Smoking, thrombomodulin oxidation, and thrombosis.

说明(申请人提供)：血栓调节蛋白是一种关键的血液凝结调节因子。血栓调节蛋白中蛋氨酸388的氧化大大减缓了血栓调节蛋白-凝血酶复合体激活蛋白C的速度，蛋白C一旦被激活，就会减缓凝块的形成。将要检验的假设是，吸烟者的蛋氨酸氧化水平高于非吸烟者。最终目的是确定这是否是吸烟者心血管疾病的重要分子原因。众所周知，烟草烟雾会对人体造成氧化应激。吸烟者过早死亡的主要原因是吸烟引起的心血管疾病。这项工作将从开发一种或多种针对还原或氧化蛋氨酸388形式的血栓调节蛋白的免疫分析开始。将针对含有这种蛋氨酸的氧化和还原形式的多肽和重组蛋白产生抗体。使用这些抗体开发的免疫分析的准确性将通过对氧化和还原蛋白质的直接化学分析来证实。在这一分析中，纯化的蛋白质将被蛋白酶消化。然后将得到的多肽混合物进行层析，以分离包含感兴趣的蛋氨酸的氧化和还原多肽。经过验证后，这些工具将用于对吸烟者和非吸烟者中血栓调节蛋白388氧化的差异进行小规模研究。免疫分析将被用来比较尿液中发现的血栓调节蛋白中还原和氧化的蛋氨酸388与血浆中发现的蛋白质的比率，这一信息将与其他对血液凝固重要的因素相关联。在一小群志愿者中，也将跟踪血栓调节蛋白氧化随时间的变化。

DESCRIPTION (provided by applicant): Thrombomodulin is a key regulator of blood clotting. Oxidation of methionine 388 in thrombomodulin greatly slows the rate at which the thrombomodulin-thrombin complex activates protein C, which, upon activation, slows clot formation. The hypothesis that will be tested is that oxidation of this methionine is elevated in smokers relative to non-smokers. The ultimate aim is to establish if this is an important molecular cause of cardiovascular disease in smokers. Tobacco smoke is known to impose an oxidative stress on the body. The leading cause of premature death in smokers is cardiovascular disease due to smoking. The work will begin with development of one or more immunoassays that are specific for either the reduced or oxidized methionine 388 forms of thrombomodulin. Antibodies will be raised against peptides and recombinant proteins containing the oxidized and reduced forms of this methionine. The accuracy of the immunoassays developed using these antibodies will be confirmed by direct chemical analysis of oxidized and reduced proteins. In this analysis, the purified protein will be digested with proteases. The resulting peptide mixture will then be subjected to chromatography to separate the oxidized and reduced peptides containing the methionine of interest. After validation, these tools will be used to conduct a small scale study of the differences in thrombomodulin 388 oxidation in smokers and non-smokers. The immunoassays will be used to compare the ratio of reduced and oxidized methionine 388 in thrombomodulin found in the urine to the ratio in protein found in the plasma and this information will be correlated with other factors important to blood coagulation. Variation of thrombomodulin oxidation over time will also be followed in a small group of volunteers.