Thrombomodulin (TM) binds thrombin on the surface of the endothelium and accelerates the activation of protein C, a natural anticoagulant. The long term goal of this project is to understand the nature of the regulation of TM function, and in a broad sense, how the regulatory proteins of the coagulation system function. Currently, the relationship between TM structure and function is poorly understood, but some of the properties of the system make it an ideal model for other coagulation complexes. This application is designed to determine: 1) the number and the nature of the membrane interaction sites for both TM and protein C, 2) the sites of interaction of thrombin with TM, the orientation of the membrane bound complex and the influence of other coagulation factors on this orientation and conformation of thrombin in the membrane bound complex, 3) whether heparin is associated with TM and, if so, to analyze the function of the associated heparin, 4) the molecular basis of why human TM appears to be a weaker direct inhibitor of thrombin procoagulant activity than other species, 5) whether, when and how lupus anticoagulants, known to be associated with thrombosis, inhibit TM activity in purified systems and on cell surfaces, 6) which of the vitamin K dependent Gla residues are required for protein C activation and for expression of anticoagulant activity, 7) where and how Ca2+ contributes to protein C activation in the soluble and membrane associated complexes, and 8) whether the active center of thrombin in the thrombin-TM complex resembles that of gamma thrombin. These studies should aid in our understanding of how the thrombin-TM complex functions, and increase our understanding of both the structure and the topography of the complex. Furthermore, the approach will allow a more complete understanding of the role of posttranslational modifications in the unique properties of TM and establish a direct experimental foundation for comparing and contrasting TM to other cellular receptors such as the LDL receptor.
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