Difluorinated TxA2 reveals crosstalk between platelet activatory and inhibitory pathways by targeting both the TP and IP receptor

Megan F Allen, J L Hutchinson, Michael R W 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

Research output: Contribution to journalArticle (Academic Journal)peer-review

Abstract

Background and Purpose
Thromboxane A2 (TxA2) is a prostanoid produced during platelet activation and is important in enhancing platelet reactivity by activation of the TxA2 receptor (TPR). However, due to the short half-life, studying TxA2 signalling is challenging. To enhance our understanding of TPR-mediated platelet biology, we therefore synthesised mono and difluorinated TxA2 analogues and explored their pharmacology on heterologous and endogenously expressed TPR function.

Experimental approach
Platelet functional and signalling responses were studied using aggregometry, Ca2+ mobilisation experiments and immunoblotting and compared to 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.

Key Results
BRET studies revealed that F-TxA2 and F2-TxA2 promoted receptor-stimulated TPR 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 (IPR) switched F2-TxA2 -mediated reversible aggregation into sustained aggregation. Further BRET studies confirmed F2-TxA2-mediated IPR activation. F2-TxA2 rapidly and potently stimulated platelet TP-mediated protein kinase C/P-pleckstrin, whereas IP-mediated protein kinase A/P-vasodilator-stimulated phosphoprotein was more delayed.

Conclusion and Implications
F-TxA2 is a close analogue to TxA2 and can be used as a selective tool for TPR platelet activation. In contrast, F2-TxA2 acts on both TPR and IPR differently over time, resulting in an initial wave of TPR-mediated platelet aggregation followed by IPR-induced reversibility of aggregation. This study reveals the potential difference in the temporal aspects of stimulatory and inhibitory pathways involved in platelet activation.
Original languageEnglish
JournalBritish Journal of Pharmacology
Publication statusAccepted/In press - 2024

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