The Conformation Changes of the Finger Domain of Tissue Type Plasminogen Activator during the Activator-lnhibitor Reaction

Summary

A peptide fragment of tissue plasminogen activator (tPA) corresponding to amino acid residues 4-8 (tPA₄₋₈) was synthesized, coupled to thyroglobulin and injected into rabbits. Antibodies specific to the peptide tPA₄₋₈ were purified immunochemically on the pentapeptide coupled to CNBr-Sepha rose 4B. Anti-tPA₄₋₈ antibodies, reacted with iodinated peptide tPA₄₋₈, showing a relatively high binding affinity (K_D = 2.3 × 10⁻⁸)

There was no interaction between anti-tPA_{4 − 8} antibodies and native one- or two-chain tPA. However, reduction of disulfide bonds unmasked the epitope on the heavy chain of tPA which became accessible to anti-tPA₄₋₈ antibodies. Similarly, complex-ing of tPA with Α₁arantitrypsin inhibitor resulted in unmasking of the epitope formed by amino acid residues in the positions 4-8.

Presented data suggest that complexing of tPA with inhibitors results in conformational changes occurring in the “finger” domain of tPA molecule and such conformational transition can be detected by antipeptide antibodies. Therefore, anti-tPA₄₋₈ antibodies may be employed as sequence-specific reporter molecules to monitor local conformational changes in tPA molecule.

• References

• 1 Rånby M, Begsdorf N, Nilsson T. Enzymatic properties of the one and two chain form of tissue plasminogen activator. Thromb Res 1982; 27: 175-183
• 2 Rijken DC, Hoylaerts M, Collen D. Fibrinolytic properties of one-chain and two-chain human extrinsic (tissue type) plasminogen activator. J Biol Chem 1982; 257: 2920-2925
• 3 Wallen P, Bergsdorf N, Rånby M. Purification and identification of two structural variants of porcine tissue plasminogen activator by affinity adsorption on fibrin. Biochim Biophys Acta 1982; 719: 318-328
• 4 Hoylaerts M, Rijken DC, Lijnen HR, Collen D. Kinetics of the activation of plasminogen by human tissue plasminogen activator. J Biol Chem 1982; 257: 2912-2919
• 5 Rånby M. Studies on the kinetics of plasminogen by tissue plasminogen activator. Biochim Biophys Acta 1982; 704: 461-469
• 6 Wallen P, Pohl G, Bergsdorf N, Rånby M, Ny T, Jornvall H. Purification and characterization of a melanoma cells plasminogen activator. Eur J Biochem 1983; 132: 681-686
• 7 Ny T, Elgh F, Lund B. The structure of the human tissue-type plasminogen activator gene: correlation of intron and exon structures to functional and structural domains. Proc Natl Acad Sci USA 1984; 81: 5355-5359
• 8 Banay L, Varadi A, Patthy L. Common evolutionary origin of the fibrin-binding structures of fibronectin and tissue-type plasminogen activator. FEBS Lett 1983; 163: 37-41
• 9 van Zonnenfeld AJ, Veerman H, Pannekoek H. On the interaction of the finger and kringle-2 domain of tissue-type plasminogen activator with fibrin. J Biol Chem 1986; 251: 14214-14218
• 10 Dolittle RF, Feng DF, Johnson MS. Computer-based characterization of epidermal growth factor percursor. Nature 1984; 307: 558-560
• 11 Appella E, Robinson EA, Ullrich SJ, Stoppeli JP, Corti A, Cassani G, Blasi F. The receptor-binding sequence of urokinase. A biological function for the growth-factor module of proteases. J Biol Chem 1987; 262: 4437-4440
• 12 Malinowsky DP, Sadler JE, Davie EW. Characterization of a complementary deoxyrybonucleic acid coding for human and bovine plasminogen. Biochemistry 1984; 23: 4243-4250
• 13 Vali Z, Patthy L. The fibrin-binding site of human plasminogen. J Biol Chem 1984; 259: 13690-13694
• 14 Park CH, Tulinsky A. Three-dimensional structure of a kringle sequence: structure of prothrombin fragment 1. Biochemistry 1986; 25: 3977-3982
• 15 Verde P, Stoppelli MP, Galeffi P, Nocera PD, Blasi F. Identification and primary sequence of an unsplied human urokinase poly (A) RNA. Proc Natl Acad Sci USA 1984; 81: 4727-4731
• 16 Ichinose A, Takio K, Fujikawa K. Localization of the binding site of tissue-type plasminogen activator to fibrin. J Clin Invest 1986; 78: 163-169
• 17 Verheijen JH, Caspers MP M, Chang GT G, de Munk GA W, Pouwells PH, Enger-Valk BE. Involvement of finger domain and kringle 2 domain of tissue-type plasminogen activator in fibrin binding and stimulation of affinity by fibrin. EMBO J 1986; 5: 3525-3530
• 18 Pennica D, Holmes WE, Kohr WJ, Harkins RN, Vehar GA, Ward CA, Bennet WF, Yelverton C, Seeburg PH, Heynecker HL, Goeddoel DV, Collen D. Cloning and expression of human tissue-type plasminogen activator cDNA. Nature 1983; 301: 214-221
• 19 Rijken D, Groeneveld G. Isolation and functional characterization of the heavy and light chains of human tissue-type plasminogen activator. J Biol Chem 1986; 261: 3098-3102
• 20 Strasburger W, Wollmer A, Pitts JE, Glover ID, Tickle IJ, Blundell TL, Steffens GJ, Glunzler WA, Otting F, Flohe L. Adaption of plasminogen activator sequences to known protease structure. FEBS Lett 1983; 157: 219-223
• 21 Rijken DC, Juhan-Vaque I, De Cock F, Collen D. Measurement of human tissue-type plasminogen activator by a two-site immuno-radiometric assay. J Lab Clin 1983; 101: 274-284
• 22 Korninger C, Collen D. Neutralization of human extrinsic (tissue-type) plasminogen activator in human plasma: No evidence for a specific inhibitor. Thromb Haemostas 1981; 46: 662-665
• 23 Levin EG. Latent tissue plasminogen produced by human endothelial cells in culture: Evidence for an enzyme-inhibitor complex. Proc Natl Acad Sci USA 1983; 80: 6804-6808
• 24 Booth NA, Walker E, Maughan R, Bennett B. Plasminogen activator in normal subject after exercise and venous occlusion: tPA circulates as a complexes with C₁ inhibitor and PAI-1. Blood 1987; 69: 1600-1604
• 25 Booth NA, Anderson JA, Bennett B. The plasma inhibitors of plasminogen activator, studies by zymographic technique. Thromb Res 1985; 38: 261-265
• 26 Dosene AM, Dupuy E, Bodevin E. Production of fibrinolytic inhibitor by cultured human endothelial cells derived from human umbilical vein. Thromb Res 1978; 12: 377-378
• 27 Philips M, Juul AG, Thorsen S. Human endothelial cells produce a plasminogen activator inhibitor and tissue-type plasminogen activator-inhibitor complex. Biochim Biophys Acta 1984; 802: 99-110
• 28 Kruithof EK O, Tran-Thang C, Ransjin A, Beckman F. Demonstration of a fast-acting inhibitor of a plasminogen activators in human plasma. Blood 1984; 64: 907-913
• 29 Thorsen S, Philips M. Isolation of tissue-type plasminogen activator-inhibitor complexes from human plasma. Evidence for a rapid plasminogen activator inhibitor. Biochim Biophys Acta 1984; 802: 111-113
• 30 Astedt B, Lecander I, Brodin T, Lundblad A, Löw K. Purification of a specific placental plasminogen activator inhibitor by monoclonal antibody and its complex formation with plasminogen activator. Thromb Haemostas 1985; 53: 122-125
• 31 Sprengers ED, Kluft C. Plasminogen activator inhibitors. Blood 1987; 381-387
• 32 Kruithof EK O, Tran-Thang C, Gudinchet A, Hauhert J, Niciloso G, Genton C, Welti H, Bachmann F. Fibrinolysis in pregnancy: A study of plasminogen activator inhibitors. Blood 1987; 69: 460-466
• 33 Nilsson IM, Liunger H, Tengborn L. Two different mechanism with venous thrombosis and defective fibrinolysis: Low concentration of plasminogen activator or increased concentration of plasminogen activator inhibitor. Br Med J 1985; 290: 1453-1456
• 34 Hamster AB, Wiman B, de Faire V, Blombäck M. Increased plasma levels of a rapid inhibitor of tissue plasminogen activator in young survivors of myocardial infarction. N Engl J Med 1986; 313: 1557-1563
• 35 Lucore CL, Sobel BE. Interaction of tissue-type plasminogen activator with plasma inhibitors and their pharmacologic implications. Circulation 1988; 77: 660-669
• 36 Avrameas S, Trynck T. The cross-linking of proteins with glutaral-dehyde and its use for the preparation of immunoabsorbents. Immunochemistry 1969; 6: 53-66
• 37 Geiger M, Binder BR. Plasminogen activator in diabetes mellitus. J Biol Chem 1984; 259: 2976-2981
• 38 Cierniewski CS, Niewiaroswki S, Hershock D, Rucinski B, Schmaier AH. Quantitation and characterization of human platelet glycoprotein Ilia by radioommunoassay. Biochim Biophys Acta 1987; 924: 216-224
• 39 Husain SS, Lipinski B, Gurevich V. Rapid purification of a high-affinity plasminogen activator from human blood plasma by a specific adsorption on fibrin-cellite. Proc Natl Sci USA 1981; 78: 4265-4269
• 40 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
• 41 Towbin H, Staehelin T, Gordon J. Electrophoretic from polyacrylamide gel to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350-4354
• 42 Cierniewski CS, Poniatowski J, Urbanczyk J. The peptide β43–47 which increases microvascular permeability is released by plasmin during cleavage of fragment Y of fibrinogen. Biochim Biophys Acta 1986; 884: 594-597
• 43 Schleef RR, Wagner NV, Sinha M, Loskutoff DJ. A monoclonal antibody that does not recognize tissue-type plasminogen activator bound to its naturally occurring inhibitor. Thromb Haemostas 1986; 56: 328-332
• 44 Kaizu T, Kojima K, Iwasaki K, Yamashita T. One-step sandwich enzyme-linked immunoadsorbent assay of human tissue plasminogen activator using monoclonal antibodies. Thromb Res 1985; 40: 91-99
• 45 Holvoet P, Boes J, Collen D. Measurement of free, one-chain tissue-type plasminogen activator in human plasma with an enzyme-linked immunoadsorbent assay based on an active site-specific murine monoclonal antibody. Blood 1987; 69: 284-289