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. 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.

Synthesis and pharmacological characterisation of arctigenin analogues as antagonists of AMPA and kainate receptors.

(-)-Arctigenin and a series of new analogues have been synthesised and then tested for their potential as AMPA and kainate receptor antagonists of human homomeric GluA1 and GluK2 receptors expressed in HEK293 cells using a Ca2+ influx assay. In general, these compounds showed antagonist activity at both receptors with greater activity evident at AMPARs. Schild analysis indicates that a spirocyclic analogue 6c acts as a non-competitive antagonist. Molecular docking studies in which 6c was docked into the X-ray crystal structure of the GluA2 tetramer suggest that (-)-arctigenin and its analogues bind in the transmembrane domain in a similar manner to the known AMPA receptor non-competitive antagonists GYKI53655 and the antiepileptic drug perampanel. The arctigenin derivatives described herein may serve as novel leads for the development of drugs for the treatment of epilepsy.

Synthesis, Stability, and Biological Studies of Fluorinated Analogues of Thromboxane A2.

Platelet activation results in the generation of thromboxane A2 (TxA2), which promotes thrombus formation by further amplifying platelet function, as well as causing vasoconstriction. Due to its role in thrombus formation and cardiovascular disease, its production is the target of antiplatelet drugs such as aspirin. However, the study of TxA2-stimulated cellular function has been limited by its instability (t 1/2 = 32 s, pH = 7.4). Although more stable analogues such as U46619 and difluorinated 10,10-F2-TxA2 have been prepared, we targeted a closer mimic to TxA2 itself, monofluorinated 10-F-TxA2, since the number of fluorine atoms can affect function. Key steps in the synthesis of F-TxA2 included α-fluorination of a lactone bearing a ß-alkoxy group, and a novel synthesis of the strained acetal. F-TxA2 was found to be 105 more stable than TxA2, and surprisingly was only slightly less stable than F2-TxA2. Preliminary biological studies showed that F-TxA2 has similar potency as TxA2 toward inducing platelet aggregation but was superior to F2-TxA2 in activating integrin αIIbß3.

Redundant role of ASK1-mediated p38MAPK activation in human platelet function.

Apoptosis signal-regulating kinase 1 (ASK1) is a member of mitogen-activated protein kinase kinase kinase (MAP3K) family, which recently has been implicated in the regulation of p38 MAPK/PLA2/thromboxane (TxA2) generation, as well as P2Y12 signalling in murine platelets. ASK1 has therefore been proposed as a potential target for anti-thrombotic therapy. At present it is unknown whether ASK1 also contributes to TxA2 formation and platelet function in human. In this study we therefore examined the role of ASK1 using the ASK1 inhibitor selonsertib (GS-4997). We established that ASK1 is responsible for p38 phosphorylation and TxA2 formation in murine platelets, with both GS4997 and p38 inhibitors reducing TxA2 formation. Similar to murine platelets, activation of human platelets resulted in the rapid and transient phosphorylation of ASK1 and the MAP2Ks MMK3/4/6. In contrast, phosphorylation of p38 and its substrate; MAPKAP-kinase2 (MAPKAPK2) was much more sustained. In keeping with these findings, inhibition of ASK1 blocked early, but not later p38/MAPKAPK2 phosphorylation. The latter was dependent on non-canonical autophosphorylation as it was blocked by the p38 inhibitor; SB203580 and the SYK inhibitor; R406. Furthermore, ASK1 and p38 inhibitors had no effect on PLA2 phosphorylation, TxA2 formation and platelet aggregation, demonstrating that this pathway is redundant in human platelets. Together, these results demonstrate that ASK1 contributes to TxA2 formation in murine, but not human platelets and highlight the importance of confirming findings from genetic murine models in humans.