RXR ligands inhibit human platelet aggregation and GQ signalling

Leonardo A. Moraes, Timothy D. Warner and David Bishop-Bailey. Department of Cardiac, Vascular and Inflammation Research, William Harvey Research Institute, Bart’s and the London, Queen Mary University of London, Charterhouse Square, London, EC1M 6BQ, UK.

The retinoid X receptors (RXRs) consist of a family of 3 nuclear receptors (α, β and γ ) that target and regulate multiple signalling pathways and activate transcription as homodimers or as obligate heterodimeric partners of other nuclear receptors (Bastien and Rochette-Egly, 2004). We evaluated whether human platelets and megakaryocytes express functional RXR isoforms.

Human platelets were isolated from citrated blood by centrifugation. Meg-01 megakaryoblast cells were from ECACC. RXR isoforms were determined by Western blotting. Light transmission aggregometry was used to measure platelet responses with the prostaglandin endoperoxide mimetic, U46619, being used as a standard agonist. U46619-induced aggregation is mediated by TP receptors linked to Gq, PLC and PKC. To activate PKC directly we used PMA. Gq activation was measured by surrogate Rac activation (Upstate).

Platelets and Meg-01 cells expressed RXRα and RXRβ but no detectable RXRγ (Fig 1A). U46619 (1 µM; 85 ± 3.5% max) or PMA (300nM; 70 ± 3% max) induced aggregation of human platelets. U46619-induced, and to a much lesser extent PMA-induced, platelet aggregation were inhibited by 3 min pre-incubation with the RXR ligand 9 cis retinoic acid (9cRA, 1-20 µM; Fig. B). Rac activation induced by U46619 was also inhibited by 9cRA (Fig. C).

Figure (A) Western blot for RXR α, β and γ in platelet rich plasma (PRP), washed platelets (WP) and Meg-01 cells.(B) Inhibitory effects of RXR ligand (9cRA) on platelet aggregation induced by U46619 (1µM) or PMA (300nM). Data is expressed as % inhibition of aggregation. (C) Western blot of Rac activation in platelet rich plasma. All experiments represent data from at least 3 separate donors.

In summary, human platelets express RXR isoforms and RXR ligands inhibit aggregation and Gq signaling. These results highlight a potentially novel mechanism for rapid and functional non-genomic signaling of a nuclear receptor.

Bastien J and Rochette-Egly C. (2004), Gene. 2004 Mar 17; 328:1-16.

This work was funding by the Joint Research Board of St Bartholomew’s Hospital (XMNR) and the British Heart Foundation (BS/02/002).