Difluorinated thromboxane A2 reveals crosstalk between platelet activatory and inhibitory pathways by targeting both the TP and IP receptors.

Thromboxane A2 (TXA2) is a prostanoid produced during platelet activaton, important in enhancing platelet reactivity by activation of TP receptors. However, due to the short half-life, studying TXA2 signalling is challenging. To enhance our understanding of TP receptor-mediated platelet biology, we therefore synthesised mono and difluorinated TXA2 analogues and explored their pharmacology on heterologous and endogenously expressed TP receptor function. Platelet functional and signalling responses were studied using aggregometry, Ca2+ mobilisation experiments and immunoblotting and compared with an analogue of the TXA2 precursor prostaglandin H2, U46619. Gαq/Gαs receptor signalling was determined using a bioluminescence resonance energy transfer (BRET) assay in a cell line overexpression system. BRET studies revealed that F-TXA2 and F2-TXA2 promoted receptor-stimulated TP receptor G-protein activation similarly to U46619. Unexpectedly, F2-TXA2 caused reversible aggregation in platelets, whereas F-TXA2 and U46619 induced sustained aggregation. Blocking the IP receptor switched F2-TXA2-mediated reversible aggregation into sustained aggregation. Further BRET studies confirmed F2-TXA2-mediated IP receptor activation. F2-TXA2 rapidly and potently stimulated platelet TP receptor-mediated protein kinase C/P-pleckstrin, whereas IP-mediated protein kinase A/P-vasodilator-stimulated phosphoprotein was more delayed. F-TXA2 is a close analogue to TXA2 used as a selective tool for TP receptor platelet activation. In contrast, F2-TXA2 acts on both TP and IP receptors differently over time, resulting in an initial wave of TP receptor-mediated platelet aggregation followed by IP receptor-induced reversibility of aggregation. This study reveals the potential difference in the temporal aspects of stimulatory and inhibitory pathways involved in platelet activation. © 2024 The Author(s). British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.

Citation

Megan F Allen, James L Hutchinson, Michael Keith, Shahida Mallah, Robin A Corey, Justin S Trory, Changcheng Jing, Huaquan Fang, Liang Wei, Steven H Bennett, Varinder K Aggarwal, Stuart J Mundell, Ingeborg Hers. Difluorinated thromboxane A2 reveals crosstalk between platelet activatory and inhibitory pathways by targeting both the TP and IP receptors. British journal of pharmacology. 2024 Oct;181(19):3685-3699

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PMID: 38840293

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