PB0763 - Rgl1, an activator of the Ral signalling pathway, is a novel regulator of platelet activation and thrombosis

Background: Rgl1 (Ral guanine nucleotide dissociation stimulator-like 1) is a nucleotide exchange factor which is activated by the Ras signalling cascade and links it to the Ral effector pathway. We have identified Rgl1 and Ral signalling as key mediators of endothelial and macrophage activation in inflammation and therefore as a potential therapeutic target in atherothrombotic disease. It is already established that the Ral pathway is also stimulated in activated platelets where it may have roles in activation and alpha-granule release but the role of Rgl1 in platelet function and endothelial anti-platelet hormone biology is unknown.

Aims: To determine the effect of Rgl1-deficiency on platelet and endothelial function in vitro and thrombosis in vivo.

Methods: Heterozygous Rgl1-deficient mice were studied and compared to wild-type littermate controls. Aortic Rgl1 expression was measured by RT-qPCR, prostacyclin release by ELISA and nitric oxide release bioassayed as acetylcholine-induced vasodilation by wire myography. Platelet aggregation in whole blood was measured using Multiplate impedance aggregometry and thrombosis in vivo using the FeCl3-induced carotid artery injury model.

Results: Heterozygous Rgl1-deficiency resulted in ~50% reduction in Rgl1 gene expression but no change in platelet count. Prostacyclin release and nitric oxide bioactivity were unchanged in heterozygous Rgl1 knockout mouse aorta but platelets from these mice showed blunted aggregation responses to the thromboxane mimetic, U46619 in whole blood compared to controls. In line with this, thrombotic occlusion after carotid artery injury in vivo was delayed when Rgl1 was disrupted.

Conclusion(s): Rgl1 is an important driver of platelet activation and thrombosis, which likely reflects its role in Ral activation. These data add to other work from our group in inflammatory systems which together support the idea that suppression of Rgl1 activity may be a useful therapeutic target to limit vascular inflammation and platelet reactivity in atherothrombotic disease.