Genetic evidence for a predominant role of PI3Kβ catalytic activity in ITAM- and integrin-mediated signaling in platelets

Abstract

Phosphatidylinositol 3-kinase (PI3K) isoforms PI3K␤ and PI3K␥ are implicated in platelet adhesion, activation, and aggregation, but their relative contribution is still unclear or controversial. Here, we report the first comparative functional analysis of platelets from mice expressing a catalytically inactive form of PI3K␤ or PI3K␥. We demonstrate that both isoforms were similarly required for maximal activation of the small GTPase Rap1b and for complete platelet aggregation upon stimulation of G protein-coupled receptors for adenosine 5-diphosphate (ADP) or U46619. Their contribution to these events, however, was largely redundant and dispensable. However, PI3K␤, but not PI3K␥, enzymatic activity was absolutely required for Akt phosphorylation, Rap1 activation, and platelet aggregation downstream of the immunoreceptor tyrosinebased activation motif (ITAM)-bearing receptor glycoprotein VI (GPVI). Moreover, PI3K␤ was a major essential regulator of platelet adhesion to fibrinogen and of integrin ␣ IIb ␤ 3 -mediated spreading. These results provide genetic evidence for a crucial and selective role of PI3K␤ in signaling through GPVI and integrin ␣ IIb IntroductionThe phosphatidylinositol 3-kinase (PI3K) is implicated in platelet activation downstream of G protein-coupled receptors (GPCRs), immunoreceptor tyrosine-based activation motif (ITAM)-bearing receptors, as well as integrins. 1 The different PI3K isoforms are grouped in 3 classes, I, II, and III. Class I PI3K includes the p85-associated members PI3K␣, PI3K␤, and PI3K␦ (class IA), as well as the PI3K␥ isoform (class IB). Although all class I members are expressed in platelets, PI3K␥ and PI3K␤ are considered to play a major role in platelet physiology, 1,2 but their relative contribution is still controversial.PI3K␥ is activated by G protein ␤␥ dimers, and, in platelets, it has been implicated in Rap1b activation and integrin ␣ IIb ␤ 3 -supported aggregation downstream of the G i -coupled P2Y12 adenosine 5Ј-diphosphate (ADP) receptor. 3,4 This model has however been challenged by other studies, proposing a predominant role of PI3K␤ rather than PI3K␥ in ADP-induced platelet activation. 5,6 In addition, the finding that PI3K␥ also functions as a scaffold protein 7 supported a model for a kinaseindependent contribution of PI3K␥ in ADP-mediated platelet activation. 8 In platelets, PI3K is also activated upon stimulation of the ITAM-bearing collagen receptor glycoprotein VI (GPVI)/FcR ␥-chain. 9 Although there is evidence that a class IA member is involved, 10 its identity is still unknown. Similarly, integrin ␣ IIb ␤ 3 recruitment activates PI3Ks. The involvement of a class II member has been hypothesized, 11 but a possible role for class I isoforms clearly emerges from a more recent study. 12 The specific contribution of PI3K␥ and PI3K␤ in platelet activation has remained largely elusive, mainly because of the early embryonic lethality caused by genetic ablation of PI3K␤ in mice, 13 and because the interpretation of the analysis of PI3K␥ knockout mice turned out...

Role and regulation of phosphatidylinositol 3-kinase β in platelet integrin α2β1 signaling

“…4 In the present study, we investigated the activation of PI3K on platelet adhesion through integrin ␣2␤1 by measuring the phosphorylation of the downstream effector Akt. Washed human platelets were allowed to adhere to immobilized monomeric type I collagen or to GFOGER peptide in the presence of 2mM MgCl 2 for increasing times.…”

“…7 Similarly, several reports have documented that, in addition to PI3K␤, PI3K␥ is also implicated in GPCR-mediated platelet activation. 4,9,11,12 These observations are indicative of a still poorly appreciated interplay between the different PI3K isoforms in selected contexts of platelet activation. PI3K␤ has also been proposed to be involved in integrin ␣IIb␤3-mediated outside-in signaling, a process essential for platelet spreading, stable thrombus formation, and clot retraction.…”

“…PI3K␤ has also been proposed to be involved in integrin ␣IIb␤3-mediated outside-in signaling, a process essential for platelet spreading, stable thrombus formation, and clot retraction. [3][4][5]13 In contrast, very little is known about the role and regulation of PI3K downstream of the other major platelet integrin, integrin ␣2␤1.Together with GPVI, integrin ␣2␤1 is a platelet receptor for collagen, 14 but it can also interact with other ligands, including decorin and tenascin. 15,16 Although some controversies persist, the role of integrin ␣2␤1 in adhesion to collagen, platelet activation, and thrombus formation is well documented.…”

“…We showed previously that adhesion and spreading on immobilized fibrinogen was severely compromised in PI3K␤ KD platelets. 4 Therefore, it is clear that PI3K␤ plays different roles downstream of integrins ␣2␤1 and ␣IIb␤3. This conclusion is not related to a difference in ligand density, which has been shown, for example, to affect outside-in signaling through integrin ␣IIb␤3, 42,43 because we observed normal adhesion and spreading in the presence of PI3K inhibitors even after coating with a much lower concentration of monomeric collagen (eg, 0.1g/mL; data not shown).…”

“…PI3K␤ also mediates Rap1b activation downstream of the P2Y12 ADP receptor. 4,44 Although P2Y12 receptor is unable to increase intracellular Ca 2ϩ , activation of Rap1b by ADP is still dependent on CalDAG-GEFI. 45 Our results actually support the model of a strict cooperation between Ca 2ϩ and PI3K␤ for maximal activation of CalDAG-GEFI leading to GTP-Rap1b accumulation.…”