It is proposed to continue research on the conformational and dynamic properties of plasminogen (92,000 Mr), with emphasis in understanding the structrure, function and biochemical regulation of the plasminogen heavy chain, in particular of its five kringles. Plasminogen kringles are 10,000 Mr strutural and folding domains constrained by three Cys-Cys bridges; they are responsible for anchoring plasminogen to fibrin and to blood clots, and probably mediate interactions with alpha 2-antiplasmin and with the plasminogen pre-activation peptide (haemostasis control factors). These interactions are thought to involve a lysine-binding site (LBS), known to be carried to kringle 1 (K1), K2 and, in all likelihood, by K5. K1, K4, K5 and the combined K2+3 are isolated by proteolytic cleavage of plasminogen extracted from human blood plasma. The kringles are then subjected to 1-D and 2-D NMR spectroscopy at 300 and 600 MHz (1H frequencies) both in 1H2O and in 2H2O. Particular emphasis will be placed on systematically characterizing the structural/steric requirements of ligand drugs and of short model synthetic polypeptide chains containing C-terminal L-Lys. From Overhauser studies and other chemical evidence on K4, it is proposed that the LBS is structured by the Cys51 -Cys75 loop, and involves polar residues Asp57 and Arg71, and the aromatic side chains of Phe 64 and Trp 72. The project involves extensive NMR experimentation aimed at identifying and assigning the complete kringles' spectra and at characterizing the conformational dynamics including H-exchange and ligand on-off binding kinetics. Since kringles are present in other blood plasma proteins such as prothrombin, the tissue plasminogen activator, fibronectin and haptoglobin, and in urokinase, a kidney plasminogen activator, it is clear that the proposed studies have implications in the understanding of key kringle-mediated protein-protein interactions in haemostasis. In this context, current efforts to develop emergency thrombosis therapy via bio-engineered plasminogen activators ought to be greatly assisted if the cloned kringles carried structural components which enhance their binding affinity to the fibrin clot. Furthermore, the proposed studies have direct bearing in the design of antifibrinolytic drugs aimed at promoting healing in the treatment of haemorragic disorders.
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