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. Gα_q/Gα_s 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.