Inverse Agonism of SQ 29,548 and Ramatroban on Thromboxane A2 Receptor

Raja Chakraborty,John Hwa,Prashen Chelikani,Shyamala Dakshinamurti,Rajinder P Bhullar,James Porter

doi:10.1371/journal.pone.0085937

Abstract

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.

Highlights

Thromboxane A2 (TXA2) is a major product of arachidonic acid metabolism and is known to be the key mediator of platelet aggregation and smooth muscle contraction [1,2,3]
The action of TXA2 is mediated by its cognate G protein-coupled receptor (GPCR) thromboxane A2 receptor (TP), which exists in two isoforms, TPa and TPb, differing only in their C- terminal region
Our results suggest that SQ 29,548 and Ramatroban are inverse agonists for TP, whereas, L-670596 and Diclofenac are neutral antagonists

Summary

Introduction

Thromboxane A2 (TXA2) is a major product of arachidonic acid metabolism and is known to be the key mediator of platelet aggregation and smooth muscle contraction [1,2,3]. GPCRs are known to function even in the absence of an agonist molecule and this phenomenon is known as constitutive receptor activity. It can be explained using the multiple state model of receptor activation [6,7]. Over the last decade considerable number of GPCRs were shown to have constitutive activity [8,9] This phenomenon of GPCRs became the most important tool in discriminating between inverse agonists and neutral antagonists [10,11]. Inverse agonists are compounds or drugs known to reduce the constitutive GPCR activity and are often defined to have a (21) efficacy whereas neutral antagonists do not affect the basal GPCR activity and have (0) efficacy [12]. A number of drugs currently in use that target

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