Deletion of the p110β isoform of phosphoinositide 3-kinase in platelets reveals its central role in Akt activation and thrombus formation in vitro and in vivo

Abstract

During platelet activation, phosphoinositide 3-kinases (PI3Ks) produce lipid second messengers participating in the regulation of functional responses. Here, we generated a megakaryocyte-restricted p110␤ null mouse model and demonstrated a critical role of PI3K␤ in platelet activation via an immunoreceptor tyrosine-based activation motif, the glycoprotein VI-Fc receptor ␥-chain complex, and its contribution in response to Gprotein-coupled receptors. Interestingly, the production of phosphatidylinositol 3,4,5-trisphosphate and the activation of protein kinase B/Akt were strongly inhibited in p110␤ null platelets stimulated either via immunoreceptor tyrosine-based activation motif or G-protein-coupled receptors. Functional studies showed an important delay in fibrin clot retraction and an almost complete inability of these platelets to adhere onto fibrinogen under flow condition, suggesting that PI3K␤ is also acting downstream of ␣ IIb ␤ 3 . In vivo studies showed that these mice have a normal bleeding time and are not protected from acute pulmonary thromboembolism but are resistant to thrombosis after FeCl 3 injury of the carotid, suggesting that PI3K␤ is a potential target for antithrombotic drugs. IntroductionPlatelet activation is a highly regulated process involving various signaling pathways initiated by specific receptors coupled to heterotrimeric G proteins (GPCRs), integrins, or immunoreceptor tyrosine-based activation motif (ITAM)-containing proteins. In all cases, key signaling enzymes, such as phospholipases C (PLC) and phosphoinositide 3-kinase (PI3K) isoforms, are activated. If the situation is clear for PLC (ie, the PLC␤ isoforms are activated by heterotrimeric Gq proteins, whereas the ␥ isoforms are stimulated via tyrosine phosphorylation and ITAM signaling), the implication of the different PI3K isoforms downstream of the major platelet receptors is still poorly known. Class Ia PI3Ks (␣,␤,␦), composed of a catalytic subunit (p110) and a regulatory subunit, are classically activated by their association with phosphotyrosine residues containing sequences via the SH2 domains of their regulatory subunit. 1 However, the p110␤ isoform may not follow this rule because its activation has been proposed to involve both G␤␥ and phosphotyrosyl peptides. 2-5 Using a selective inhibitor of p110␤, Jackson et al 6 have proposed a role of p110␤ in the regulation of ␣ IIb ␤ 3 integrin in a shear-dependent manner. Interestingly, this inhibitor prevented the formation of an occlusive thrombus generated in vivo. To firmly establish the role of the PI3K␤ in platelet activation and evaluate its impact on hemostasis in vivo, we created a mouse line in which this isoform has been inactivated by gene targeting selectively in the megakaryocyte lineage. Methods MaterialsCollagen was from Nycomed, U46619 from QBiogen Inc; integrilin from Glaxo Group Ltd; p110␣, ␤, ␥, and ␦ antibodies from Santa Cruz Biotechnology; p85 antibody from Upstate Biotechnology; TGX-221 from Cayman Chemical; and other reagents from Sigma-Aldrich. An...

PI3K isoform dependence of PTEN-deficient tumors can be altered by the genetic context

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The p110α and p110β isoforms of PI3K play divergent roles in mammary gland development and tumorigenesis

“…In the case of p110b, its negative effect lies at least in part in its role as a weak kinase competing with the more potent p110a isoform for binding sites on RTKs. Its positive function in certain tumors arising from PTEN loss may arise from a requirement for p110b in mediating signals downstream from particular activating inputs such as certain GPCRs or integrins (Hirsch et al 2000;Guillermet-Guibert et al 2008;Jia et al 2008;Ciraolo et al 2010;Martin et al 2010). Thus, it has been hypothesized that, in at least some PTEN loss-driven tumors, the signal activating PI3K comes from a GPCR.…”

P2 receptors and platelet function

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