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

Background: The thromboxane A2 receptor (TPαR) plays an important role in the amplification of platelet responses during thrombosis. Receptor activity is regulated by internalisation and receptor desensitisation. The mechanism by which constitutive surface expression of the TPαR is regulated is unknown. Recently it has been demonstrated that Pim kinase inhibitors reduce platelet functional responses in a TPαR-dependent manner.

Objective: To investigate whether Pim kinases regulate constitutive TPαR surface expression.

Methods: TPαR surface expression was measured in platelets and 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-4AM. Site prediction modelling, and site directed mutagenesis was 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 internalisation in a dynamin and β-arrestin dependent manner. 4 putative Pim kinase phosphorylation sites in the TPαR were mutated and serine 57 in the first intracellular loop of TPαR 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.