Blood coagulation derives from a series of specific proteolytic activation reactions that are catalyzed with narrow and defined specificity by trypsin-like serine proteinases. Many of these proteinases function in membrane assembled enzyme complexes. There are major gaps in the current understanding of the molecular bases for the distinctive protein substrate specificities and the modulation of enzymic function by interactions with membranes and cofactors that are hallmarks of the proteolytic reactions of blood coagulation. This program proposes an integrated approach to focus on the contributions of macromolecular interactions to specificity and the modulation of enzymic function to explain the action of the coagulation enzymes in vivo. Project 1 (Krishnaswamy) uses the prothrombinase complex as a paradigm to investigate the role of extended macromolecular interactions between the protein substrate and the enzyme complex in explaining protein substrate specificity and cofactor function. Project 2 (Camire) will investigate the relationship between procofactor and zymogen proteolysis and the development of discrete macromolecular binding interactions that lead to the assembly of the prothrombinase complex and the expression of enzymic function. Using the x-ray structure of the catalytic domain of XIa, Project 3 (Walsh) proposes structure function studies to examine the contributions of liganding interactions at extended macromolecular recognition surfaces to XIa function. These approaches in basic biochemistry and enzymology are complemented by Project 4 (High) that proposes to use genetic mouse models to investigate the roles of factors X and Xa in vivo and explore the relative contributions of Xa formation via the extrinsic and intrinsic pathways to hemostasis and thrombosis. The objectives of the four projects will be supported by an administrative core and a core that provides support for molecular biology and protein expression. Overall, this program applies the expertise of the individual investigators towards addressing major unanswered questions in the coagulation field. The proposed approaches will provide new insights into the chemistry and biology of the blood coagulation reactions with implications for an understanding of normal hemostasis and will suggest novel therapeutic strategies for targeting thrombosis and vascular disease.
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