Development of an immunoassay for oxidized or unoxidized forms of thrombomodulin

Abstract

Cardiovascular disease is the leading cause of premature death in smokers, and this elevated risk of death is thought to be linked to the hypercoagulability of smokers. Although it is well known that smokers are in a prothrombotic state the underlying molecular mechanisms for this state remain obscure. Thrombomodulin is a key component in the blood coagulation pathway. The thrombomodulin-thrombin complex activates protein C, which in turn inhibits clot formation. Oxidation of methionine 388 in thrombomodulin is known to greatly decrease the activation of protein C. Smoking imposes an oxidative stress on the body and is known to oxidize methionine in other proteins. We hypothesized that the oxidation of methionine 388 is elevated in smokers relative to non-smokers, and this may be a root molecular cause of elevated cardiovascular disease in smokers. The aim of this study is to develop an immunoassay specific for either the oxidized or unoxidised form of thrombomodulin. The accuracy of the immunoassay can be assessed by chemical analysis of the oxidized and unoxidised forms of the protein. These tools will be utilized to conduct a small scale study of the difference in oxidation state of thrombomodulin in smokers and non-smokers. In addition, these tools will be used to compare the oxidation status of thrombomodulin in plasma and urine.