title: Structural elements that govern the substrate specificity of the clot-dissolving enzyme plasmin.
abstract: There is remarkable homology between the core structures of plasmin, a fibrin clot-degrading enzyme, and factor D, a complement-activating enzyme, despite markedly different biological functions. We postulated that sequence divergence in the loop structures between these two enzymes mediated the unique substrate and inhibitor interactions of plasmin. Recombinant microplasminogens chimerized with factor D sequences at loops 3, 5, and 7 were cleaved by the plasminogen activator urokinase and developed titratable active sites. Chimerization abolished functional interactions with the plasminogen activator streptokinase but did not block complex formation. The microplasmin chimeras showed enhanced resistance (k(i) decreased up to two to three times) to inactivation of microplasmin by alpha(2)-antiplasmin. Microplasmin chimerization had minimal ( approximately 2 fold) effects on the catalytic efficiency for cleavage of small substrates and did not alter the cleavage of fibrin. However, microplasmin and the microplasmin chimeras showed enhanced abilities to degrade fibrin in plasma clots suspended in human plasma. These studies indicate that loop regions of the protease domain of plasmin are important for interactions with substrates, regulatory molecules, and inhibitors. Because modification of these regions affected substrate and inhibitor interactions, loop chimerization may hold promise for improving the clot dissolving properties of this enzyme.
authors.affiliation: Cardiovascular Biology Laboratory, Harvard School of Public Health and the Massachusetts General Hospital, Boston, Massachusetts 02114, USA.
authors.forename: Ryan B
authors.initials: RB
authors.lastname: Turner
authors.forename: Lin
authors.initials: L
authors.lastname: Liu
authors.forename: Irina Y
authors.initials: IY
authors.lastname: Sazonova
authors.forename: Guy L
authors.initials: GL
authors.lastname: Reed
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D011416:Complement Factor D / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D005341:Fibrinolysin / Q000737:chemistry* / Q000378:metabolism*; D005343:Fibrinolytic Agents / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010958:Plasminogen / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013300:Streptokinase / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors; D000979:alpha-2-Antiplasmin / Q000378:metabolism
chemical_list: D005343:Fibrinolytic Agents; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000979:alpha-2-Antiplasmin; D005337:Fibrin; D005340:Fibrinogen; D010958:Plasminogen; D013300:Streptokinase; C493488:CFD protein, human; D011416:Complement Factor D; D005341:Fibrinolysin