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Critical roles for the phosphatidylinositide 3-kinase isoforms p110? and p110? in thrombopoietin-mediated priming of platelet function.

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 p110? plays an essential role in TPO-mediated (i) priming of protease-activated receptor (PAR)-mediated integrin ?_IIb?₃ activation and ?-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 p110? and p110?. p110? inhibition/deletion, or inhibition of p110?, resulted in a partial reduction, whereas inhibiting both p110? and p110? 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 p110?, whereas p110? and p110? contribute to the effect of TPO on ERK1/2 phosphorylation and TxA₂ formation.

SUBMITTER: Moore SF

PROVIDER: S-EPMC6365529 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Critical roles for the phosphatidylinositide 3-kinase isoforms p110β and p110γ in thrombopoietin-mediated priming of platelet function.

Moore Samantha F SF Smith Nina R NR Blair Thomas A TA Durrant Tom N TN Hers Ingeborg I

Scientific reports 20190206 1

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 α<sub>IIb</sub>β<sub>3</sub> activation and α-granule secre ...[more]

PMID: 30728366

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Project description:BackgroundPhosphoinositide 3-kinase ? (PI3K?) and PI3K? are second messenger-generating enzymes with key roles in proliferation, differentiation, survival, and function of leukocytes. Deficiency of the catalytic subunits p110? and p110? of PI3K? and PI3K? in p110?/?-/- mice leads to defective B- and T-cell homeostasis. Here we examined the role of p110? and p110? in the homeostasis of neutrophils by analyzing p110?-/-, p110?-/- and p110?/?-/- mice.MethodsNeutrophils and T cells in leukocyte suspensions from the bone marrow (BM), blood, spleen and lung were analyzed by flow cytometry. Serum concentrations of IL-17, of the neutrophilic growth factor G-CSF, and of the neutrophil mobilizing CXC chemokines CXCL1/KC and CXCL2/MIP-2 were measured by Bio-Plex assay. Production of G-CSF and CXCL1/KC by IL-17-stimulated primary lung tissue cells were determined by ELISA, whereas IL-17-dependent signaling in lung tissue cells was analyzed by measuring Akt phosphorylation using immunoblot.ResultsWe found that in contrast to single knock-out mice, p110?/?-/- mice exhibited significantly elevated neutrophil counts in blood, spleen, and lung. Increased granulocytic differentiation stages in the bone marrow of p110?/?-/- mice were paralleled by increased serum concentrations of G-CSF, CXCL1/KC, and CXCL2/MIP-2. As IL-17 induces neutrophilia via the induction of G-CSF and CXC chemokines, we measured IL-17 and IL-17-producing T cells. IL-17 serum concentrations and frequencies of IL-17+ splenic T cells were significantly increased in p110?/?-/- mice. Moreover, IFN-?+, IL-4+, and IL-5+ T cell subsets were drastically increased in p110?/?-/- mice, suggesting that IL-17+ T cells were up-regulated in the context of a general percentage increase of other cytokine producing T cell subsets.ConclusionsWe found that p110?/? deficiency in mice induces complex immunological changes, which might in concert contribute to neutrophilia. These findings emphasize a crucial but indirect role of both p110? and p110? in the regulation of neutrophil homeostasis.

Project description:RAP GTPases, important regulators of cellular adhesion, are abundant signaling molecules in the platelet/megakaryocytic lineage. However, mice lacking the predominant isoform, RAP1B, display a partial platelet integrin activation defect and have a normal platelet count, suggesting the existence of a RAP1-independent pathway to integrin activation in platelets and a negligible role for RAP GTPases in megakaryocyte biology. To determine the importance of individual RAP isoforms on platelet production and on platelet activation at sites of mechanical injury or vascular leakage, we generated mice with megakaryocyte-specific deletion (mKO) of Rap1a and/or Rap1b Interestingly, Rap1a/b-mKO mice displayed a marked macrothrombocytopenia due to impaired proplatelet formation by megakaryocytes. In platelets, RAP isoforms had redundant and isoform-specific functions. Deletion of RAP1B, but not RAP1A, significantly reduced α-granule secretion and activation of the cytoskeleton regulator RAC1. Both isoforms significantly contributed to thromboxane A2 generation and the inside-out activation of platelet integrins. Combined deficiency of RAP1A and RAP1B markedly impaired platelet aggregation, spreading, and clot retraction. Consistently, thrombus formation in physiological flow conditions was abolished in Rap1a/b-mKO, but not Rap1a-mKO or Rap1b-mKO, platelets. Rap1a/b-mKO mice were strongly protected from experimental thrombosis and exhibited a severe defect in hemostasis after mechanical injury. Surprisingly, Rap1a/b-mKO platelets were indistinguishable from controls in their ability to prevent blood-lymphatic mixing during development and hemorrhage at sites of inflammation. In summary, our studies demonstrate an essential role for RAP1 signaling in platelet integrin activation and a critical role in platelet production. Although important for hemostatic/thrombotic plug formation, platelet RAP1 signaling is dispensable for vascular integrity during development and inflammation.

Dataset Information

Critical roles for the phosphatidylinositide 3-kinase isoforms p110? and p110? in thrombopoietin-mediated priming of platelet function.

Publications

Critical roles for the phosphatidylinositide 3-kinase isoforms p110β and p110γ in thrombopoietin-mediated priming of platelet function.

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