title: Tissue-type plasminogen activator and its substrate Glu-plasminogen share common binding sites in limited plasmin-digested fibrin.
abstract: The enzyme tissue-type plasminogen activator (t-PA) and its substrate Glu-plasminogen can both bind to fibrin. The assembly of these three components results in about a 1000-fold acceleration of the conversion of Glu-plasminogen into plasmin. Fibrin binding of t-PA is mediated both by its finger (F) domain and its kringle-2 domain. Fibrin binding of Glu-plasminogen involves its kringle structures (K1-K5). It has been suggested that particular kringles contain lysine-binding sites and/or aminohexyl-binding sites, exhibiting affinity for specific carboxyl-terminal lysines and intrachain lysines, respectively. We investigated the possibility that t-PA and Glu-plasminogen kringles share common binding sites in fibrin, limitedly digested with plasmin. For that purpose we performed competition experiments, using conditions that exclude plasmin formation, with Glu-plasminogen and either t-PA or two deletion mutants, lacking the F domain (t-PA del.F) or lacking the K2 domain (t-PA del.K2). Our data show that fibrin binding of t-PA, mediated by the F domain, is independent of Glu-plasminogen binding. In contrast, partial inhibition by Glu-plasminogen of t-PA K2 domain-mediated fibrin binding is observed that is dependent on carboxyl-terminal lysines, exposed in fibrin upon limited plasmin digestion. Half-maximal competition of fibrin binding of both t-PA and t-PA del.F is obtained at 3.3 microM Glu-plasminogen. The difference between this value and the apparent dissociation constant of Glu-plasminogen binding to limitedly digested fibrin (12.1 microM) under these conditions is attributed to multiple, simultaneous interactions, each having a separate affinity. It is concluded that t-PA and Glu-plasminogen can bind to the same carboxyl-terminal lysines in limitedly digested fibrin, whereas binding sites composed of intrachain lysines are unique both for the K2 domain of t-PA and the Glu-plasminogen kringles.
authors: Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Department of Molecular Biology, Amsterdam.
authors: C
authors: C
authors: de Vries
authors: H
authors: H
authors: Veerman
authors: E
authors: E
authors: Koornneef
authors: H
authors: H
authors: Pannekoek
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D002872:Chromosome Deletion; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism*; D014644:Genetic Variation; D006801:Humans; D007531:Isoflurophate / Q000494:pharmacology; D007700:Kinetics; D009154:Mutation; D010958:Plasminogen / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D007531:Isoflurophate; D005337:Fibrin; D010958:Plasminogen; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin