Opposing Roles of GSK3α and GSK3ß Phosphorylation in Platelet Function and Thrombosis.

One of the mechanisms by which PI3 kinase can regulate platelet function is through phosphorylation of downstream substrates, including glycogen synthase kinase-3 (GSK3)α and GSK3ß. Platelet activation results in the phosphorylation of an N-terminal serine residue in GSK3α (Ser21) and GSK3ß (Ser9), which competitively inhibits substrate phosphorylation. However, the role of phosphorylation of these paralogs is still largely unknown. Here, we employed GSK3α/ß phosphorylation-resistant mouse models to explore the role of this inhibitory phosphorylation in regulating platelet activation. Expression of phosphorylation-resistant GSK3α/ß reduced thrombin-mediated platelet aggregation, integrin αIIbß3 activation, and α-granule secretion, whereas platelet responses to the GPVI agonist collagen-related peptide (CRP-XL) were significantly enhanced. GSK3 single knock-in lines revealed that this divergence is due to differential roles of GSK3α and GSK3ß phosphorylation in regulating platelet function. Expression of phosphorylation-resistant GSK3α resulted in enhanced GPVI-mediated platelet activation, whereas expression of phosphorylation-resistant GSK3ß resulted in a reduction in PAR-mediated platelet activation and impaired in vitro thrombus formation under flow. Interestingly, the latter was normalised in double GSK3α/ß KI mice, indicating that GSK3α KI can compensate for the impairment in thrombosis caused by GSK3ß KI. In conclusion, our data indicate that GSK3α and GSK3ß have differential roles in regulating platelet function.

Characterisation of the Ral GTPase inhibitor RBC8 in human and mouse platelets.

The Ral GTPases, RalA and RalB, have been implicated in numerous cellular processes, but are most widely known for having regulatory roles in exocytosis. Recently, we demonstrated that deletion of both Ral genes in a platelet-specific mouse gene knockout caused a substantial defect in surface exposure of P-selectin, with only a relatively weak defect in platelet dense granule secretion that did not alter platelet functional responses such as aggregation or thrombus formation. We sought to investigate the function of Rals in human platelets using the recently described Ral inhibitor, RBC8. Initial studies in human platelets confirmed that RBC8 could effectively inhibit Ral GTPase activation, with an IC50 of 2.2 µM and 2.3 µM for RalA and RalB, respectively. Functional studies using RBC8 revealed significant, dose-dependent inhibition of platelet aggregation, secretion (α- and dense granule), integrin activation and thrombus formation, while α-granule release of platelet factor 4, Ca2+ signalling or phosphatidylserine exposure were unaltered. Subsequent studies in RalAB-null mouse platelets pretreated with RBC8 showed dose-dependent decreases in integrin activation and dense granule secretion, with significant inhibition of platelet aggregation and P-selectin exposure at 10 µM RBC8. This study strongly suggests therefore that although RBC8 is useful as a Ral inhibitor in platelets, it is likely also to have off-target effects in the same concentration range as for Ral inhibition. So, whilst clearly useful as a Ral inhibitor, interpretation of data needs to take this into account when assessing roles for Rals using RBC8.

Small GTPases in platelet membrane trafficking.

Our understanding of fundamental biological processes within platelets is continually evolving. A critical feature of platelet biology relates to the intricate uptake, packaging and release of bioactive cargo from storage vesicles, essential in mediating a range of classical (haemostasis/thrombosis) and non-classical (regeneration/inflammation/metastasis) roles platelets assume. Pivotal to the molecular control of these vesicle trafficking events are the small GTPases of the Ras superfamily, which function as spatially distinct, molecular switches controlling essential cellular processes. Herein, we specifically focus on members of the Rab, Arf and Ras subfamilies, which comprise over 130 members and platelet proteomic datasets suggest that more than half of these are expressed in human platelets. We provide an update of current literature relating to trafficking roles for these GTPases in platelets, particularly regarding endocytic and exocytic events, but also vesicle biogenesis and provide speculative argument for roles that other related GTPases and regulatory proteins may adopt in platelets. Advances in our understanding of small GTPase function in the anucleate platelet has been hampered by the lack of specific molecular tools, but it is anticipated that this will be greatly accelerated in the years ahead and will be crucial to the identification of novel therapeutic targets controlling different platelet processes.

Mouse Platelet Ral GTPases Control P-Selectin Surface Expression, Regulating Platelet-Leukocyte Interaction.

OBJECTIVE: RalA and RalB GTPases are important regulators of cell growth, cancer metastasis, and granule secretion. The purpose of this study was to determine the role of Ral GTPases in platelets with the use of platelet-specific gene-knockout mouse models. APPROACH AND RESULTS: This study shows that platelets from double knockout mice, in which both GTPases have been deleted, show markedly diminished (≈85% reduction) P-selectin translocation to the surface membrane, suggesting a critical role in α-granule secretion. Surprisingly, however, there were only minor effects on stimulated release of soluble α- and δ-granule content, with no alteration in granule count, morphology, or content. In addition, their expression was not essential for platelet aggregation or thrombus formation. However, absence of surface P-selectin caused a marked reduction (≈70%) in platelet-leukocyte interactions in blood from RalAB double knockout mice, suggesting a role for platelet Rals in platelet-mediated inflammation. CONCLUSIONS: Platelet Ral GTPases primarily control P-selectin surface expression, in turn regulating platelet-leukocyte interaction. Ral GTPases could therefore be important novel targets for the selective control of platelet-mediated immune cell recruitment and inflammatory disease.