Constitutive surface expression of the thromboxane A2 receptor is Pim kinase-dependent

Nock, Sophie H, Hutchinson, James L, Blanco-Lopez, Maria, Naseem, Khalid, Jones, Sarah ORCID: https://orcid.org/0000-0001-8154-3928, Mundell, Stuart J and Unsworth, Amanda J ORCID: https://orcid.org/0000-0003-3809-5984 (2025) Constitutive surface expression of the thromboxane A2 receptor is Pim kinase-dependent. Journal of Thrombosis and Haemostasis, 23 (1). pp. 293-305. ISSN 15387836

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Abstract

Background The thromboxane A2 receptor (TPαR) plays an important role in the amplification of platelet responses during thrombosis. Receptor activity is regulated by internalization and receptor desensitization. The mechanism by which constitutive surface expression of the TPαR is regulated is unknown. Recently, it has been demonstrated that proviral insertion in murine lymphoma (Pim) kinase inhibitors reduce platelet functional responses in a TPαR-dependent manner. Objectives To investigate whether Pim kinases regulate constitutive TPαR surface expression. Methods TPαR surface expression was measured in platelets, and human embryonic kidney 293T (HEK293T) cells transfected with tagged TPαRs in the presence and absence of Pim kinase inhibitors using flow cytometry and confocal microscopy. TPαR-dependent calcium flux was assessed using Fluo-4 AM. Site prediction modeling and site-directed mutagenesis were used to identify the TPαR PIM kinase phosphorylation site. Results Surface expression of TPαR and calcium responses to U46619 were reduced in platelets and HEK293T cells following Pim kinase inhibition. Overexpression of kinase-dead Pim-1 also reduced TPαR surface expression on HEK293T cells. Reduced surface expression of the TPαR was found to be mediated by increased receptor internalization in a dynamin and β-arrestin-dependent manner. Four putative Pim kinase phosphorylation sites in the TPαR were mutated, and serine 57 in the first intracellular loop of TPαR was identified to be a novel regulatory site important for maintaining TPαR surface expression and thromboxane A2-dependent functional responses. Conclusion Pim kinase inhibition may offer a novel therapeutic approach to limit cellular responses to thromboxane A2, independent of cyclooxygenase inhibition and direct antagonism of the receptor.