In vitro
studies have suggested that reactive oxygen species such as superoxide can produce prothrombotic effects, including enhanced platelet activation, increased tissue factor (TF) expression, and an oxidative modification in thrombomodulin impairing its capacity to enhance the generation of activated protein C (APC) by thrombin. It is not known, however, if elevated levels of superoxide accelerate susceptibility to experimental thrombosis
in vivo
. We used mice genetically deficient in superoxide dismutase-1 (SOD1, an antioxidant enzyme that dismutates superoxide to hydrogen peroxide), to test the hypothesis that lack of SOD1 enhances susceptibility to thrombosis. Susceptibility to carotid artery thrombosis in a photochemical injury model demonstrated that
Sod1-/-
mice formed stable occlusions significantly faster than
Sod1+/+
mice (P<0.05). In an inferior vena cava (IVC) stasis model
Sod1-
/- mice developed significantly larger thrombi 48 hours after IVC ligation (P<0.05 vs.
Sod1+/+
mice). After activation with thrombin (0.5 U/ml) or convulxin (200 ng/ml), no differences in surface expression of P-selectin or binding of fibrinogen were observed between platelets from
Sod1-/-
and
Sod1+/+
mice. The expression of TF mRNA in lung measured by real time qPCR showed similar levels in
Sod1-/-
and
Sod1
+/+ mice. However, the activation of exogenous protein C by thrombin in lung homogenates was decreased in
Sod1
-/- mice (P<0.05 vs.
Sod1
+/+ mice). Further,
in vivo
generation of activated protein C in response to thrombin (40 U/Kg) infusion was significantly lower in
Sod1-/-
mice (P<0.05
vs. Sod1+/+
mice). No differences in mRNA levels for thrombomodulin or endothelial protein C receptor were detected in
Sod1
-/- mice vs.
Sod1
+/+ mice, suggesting that altered generation of activated protein C in
Sod1-/-
mice may be related to a direct oxidative effect on thrombomodulin. In accordance, thrombomodulin treated with xanthine/hypoxanthine showed 40% loss of ability to activate protein C that was overcome by addition of SOD and catalase (P<0.05). We conclude that endogenous SOD1 in mice protects from impaired generation of activated protein C and accelerated thrombosis.
Conjugates of Superoxide Dismutase 1 with Amphiphilic Poly(2-oxazoline) Block Copolymers for Enhanced Brain Delivery: Synthesis, Characterization and Evaluation in Vitro and in Vivo
