Poster Board III-955
Despite the well-documented involvement of thromboxane A2 receptor (TPR) signaling in the pathogenesis of thrombotic diseases, there are currently no rationally designed antagonists available for clinical use. To a large extent this derives from a lack of knowledge regarding the topography of the TPR ligand binding pocket. To this end, while previous work has defined the C-terminal segment of the second extracellular loop (C-EL2) of TPR as the ligand binding site, we have recently mapped the amino acid residues, within this region, which mediate ligand interaction. Since these studies employed a polyclonal antibody targeting the EL2 region of the receptor protein, and since EL2 has been shown to contain the ligand binding domain, we hypothesized that this antibody (referred to hereafter as C-EL2Ab) would exhibit biological activity. On this basis, the purpose of the current study was to investigate the capacity of C-EL2Ab to block TPR-mediated function, using human and mouse platelets. Our initial results demonstrated the C-EL2Ab blocked human and mouse platelet aggregation triggered by 1μM of the TPR agonist U46619, or 0.5mM of the thromboxane A2 mimetic arachidonic acid, in a dose-dependent fashion (150-250nM). On the other hand, control experiments revealed that C-EL2Ab, even at 250 nM, did not produce any detectable effects on platelet activation by ADP (15μM) or the thrombin receptor activating-peptide 4 (40μM). Together, these findings suggest that C-EL2Ab acts as a selective antagonist for TPRs. Consistent with this notion, we found that C-EL2Ab has the ability to displace a radio-labeled TPR antagonist (i.e., [3H]SQ29,548) from its TPR binding sites, both in human and mouse platelets. We next examined the anti-platelet activity of C-EL2Ab under ex vivo experimental settings. It was found that intravenous (IV) tail injections of 250nM of C-EL2Ab resulted in significant blockade of platelet aggregation by 1μM U46619, but not by 15μM ADP. Finally, using a mouse carotid artery thrombosis model, our data demonstrated that C-EL2Ab (at 250 nM; administered IV) prolonged the time for occlusion. Thus, C-EL2Ab has the potential to protect against thrombosis development; which is consistent with the established link for TPRs in thrombotic disorders. In summary, these studies show that C-EL2Ab dose-dependently blocks platelet aggregation induced by the TPR pathway, under both in vitro and ex vivo conditions, and that these effects are mediated via direct interaction with the receptor protein. Furthermore, IV injections of C-EL2Ab are found to confer protection against thrombosis development, in mice. Collectively, these results clearly demonstrate that C-EL2Ab has anti-platelet/anti-thrombotic effects, mediated via its binding to platelet TPRs, making it the first function-blocking antibody against these receptors. Moreover, the identification of a functionally active TPR sequence will significantly aid molecular modeling study predictions for organic derivatives which possess in vivo activity.
No relevant conflicts of interest to declare.