Megan F. Allen, James L. Hutchinson, Michael Keith, Shahida Mallah, Robin A. Corey, Justin S. Trory, Changcheng Jing, Huaquan Fang, Liang Wei, Steven H. Bennett, Varinder K. Aggarwal, Stuart J. Mundell, Ingeborg Hers
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To enhance our understanding of TP receptor-mediated platelet biology, we therefore synthesised mono and difluorinated TXA<sub>2</sub> analogues and explored their pharmacology on heterologous and endogenously expressed TP receptor function.</p>\n </section>\n \n <section>\n \n <h3> Experimental Approach</h3>\n \n <p>Platelet functional and signalling responses were studied using aggregometry, Ca<sup>2+</sup> mobilisation experiments and immunoblotting and compared with an analogue of the TXA<sub>2</sub> precursor prostaglandin H<sub>2</sub>, U46619. Gα<sub>q</sub>/Gα<sub>s</sub> receptor signalling was determined using a bioluminescence resonance energy transfer (BRET) assay in a cell line overexpression system.</p>\n </section>\n \n <section>\n \n <h3> Key Results</h3>\n \n <p>BRET studies revealed that F-TXA<sub>2</sub> and F<sub>2</sub>-TXA<sub>2</sub> promoted receptor-stimulated TP receptor G-protein activation similarly to U46619. Unexpectedly, F<sub>2</sub>-TXA<sub>2</sub> caused reversible aggregation in platelets, whereas F-TXA<sub>2</sub> and U46619 induced sustained aggregation. Blocking the IP receptor switched F<sub>2</sub>-TXA<sub>2</sub>-mediated reversible aggregation into sustained aggregation. Further BRET studies confirmed F<sub>2</sub>-TXA<sub>2</sub>-mediated IP receptor activation. F<sub>2</sub>-TXA<sub>2</sub> 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.</p>\n </section>\n \n <section>\n \n <h3> Conclusion and Implications</h3>\n \n <p>F-TXA<sub>2</sub> is a close analogue to TXA<sub>2</sub> used as a selective tool for TP receptor platelet activation. 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引用次数: 0
摘要
背景和目的：血栓素 A2（TXA2）是血小板活化过程中产生的一种类固醇，对通过激活 TP 受体增强血小板反应性非常重要。然而，由于血栓素 A2 的半衰期很短，研究 TXA2 的信号传导具有挑战性。为了加深我们对 TP 受体介导的血小板生物学的了解，我们合成了单氟和二氟 TXA2 类似物，并探索了它们对异源和内源性表达的 TP 受体功能的药理作用：实验方法：使用聚集测定法、Ca2+动员实验和免疫印迹法研究血小板的功能和信号反应，并与TXA2前体前列腺素H2的类似物U46619进行比较。在细胞系过表达系统中使用生物发光共振能量转移（BRET）测定法确定了 Gαq/Gαs 受体信号：BRET 研究显示，F-TXA2 和 F2-TXA2 与 U46619 类似，都能促进受体刺激的 TP 受体 G 蛋白活化。意想不到的是，F2-TXA2 会引起血小板的可逆性聚集，而 F-TXA2 和 U46619 则会引起持续性聚集。阻断 IP 受体可将 F2-TXA2 介导的可逆性聚集转变为持续性聚集。进一步的 BRET 研究证实了 F2-TXA2 介导的 IP 受体激活。F2-TXA2 能快速有效地刺激血小板 TP 受体介导的蛋白激酶 C/P-pleckstrin，而 IP 介导的蛋白激酶 A/P-血管扩张剂刺激的磷蛋白则更为延迟：F-TXA2是TXA2的近似物，可作为TP受体血小板活化的选择性工具。相反，F2-TXA2 对 TP 和 IP 受体的作用随着时间的推移而不同，导致最初一波 TP 受体介导的血小板聚集，随后是 IP 受体诱导的可逆性聚集。这项研究揭示了参与血小板活化的刺激和抑制途径在时间上的潜在差异。
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.
期刊介绍：
The British Journal of Pharmacology (BJP) is a biomedical science journal offering comprehensive international coverage of experimental and translational pharmacology. It publishes original research, authoritative reviews, mini reviews, systematic reviews, meta-analyses, databases, letters to the Editor, and commentaries.
Review articles, databases, systematic reviews, and meta-analyses are typically commissioned, but unsolicited contributions are also considered, either as standalone papers or part of themed issues.
In addition to basic science research, BJP features translational pharmacology research, including proof-of-concept and early mechanistic studies in humans. While it generally does not publish first-in-man phase I studies or phase IIb, III, or IV studies, exceptions may be made under certain circumstances, particularly if results are combined with preclinical studies.
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