with platelets isolated from at least 3 donors.…”

Platelet proteomic progress and restraining mechanisms in glycoprotein VI-mediated thrombus formation

“…First studies using gel spots to identify phosphorylated signaling targets resulted in an only small sets of differentially regulated proteins linked to GPVI-induced platelet activation (Suppl. Table 1) 57,58 . However, in a recent GPVI phosphoproteome paper, >3.0k phosphorylation events (>1.3k proteins) were identified by tandem mass tag (TMT) labelling and triple stage tandem mass spectrometry.…”

“…For optimal, diffusion-limited mixing, the speed of agonist injection was set at 125 μL/s 41 . Calibrated, nanomolar changes in cytosolic [Ca 2+ ] i were calculated as before 57,58 . Measurements were performed in triplicate wells, with platelets isolated from at least 3 donors.…”