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Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIβ (CaMKIIβ). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIβ, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.

Platelet activation at sites of vascular injury is essential for the arrest of bleeding; however, excessive platelet accumulation at regions of atherosclerotic plaque rupture can result in the development of arterial thrombi, precipitating diseases such as acute myocardial infarction and ischemic stroke. Rheological disturbances (high shear stress) have an important role in promoting arterial thrombosis by enhancing the adhesive and signaling function of platelet integrin [alpha][sub.IIb][beta][sub.3] (GPIIb-IIIa). In this study we have defined a key role for the Type Ia phosphoinositide 3-kinase (PI3K) p110[beta] isoform in regulating the formation and stability of integrin [alpha][sub.IIb][beta][sub.3] adhesion bonds, necessary for shear activation of platelets. Isoform-selective PI3K p110[beta] inhibitors have been developed which prevent formation of stable integrin [alpha][sub.IIb][beta][sub.3] adhesion contacts, leading to defective platelet thrombus formation. In vivo, these inhibitors eliminate occlusive thrombus formation but do not prolong bleeding time. These studies define PI3K p110[beta] as an important new target for antithrombotic therapy., Author(s): Shaun P Jackson (corresponding author) [1]; Simone M Schoenwaelder [1, 5]; Isaac Goncalves [1, 5]; Warwick S Nesbitt [1]; Cindy L Yap [1]; Christine E Wright [2]; Vijaya Kenche [...]

This corrects the article DOI: 10.1038/ncomms12862 [ABSTRACT FROM AUTHOR]

Phosphoinositide (PI) 3-kinases play an important role in regulating the adhesive function of a variety of cell types through affinity modulation of integrins. Two type I PI 3-kinase isoforms (P110β and p110γ) have been implicated in Gi-dependent integrin αIIbβ3 regulation in platelets, however, the mechanisms by which they coordinate their signaling function remains unknown. By employing isoform-selective PI 3-kinase inhibitors and knock-out mouse models we have identified a unique mechanism of PI 3-kinase signaling co-operativity in platelets. We demonstrate that p110β is primarily responsible for G1-dependent phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) production in ADP-stimulated platelets and is linked to the activation of Rap1b and AKT. In contrast, defective integrin αIIbβ3 activation in p110γ-/- platelets was not associated with alterations in the levels of PI(3,4)P2 or active Rap1b/AKT. Analysis of the effects of active site pharmacological inhibitors confirmed that P110γ principally regulated integrin αIIbβ3 activation through a non-catalytic signaling mechanism. Inhibition of the kinase function of PI 3-kinases, combined with deletion of p110γ, led to a major reduction in integrin αIIbβ3 activation, resulting in a profound defect in platelet aggregation, hemostatic plug formation, and arterial thrombosis. These studies demonstrate a kinase-independent signaling function for p110γ in platelets. Moreover, they demonstrate that the combined catalytic and non-catalytic signaling function of p110β and p110γ is critical for P2Y12/Gi-dependent integrin αIIbβ3 regulation. These findings have potentially important implications for the rationale design of novel antiplatelet therapies targeting PI 3-kinase signaling pathways. [ABSTRACT FROM AUTHOR]

The Dok proteins are a family of adaptor molecules that have a well defined role in regulating cellular migration, immune responses, and tumor progression. Previous studies have demonstrated that Doks-1 to 3 are expressed in platelets and that Dok-2 is tyrosine-phosphorylated downstream of integrin αIIbβ3, raising the possibility that it participates in integrin αIIbβ3 outside-in signaling. We demonstrate that Dok-2 in platelets is primarily phosphorylated by Lyn kinase. Moreover, deficiency of Dok-2 leads to dysregulated integrin αIIbβ3-dependent cytosolic calcium flux and phosphatidylinositol(3,4)P2 accumulation. Although agonist-induced integrin αIIbβ3 affinity regulation was unaltered in Dok-2-/- platelets, Dok-2 deficiency was associated with a shear-dependent increase in integrin αIIbβ3 adhesive function, resulting in enhanced platelet-fibrinogen and platelet-platelet adhesive interactions under flow. This increase in adhesion was restricted to discoid platelets and involved the shear-dependent regulation of membrane tethers. Dok-2 deficiency was associated with an increased rate of platelet aggregate formation on thrombogenic surfaces, leading to accelerated thrombus growth in vivo. Overall, this study defines an important role for Dok-2 in regulating biomechanical adhesive function of discoid platelets. Moreover, they define a previously unrecognized prothrombotic mechanism that is not detected by conventional platelet function assays. [ABSTRACT FROM AUTHOR]