This research program is aimed at elucidating the mechanisms of thrombin function and its interaction with a number of macromolecular effectors of physiological importance. A key component of the program includes a complete thermodynamic analysis of thrombin interaction with its natural substrate fibrinogen. One of our goals is to understand the nature of the specificity of thrombin-fibrinogen interaction in physico-chemical terms. We will dissect the energetics of this interaction that plays a central role in blood coagulation in terms of elementary components involving different structural domains of the enzyme. We will study how the catalytic pocket of the enzyme and the fibrinogen-recognition-site participate in fibrinogen binding and catalysis. Natural and synthetic macromolecular inhibitors of thrombin function, such as hirudin and its derivatives, will be used to study in detail the energetics of each of these structural domains. Another key aspect of this program is aimed at elucidating the nature of conformational transitions induced by Na+ and Cl- binding to the enzyme. These recently discovered effects play a key role in macromolecular recognition by thrombin and its interaction with substrates and physiological effectors and are likely to provide important new information on the role of thrombin in the patho-physiology of blood coagulation. We will explore the thermodynamic and structural basis of these transitions using thrombin derivatives and mutants perturbed at critical regions of the enzyme, hirudin and hirudin mutants, as well as a number of synthetic substrates and inhibitors. We will investigate how structural perturbations affect thrombin function and modulation under a wide variety of experimental conditions. We will encapsulate the information collected by our functional studies into a detailed molecular mechanism for thrombin interaction with fibrinogen and other effectors. A long term impact of our research program will be in its carry-over into the analysis of other enzyme systems, and particularly those involved in the coagulation cascade.
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