PB0392 - Pim kinase: A novel regulator of platelet and megakaryocyte thromboxane A2 and C-X-C-R motif receptors

Background: Pim kinases have recently been identified to play a role in the regulation of platelet function and thrombosis. Deletion or inhibition of Pim-1 results in reduced thrombus formation without altered hemostasis, suggesting it is a desirable antiplatelet target. In other cell types, Pim kinases have been shown to regulate CXCR4 and we have previously described that Pim-1 regulates platelet function via regulation of TPαR signalling.

Aims: We set out to investigate the mechanism by which Pim-1 regulates TPαR and CXCR4 signalling in platelets and megakaryocytes.

Methods: HEK293 cells transfected with Flag or GFP-tagged TPαR and MEG01 cells (expressing CXCR4) were treated with Pim kinase inhibitors (AZD1208, LGH447) for 10 minutes. Receptor levels (surface and total) were then assessed using both flow cytometry and microscopy. TxB2 generation was measured by ELISA and calcium mobilisation was measured to determine effects on downstream signalling

Results: No alteration in TxB2 generation indicates Pim kinase regulates platelet TxA2 receptor signalling independently of COX1 regulation. Pim kinase inhibition causes internalisation of both the CXCR4 and TPαR when assessed using flow cytometry and fluorescence microscopy. Consistent with the receptors being internalised, a reduction in TxA2 and SDF1a mediated calcium mobilisation, and phospho-PKC signalling was observed following treatment of platelets, HEK293 and MEG01 cells with Pim kinase inhibitors.

Conclusion(s): Inhibition of Pim kinase attenuates TPαR and CXCR4 signalling via receptor internalisation. CXCR4 and TPαR share similar consensus sequences, containing homologous serine residues within their intracellular loops suggesting that Pim could phosphorylate TPαR in a similar mechanism to CXCR4, regulating receptor expression at the platelet and megakaryocyte surface.
Reduction of platelet and megakaryocyte TPαR and CXCR4 receptor levels and signalling offers a novel platelet targeting strategy, especially in inflammatory conditions, such as rheumatoid arthritis and SLE that are associated with increased circulating plasma levels of thromboxane A2 and SDF-1a.