Effects of alpha-thrombin on superoxide dismutase levels in human cerebralmicrovascular endothelial cells

Background: Sequelae of traumatic brain injury include generation of oxygen -free radicals and fibrin deposition, which worsen the initial injury, Supe roxide dismutases (SODs) scavenge and bind to the free-radical superoxide a nion (O-2(-)), potentially defending against oxidative stress. rn the prese nt study, me investigated the production of SOD within human cerebral micro vascular endothelial (HCME) cells after exposure to alpha-thrombin, hypothe sizing that manganese SOD (MnSOD) expression is increased. Our aims were to determine whether alterations in SOD are observed at the mRNA level, to ex amine whether a particular species is preferentially expressed, and to dete rmine the requirement of the active site of alpha-thrombin. Methods: HCME cells were characterized and grown to confluence, Control cel ls and cells exposed to 10 nmol/L alpha-thrombin were harvested for mRNA is olation using reverse transcriptase-polymerase chain reaction. Quantitation of mRNA production determined the levels of copper-zinc SOD and MnSOD, Act ive site blocked alpha-thrombin was used as a negative control and determin ed the specificity of the response. Results: The cells in culture were identified as endothelial after fulfilli ng criteria, such as positive immunocytochemical staining for factor VIII/v on Willebrand factor antigen and binding of Ulex europaeus agglutinin-1 lec tin, Levels of MnSOD mRNA were elevated at all time points in response to a lpha-thrombin, whereas the cytosolic form was undetectable. HCME cells that were exposed to active site-blocked alpha-thrombin produced mRNA levels of MnSOD that were increased above those of controls, but this increase was h alf that of mRNA levels of MnSOD produced by HCME cells that were exposed t o alpha-thrombin. Conclusion: Our study showed for the first time that alpha-thrombin partial ly modulates SOD in HCME cells, causing a preferential increase in MnSOD, F urther investigation into secondary brain injury will provide insights into the role of alpha-thrombin in the mechanism of free radical-induced altera tions, potentially improving the outcome of patients with head injury.