Continuous signaling via PI3K isoforms β and γ is required for platelet ADP receptor function in dynamic thrombus stabilization

Department of Biochemistry and Human Biology, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, PO Box 616, 6200 MD Maastricht, the Netherlands.
Blood (Impact Factor: 10.45). 12/2006; 108(9):3045-52. DOI: 10.1182/blood-2006-03-006338
Source: PubMed

ABSTRACT Signaling from collagen and G protein-coupled receptors leads to platelet adhesion and subsequent thrombus formation. Paracrine agonists such as ADP, thromboxane, and Gas6 are required for platelet aggregate formation. We hypothesized that thrombi are intrinsically unstable structures and that their stabilization requires persistent paracrine activity and continuous signaling, maintaining integrin alpha(IIb)beta3 activation. Here, we studied the disassembly of human and murine thrombi formed on collagen under high shear conditions. Platelet aggregates rapidly disintegrated (1) in the absence of fibrinogen-containing plasma; (2) by blocking or inhibiting alpha(IIb)beta3; (3) by blocking P2Y12 receptors; (4) by suppression of phosphoinositide 3-kinase (PI3K) beta. In murine blood, absence of PI3Kgamma led to formation of unstable thrombi, leading to dissociation of multiplatelet aggregates. In addition, blocking PI3Kbeta delayed initial thrombus formation and reduced individual platelet-platelet contact. Similarly without flow, agonist-induced aggregation was reversed by late suppression of P2Y12 or PI3K isoforms, resulting in single platelets that had inactivated alpha(IIb)beta3 and no longer bound fibrinogen. Together, the data indicate that continuous outside-in signaling via P2Y12 and both PI3Kbeta and PI3Kgamma isoforms is required for perpetuated alpha(IIb)beta3 activation and maintenance of a platelet aggregate. This novel concept of intrinsic, dynamic thrombus instability gives possibilities for the use of antiplatelet therapy.

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Available from: Johan Heemskerk, Sep 28, 2015
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    • "Eur J Pharmacol (2014), Several studies showed that PI3K/Akt plays an important role in regulating platelet aggregation and thrombus formation (Cosemans et al., 2006; O'Brien et al., 2011). Our data revealed that MK571 also inhibits Akt phosphorylation. "
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    ABSTRACT: The multidrug resistance protein 4 (MRP4) is a member of the ABCC subfamily of the adenosine triphosphate-binding cassette transporters that remove cyclic nucleotides from platelets and uptake ADP into dense granule in platelets. However, whether MRP4 directly involves platelet activation remains unclear. Thus, the aim of our study was to determine the detailed mechanisms underlying the regulation of MRP4 in platelet activation. Our results revealed that the MRP4 inhibitor MK571 inhibited collagen-induced platelet aggregation which was partially reversed by the PKA inhibitor H89, but not by the adenylyl cyclase (AC) inhibitor SQ22536 and the guanylyl cyclase (GC) inhibitor ODQ, suggesting that MK571 can prevent collagen-induced aggregation via a route independent of cyclic nucleotide production. In the present study, we found that MK571 inhibited collagen-induced ATP release and calcium mobilization. The phosphorylation of protein kinase C, JNK, and Akt was also inhibited by MK571, and electron spin resonance experiment showed that MK571 significantly reduced hydroxyl radical formation. Moreover, MK571 delayed platelet plug formation in vitro by a PFA-100 device, and delayed thrombus formation in mesenteric venules of mice irradiated by fluorescein sodium. However, previous studies have reported that MK571 also blocks MRP1 and leukotriene D4 (LTD4) receptor. Therefore, whether MK571 inhibits platelet activation through MRP1 or LTD4 receptor needs to be considered and further defined. In conclusion, in addition to blocking the transport of cyclic nucleotides, MRP4 inhibition may prevent thrombus formation in vitro and in vivo. Our findings also support the idea that MRP4 may represent a potential target for the development of novel therapeutic interventions for the treatment of thromboembolic disorders.
    European Journal of Pharmacology 05/2014; 737. DOI:10.1016/j.ejphar.2014.05.001 · 2.53 Impact Factor
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    • "In this study, we demonstrated that the activation of MAPKs is inhibited by amarogentin, suggesting that amarogentin attenuated platelet activation and thrombus formation, at least in part, through MAPK cell-signaling pathway. In addition, several studies showed that PI3K/Akt plays an important role in regulating platelet aggregation and thrombus formation [15, 21, 22]. Thus, we also observed the influence of amarogentin on Akt and found that Akt was not associated with amarogentin-mediated inhibition of platelet activation. "
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    ABSTRACT: Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60 μ M) inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLC) γ 2, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs). It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLC γ 2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders.
    BioMed Research International 04/2014; 2014:728019. DOI:10.1155/2014/728019 · 3.17 Impact Factor
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    • "Potentiation of platelet activation through IGF-1 receptor (Hers, 2007; Kim et al., 2007; Motani et al., 1996) Cooperative and non-redundant role with PI3Kb downstream of GPVI receptor (Gilio et al., 2009; Kim et al., 2009) Slight implication on Akt activation (Kim et al., 2009) PI3Kb Thrombus growth and stability in vivo and ex vivo (Jackson et al., 2005; Martin et al., 2010; Laurent et al., submitted for publication) Control of Akt/GSK3 axis (sustained aIIbb3 activation) (Gilio et al., 2009; Kim et al., 2009; Martin et al., 2010; Laurent et al., submitted for publication) Regulation of PLCg2 and Ca 2þ mobilization downstream of GPVI and aIIbb3 (Jackson et al., 2005; Martin et al., 2010) Regulation of Rap1b for integrin activation (Canobbio et al., 2009; Consonni et al., 2012) Cooperative and non-redundant role with PI3Ka downstream of GPVI receptor (Gilio et al., 2009; Kim et al., 2009) Cooperative role with PI3Kg in P2Y 12 signaling (Canobbio et al., 2009; Cosemans et al., 2006; Schoenwaelder et al., 2007; van der Meijden et al., 2008; Woulfe et al., 2002) PI3Kg Role in ADP-induced thromboembolism (Hirsch et al., 2001) Cooperative role with PI3Kb in P2Y 12 signaling (Canobbio et al., 2009; Cosemans et al., 2006; Schoenwaelder et al., 2007; van der Meijden et al., 2008; Woulfe et al., 2002) PI3Kd Minor role downstream of GPVI receptor (Senis et al., 2005; Zhang et al., 2002) "
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    ABSTRACT: Class I phosphoinositide 3-kinases (PI3K) have been extensively studied in different models these last years and several isoforms are now promising drug targets to treat cancer and immune diseases. Blood platelets are non-nucleated cells critical for hemostasis and strongly involved in arterial thrombosis, a leading cause of death worldwide. Besides their role in hemostasis and thrombosis, platelets provide an interesting model to characterize the implication of the different isoforms of PI3K in signaling. They are specialized for regulated adhesion, particularly under high shear stress conditions found in arteries and use highly regulated signaling mechanisms to form and stabilize a thrombus. In this review we will highlight the role of class I PI3K in these processes and the pertinence of targeting them in the context of antithrombotic strategies but also the potential consequences on the bleeding risk of inhibiting the PI3K signaling in cancer therapy. The implication of upstream regulators of the most important isoforms of PI3K in platelets and their downstream effectors such as protein kinase B (PKB or Akt) and its target glycogen synthase kinase 3 (GSK3) will be discussed as well as the impact of PTEN and SHIP phosphatases as modulators of this pathway.
    09/2013; 54(1). DOI:10.1016/j.jbior.2013.09.006
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