The long range objective of our research is to understand the structure-function relationships of proteins that are responsible for blood clot dissolution. In this application we are focusing on the protein chemistry, enzymology, immunochemistry, spectroscopic properties and molecular biology of human plasminogen, tissue plasminogen activator, urokinase, streptokinase and fibrin(ogen). The above proteins have been proposed to contain regions that can be classified as domains,which have residing within them the different functions of the proteins. The central hypothesis of this proposal is that specific properties of these molecules, such as fibrin binding, epsilon-aminocaproic acid binding, inhibitor binding, anion binding, protease sites that regulate function, etc., can be integrally removed and/or inserted into other proteins by recombinant DNA methodology to provide variant proteins with strategically altered properties. We first propose to examine, by calorimetric, immunologic and NMR methodology, the degree of independence of the domain regions (e.g., kringles, growth factor, finger) in the recombinant proteins and in a variety of mutant proteins. The conformational determinants and individual ligand binding properties of these domains will be investigated through a powerful combination of scanning and titration calorimetry, circular dichroism, high-field NMR, structural predictive techniques, synthesis of functional peptides and evaluation of the functional consequences of interaction of the proteins with antibodies to these synthetic peptides. In order to assess some aspects of structure-function relationships in these proteins, advantage will be taken of strategically-generated variant forms of the proteins, obtained through oligonucleotide-directed mutagenesis of their respective cDNAs, which lack, or have inserted within them, selected regions. Identification of important specific amino acids residues of the relevant proteins that are involved in their structural stabilities and interactions of interest will be undertaken by investigating the properties of synthetic peptides, and their corresponding antibodies, lacking or having inserted into them, specific amino acid alterations. The study will also include investigations on proteins and protein fragments with these same specific amino acid changes. Execution of this strategy will allow an increased understanding of the regions of the proteins that are responsible for their functions, and will provide the opportunity to generate proteins with engineered functions that will enhance or diminish their abilities to contribute to the reactions that lead to blood clot dissolution. Important new thrombolytic agents, and/or strategies for application of thrombolytic therapy to human vascular disease, is expected to directly or indirectly result from the basic knowledge provided by this work.
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