Thrombin is a serine protease that plays a critical role in hemostasis, with both procoagulant and anticoagulant functions. The procoagulant effects of thrombin include cleavage of fibrinogen, activation of factors V, VIII, XIII; and activation of platelets. Thrombin is subject to allosteric control by thrombomodulin, an endothelial cell surface protein that stimulates the activation of the protein C, a natural anticoagulant. Thrombin also has a multitude of reported effects on macrophages, endothelium, fibroblasts, and smooth muscle cells, suggesting a role in the response to vascular injury. The proposed research will employ site- directed mutagenesis to define thrombin structures important for interaction with macromolecules that regulate thrombin activity. The role of specific exosites in binding to glycosaminoglycans, thrombomodulin, fibrin, and extracellular matrix will be determined by direct and competition binding assays. The effects of specific mutations on thrombin substrate specificity and the rate of thrombin inhibition by serpins will be determined by kinetic methods. The interaction of thrombin with heparin and related glycosaminoglycans is especially important to understand because of the prominent role of heparin as a therapeutic anticoagulant. These studies will illuminate the physiologic and pathologic roles of thrombin in blood coagulation and inflation. The study of protein structure-function by a site-directed mutagenesis approach encompasses a wide range of molecular biology and biochemistry methods thus making it an excellent vehicle for research training. These techniques are generally applicable to the study of proteins in many experimental systems. The environment for training is excellent; the sponsor is an established investigator in the field of blood coagulation with extensive experience in both molecular biology and protein chemistry. In addition, the Hematology-Oncology Division is committed to the support of basic research with adequate space, financial resources and a SCOR in Thrombosis. Through the study of protein structure-function relationships important for the regulation of thrombin activity, the candidate will acquire the necessary knowledge and tools to pursue independent investigation. The candidate's long term goal is to understand how the interaction between blood coagulation and the inflammatory response contributes to endothelial cell injury, pathologic thrombosis and atherosclerosis.
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