Protein S controls hypoxic/ischemic blood-brain barrier disruption through the TAM receptor Tyro3 and sphingosine 1-phosphate receptor

The anticoagulant factor protein S (PS) has direct cellular activities. Lack of PS in mice causes lethal coagulopathy, ischemic/thrombotic injuries, vascular dysgenesis, and blood-brain barrier (BBB) disruption with intracerebral hemorrhages. Thus, we hypothesized that PS maintains and/or enhances the BBB integrity. Using a BBB model with human brain endothelial cells, we show PS inhibits time- and dose-dependently (half maximal effective concentration [EC₅₀] = 27 ± 3 nM) oxygen/glucose deprivation-induced BBB breakdown, as demonstrated by measurements of the transmonolayer electrical resistance, permeability of endothelial monolayers to dextran (40 kDa), and rearrangement of F-actin toward the cortical cytoskeletal ring. Using Tyro-3, Axl, and Mer (TAM) receptor, tyrosine kinase silencing through RNA interference, specific N-terminus-blocking antibodies, Tyro3 phosphorylation, and Tyro3-, Axl- and Mer-deficient mouse brain endothelial cells, we show that Tyro3 mediates PS vasculoprotection. After Tyro3 ligation, PS activated sphingosine 1-phosphate receptor (S1P₁), resulting in Rac1-dependent BBB protection. Using 2-photon in vivo imaging, we show that PS blocks postischemic BBB disruption in Tyro3^+/+, Axl^-/-, and Mer^-/- mice, but not in Tyro3^-/- mice or Tyro3^+/+ mice receiving low-dose W146, a S1P₁-specific antagonist. Our findings indicate that PS protects the BBB integrity via Tyro3 and S1P₁, suggesting potentially novel treatments for neurovascular dysfunction resulting from hypoxic/ischemic BBB damage.