PB0410 - Difluorinated Thromboxane A2 Reveals Crosstalk Between Platelet Activatory and Inhibitory Pathways by Targeting Both the TP and IP Receptor.

Background: Thromboxane A2 (TxA2) is a prostanoid produced during platelet activation and is important in enhancing platelet responses. However, due to its short half-life of 32 seconds, studying TxA2 signalling using its endogenous structure is challenging. We recently synthesised a mono (F-TxA2) and difluorinated (F2-TxA2) TxA2 analogue with enhanced half-life.

Aims: Explore the effect of these fluorinated analogues on human platelet function and signalling.

Methods: We employed light transmission aggregometry, calcium mobilisation and western blotting assays to study human platelet function and signalling. Bioluminescence resonance energy transfer (BRET) in transfected HEK-293T cells was used to study direct receptor activation of the TP and IP receptor individually. Statistical significance was determined using appropriate analysis (P < 0.05 as significant, n=5).

Results: F-TxA2 induced sustained aggregation to the same extent as the prostaglandin H2 analogue U46619. In contrast, F2-TxA2 induced initial aggregation followed by a reversal in aggregation, resulting in a reduction in area under the curve. IP receptor, but not EP2 or EP4 receptor antagonism, converted this transient response into irreversible, sustained aggregation. Through western blotting, F2-TxA2 rapidly activated platelet TP-mediated protein kinase C (PKC) activation, whereas onset of IP-mediated protein kinase A (PKA) activation was delayed. Moreover, F2-TxA2 was more potent in inducing PKC signalling compared to PKA signalling yielding EC50 values of 32.34 nM and 1.16 µM respectively (P < 0.05). BRET assays confirmed that F2-TxA2 behaved as a full agonist at the TP receptor but a partial agonist at the IP receptor.

Conclusion(s): F-TxA2 is a close analogue of TxA2 and can be used as a tool to activate platelets through the TP receptor. In contrast F2-TxA2 acts on both the TP and IP receptor as a full and partial agonist, respectively. This results in initial TP-mediated platelet aggregation followed by IP-induced reversibility of aggregation.

This work was funded by the British Heart Foundation.