Abstract
Thrombopoietin (TPO) enhances platelet activation through activation of the tyrosine kinase; JAK2 and the lipid kinase phosphatidylinositide 3-kinase (PI3K). The aim of our study was to identify the PI3K isoforms involved in mediating the effect of TPO on platelet function and elucidate the underlying mechanism. We found that p110β plays an essential role in TPO-mediated (i) priming of protease-activated receptor (PAR)-mediated integrin αIIbβ3 activation and α-granule secretion, (ii) synergistic enhancement of PAR-mediated activation of the small GTPase RAP1, a regulator of integrin activation and (iii) phosphorylation of the PI3K effector Akt. More importantly, the synergistic effect of TPO on phosphorylation of extracellular-regulated kinase (ERK1/2) and thromboxane (TxA2) synthesis was dependent on both p110β and p110γ. p110β inhibition/deletion, or inhibition of p110γ, resulted in a partial reduction, whereas inhibiting both p110β and p110γ completely prevented the synergistic effect of TPO on ERK1/2 phosphorylation and TxA2 synthesis. The latter was ablated by inhibition of MEK, but not p38, confirming a role for ERK1/2 in regulating TPO-mediated increases in TxA2 synthesis. In conclusion, the synergistic effect of TPO on RAP1 and integrin activation is largely mediated by p110β, whereas p110β and p110γ contribute to the effect of TPO on ERK1/2 phosphorylation and TxA2 formation.
Highlights
phosphatidylinositide 3-kinase (PI3K) isoforms involved in mediating the effect of TPO on platelet function and elucidate the underlying mechanism
To confirm that TPO increases platelet function in a PI3K-dependent manner, we examined proteaseactivated receptor (PAR)-mediated (SFLLRN) integrin αIIbβ[3] activation and α-granule secretion in washed platelets pre-treated with TPO in the absence and presence of the panPI3K inhibitor wortmannin
When using sub-threshold concentrations of SFLLRN, TPO mediated two distinct waves of aggregation with the secondary but not primary wave being blocked by PD184352 (Fig. 6e). This is the first study to demonstrate critical roles for the PI3K isoforms p110β and p110γ in supporting platelet priming by TPO. Using both pharmacological tools and genetic models we have found that p110β is the predominant PI3K isoform involved in mediating the priming effect of TPO on PAR-mediated integrin αIIbβ[3] activation and α-granule secretion, and the concurrent synergistic activation of RAP1 (Fig. 7)
Summary
PI3K isoforms involved in mediating the effect of TPO on platelet function and elucidate the underlying mechanism. The endogenous myeloproliferative leukaemia protein (c-MPL) agonist; thrombopoietin (TPO) is a cytokine primarily involved in regulating platelet production, but it can act as a platelet primer by enhancing platelet activation and function[1,2,3] This cytokine can play a pro-thrombotic and potentially pathogenic role in clinical conditions where it is found to be elevated. We have evidence that TPO/c-MPL in platelets can signal through the PI3K isoform p110γ; an isoform more commonly associated with being activated downstream of G-protein-coupled receptors (GPCR) Activation of this isoform in combination with p110β by TPO, is a requirement for the synergistic activation of the mitogen-activated protein kinase (MAPK) extracellular signal–regulated kinase (ERK1/2) and subsequent priming of TxA2 production
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