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

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.

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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.