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Aggregated, conformationally changed fibrinogen exposes the stimulating sites for t-PA-catalysed plasminogen activation

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Author: Haddeland, U. · Sletten, K. · Bennick, A. · Nieuwenhuizen, W. · Brosstad, F.
Type:article
Date:1996
Institution: Gaubius Instituut TNO
Source:Thrombosis and Haemostasis, 2, 75, 326-331
Identifier: 233211
Keywords: Biology · Gel permeation chromatography · Plasminogen activation · Protein aggregation · Amino Acid Sequence · Animals · Antibodies, Monoclonal · Binding Sites · Biopolymers · Chromatography, Gel · Enzyme-Linked Immunosorbent Assay · Epitopes · Fibrinogen · Heat · Humans · Kinetics · Molecular Sequence Data · Oligopeptides · Plasminogen · Protein Conformation · Rabbits · Tissue Plasminogen Activator

Abstract

The present paper shows that conformationally changed fibrinogen can expose the sites Aα-(148-160) and γ-(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°C for 25 min) demonstrated the presence of both aggregated and monomeric fibrinogen. The aggregated fibrinogen, but not the monomeric fibrinogen, had exposed the epitopes Aα-(148-160) and γ-(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 Aα-(148-160) and γ-(312-324) involved in t-PA-stimulation, whereas dilution of GPRP to a concentration that is no 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. Chemicals/CAS: Antibodies, Monoclonal; Biopolymers; Epitopes; Fibrinogen, 9001-32-5; glycyl-prolyl-arginyl-proline, 67869-62-9; Oligopeptides; Plasminogen, 9001-91-6; Tissue Plasminogen Activator, EC 3.4.21.68