Opposing Roles of GSK3α and GSK3β Phosphorylation in Platelet Function and Thrombosis.

Samantha F Moore,Andreas Wersäll,Ejaife O Agbani,Chris M Williams,Alastair W Poole,Xiaojuan Zhao,Yong Li,James L Hutchinson,Roger W Hunter,Ingeborg Hers

doi:10.3390/ijms221910656

Abstract

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 GSK3β. Platelet activation results in the phosphorylation of an N-terminal serine residue in GSK3α (Ser21) and GSK3β (Ser9), which competitively inhibits substrate phosphorylation. However, the role of phosphorylation of these paralogs is still largely unknown. Here, we employed GSK3α/β phosphorylation-resistant mouse models to explore the role of this inhibitory phosphorylation in regulating platelet activation. Expression of phosphorylation-resistant GSK3α/β reduced thrombin-mediated platelet aggregation, integrin αIIbβ3 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 GSK3β phosphorylation in regulating platelet function. Expression of phosphorylation-resistant GSK3α resulted in enhanced GPVI-mediated platelet activation, whereas expression of phosphorylation-resistant GSK3β 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/β KI mice, indicating that GSK3α KI can compensate for the impairment in thrombosis caused by GSK3β KI. In conclusion, our data indicate that GSK3α and GSK3β have differential roles in regulating platelet function.

Highlights

Phosphoinositide 3-kinase (PI3K) activation occurs downstream of multiple receptors involved in regulating platelet activation, controlling both thrombosis and haemostasis [1,2,3]
Immunoblotting for GSK3α and GSK3β with isoform-specific and dual-specificity antibodies confirmed the presence of both GSK3α and GSK3β in both human and mouse platelets (Figure 1A)
Due to the alterations in platelet function observed in platelets expressing either phosphorylation-resistant GSK3α or GSK3β, we examined whether thrombin and/or CRP-XL-mediated increases in intracellular [Ca2+] were altered using the intracellular Ca2+ indicator FURA-PE3

Summary

Introduction

Phosphoinositide 3-kinase (PI3K) activation occurs downstream of multiple receptors involved in regulating platelet activation, controlling both thrombosis and haemostasis [1,2,3] This has led to an interest in examining how this pathway can be targeted for anti-thrombotic therapies but how platelet function may be altered in the clinic in patients using drugs directed against this pathway for the treatment of cancer and neurological diseases. The GSK3 gene family encodes two highly conserved kinases, 47kD GSK3β and 51kD GSK3α Both are ubiquitously expressed, multifunctional serine/threonine kinases [13] with both common and non-overlapping cellular functions, making them key regulators of numerous signalling pathways [14]. GSK3α/β are unusual in that phosphorylation of an Nterminal serine results in a decrease in kinase activity towards substrates [16,17] This occurs due to GSK3α/βs’ preference for target proteins that are pre-phosphorylated at a “priming” residue. Their requirement for priming phosphorylation means that substrate phosphorylation is regulated by the activity of the priming kinase

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