The Thromboxane Receptor Antagonist PBT-3, a Hepoxilin Stable Analog, Selectively Antagonizes the TPα Isoform in Transfected COS-7 Cells

Abstract

The hepoxilin analog PBT-3 [10(S)-hydroxy-11,12-cyclopropyleicosa-5Z,8Z,14Z-trienoic acid methyl ester] was previously shown to inhibit the aggregation of human platelets and to antagonize the binding of the thromboxane receptor agonist I-BOPin human platelets (Pace-Asciak et al., 2002). We show herein that PBT-3 inhibits, to different degrees, binding of the TP receptor antagonist, to the TP receptor isoforms in TP␣-and TP␤-transfected COS-7 cells. These isoforms possess a different tail length, the ␣ being shorter than the ␤ isoform. In contrast, SQ 29,548 shows no selection for the two TP isoforms. The IC 50 value for PBT-3 ϭ 2.0 Ϯ 0.3 ϫ 10 Ϫ7 M was observed for TP␣, whereas this was one-sixth less active on the TP␤ isoform (IC 50 ϭ 1.2 Ϯ 0.2 ϫ 10 Ϫ6 M), suggesting selectivity for the TP␣ isoform. To investigate whether the tail contributes to the difference in competition binding by PBT-3, we investigated the tailless TP isoform expressed in transfected COS-7 cells. Its IC 50 was similar to that of the TP␣ isoform. In additional studies, we investigated the effect of PBT-3 on the collagen and I-BOP evoked intracellular calcium release and on the collagen and I-BOP evoked phosphorylation of pleckstrin. PBT-3 blocked both pathways further demonstrating its TP receptor antagonist activity. These results demonstrate that the action of PBT-3 in inhibiting platelet aggregation is mediated via inhibition of the TP␣ isoform of the thromboxane receptor and that the tail may play an important role in recognition of this TP receptor antagonist.

Vascular hepoxilin and trioxilins mediate vasorelaxation through TP receptor inhibition in mouse arteries

“…showed that the hepoxilin analogue, PBT‐3 [10(S)‐hydroxy‐11,12‐cyclopropyleicosa‐5(Z),8(Z),14(Z)‐trienoic acid methyl ester] inhibited binding of the TP antagonist [ 3 H]‐SQ29548 to TP α , but not TP β , receptors in transiently transfected COS‐7 cells (Qiao et al . ). These data and ours indicate that the structure around carbon 11 and 12 may vary greatly and still inhibit TP receptors.…”

In Vitro and in Vivo Pharmacological Characterization of BM-613 [N-n-Pentyl-N′-[2-(4′-methylphenylamino)-5-nitrobenzenesulfonyl]urea], a Novel Dual Thromboxane Synthase Inhibitor and Thromboxane Receptor Antagonist

“…Moreover, the exact physiological significance of the existence of two iso- forms is still unclear, although it has been shown that TP␣ was the only isoform expressed in platelets (Habib et al, 1999) and that both TP␣ and TP␤ were expressed in smooth muscle cells (Miggin and Kinsella, 1998). Recently, Qiao et al (2003) showed for the first time that the hepoxilin-stable analog PBT-3 could selectively bind the TP␣ isoform in transfected COS-7 cells. Thus, we postulated that BM-613 could have a greater affinity for TP␣ than TP␤ and that this could be coupled with higher activity as an antiplatelet agent.…”

Hepoxilin Analogs, Potential New Therapeutics in Disease

“…PBT-3 was the most active of the PBTs showing 5-10 fold greater activity in inhibiting platelet aggregation than the next best PBTs. Further studies using COS-7 cells in which transient expression of the TPα and TPβ receptor were carried out, showed a greater specificity for PBT-3 for the TPα receptor, the isoform abundantly present in human platelets [39]. These findings demonstrate a potentially important and powerful action of PBT-3 as an antagonist of the TP receptor revealing a likely mechanism for its inhibitory actions on platelet aggregation.…”