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...

Dasatinib is an oral potent adenosine triphosphate (ATP)-competitive inhibitor of BCR-ABL, cKIT, platelet-derived growth factor receptor, and SRC family kinases (SFKs), which has demonstrated high efficiency in patients with imatinib-resistant chronic myelogenous leukemia. Here, we show that dasatinib weakly affects platelet activation by thrombin or adenosine diphosphate but is a potent inhibitor of platelet signaling and functions initiated by collagen or Fc␥RIIA cross-linking, which require immunoreceptor tyrosinebased activation motif phosphorylation by SFKs. Accordingly, dasatinib treatment rapidly decreases the volume of thrombi formed under arterial flow conditions in whole blood from patients or mice perfused over a matrix of collagen. Moreover, treatment of mice with dasatinib increases the tail bleeding time in a dose-dependent manner. Interestingly, these effects are rapidly reversible after interruption of the treatment. Our data clearly demonstrate that, in contrast to imatinib, dasatinib affects platelet functions in vitro and in vivo, which has important implications in clinic and could explain increased risks of bleeding observed in patients. Moreover, dasatinib efficiently prevents platelet activation mediated by Fc␥RIIA cross-linking and by sera from patients with heparin-induced thrombocytopenia, suggesting that reversible antiplatelet agents acting as ATP-competitive inhibitors of SFKs may be of therapeutic interest in the treatment of this pathology.