Physiological hemostasis involves the expedient removal of blood clots (""""""""thrombi"""""""") deposited on the wall of blood vessels. Fibrin is a polymeric, highly crosslinked, meshwork protein which structures the clot. Plasminogen, a glycoprotein of ~ 92,000 Mr, is the proenzyme of plasmin, the serine protease responsible for the dissolution of blood clots via hydrolysis of fibrin. The heavy chain of plasmin(ogen) is structured by the N-terminal """"""""preactivation"""""""" peptide plus a tandem array of five """"""""kringle"""""""" domains of ~ 80 residues each. The kringles are thought to mediate fibrin binding. The long term goal of this proposal is to achieve a complete description of the structure, dynamics and function of the plasminogen molecule, with particular emphasis on its kringle domains. The experimental approach is mostly based on high-resolution 1H-NMR spectroscopy at 300, 500 and 620 MHz, and associated computational methods. A secondary goal is to clone and express individual plasminogen kringles in order to make them available in sufficient quantities for the NMR research and also to generate site- specific mutants of structural and functional interest. We also plan to study the kringles present in the tissue plasminogen activator (t-PA) and in urokinase, a kidney plasminogen activator (u-PA). While the project will maintain its focus on both the structure of the binding site and the affinity of kringles for small molecules (analogs of L-lysine that exhibit potent antifibrinolytic properties, such as epsilon-aminocaproic acid or rho-benzyl-aminesulfonic acid) we also plan to investigate in depth the interaction of kringles with peptides that duplicate or mimic functional segments of fibrin, alpha2-antiplasmin and the N-terminal plasminogen preactivation peptide. Such studies will have implications for our understanding of the basic chemistry of thrombolysis and in the rational design of both improved antifibrinolytic drugs and novel fibrinolytic chimeric proteins of clinical potential for the emergency therapy of myocardial infarction, brain stroke and deep vein thrombosis.
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