Importantly other aspects of platelet function aggregation s

Importantly, other aspects of platelet function (aggregation, spreading, in vitro and in vivo thrombosis) important for haemostasis and thrombosis were unaltered in Ral DKO platelets and mice. Beyond haemostasis and thrombosis however, platelets have been implicated in numerous pathophysiological processes, in which secreted biomolecules from platelet α-granules are likely to play major roles [18,19]. For instance, platelet-expressed P-selectin is crucial in mediating platelet-leukocyte/immune cell and platelet-endothelial cell interactions involved in inflammatory responses [20]. Therefore, since we have shown them to be critically selective for the regulation of surface expression of P-selectin, platelet Ral GTPases may represent a possible candidate for targeting inflammatory diseases in which platelets have well-established roles in, in particular atherosclerosis and cancer [21,22]. We recently showed that the platelet-specific RalAB DKO mice have delayed onset of clinical signs in an inflammatory bowel disease model [17]. However, despite similarities in relative abundance of both Rals between mouse and human platelets, it is still important to clarify if Rals perform similar roles in human platelets, which could pave the way for future therapeutic approaches targeting platelet P-selectin release [9,23]. The development of selective inhibitors targeting small GTPases has posed significant challenges for researchers. There is substantial interested in targeting these molecules, most notably in the field of oncology where the development of direct Ras inhibitors are often referred to as the “Holy Grail” of cancer therapeutics [24]. Numerous targeting strategies have been sought to overcome this, including the specific targeting of downstream effectors of Ras [25]. Being downstream of Ras, RalA and RalB have gained increasing interest as potential targets in the treatment of cancer, leading to the development of the first commercially available Ral inhibitor, RBC8 [26]. This non-competitive ESI-09 binds to an allosteric site on GDP-bound Rals, locking them in an inactive state, which blocks their interaction with the effector protein, Ral binding protein 1 (RalBP1). Importantly, RBC8 showed selectivity over GTPases Ras and Rho, and functionally it could suppress tumor xenograft growth in mice, which is consistent with a previous publication describing redundant roles for RalA and RalB in tumorigenesis [13,26]. Based on our findings in RalAB DKO platelets, we sought to investigate the role of Ral GTPases in human platelets using RBC8 as a molecular tool to block Ral function, while utilising RalAB DKO platelets to assess the specificity of RBC8 in similar functional assays. We confirm that RBC8 effectively reduces RalA and RalB activation in human platelets, but the compound was able to inhibit also functional responses that were not seen to be inhibited by gene deletion in the DKO mouse. Furthermore, RBC8 inhibited various aspects of platelet function in RalAB-null mouse platelets, suggesting RBC8 also has off target activity in platelets, in the concentration range where Ral is inhibited. We conclude therefore that, although clearly useful as a reagent to effectively inhibit RalA and RalB activity, in platelets at least there are likely to be other targets for RBC8. So, whilst clearly useful as a Ral inhibitor, interpretation of data needs to take this into account when assessing roles for Rals in platelets using RBC8.
Materials and methods
Results & discussion Targeting specific signalling molecules in anucleate human platelets is often hampered by the availability of selective pharmacological inhibitors. As a result, the field of platelet biology is critically reliant on studies using genetically modified mice or blood from patients with inherited bleeding disorders due to mutations in genes regulating haemostasis [32]. Our recent discovery in mouse platelets suggested a critical role for the Ral GTPases, RalA and RalB, in regulating secretion of P-selectin [17]. This finding opens therapeutic avenues for targeting platelet-mediated inflammatory disorders requiring platelet expression of P-selectin, and in our study we showed that platelet-specific deletion of RalA and RalB significantly slowed the onset of symptoms in a mouse model of inflammatory bowel disease. We therefore set out to assess the role of Rals in human platelets using the recently described Ral inhibitor RBC8 [26].