Removal of blood clots (thrombi) is an important aspect of hemostasis, the regulation of the physiological steady state. As exemplified by infarct or brain stroke, thrombolytic failure can lead to fatal outcomes. The main protein structuring the clot meshwork is fibrin, a nicked, crosslinked derivative of fibrinogen (Mr approximately 350K). Thus, defradation of the fibrin polymer, or fibrinolysis, is a key component of the hemostatic response. Fibrinolysis involves a cascade of enzyme- catalyzed reactions whereby plasminogen (Pgn), a glycoprotein of Mr approximately 93K, becomes activated to plasmin, a proteinase which effectively digests the clot fibrin matrix. Two physiological Pgn activators are known, the tissue-type plasminogen activator (tPA; Mr approximately 70K) and the kidney-type plasminogen activator (urokinase, uPA; Mr approximately 54K). TPA is secreted by the vessel endothelial cells in response to fibrin deposition, and its activity is highly fibrin-dependent. UPA, in contrast, is not fibrin-dependent but activates Pgn wit 100-fold higher efficiency when complexed to a receptor, uPAR (Mr approximately 58K). Pgn activation via the uPA- uPAR complex is important for cell migration and metastasis as the generated plasmin plays an important role in degrading tissue matrix proteins.Pgn, tPA, uPA are mosaic proteins composed of various modules known as finger, growth factor (GF), kringle (K) and protease. Similarily, the uPAR extracellular fragment contains three repeats homologous to snake venom neurotoxins, which bind the uPA/GF unit. This proposal addresses the questions of the structure of (a) Pgn/K2+3 and how kringles interact with alpha2-antiplasmin, its physiological inhibitor, and with thePgn N-terminal peptide domain (NTP), with particular focus on the NTP-K1 construct; (b); the Pgn K1+2+3 and K1+2+3+4 tandem arrays ('angiostatin') which have been proposed to inhibit vascularization of tumors; ' tPA/K1, protease and a K2 protease consruct (BM 06.022) and how the latter interact with the Pgn activation loop, (d) uPA/K and how it interacts with the Pgn kringles and heparin-like polyanions, (e) uPAR and how its various modules interact with the uPA/GF domain. We also propose to investigate the way Pgn kringles interact with fibrinopeptides and lysine containing model peptides. Except for the protease (Mr approximately 28K), the modules are rather small (Mr approximately 10K). Our approach is to generate the units via proteolytic fragmentation of the parent proteins and/or microbial expression of the recombinant polypeptides, and analysis of the various constructs via heternuclear, multidimensional NMR spectroscopy. By studying how the modular arrays interact with their substrates, effectors and drugs of clinical interest we expect to gain understanding of the way the Pgn molecular system works while helping generate models of use in fibrinolytic therapy, the control of tissue vascularization and the regulation of malignant cell proliferation.
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