Phosphoinositide 3-kinases (PI3K) have been implicated in platelet activation downstream of G protein-coupled receptors (GPCRs), ITAM-bearing receptors, as well as integrins. Among the multiple PI3K isoforms expressed in platelets, PI3Kγ and PI3Kβ are considered to be involved in GPCRs-mediated signaling leading to Rap1b activation and cell aggregation. However, a definitive genetic analysis of the relative contribution of PI3Kβ versus PI3Kγ in the different contexts of platelet activation has not been provided yet. PI3Kγ knockout mice have been widely investigated, but PI3Kγ has been recognized to mediate also kinase-independent effects. Moreover, deletion of p110β is embryonic lethal, and thus PI3Kβ knockout platelets have not been previously available for analysis. Knockin mice carrying a kinase dead form of either PI3Kβ or PI3Kγ have been recently generated by introducing K833R mutation in p110γ, or K805R mutation in p110β, respectively (
Patrucco et al, Cell 2004:118,375
; Ciraolo et al, 2008, in press). In this study, we have compared platelet activation in mice homozygous for the kinase dead forms of PI3Kβ (PI3KβKD) or PI3Kγ (PI3KγKD). Platelet count and tail bleeding time were not altered in either PI3KγKD or PI3KβKD mice. Platelet aggregation induced by the thromboxane A2 analogue U46619, which stimulates GPCRs, was only slightly reduced in PI3KγKD platelets, and was not further inhibited by treatment with the selective PI3Kβ inhibitor TGX221. Accordingly, catalytic inactivation of PI3Kβ caused only a modest reduction of platelet aggregation induced by U46619. Similar results were also obtained upon stimulation of GPCRs for ADP or thrombin. ADP- and U46619-induced activation of the small GTPase Rap1b, a key regulator of platelet aggregation, was only reduced in PI3KγKD, but was almost completely suppressed in PI3KβKD, pointing to a major role for this isoform in the GPCRs-mediated regulation of the GTPase. Rap1b was normally activated upon stimulation of PI3KγKD platelets with the GPVI ligand convulxin (CVX), both in the absence and presence of the ADP scavenger apyrase. Moreover, CVX-induced aggregation of PI3KγKD platelets was only slightly reduced. By contrast, PI3KβKD platelets completely failed to aggregate in response to a wide range of concentrations of CVX, and accumulation of active GTP-bound Rap1b was almost undetectable. Accordingly, the PI3Kβ inhibitor TGX221 completely suppressed both Rap1b activation and aggregation induced by GPVI stimulation in either wild type or PI3KγKD platelets, both in the presence and absence of apyrase. PI3KγKD platelets adhered normally to immobilize type I collagen or fibrinogen, and displayed only a small defect of spreading on fibrinogen. Similarly, adhesion to collagen was comparable in PI3KβKD and wild type platelets. However, PI3KβKD revealed a severely impaired adhesion to fibrinogen. In addition, adherent cells completely failed to spread, and virtually no lamellipodia-forming platelets were observed. Altogether these results indicate both PI3Kγ and PI3Kβ are both involved in Rap1b activation and platelet aggregation upon stimulation of GPCRs, but provide the first genetic evidence that PI3Kβ, rather than PI3Kγ, plays an essential role in platelet activation downstream of the ITAM-bearing receptor GPVI, as well as in integrin αIIbβ3 outside-in signaling.
Disclosures: No relevant conflicts of interest to declare.
