Characterization of isoprostane signaling: Evidence for a unique coordination profile of 8-iso-PGF2α with the thromboxane A2 receptor, and activation of a separate cAMP-dependent inhibitory pathway in human platelets

Abstract

Since isoprostanes are thought to participate in the pathogenesis of thrombosis, presumably through their interaction with thromboxane receptors (TPRs), we examined the ability of 8-iso-PGF 2α to bind/signal through TPRs. Using TPR expressing HEK cells, it was found that 8-iso-PGF 2α mobilized calcium and bound TPRs with a dissociation constant (K d ) of 57 nM. Interestingly, site-directed-mutagenesis revealed that 8-iso-PGF 2α has a unique coordination profile with TPRs. Thus, while Phe 184 and Asp 193 are shared by both 8-iso-PGF 2α and classical TPR ligands, Phe 196 was found to be required only for 8-iso-PGF 2α binding. Functional studies also revealed interesting results. Namely, that 8-iso-PGF 2α signals in human platelets through both a stimulatory (TPR-dependent) and an inhibitory (cAMP-dependent) pathway. Consistent with the existence of two signaling pathways, platelets were also found to possess two separate binding sites for 8-iso-PGF 2α . While the stimulatory site is represented by TPRs, the second cAMP inhibitory site is presently unidentified, but does not involve receptors for PGI 2 , PGD 2 or PGE 2 . In summary, these studies provide the first documentation that: (1) 8-iso-PGF 2α coordinates with Phe 184 , Asp 193 and Phe 196 on platelet TPRs; (2) Phe 196 serves as a unique TPR binding site for 8-iso-PGF 2α ; (3) 8-iso-PGF 2α signals through both stimulatory and inhibitory pathways in platelets; (4) 8-iso-PGF 2α inhibits human platelet activation through a cAMP-dependent mechanism; (5) 8-iso-PGF 2α interacts with platelets at two separate binding sites. Collectively, these results provide evidence for a novel isoprostane function in platelets which is mediated through a cAMP-coupled receptor.

Investigation of a Thromboxane A 2 Receptor–Based Vaccine for Managing Thrombogenesis

“…This approach, unlike aspirin, would not be expected to be associated with gastric ulcers or promote the production of isoprostanes (eg, 8‐iso Prostaglandin F 2α ) 67, 68. The latter were found to be potential markers of oxidative stress and mediators of platelet activation 30, 69. In terms of other measures of safety, there was no apparent toxicity in the mice vaccinated with the C‐EL2 peptide (and controls).…”

“…However, although the cyclooxygenase enzyme inhibitor aspirin is in clinical use, it is associated with inherent limitations 26, 27, 28. Thus, aspirin: was found to (1) lack selectivity to TXA 2 because it also inhibits prostaglandin I 2 synthesis25; (2) cause bleeding29 and gastric ulcers,28 adverse effects that in some instances mandate its discontinuation; and (3) redirect arachidonic acid metabolism to isoprostanes, which themselves modulate platelet function30; and is (4) associated with sensitivity; and (5) an increasing rate of resistance worldwide 26, 27, 28…”

“…One apparent reason for this failure is because these agents were empirically designed on the basis of the complex structures of prostaglandin H 2 and/or TXA 2 , with little information on the actual TPR binding domains. In this connection, research efforts11, 30, 39, 40, 41 by us and others to map the TPR ligand‐binding domain revealed the following: (1) the ligand‐binding domain resides in the C‐terminus of the second extracellular loop (C‐EL2; C 183 ‐D 193 ) of the receptor protein; (2) this extracellular segment contains ligand–amino acid coordination sites; and (3) an antibody raised against this sequence (ie, abbreviated C‐EL2Ab) inhibits TPR ligand binding and platelet aggregation and protects from thrombogenesis without any apparent bleeding diathesis, making it the first functional antibody against platelet TPRs. Based on these considerations, we sought to further assess the contributions of the C‐EL2 domain to in vivo TPR‐dependent platelet activation (eg, hemostasis/bleeding time) and the genesis of thrombosis, by using a vaccine‐based approach using the cognate TPR C‐EL2 peptide as an immunogen.…”

Repurposing an old drug for a new use: glybenclamide exerts antiplatelet activity by interacting with the thromboxane A2 receptor

“…Consequently, glybenclamide has the potential to serve as a therapeutic agent, and can be added to the arsenal of interventions available for treating thrombotic disease. Furthermore, based on recent evidence [46] of a novel inhibitory function for isoprostanes in platelets, which was shown to prevail under conditions of TPR antagonism, concurrent therapy of a TPR antagonist (eg, glybenclamide) and aspirin might be superior to either therapy alone. Thus, glybenclamide may prove beneficial in combination therapy approaches.…”

Thromboxane A₂ Mediates Iron-Overload Cardiomyopathy in Mice Through Calcineurin-Nuclear Factor of Activated T Cells Signaling Pathway

“…TXA2 is highly expressed in platelets and exerts potent effects on platelet aggregation and smooth muscle contraction in vessels. 12 Although the role of TXA2 in vascular disorders, such as atherosclerosis and thrombus formation, is clear, its direct action on the heart is less certain. 13 In the present study, we set out to elucidate the role of TXA2 in iron-overload cardiomyopathy using a mouse model, together with TXAS-deleted mice.…”