Chakraborty Raja, Bhullar Rajinder P, Dakshinamurti Shyamala, Hwa John, Chelikani Prashen
Department of Oral Biology, University of Manitoba, Winnipeg, Manitoba, Canada ; Biology of Breathing Group- Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada.
Department of Oral Biology, University of Manitoba, Winnipeg, Manitoba, Canada.
PLoS One. 2014 Jan 23;9(1):e85937. doi: 10.1371/journal.pone.0085937. eCollection 2014.
G protein-coupled receptors (GPCRs) show some level of basal activity even in the absence of an agonist, a phenomenon referred to as constitutive activity. Such constitutive activity in GPCRs is known to have important pathophysiological roles in human disease. The thromboxane A2 receptor (TP) is a GPCR that promotes thrombosis in response to binding of the prostanoid, thromboxane A2. TP dysfunction is widely implicated in pathophysiological conditions such as bleeding disorders, hypertension and cardiovascular disease. Recently, we reported the characterization of a few constitutively active mutants (CAMs) in TP, including a genetic variant A160T. Using these CAMs as reporters, we now test the inverse agonist properties of known antagonists of TP, SQ 29,548, Ramatroban, L-670596 and Diclofenac, in HEK293T cells. Interestingly, SQ 29,548 reduced the basal activity of both, WT-TP and the CAMs while Ramatroban was able to reduce the basal activity of only the CAMs. Diclofenac and L-670596 showed no statistically significant reduction in basal activity of WT-TP or CAMs. To investigate the role of these compounds on human platelet function, we tested their effects on human megakaryocyte based system for platelet activation. Both SQ 29,548 and Ramatroban reduced the platelet hyperactivity of the A160T genetic variant. Taken together, our results suggest that SQ 29,548 and Ramatroban are inverse agonists for TP, whereas, L-670596 and Diclofenac are neutral antagonists. Our findings have important therapeutic applications in the treatment of TP mediated pathophysiological conditions.
G蛋白偶联受体（GPCRs）即使在没有激动剂的情况下也表现出一定水平的基础活性，这种现象被称为组成性活性。已知GPCRs中的这种组成性活性在人类疾病中具有重要的病理生理作用。血栓素A2受体（TP）是一种GPCR，它在与前列腺素血栓素A2结合后促进血栓形成。TP功能障碍广泛涉及诸如出血性疾病、高血压和心血管疾病等病理生理状况。最近，我们报道了TP中一些组成性活性突变体（CAMs）的特征，包括一个基因变体A160T。现在，我们以这些CAMs作为报告分子，在HEK293T细胞中测试TP的已知拮抗剂SQ 29548、雷马曲班、L-670596和双氯芬酸的反向激动剂特性。有趣的是，SQ 29548降低了野生型TP和CAMs的基础活性，而雷马曲班仅能降低CAMs的基础活性。双氯芬酸和L-670596在野生型TP或CAMs的基础活性方面未显示出统计学上的显著降低。为了研究这些化合物对人类血小板功能的作用，我们在基于人类巨核细胞的血小板激活系统中测试了它们的效果。SQ 29548和雷马曲班都降低了A160T基因变体的血小板过度活性。综上所述，我们的结果表明，SQ 29548和雷马曲班是TP的反向激动剂，而L-670596和双氯芬酸是中性拮抗剂。我们的发现对于TP介导的病理生理状况的治疗具有重要的治疗应用价值。
