Abstract

The anticoagulant factor protein S (PS) protects neurons from hypoxic/ischemic injury. However, molecular mechanisms mediating PS protection in injured neurons remain unknown. Here, we show mouse recombinant PS protects dose-dependently mouse cortical neurons from excitotoxic NMDA-mediated neuritic bead formation and apoptosis by activating the phosphatidylinositol 3-kinase (PI3K)–Akt pathway (EC₅₀ = 26 ± 4 nm). PS stimulated phosphorylation of Bad and Mdm2, two downstream targets of Akt, which in neurons subjected to pathological overstimulation of NMDA receptors (NMDARs) increased the antiapoptotic Bcl-2 and Bcl-X_L levels and reduced the proapoptotic p53 and Bax levels. Adenoviral transduction with a kinase-deficient Akt mutant (Ad.Akt^K179A) resulted in loss of PS-mediated neuronal protection, Akt activation, and Bad and Mdm2 phosphorylation. Using the TAM receptors tyrosine kinases Tyro3-, Axl-, and Mer-deficient neurons, we showed that PS protected neurons lacking Axl and Mer, but not Tyro3, suggesting a requirement of Tyro3 for PS-mediated protection. Consistent with these results, PS dose-dependently phosphorylated Tyro3 on neurons (EC₅₀ = 25 ± 3 nm). In an in vivo model of NMDA-induced excitotoxic lesions in the striatum, PS dose-dependently reduced the lesion volume in control mice (EC₅₀ = 22 ± 2 nm) and protected Axl^−/− and Mer^−/− transgenic mice, but not Tyro3^−/− transgenic mice. Using different structural PS analogs, we demonstrated that the C terminus sex hormone-binding globulin-like (SHBG) domain of PS is critical for neuronal protection in vitro and in vivo. Thus, our data show that PS protects neurons by activating the Tyro3–PI3K–Akt pathway via its SHGB domain, suggesting potentially a novel neuroprotective approach for acute brain injury and chronic neurodegenerative disorders associated with excessive activation of NMDARs.