title: Aggregated, conformationally changed fibrinogen exposes the stimulating sites for t-PA-catalysed plasminogen activation.
abstract: The present paper shows that conformationally changed fibrinogen can expose the sites A alpha-(148-160) and gamma-(312-324) involved in stimulation of the tissue-type plasminogen activator (t-PA)-catalysed plasminogen activation. The exposure of the stimulating sites was determined by ELISA using mABs directed to these sites, and was shown to coincide with stimulation of t-PA-catalysed plasminogen activation as assessed in an assay using a chromogenic substrate for plasmin. Gel permeation chromatography of fibrinogen conformationally changed by heat (46.5 degrees C for 25 min) demonstrated the presence of both aggregated and monomeric fibrinogen. The aggregated fibrinogen, but not the monomeric fibrinogen, has exposed the epitopes A alpha-(148-160) and gamma-(312-324) involved in t-PA-stimulation. Fibrinogen subjected to heat in the presence of 3 mM of the tetrapeptide GPRP neither aggregates nor exposes the rate-enhancing sites. Thus, aggregation and exposure of t-PA-stimulating sites in fibrinogen seem to be related phenomena, and it is tempting to believe that the exposure of stimulating sites is a consequence of the conformational changes that occur during aggregation, or self-association. Fibrin monomers kept in a monomeric state by a final GPRP concentration of 3 mM do not expose the epitopes A alpha-(148-160) and gamma-(312-324) involved in t-PA-stimulation, whereas dilution of GPRP to a concentration that is not longer anti-polymerizing, results in exposure of these sites. Consequently, the exposure of t-PA-stimulating sites in fibrin as well is due to the conformational changes that occur during self-association.
authors.affiliation: Research Institute for Internal Medicine, Rikshospitalet, Oslo, Norway.
authors.forename: U
authors.initials: U
authors.lastname: Haddeland
authors.forename: K
authors.initials: K
authors.lastname: Sletten
authors.forename: A
authors.initials: A
authors.lastname: Bennick
authors.forename: W
authors.initials: W
authors.lastname: Nieuwenhuizen
authors.forename: F
authors.initials: F
authors.lastname: Brosstad
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D001704:Biopolymers; D002850:Chromatography, Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D005340:Fibrinogen / Q000737:chemistry* / Q000276:immunology; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D010958:Plasminogen / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D010959:Tissue Plasminogen Activator / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D001704:Biopolymers; D000939:Epitopes; D009842:Oligopeptides; C034585:glycyl-prolyl-arginyl-proline; D005340:Fibrinogen; D010958:Plasminogen; D010959:Tissue Plasminogen Activator