Aggregated, Conformationally Changed Fibrinogen Exposes the Stimulating Sites for t-PA-Catalysed Plasminogen Activation

Summary

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 selfassociation.

• References

• 1 Kudryk BJ, Collen D, Woods KR, Blombäck B. Evidence for localization of polymerization sites in fibrinogen. J Biol Chem 1974; 249: 3322-3225
• 2 Olexa SA, Budzynski AZ. Localization of a fibrin polymerization site. J Biol Chem 1981; 256: 3544-3549
• 3 Olexa SA, Budzynski AZ. Evidence for four different polymerization sites involved in human fibrin formation. Proc Natl Acad Sci USA 1980; 77: 1374-1378
• 4 Rozenfeld MA, Vasileva MV. Mechanism of aggregation of fibrinogen molecules: the influence of fibrin-stabilising factor. Biomed Sci 1991; 2: 155-161
• 5 Gollwitzer R, Bode W, Karges HE. On the aggregation of fibrinogen molecules. Thromb Res 1983; suppl 5: 41-53
• 6 Cohen G, Stayter H, Kucera J, Hall C. Polymorphism in fibrinogen aggregates. J Mol Biol 1966; 22: 385-388
• 7 Becker CM. Bovine fibrinogen aggregate: electron microscopic observations of quasi-globular structures. Thromb Res 1987; 48: 101-110
• 8 Laudano AP, Doolittle RF. Synthetic peptide derivatives that bind to fibrinogen and prevent the polymerization of fibrin monomers. Proc Natl Acad Sci USA 1978; 75: 3085-3089
• 9 Suenson E, Petersen LC. Fibrin and plasminogen structures essential to stimulation of plasmin formation by tissue-type plasminogen activator. Biochim Biophys Acta 1986; 870: 510-519
• 10 Verheijen JH, Nieuwenhuizen W, Wijngaards G. Activation of plasminogen by tissue activator is increased specifically in the presence of certain soluble fibrin(ogen) fragments. Thromb Res 1982; 27: 377-385
• 11 Dunn FW, Deguchi K, Soria J, Soria C, Lijnen HR, Tobelem G, Caen J. Importance of the interaction between plasminogen and fibrin for plasminogen activation by tissue-type plasminogen activator. Thromb Res 1984; 36: 345-351
• 12 Ranby M. Studies on the kinetics of plasminogen activation by tissue plasminogen activator. Biochim Biophys Acta 1982; 704: 461-469
• 13 Fisher BE, Will H. Effects of intact fibrin and partially plasmin-degraded fibrin on kinetic properties of one-chain tissue-type plasminogen activator. Biochim Biophys Acta 1990; 1041: 48-54
• 14 Nieuwenhuizen W, Verheijen JH, Vermond A, Chang GTG. Plasminogen activation by tissue activator is accelerated in the presence of fibrin(ogen) cyanogen bromide fragment FCB-2. Biochim Biophys Acta 1983; 755: 531-533
• 15 Yonekawa O, Voskuilen M, Nieuwenhuizen W. Localization in the fibrinogen a-chain of a new site that is involved in the acceleration of the tissue-type plasminogen activator-catalysed activation of plasminogen. Biochem J 1992; 283: 187-191
• 16 Yonekawa O, Vermond A, Nieuwenhuizen W. Localization of a new site in fibrin involved in the acceleration of the tissue-type plasminogen activator (t-PA)-catalysed activation of plasminogen. In: Fibrinogen. Current basic and clinical aspects Matsuda M, Iwanaga S, Takada A, Henschen A. eds Exerpta Medica, Amsterdam; 1990: 111-116
• 17 Voskuilen M, Vermond A, Veeneman GH, Van Boom JH, Klasen EA, Zegers ND, Nieuwenhuizen W. Fibrinogen lysine residue A alpha 157 plays a crucial role in the fibrin induced acceleration of plasminogen activation, catalysed by tissue-type plasminogen activator. J Biol Chem 1987; 262: 5944-5946
• 18 Schielen WJG, Adams HPHM, van Leuven K, Voskuilen M, Tesser GI, Nieuwenhuizen W. The sequence y-(312-324) is a fibrin-specific epitope. Blood 1991; 77: 2169-2173
• 19 Schielen WJG, Voskuilen M, Tesser GI. Nieuwenhuizen. The sequence Aa-(148-160) in fibrin, but not in fibrinogen, is accessible to monoclonal antibodies. Proc Natl Acad Sci USA 1989; 86: 8951-8954
• 20 Procyk R, Medved L, Engelke KJ, Kudryk B, Blombäck B. Non clottable Fibrin Obtained from Partially Reduced Fibrinogen: Characterization and Tissue Plasminogen Activator Stimulation. Biochemistry 1992; 31: 2273-2278
• 21 de Serrano VS, Urano T, Gaffney PJ, Castellino FJ. Influence of Various Structural Domains of Fibrinogen and Fibrin on the Potentiation of Plasminogen Activation by Recombinant Tissue Plasminogen Activator. J Protein Chemistry 1989; 8: 61-77
• 22 Haddeland U, Sletten K, Bennick A, Brosstad F. Freeze-dried fibrinogen or fibrinogen in EDTA stimulate the tissue plasminogen activator-catalysed conversion of plasminogen to plasmin. Blood Coag Fibrinol 1994; 5: 575-581
• 23 Hoegee-De Nobel E, Voskuilen M, Brommer EJP, Nieuwenhuizen W. A monoclonal antibody-based quantitative enzyme immunoassay for the determination of plasma fibrinogen concentration. Thromb Haemost 1988; 60: 415-418
• 24 Jakobsen E, Kierulf P. A modified beta-alanine precipitation procedure to prepare fibrinogen free of anti-thrombin III and plasminogen. Thromb Res 1973; 3: 145-159
• 25 Jacobsson K. Studies on the determination of fibrinogen in human blood plasma. Scand J Clin Lab Invest 1955; 7: 1-54
• 26 Blombäck B. On the properties of fibrinogen and fibrin. Arkiv for kemi 1958; 12: 99-113
• 27 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
• 28 Burnette WN. “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulphate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 1981; 112: 195-203
• 29 Grpn B, Filion-Myklebust C, Bennick A, Nieuwenhuizen W, Matsueda GR, Brosstad F. Early cross-linked fibrin in human plasma contains a-polymers with intact fibrinopeptide A. Blood Coag Fibrinol 1992; 3: 731-736
• 30 Nieuwenhuizen W, Voskuilen M, Matsuda M, Haverkate F, Koopman J, Briet E, Janzarik H. Abnormal fibrins defective in the exposure of sites involved in the acceleration of t-PA mediated plasminogen activation. Fibrinolysis 1994 8. 01 suppl abstract 265
• 31 Radcliffe R, Heinze T. Stimulation of tissue plasminogen activator by denatured proteins and fibrin clots: a possible additional role for plasminogen activator. Arch Biochem Biophys 1981; 211: 750-761
• 32 Soeda S, Shimeno H, Nagamatsu A. A comparison of bovine serum albumins, modified with a variety of carboxyl group agents, as stimulators of tissue-type plasminogen activator-catalysed activation of plasminogen. Chem Pharm Bull 1989; 37: 723-728
• 33 Mosesson MW, Siebenlist KR, Hainfeld JF, Wall JS. The covalent structure of factor XHIa-crosslinked fibrinogen fibrils. In press, Jour Structural Biol 1995
• 34 Haddeland U, Bennick A, Brosstad F. Stimulating effect on tissue-type plasminogen activator - a new and sensitive indicator of denatured fibrinogen. Thromb Res 1995; 77: 329-336