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. 2024 Oct;181(19):3685-3699.
doi: 10.1111/bph.16435. Epub 2024 Jun 5.

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

Megan F Allen  1 , James L Hutchinson  1 , Michael Keith  1 , Shahida Mallah  1 , Robin A Corey  1 , Justin S Trory  1 , Changcheng Jing  2 , Huaquan Fang  2 , Liang Wei  2 , Steven H Bennett  2 , Varinder K Aggarwal  2 , Stuart J Mundell  1 , Ingeborg Hers  1
Affiliations

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

Megan F Allen et al. Br J Pharmacol. 2024 Oct.

Abstract

Background and purpose: 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.

Experimental approach: 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.

Key results: 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.

Conclusion and implications: 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.

Keywords: BRET; F2‐TXA2; F‐TXA2; G‐protein coupled receptor; IP receptor; TP receptor; TXA2; platelets; prostanoids; thromboxane A2.

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