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

BACKGROUND AND PURPOSE

Thromboxane A (TXA) 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 TXA signalling is challenging. To enhance our understanding of TP receptor-mediated platelet biology, we therefore synthesised mono and difluorinated TXA analogues and explored their pharmacology on heterologous and endogenously expressed TP receptor function.

EXPERIMENTAL APPROACH

Platelet functional and signalling responses were studied using aggregometry, Ca mobilisation experiments and immunoblotting and compared with an analogue of the TXA precursor prostaglandin H, U46619. Gα/Gα 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-TXA and F-TXA promoted receptor-stimulated TP receptor G-protein activation similarly to U46619. Unexpectedly, F-TXA caused reversible aggregation in platelets, whereas F-TXA and U46619 induced sustained aggregation. Blocking the IP receptor switched F-TXA-mediated reversible aggregation into sustained aggregation. Further BRET studies confirmed F-TXA-mediated IP receptor activation. F-TXA 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-TXA is a close analogue to TXA used as a selective tool for TP receptor platelet activation. In contrast, F-TXA 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.