title: Kinetic characterization of tissue-type plasminogen activator (t-PA) and t-PA deletion mutants.
abstract: The binding of t-PA to fibrin is mediated both by its "finger" (F) and its "kringle 2" (K2) domain. In addition, these domains are involved in the stimulation of t-PA activity by fibrin. We analyzed the kinetic characteristics of Glu-plasminogen activation by t-PA and a set of t-PA deletion mutants in the absence and the presence of desA-fibrin. In the absence of desA-fibrin, the activity of t-PA (variants) is determined by the presence of the protease domain, irrespective of the composition of the amino-terminal heavy chain. In the presence of the cofactor desA-fibrin, the activity of t-PA (variants) is dependent on the domain composition of the heavy chain. The activity of t-PA is stimulated 2,400 fold by desA-fibrin, whereas the activity of the mutant lacking the K1 domain (del. K1) increases 936 fold in the presence of this cofactor. Mutants lacking either the K2 domain (del. K2) or the F domain (del. F) exhibit an enhanced activity upon desA-fibrin addition of 200 and 210 fold, respectively. DesA-fibrin has no stimulatory effect on the activity of the mutant containing only the serine-protease domain (del.FE K1 K2) nor on the activity of the variant containing only the K1 domain and the serine-protease domain (del. FE K2). Furthermore, we determined the relative fibrin affinity of each t-PA variant, which is similarly dictated by the composition of the heavy chain.
authors.affiliation: Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Department of Molecular Biology, Amsterdam.
authors.forename: C
authors.initials: C
authors.lastname: de Vries
authors.forename: H
authors.initials: H
authors.lastname: Veerman
authors.forename: M E
authors.initials: ME
authors.lastname: Nesheim
authors.forename: H
authors.initials: H
authors.lastname: Pannekoek
mesh_terms: D000595:Amino Acid Sequence; D005337:Fibrin / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010958:Plasminogen / Q000187:drug effects*; D011485:Protein Binding; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D005337:Fibrin; D010958:Plasminogen; D010959:Tissue Plasminogen Activator