Ultra High-throughput Screening Assay to Identify New Antiplatelet Drugs with Discriminative Mode of Receptor-dependent Action


Platelet G protein–coupled receptors (GPCRs) influence platelet function by mediating the response to various agonists, including ADP, thromboxane A2, and thrombin. Blockade of the ADP receptor, P2Y12, in combination with cyclooxygenase-1 inhibition by aspirin has been among the most widely used pharmacological strategies to reduce the occurrence of cardiovascular events in high-risk patients. This dual antiplatelet therapy is one of the greatest advances in the field of cardiovascular medicine. While antiplatelet drugs targeting GPCRs are currently used for the secondary prevention of atherothrombotic events, they do come with an inherent challenge: there is an increased propensity of bleeding risk. Clinical studies suggest that targeting of immunoreceptor tyrosine-based activation motif (ITAM)-linked receptors, such as glycoprotein VI (GPVI), provides an improved antithrombotic-hemostatic profile. This urges for high-throughput assays to screen for potential inhibitory drugs of the GPVI receptor pathways. This was the goal of Delia Irene Fernández, Ph.D., of Maastricht University in the Netherlands, as she stated on Monday during the Diagnostics and OMIC virtual session. She sought to develop an ultra-high-throughput (UTH) screening assay for finding new inhibitory molecules that differentiate between ITAM-linked receptor and GPCR-induced platelet activation.

This UTH assay was based on intracellular calcium signaling because, as Fernández remarked, platelet activation via GPVI or GPCR elicits distinct calcium responses. The GPVI agonist c-reactive protein showed time-prolonged [Ca2+]i increases, whereas the protease-activated receptors 1 and 4 (PAR1/4) agonist thrombin evoked quick monophasic transient [Ca2+]i rises. To identify agents that maximally differentiated in the suppression of GPVI and PAR1/4 responses, the effects of a panel of well-known platelet inhibitors on intracellular calcium signaling were studied. The inhibitory panel included in the study differed in their respective effects on CRP- or thrombin-induced parameters. Pathway analysis revealed a wider pathway contribution of GPVI-induced calcium signaling in comparison with PAR1/4 as well as separate signaling domains downstream of ITAM-linked or G protein–coupled receptors, which suggested that each pathway has potential targetable signaling domains. This separate mode of inhibition in response to CRP or thrombin was used to analyze a screening of publicly available small molecule compounds and suggests that the UTH screening assay of agonist-induced [Ca2+]i rises has the potential to identify new antiplatelet drugs with a discriminative mode of receptor-dependent action.

Read the full abstract here.

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