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Thromb Haemost 1992; 67(02): 226-232
DOI: 10.1055/s-0038-1648417
Original Articles
Schattauer GmbH Stuttgart

Thrombomodulin Is a Cofactor for Thrombin Degradation of Recombinant Single-Chain Urokinase Plasminogen Activator "In Vitro" and in a Perfused Rabbit Heart Model

Antonio Molinari
The Haemostasis Unit, Cardiovascular Line, IRCCS Maggiore Hospital and University of Milano, Italy
,
Carla Giorgetti
The Haemostasis Unit, Cardiovascular Line, IRCCS Maggiore Hospital and University of Milano, Italy
,
Jacqueline Lansen
The Haemostasis Unit, Cardiovascular Line, IRCCS Maggiore Hospital and University of Milano, Italy
,
Fabrizio Vaghi
The Haemostasis Unit, Cardiovascular Line, IRCCS Maggiore Hospital and University of Milano, Italy
,
Gaetano Orsini
*   Biotechnology Department, R & D, Farmitalia Carlo Erba, Erbamont Group, IRCCS Maggiore Hospital and University of Milano, Italy
,
Elena Maria Faioni
**   Angelo Bianchi Bonomi, Haemophilia and Thrombosis Center and Institute of Internal Medicine, IRCCS Maggiore Hospital and University of Milano, Italy
,
Pier Mannuccio Mannucci
**   Angelo Bianchi Bonomi, Haemophilia and Thrombosis Center and Institute of Internal Medicine, IRCCS Maggiore Hospital and University of Milano, Italy
› Author Affiliations
Further Information

Publication History

Received 13 March 1991

Accepted after revision 21 August 1991

Publication Date:
02 July 2018 (online)

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Summary

Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) to a two-chain derivative (tcu-PA) fibrinolyti-cally inactive. This reaction was accelerated in vitro by purified rabbit lung thrombomodulin in equimolar complex with thrombin. Polyclonal antibodies to rabbit thrombomodulin prevented this effect.

We also observed that heparin and other sulfated polysaccharides had an accelerating effect on thrombin cleavage of recombinant scu-PA. Their effect was concentration-dependent and then reversed at high levels. The effect of heparin and heparan sulfate was independent and synergic with respect to thrombomodulin. All observations except the effect of heparin, could be confirmed in a Langendorff isolated rabbit heart model. From competition experiments carried out with scu-PA derivatives and mutants, we postulate that the amino-terminal sequence of rscu-PA, containing the epidermal growth factor (EGF)-like and the kringle domains is involved in the cofactor effect of thrombomodulin on scu-PA inactivation by thrombin. We conclude that a regulatory mechanism of scu-PA inactivation is present at the cell surface.

 

  • References

  • 1 Nolan C, Hall LS, Barlow GH, Tribby I. I. Plasminogen activator from human embryonic kidney cell cultures evidence for a proactivator. Biochem Biophys Acta 1977; 496: 384-400
    CrossrefPubMedGoogle Scholar
  • 2 Husain SS, Gurewich V, Lipinski B. Purification and partial characterization of a single-chain high-molecular weight form of urokinase from human urine. Arch Biochem Biophys 1983; 22: 31-38
    PubMedGoogle Scholar
  • 3 Collen D. Report of the Subcommittee on Fibrinolysis. Thromb Haemostas 1985; 54: 893
    PubMedGoogle Scholar
  • 4 Stump DC, Thienpont M, Collen D. Urokinase-related proteins in human urine. J Biol Chem 1986; 261: 1267-1273
    PubMedGoogle Scholar
  • 5 Wun TC, Schleiuning WD, Reich E. Isolation and characterization of urokinase from human plasma. J Biol Chem 1982; 257: 3276-3283
    PubMedGoogle Scholar
  • 6 Wijngaards G, Rijken DC, Van Wezel AL, Groeneveld E, Van Der Velden CAM. Characterization and fibrin-binding properties of different molecular forms of pro-urokinase from a monkey kidney cell culture. Thromb Res 1986; 42: 749-760
    CrossrefPubMedGoogle Scholar
  • 7 Gunzler WA, Steffans GJ, Otting F, Buse G, Flohe L. Structural relationship between high and low molecular mass urokinase. Hoppe-Seyler’s Physiol Chem 1982; 363: 133-141
    PubMedGoogle Scholar
  • 8 Verde P, Stoppelli MP, Galeffi P, Dinnocera P, Blasi F. Identification and primary sequence of an unspliced human urokinase poly (A) + RNA. Proc Natl Acad Sci USA 1984; 81: 4727-4731
    CrossrefPubMedGoogle Scholar
  • 9 Kasai S, Arimura H, Nishida M, Suyama T. Primary structure of single-chain pro-urokinase. J Biol Chem 1985; 260: 12382-12389
    PubMedGoogle Scholar
  • 10 Holmes WE, Pennica D, Blaber M, Rey MW, Gunzler WA, Steffans GJ, Heynecher HI. Cloning and expression of the gene for prourokinase in Escherichia coli . Biotechnology 1985; 3: 923-929
    CrossrefPubMedGoogle Scholar
  • 11 Zamarron C, Lijnen HR, Van Hoef B, Collen D. Biological and thrombolytic properties of proenzyme and active forms of human urokinase. I. Fibrinolytic and fibrinogenolytic properties in human plasma in vitro of urokinases obtained from human urine or by recombinant DNA technology. Thromb Haemostas 1984; 52: 19-23
    PubMedGoogle Scholar
  • 12 Gurewich V, Pannell R, Louie S, Kelley P, Suddith RL, Greenlee R. Effective and fibrin-specific clot lysis by a zymogen precursor form of urokinase (pro-urokinase). A study in vitro and in two animal species. J Clin Invest 1984; 73: 1731-1739
    CrossrefPubMedGoogle Scholar
  • 13 Husain SS, Gurewich V, Lipinski B. Purification and partial characterization of a single-chain high-molecular-weight form of urokinase from human urine. Arch Biochem Biophys 1983; 220: 6231-6238
    PubMedGoogle Scholar
  • 14 Eaton DL, Scott RW, Baker JB. Purification of human fibroblast urokinase proenzyme and analysis of its regulation by proteases and protease nexin. J Biol Chem 1984; 259: 6241-6247
    PubMedGoogle Scholar
  • 15 Ichinose A, Fujikawa K, Suyama T. The activation of pro-urokinase by plasma kallikrein and its inactivation by thrombin. J Biol Chem 1986; 261: 3486-3489
    PubMedGoogle Scholar
  • 16 Gurewich V, Pannell R. Inactivation of single-chain urokinase (prourokinase) by thrombin and thrombin-like enzymes: relevance of the findings to the interpretation of fibrin-binding experiments. Blood 1987; 69: 769-772
    PubMedGoogle Scholar
  • 17 Conforti G, Loskutoff DF. Plasmin and thrombin modulate plasminogen activation by fibro-sarcoma cells. Thromb Haemostas 1985; 54: 171 (Abstr)
    PubMedGoogle Scholar
  • 18 Lijnen HR, Van Hoef B, Collen D. Activation with plasmin of two-chain urokinase-type plasminogen activator derived from single-chain urokinase-type plasminogen activator by treatment with thrombin. Eur J Biochem 1987; 169: 359-364
    CrossrefPubMedGoogle Scholar
  • 19 De Munk GAW, Groeneveld D, Rijken DC. Acceleration of the thrombin inactivation of single-chain urokinase-type plasminogen activator (pro-urokinase) by thrombomodulin. Fibrinolysis 1990; 4: 161 (Abstr)
    PubMedGoogle Scholar
  • 20 Molinari A, Cauet G, Valenti R, Giorgetti C, Lansen J. Effect of various glycosaminoglycans and thrombomodulin on thrombin-mediated conversion of single-chain to two-chain urokinase-type plasminogen activator. Fibrinolysis 1990; 4: 34 (Abstr)
    PubMedGoogle Scholar
  • 21 Orsini G, Brandazza A, Sarmientos P, Molinari A, Lansen J, Cauet G. Efficient renaturation and fibrinolytic properties of pro-urokinase and deletion mutant expressed in E. coli as inclusion bodies. Eur J Biochem 1991; 195: 691-697
    CrossrefPubMedGoogle Scholar
  • 22 Molinari A, Giorgetti C, Bonomini L, Gerna M, Lansen J, Gurewich V. Differential detection of single-chain urokinase-type plasminogen activator and two-chain urokinase-type plasminogen activator in human and monkey plasma. Fibrinolysis. 1992 in press
    PubMedGoogle Scholar
  • 23 Laemmly UK. Cleavage of structural proteins during assembly of the head of bacteriophage T. Nature 1970; 227: 680
    CrossrefPubMedGoogle Scholar
  • 24 Lijnen HR, Zamarron C, Blaber M, Winkler ME, Collen D. Activation of plasminogen by pro-urokinase. I. Mechanism. J Biol Chem 1986; 261: 1253-1258
    PubMedGoogle Scholar
  • 25 Owen WG, Esmon CT, Jackson CM. The conversion of prothrombin to thrombin. I. Characterization of the reaction products formed during the activation of bovine prothrombin. J Biol Chem 1974; 249: 594-605
    PubMedGoogle Scholar
  • 26 Esmon NL, Owen WG, Esmon CT. Isolation of a membrane-bound cofactor for thrombin-catalyzed activation of protein C. J Biol Chem 1982; 257: 859-864
    PubMedGoogle Scholar
  • 27 Dorsey TE, McDonald PW, Roels OA. A heated biotinfolin protein assay which gives equal absorbance with different proteins. Anal Biochem 1977; 78: 156-164
    CrossrefPubMedGoogle Scholar
  • 28 Esmon CT, Owen W. Identification of an endothelial cell cofactor for thrombin-catalyzed activation of protein C. Proc Natl Acad Sci USA 1981; 78: 2249-2252
    CrossrefPubMedGoogle Scholar
  • 29 Lundblad RL, Kingdon HS, Mann KG. Thrombin. In: Methods in Enzymology, Vol. 45: Proteolytic Enzymes. Lorand L. (ed). Academic Press; New York: 1976. pp 156-176
    Google Scholar
  • 30 Esmon CT, Esmon NL, Harris KW. Complex formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation. J Biol Chem 1982; 256: 7944-7947
    PubMedGoogle Scholar
  • 31 Andrade-Gordon P, Stricklands S. Interaction of heparin with plasminogen activators and plasminogen: effects on the activation of plasminogen. Biochemistry 1986; 25: 4033-4040
    CrossrefPubMedGoogle Scholar
  • 32 Watahiki Y, Scully MF, Ellis V, Kakkar VV. Influence of heparin and various glycosaminoglycans on activation of single chain urokinasetype plasminogen activtor (scu-PA) by plasmin. Fibrinolysis 1989; 3: 31-35
    PubMedGoogle Scholar
  • 33 Esmon NL. Thrombomodulin. Semin Thromb Hemostas 1987; 13: 454-463
    Article in Thieme ConnectPubMedGoogle Scholar
  • 34 Pepper MS, Vassalli JD, Montesano R, Orci L. Urokinase-type plasminogen activator is induced in migrating capillary endothelial cells. J Cell Biol 1987; 105: 2535-2541
    CrossrefPubMedGoogle Scholar