Date of Graduation

5-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry (PhD)

Degree Level

Graduate

Department

Chemistry & Biochemistry

Advisor

Wesley E. Stites

Committee Member

Paul Adams

Second Committee Member

Dan Davis

Third Committee Member

Charles Rosenkrans, Jr.

Fourth Committee Member

Julie Stenken

Abstract

This work tested the hypothesis that oxidation of methionine 388 in thrombomodulin is higher in cigarette smokers, and thus a likely contributor towards the hypercoagulable state in smokers. Thrombomodulin, a protein cofactor found on endothelial cell surfaces, regulates the activity of thrombin. Thrombin bound to thrombomodulin no longer converts fibrinogen to fibrin, but instead activates Protein C which, in turn, stops the coagulation cascade by inactivation of clotting factors. The oxidation of methionine 388 of thrombomodulin has been shown in vitro to dramatically decrease the anticoagulant cofactor activity of thrombomodulin. The blood of cigarette smokers is more prone to clot than that of non-smokers, a major factor in their premature deaths from cardiovascular disease. Cigarette smoke consists of many oxidizing species that impose oxidative stress on the body. These species include organic radicals and hydrogen peroxide, which can oxidize methionine. The fact that smokers are in a hypercoagulable state has been established, however the molecular origins of such a state have not been elucidated. Techniques were developed to isolate thrombomodulin cleared from the blood stream in urine, proteolytically digest it, and identify using mass spectrometry the peptide containing methionine 388 in both its oxidized and reduced forms. In many cases the oxidized version of the peptide was below the limits of detection in non-smokers and the reduced version was not detected in smokers. The intensity of these peaks in the mass spectra do not allow calculation of absolute percentages of oxidation because of differences in proton affinity of the two forms, but there is a very statistically significant difference (P=0.002 by Mann-Whitney Rank Sum test) in the apparent median reduced to oxidized ratios of >2.043 for non-smokers and of

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