Given that Akt undoubtedly plays a supporting role in platelet function in vivo
, the regulation of Akt activity is also the subject of intense study. The Akt kinase is comprised of three domains: a pleckstrin homology domain (PH), a catalytic domain and a regulatory domain [2
]. The consensus model for its activation is that the binding of 3-phosphorylated polyphosphoinositides to the PH domain localizes Akt to the membrane, where it becomes exposed to kinases that catalyze its phosphorylation at two sites: Thr308 in the kinase domain and Ser473 in the regulatory domain (reviewed in [48
]). Akt is, thus, critically regulated by the concentrations of PI(3,4)P2 and phosphatidyl inositol-3,4,5-trisphospohate (PI[3,4,5]P3) and by the kinases that phosphory-[3,4,5]P3) and by the kinases that phosphory-P3) and by the kinases that phosphorylate these two sites. Thr308 in the catalytic domain is phosphorylated by a 3-phosphoinositide-dependent kinase, PDK1 [50
], so termed because of its regulation by phosphoinositide binding. The identity of the kinase responsible for phosphorylation of Ser473 was generically termed PDK2 before the protein responsible for the activity was identified. Currently, there appear to be a number of kinases mediating this activity in different cell lines. Phosphorylation of Ser473 is catalyzed by a complex of rictor and mTOR in various cell lines [51
] or by DNA-dependent protein kinase in the nucleus [53
], but there have been reports that PKCβ may regulate phosphorylation of the Akt residue in mast cells [54
] and that PAK1 may do so in cardiomyocytes [55
]. Platelets express each of these proteins (although they express the b1 form of PKC, whereas the b2 form regulated mast cell Akt) and it is unclear which is responsible for the activation of Akt in platelets.
Upon agonist binding to platelets, D3-phosphorylated phosphoinositides are generated in a PI3K-dependent fashion [56
]. PI(3,4)P2 and PI(3,4,5)P3 both bind to the PH domain of Akt and have been shown to result in Akt activation. There is evidence that platelets stimulated with thrombin generate an initial wave of PI(3,4,5)P3, inducing Akt activation, which is then sustained by an increase in PI(3,4)P2 levels following integrin engagement [57
A number of PI3Ks are expressed in platelets and the effects of individual isoforms of PI3K are somewhat agonist dependent. Akt becomes activated upon platelet stimulation with diverse agonists, such as collagen [6
], ristocetin/vWF [7
], thrombin [3
], ADP [59
], IGF-1 [60
] and Gas6 [61
]. In each case, stimulation of Akt phosphorylation is dependent upon PI3K. However, the roles of each of the PI3K isoforms in the regulation of Akt phosphorylation by different agonists is still the subject of much study. The best-studied class of PI3Ks is designated class I and, thus far, these seem to have most relevance to platelet signaling. This class of PI3Ks is composed of two poly peptide subunits: a regulatory subunit and a catalytic subunit (for a review see [62
]). Platelets contain class IA regulatory subunits p85α, p85β and class IB p101γ. These may associate with the catalytic subunits p110α or β for the class IA enzymes, or with p110γ for the class IB. Genetic deletion of the catalytic subunit, for which the PI3K isoform is generally named, has yielded some insight into which isoforms regulate platelet function; however, results of studies conducted using subtype-selective inhibitors, gene deletion in mice or knockin of mutant forms of the PI3Ks have in some cases yielded contradictory results and suggest that the contribution of individual PI3Ks are agonist specific and, in some cases, cooperative.
In the first report addressing the role of PI3Kγ on Akt and platelet function, platelets from mice with genetic deletion of PI3Kγ displayed reduced aggregation and reduced Akt phosphorylation when stimulated with ADP; however, responses to other agonists were normal [63
]. Although two additional studies confirmed defects in ADP-dependent aggregation in platelets from PI3K
], one of these studies detected no difference in ADP-dependent phosphorylation of Akt-Ser473 accompanying the defect in ADP-dependent aggregation [65
]. This result, combined with the observation that PI3Kγ-selective kinase inhibitors had no effect on ADP-induced platelet aggregation, led the authors to conclude that PI3Kγ plays a role in ADP-dependent platelet function, and this role is not dependent on its catalytic activity or on Akt activation [65
]. However, a recent study examining the platelets of mice expressing a kinase-dead mutant of PI3Kγ found that ADP-dependent Akt phosphorylation is, indeed, selectively inhibited in platelets lacking PI3Kγ enzymatic activity [66
]. Loss of PI3Kγ expression was associated with resistance to thrombosis in an arterial injury model [64
] and a disseminated thrombosis model [63
] but the contribution of Akt signaling to PI3Kγ-dependent platelet responses in vivo
remains difficult to interpret.
While PI3Kγ signaling in platelets appears to be restricted to ADP-dependent pathways, a broader role for PI3Kβ is seen in Akt activation downstream of a number of platelet agonists. Studies using isoform-selective inhibitors suggest that PI3Kβ is required for Akt phosphorylation induced by the collagen receptor glycoprotein (GP)VI [67
], and also contributes to Akt activation induced by several agonists for G-protein-coupled receptors [68
]. Platelets from mice expressing a kinase-dead form of PI3Kβ exhibit near-complete loss of Akt phosphorylation in response to the GPVI agonist, convulxin, the thromboxane mimetic U46619, and ADP; aggregation in these platelets is impaired by approximately 30% to U46619, by approximately 50% to ADP, and is nearly blocked in response to convulxin [66
]. That ADP-dependent Akt phosphorylation is ablated in the presence of kinase-dead forms of either PI3Kβ or γ suggests a possible cooperative relationship between the two isoforms and demonstrates that Akt activation is not strictly required for aggregation, since aggregation is reduced, but not ablated, by loss of activity of either isoform. There is also some evidence for unique roles of β and γ isoforms in stabilization of thrombi formed under arterial flow conditions. Mouse platelets lacking PI3Kγ failed to form thrombi of normal height on collagen and vWF-coated surface and the platelets detached with greater frequency [69
]. As with platelets lacking PI3Kγ, mouse platelets treated with PI3Kβ inhibitor TGX-221 also detach more frequently from collagen-coated surfaces, but loss of PI3Kγ in combination with TGX-221 treatment did not yield additive effects [69
]. Thus, both β and γ isoforms appear to be required for optimal thrombus formation and for optimal Akt activation. This, coupled with the defects in platelet activation and thrombosis seen in mice lacking Akt isoforms, suggests that at least part of the contribution of PI3K to platelet activity is due to Akt activation.
PI3Kα is also expressed in platelets, although its role in platelet function is not as well defined as that of PI3Kγ or β. Akt phosphorylation in platelets can be stimulated by IGF-1, and this mode of phosphorylation is predominantly sensitive to inhibitors of PI3Kα and is partially inhibited by an inhibitor of PI3Kβ [60
]. IGF-1 potentiates platelet aggregation responses to PAR1 agonist peptide, and its potentiating effect is blocked by inhibitors of PI3Kα [60
]. PI3Kα inhibitors also block the effect of IGF-1 on Akt phosphorylation, suggesting that PI3Kα-dependent Akt activation may enhance aggregation to PAR1 agonist. In summary, agonists stimulate unique pathways leading to activation of individual PI3K isoforms and the α, β and γ isoforms all play some role in activation of Akt by various agonists. The role of PI3Kα in thrombus formation has not been evaluated directly. provides a summary of the functional effects of genetic deletion or inhibition of Akt-regulatory proteins in platelets.
Effects of genetic deletion/modification of Akt isoforms or its regulatory proteins in platelets.
The role of PI(3,4,5)P3 versus PI(3,4)P2 in Akt activation in platelets is still not entirely clear. Lova et al.
reported that exogenous application of PI(3,4)P2 was as effective in mediating Akt phosphorylation as PI(3,4,5)P3 [28
]. It has been reported that, following thrombin activation of platelets, an initial wave of PI(3,4,5) P3 is generated, followed by a later wave of PI(3,4)P3 generated upon integrin activation [57
]. While this latTer wave was initially reported to be associated with a second wave of Akt phosphorylation [58
], a more recent study detected no difference in thrombin-induced Akt phosphorylation following integrin blockade with RGDS [21
]. Ma et al.
recently reported that in B cells, levels of PI(3,4)P2 correlate with phosphorylation of Ser473, while PI(3,4,5) P3 levels regulate Thr308 phosphorylation, implying that both phosphoinositides may play important roles in regulating Akt activity [71
]. In any case, enzymes that regulate the concentration of either of these two phosphoinositides may effect activation of Akt. The best-characterized phosphoinositide phosphatase in platelets to date is Src homology 2-containing inositol phosphatase-1 (SHIP1), which dephosphorylates PI(3,4,5)P3 at the D5 position and may, therefore, be expected to regulate both basal and agonist-mediated PI(3,4,5)P3 levels. The effects of SHIP1 deletion on platelet function are controversial [72
]. In one report, deletion of SHIP1 was accompanied by an increase in adhesion under physiological flow conditions, suggesting a negative role for SHIP1 in platelet adhesion [73
]. By contrast, Severin et al.
reported reduced outside in signaling of integrin αIIb
platelets and a decrease in arterial thrombus formation in vivo
, suggesting a positive role for SHIP1 in platelet function and thrombosis [74
]. Akt phosphorylation was not directly measured in either study, so, presently, it is not clear whether the functional results of SHIP1 deletion were in part, due to effects on Akt activation. Another phosphatase, PTEN, catalyzes the hydrolysis of the inositide D3 phosphate in many cells, and its expression in platelets has been detected [4
]. The effects of PTEN deletion in platelets have not yet been reported.
There are some reports of PI3K-independent pathways to Akt activation. Kroner et al.
reported that thrombin-dependent phosphorylation of Akt-Ser473 in platelets was not completely inhibited by the PI3K inhibitor LY294002 but was substantially inhibited by inhibitors of PKC isoforms α or β; however, PKC activity was insufficient to induce Akt phosphorylation in vitro
, suggesting that another cofactor in the platelet lysate was required [3
]. Resendiz et al.
have also noted that during early time points after PAR1 or PAR4 stimulation (1 min and earlier), some platelet Akt phosphorylation is insensitive to PI3K inhibitors [21
]. These PI3K-independent pathways are sensitive to inhibitors of phospholipase C, PKC, calcium and calmodulin but the details of these pathways remain to be fully described.
As mentioned previously, the pathways to Akt phosphorylation are somewhat agonist specific; however, Akt phosphorylation induced by a number of agonists, including thrombin, collagen, convulxin and ADP, are all dependent on the P2Y12
receptor for ADP [59
]. ADP-stimulated Akt phosphorylation is entirely dependent on the Gi-coupled P2Y12
receptor, rather than the P2Y1
receptor. Thrombin-dependent signaling to Akt is also dependent on P2Y12
and is dramatically inhibited by inhibitors of P2Y12
or PKC inhibitors that block ADP secretion following thrombin exposure [59
]. This is owing to an apparent requirement for activation of the Gi-subunit coupled to P2Y12
]. Further studies by the Kunapuli laboratory suggest that selective stimulation of G12/13 by PARs potentiates Gi-dependent signaling to Akt via a Src family kinase-dependent pathway [76
]. The mechanism by which stimulation of Gi subunits leads to Akt activation is also dependent on Src family kinases, but the precise pathway remains to be elucidated. provides a summary of thrombin-mediated regulatory pathways of Akt activation in platelets.
Mechanisms of thrombin-mediated Akt activation in platelets
Taken together, studies from platelets and other cells suggest that D3 phosphorylated phosphoinositides bind to the PH domain of Akt, allowing colocalization of Akt with the PDKs mediating its phosphorylation and activation. However, there remain many questions regarding how Akt becomes activated by different agonists in platelets, and the relative roles of PI3K family members and other kinases, such as PKC, in its activation.