Cardiovascular Engineering and Technology

, Volume 4, Issue 4, pp 328–338 | Cite as

Fibrinolytic Mechanisms of tPA, prouPA, Mutant prouPA and Their Implications for Therapeutic Thrombolysis

  • Victor GurewichEmail author


Experience with thrombolysis for 25 years has been essentially limited to tissue plasminogen activator (tPA). Therefore, tPA has been perceived as synonymous with all thrombolysis. However, tPA has a unique mode of action different from that of urokinase plasminogen activator (uPA), whose native form is prouPA. At the pharmacological concentrations, both physiological activators induced unanticipated effects that were also different. Reperfusion with tPA is almost twice as effective as streptokinase, the former thrombolytic standard. Nevertheless, three mega-trials with a total of 94,740 patients were required to show a significant difference in acute myocardial infarction. Surprisingly, intracranial hemorrhage was also significantly more with tPA. These paradoxical findings can be explained by tPA’s rethrombosis rate undermining its efficacy, and lysis of hemostatic fibrin causing tPA bleeding. By contrast, prouPA has a low rethrombosis rate and it spares hemostatic fibrin by virtue of its different mechanism of fibrin-bound plasminogen activation. Unfortunately, these potential advantages are out of reach in practice since at high therapeutic concentrations in blood, prouPA is vulnerable to non-specific conversion to uPA, a non-specific plasminogen activator that induces a hemorrhagic diathesis. A single-site mutant, M5, was designed to correct this prouPA problem. In vitro and in vivo studies showed that M5 clot lysis was more effective than prouPA or tPA and was accompanied by a sparing of hemostatic fibrin. When M5 administration was preceded by a bolus of plasma C1-inhibitor, it further improved the plasma stability of M5 and permitted a maximum clot lysis rate to be achieved without fibrinogenolysis.


Plasminogen activators Mode of action tPA prouPA Mutant prouPA Stroke Acute myocardial infarction 


  1. 1.
    Alexandrov, A. V., and J. C. Grotta. Arterial reocclusion in stroke patients treated with intravenous tissue plasminogen activator. Neurology 59:862–867, 2002.CrossRefGoogle Scholar
  2. 2.
    Aoki, N. Preparation of plasminogen activator from vascular trees of human cadavers. Its comparison with urokinase. J. Biochem. 75:731–741, 1974.Google Scholar
  3. 3.
    Assessment of the Safety and Efficacy of a New Treatment Strategy with Percutaneous Coronary Intervention (ASSENT-4 PCI) Investigators. Primary versus tenecteplase-facilitated percutaneous coronary intervention in patients with ST-segment elevation acute myocardial infarction (ASSENT-4 PCI): randomised trial. Lancet 367:569–578, 2006.CrossRefGoogle Scholar
  4. 4.
    Bar, F. W., J. Meyer, F. Vermeer, R. Michels, B. Charbonnier, and for the SESAM Study Group. Comparison of saruplase and alteplase in acute myocardial infarction. Am. J. Cardiol. 79:727–732, 1997.CrossRefGoogle Scholar
  5. 5.
    Correspondence. Lancet 380:1053, 2012.Google Scholar
  6. 6.
    Gruppo Italiano Per Lo Studio Della Sopravvivenza Nell’Infarto Miocardico. GISSI-2: a factorial randomised trial of alteplase versus streptokinase and heparin versus no heparin among 12,490 patients with acute myocardial infarction. Lancet 336:65–75, 1990.Google Scholar
  7. 7.
    Gulba, D. C., M. Bartheis, G. H. Reil, and P. R. Lichtlen. Thrombin/antithrombin-III complex level as early predictor of reocclusion after successful thrombolysis. Lancet 2:97, 1988.CrossRefGoogle Scholar
  8. 8.
    Gulba, D. C., M. Barthels, M. Westhoff-Bleck, S. Jost, W. Rafflenbeul, W. G. Daniel, H. Heckler, and P. R. Lichtlen. Increased thrombin levels during thrombolytic therapy in acute myocardial infarction: relevance for the success of therapy. Circulation 83:937–944, 1991.CrossRefGoogle Scholar
  9. 9.
    Gurewich, V., and R. Pannell. Synergism of tissue-type plasminogen activator (t-PA) and single chain urokinase-type plasminogen activator (scu-PA) on clot lysis in vitro and a mechanism for this effect. Thromb. Haemost. 57(3):372–378, 1987.Google Scholar
  10. 10.
    Gurewich, V., and R. Pannell. Recombinant human C1-inhibitor prevents non-specific proteolysis by mutant prouPA during optimal fibrinolysis. Thromb. Haemost. 102(2):279–286, 2009.Google Scholar
  11. 11.
    Gurewich, V., R. Pannell, R. J. Broeze, and J. I. Mao. Characterization of the intrinsic fibrinolytic properties of pro-urokinase through a study of plasmin resistant forms produced by site specific mutagenesis of lysine 158. J. Clin. Invest. 82:1956–1962, 1988.CrossRefGoogle Scholar
  12. 12.
    Gurewich, V., R. Pannell, A. Simmons-Byrd, P. Sarmientos, J. N. Liu, and S. F. Badylak. Thrombolysis versus bleeding from hemostatic sites by a prourokinase mutant compared with tissue plasminogen activator. J. Thromb. Haemost. 4:1559–1565, 2006.CrossRefGoogle Scholar
  13. 13.
    Hacke, W., M. Kaste, E. Fieschi, D. Toni, E. Lesaffre, and for The European Cooperative Acute Stroke Study (ECASS) Investigators. Intravenous thrombolysis with recombinant tissue plasminogen activator for acute hemispheric stroke. The European Cooperative Acute Stroke Study (ECASS). JAMA 274:1017–1025, 1995.CrossRefGoogle Scholar
  14. 14.
    Harpel, P. C., T. S. Chang, and E. Verderber. Tissue plasminogen activator and urokinase mediate the binding of Glu-plasminogen to plasma fibrin I. Evidence for new binding sites in plasmin-degraded fibrin I. J. Biol. Chem. 260(7):4432, 1985 (abstr).Google Scholar
  15. 15.
    Holemans, R., J. G. Johnston, and D. McConnell. Origin and stability of blood plasminogen activator. In: Proceedings of the 10th Congress of the European Society of Haematology, Strasbourg, part II, 1965, pp. 1253–1259.Google Scholar
  16. 16.
    Hoylaerts, M., D. C. Rijken, H. R. Lijnen, and D. Collen. Kinetics of the activation of plasminogen by human tissue plasminogen activator. Role of fibrin. J. Biol. Chem. 257:2912–2919, 1982.Google Scholar
  17. 17.
    Husain, S. S., and V. Gurewich. Purification and partial characterization of a single chain, high molecular weight form of urokinase from human urine. Arch. Biochem. Biophys. 220(1):31–38, 1983.CrossRefGoogle Scholar
  18. 18.
    Husain, S. S., V. Gurewich, and B. Lipinski. Purification of a new high MW single chain form of urokinase (UK) from urine. Thromb. Haemost. 46:11, 1981 (abstr).Google Scholar
  19. 19.
    Husain, S. S., B. Lipinski, and V. Gurewich. Rapid purification of high affinity plasminogen activator from human plasma by specific adsorption on fibrin-Celite. Proc. Natl. Acad. Sci. U.S.A. 78(7):4265–4269, 1981.CrossRefGoogle Scholar
  20. 20.
    Husain, S. S., B. Lipinski, and V. Gurewich. US Patent 4,381,346, Isolation of plasminogen activators useful as therapeutic agents (single-chain urokinase). Filed 1980, issued 1983.Google Scholar
  21. 21.
    Leys, D., and C. Cordonnier. rt-PA for ischaemic stroke: what will the next question be? Lancet 379:2320–2321, 2012.CrossRefGoogle Scholar
  22. 22.
    Liu, J., and V. Gurewich. A comparative study of the promotion of tissue plasminogen activator and pro-urokinase-induced plasminogen activation by fragments D and E-2 of fibrin. J. Clin. Invest. 88:2012–2017, 1991.CrossRefGoogle Scholar
  23. 23.
    Liu, J., and V. Gurewich. Fragment E-2 from fibrin substantially enhances pro-urokinase-induced glu-plasminogen activation. A kinetic study using a plasmin-resistant mutant pro-urokinase (Ala-158-rpro-UK). Biochemistry 31:6311–6317, 1992.CrossRefGoogle Scholar
  24. 24.
    Liu, J. N., J. X. Liu, B. Liu, Z. Sun, J. L. Zuo, P. Zhang, J. Zhang, Y. Chen, and V. Gurewich. A prourokinase mutant which induces highly effective clot lysis without interfering with hemostasis. Circ. Res. 90:757–763, 2002.CrossRefGoogle Scholar
  25. 25.
    Liu, J., R. Pannell, and V. Gurewich. A transitional state of pro-urokinase which has a higher catalytic efficiency against glu-plasminogen than urokinase. J. Biol. Chem. 267:15289–15292, 1992.Google Scholar
  26. 26.
    Liu, J. N., W. Tang, Z. Y. Sun, W. Kung, R. Pannell, P. Sarmientos, and V. Gurewich. A site-directed mutagenesis of pro-urokinase which substantially reduces its intrinsic activity. Biochemistry 35:14070–14076, 1996.CrossRefGoogle Scholar
  27. 27.
    Marder, V. J., and S. Sherry. Thrombolytic therapy: current status. N. Engl. Med. 318(23):1512–1520, 1988.CrossRefGoogle Scholar
  28. 28.
    Michels, R., H. Hoffmann, J. Windeler, H. Barth, G. Hopkins, and on Behalf of the SUTAMI Investigators. A double-blind multicenter comparison of the efficacy and safety of saruplase and urokinase in the treatment of acute myocardial infarction: report of the SUTAMI Study Group. J. Thromb. Thrombolysis 2:117–124, 1995.CrossRefGoogle Scholar
  29. 29.
    Montoney, M., S. J. Gardell, and V. J. Marder. Comparison of the bleeding potential of vampire bat salivary plasminogen activator versus tissue plasminogen activator in an experimental rabbit model. Circulation 91:1540–1544, 1995.CrossRefGoogle Scholar
  30. 30.
    Pannell, R., J. Black, and V. Gurewich. The complementary modes of action of tissue plasminogen activator (t-PA) and pro-urokinase (pro-UK) by which their synergistic effect on clot lysis may be explained. J. Clin. Invest. 81:853–859, 1988.CrossRefGoogle Scholar
  31. 31.
    Pannell, R., and V. Gurewich. Pro-urokinase—a study of its stability in plasma and a mechanism for its selective fibrinolytic effect. Blood 67:1215–1223, 1986.Google Scholar
  32. 32.
    Pannell, R., and V. Gurewich. The activation of plasminogen by single-chain urokinase or by two-chain urokinase—a demonstration that single-chain urokinase has a low catalytic activity (pro-urokinase). Blood 69:22–26, 1987.Google Scholar
  33. 33.
    Pannell, R., and V. Gurewich. A comparison of the rates of clot lysis in a plasma milieu induced by tissue plasminogen activator (t-PA) and rec-pro-urokinase: evidence that t-PA has a more restricted mode of action. Fibrinolysis 6:1–5, 1992.Google Scholar
  34. 34.
    Pannell, R., W. Kung, and V. Gurewich. C1-inhibitor prevents non-specific plasminogen activation by a prourokinase mutant without impeding fibrin-specific fibrinolysis. J. Thromb. Haemost. 5:1047–1054, 2007.CrossRefGoogle Scholar
  35. 35.
    Pennica, D., W. E. Holmes, W. J. Kohr, R. N. Harkins, G. A. Vehar, X. C. A. Wad, W. F. Bennett, E. Yelverton, P. H. Seeburg, H. L. Heyneker, D. V. Goeddel, and D. Collen. Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli. Nature 301:214–221, 1983.CrossRefGoogle Scholar
  36. 36.
    PRIMI Trial Study Group. Randomised double-blind trial of recombinant pro-urokinase against streptokinase in acute myocardial infarction. Lancet 1:863–867, 1989.Google Scholar
  37. 37.
    Rao, A. K., C. Pratt, and A. Berke. Thrombolysis in myocardial infarction (TIMI) trial—phase I: hemorrhagic manifestations and changes in plasma fibrinogen and the fibrinolytic system in patients treated with recombinant tissue plasminogen activator and streptokinase. J. Am. Coll. Cardiol. 11:1–11, 1988.CrossRefGoogle Scholar
  38. 38.
    Rapold, H. J., H. Kuemmerli, M. Weiss, H. Baur, and A. Haeberli. Monitoring of fibrin generation during thrombolytic therapy of acute myocardial infarction with recombinant tissue-type plasminogen activator. Circulation 79:980–989, 1989.CrossRefGoogle Scholar
  39. 39.
    Rijken, D. C., and D. Collen. Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture. J. Biol. Chem. 256:7035–7041, 1981.Google Scholar
  40. 40.
    Rijken, D. C., M. Hoylaerts, and D. Collen. Fibrinolytic properties of one-chain and two-chain human extrinsic (tissue-type) plasminogen activator. J. Biol. Chem. 257:2920–2925, 1982.Google Scholar
  41. 41.
    Rubiera, M., J. Alvarez-Sabin, M. Ribo, J. Montaner, E. Santamarina, J. F. Arenillas, R. Huertas, P. Delgado, F. Purroy, and C. A. Molina. Predictors of early arterial reocclusion after tissue plasminogen activator-induced recanalization in acute ischemic stroke. Stroke 36:1452–1456, 2005.CrossRefGoogle Scholar
  42. 42.
    Saqqur, M., C. A. Molina, A. Salam, M. Siddiqui, M. Ribo, K. Uchino, S. Calleja, Z. Garamik, K. Khan, N. Akhtar, F. O’Rourke, A. Shuaib, A. M. Demchuk, and A. V. Alexandrov. Clinical deterioration after intravenous recombinant tissue plasminogen activator treatment: a multicenter transcranial Doppler study. Stroke 38:69–74, 2007.CrossRefGoogle Scholar
  43. 43.
    Sasahara, A. A., J. E. Cannilla, J. S. Belko, R. L. Morse, and A. J. Criss. Urokinase therapy in clinical pulmonary embolism. N. Engl. J. Med. 30:1168–1173, 1967.CrossRefGoogle Scholar
  44. 44.
    Stone, G. W., and J. Gersh. Facilitated angioplasty: paradise lost. Lancet 367:543–546, 2006.CrossRefGoogle Scholar
  45. 45.
    Sun, Z., Y. Jiang, Z. Ma, H. Wu, B. F. Liu, Y. Xu, W. Tang, Y. Chen, C. Li, D. Zhu, V. Gurewich, and J. Liu. Identification of a flexible loop (297–313) of urokinase type plasminogen activator, which helps determine its catalytic activity. J. Biol. Chem. 272:23818–23823, 1997.CrossRefGoogle Scholar
  46. 46.
    Sun, Z., B. F. Liu, Y. Chen, V. Gurewich, D. Zhu, and J. Liu. Analysis of the forces which stabilizes the active conformation of urokinase-type plasminogen activator. Biochemistry 37:2935–2940, 1998.CrossRefGoogle Scholar
  47. 47.
    Tebbe, U., R. Michels, J. Adgey, J. Boland, A. Caspi, and for The Comparison Trial of Saruplase and Streptokinase (COMASS) Investigators. Randomized, double-blind study comparing saruplase with streptokinase therapy in acute myocardial infarction: the COMPASS equivalence trial. J. Am. Coll. Cardiol. 31(3):487–493, 1998.CrossRefGoogle Scholar
  48. 48.
    The GUSTO Investigators. An international randomized trial comparing four thrombolytic strategies for acute myocardial infarction. N. Engl. J. Med. 329:673–682, 1993.CrossRefGoogle Scholar
  49. 49.
    The IST-3 Collaborative Group. The benefits and harms of intravenous thrombolysis with recombinant tissue plasminogen activator within 6 h of acute ischaemic stroke (the third international stroke trial [IST-3]): a randomised controlled trial. Lancet 379:2352–2363, 2012.CrossRefGoogle Scholar
  50. 50.
    The National Institute of Neurological Disorders and Stroke (NINDS) rt-PA Stroke Study Group. Tissue plasminogen activator for acute ischaemic stroke. N. Engl. J. Med. 333:1581–1587, 1995.CrossRefGoogle Scholar
  51. 51.
    Third International Study of Infarct Survival Collaborative Group. ISIS-3. ISIS-3: a randomised comparison of streptokinase vs tissue plasminogen activator vs anistreplase and of aspirin plus heparin vs aspirin alone among 41,299 cases of suspected acute myocardial infarction. Lancet 339:753–770, 1992.CrossRefGoogle Scholar
  52. 52.
    Thorsen, S., P. Glas-Greenwalt, and T. Astrup. Differences in the binding to fibrin of urokinase and tissue plasminogen activator. Thromb. Diathos. Haemorrh. 28:65–74, 1972.Google Scholar
  53. 53.
    Tomasi, S., P. Sarmientos, G. Giorda, V. Gurewich, and A. Vercelli. Mutant prourokinase with adjunctive C1-inhibitor is an effective and safer alternative to tPA in rat stroke. PLoS ONE 6:e21999, 2011.CrossRefGoogle Scholar
  54. 54.
    Topol, E. J., and R. M. Califf. Tissue plasminogen activator: why the backlash? J. Am. Coll. Cardiol. 13:1477–1480, 1989.CrossRefGoogle Scholar
  55. 55.
    van Zonneveld, A.-J., H. Veerman, and H. Pannekoek. On the interaction of the finger and the kringle-2 domain of tissue-type plasminogen activator with Fibrin. Inhibition of kringle-2 binding to fibrin by ε-amino caproic acid. J. Biol. Chem. 261:14214–14218, 1986.Google Scholar
  56. 56.
    Varadi, A., and L. Patthy. Beta (Leu121-Lys122) segment of fibrinogen is in a region essential for plasminogen binding by fibrin fragment E. Biochemistry 23(9):2108–2112, 1984 (abstr).CrossRefGoogle Scholar
  57. 57.
    Verheugt, F. W. A., A. Meijer, W. K. Lagrand, and M. J. van Eenige. Reocclusion: the flip side of coronary thrombolysis. J. Am. Coll. Cardiol. 27:766–773, 1996.CrossRefGoogle Scholar
  58. 58.
    Voskuilen, M., A. Vermond, G. H. Veeneman, J. H. van Boom, E. A. Klasen, N. D. Zegers, and W. Nieuwenhuizen. Fibrinogen lysine residue Aα157 plays a crucial role in the fibrin-induced acceleration of plasminogen activation, catalyzed by tissue-type plasminogen activator. J. Biol. Chem. 262(13):5944–5946, 1987.Google Scholar
  59. 59.
    Weaver, W. D., J. R. Hartmann, J. L. Anderson, P. S. Reddy, J. C. Sobolski, A. Sasahara, and for the Prourokinase Study Group. New recombinant glycosylated prourokinase for treatment of patients with acute myocardial infarction. J. Am. Coll. Cardiol. 24(5):1242–1248, 1994.CrossRefGoogle Scholar
  60. 60.
    Wun, T. C., L. Ossowski, and E. Reich. A proenzyme form of human urokinase. J. Biol. Chem. 257(12):7262–7268, 1982.Google Scholar
  61. 61.
    Yakovlev, S., E. Makogonenko, N. Kurochkina, W. Nieuwenhuizen, K. Ingham, and L. Medved. Conversion of fibrinogen to fibrin: mechanism of exposure of tPA- and plasminogen-binding sites. Biochemistry 39:15730–15741, 2000.CrossRefGoogle Scholar
  62. 62.
    Zarich, S. W., G. J. Kowalchuk, W. D. Weaver, J. Loscalzo, M. Sassower, K. Manzo, C. Byrnes, J. E. Muller, V. Gurewich, and for the PATENT Study Group. Sequential combination thrombolytic therapy for acute myocardial infarction: results of the pro-urokinase and t-PA enhancement of thrombolysis (PATENT) trial. J. Am. Coll. Cardiol. 26:374–379, 1995.CrossRefGoogle Scholar
  63. 63.
    Zinkstok, S. M., Y. B. Roos, and for the ARTIS Investigators. Early administration of aspirin in patients treated with alteplase for acute ischaemic stroke: a randomised controlled trial. Lancet 380:731–737, 2012.CrossRefGoogle Scholar

Copyright information

© Biomedical Engineering Society 2013

Authors and Affiliations

  1. 1.Mount Auburn HospitalHarvard Medical SchoolCambridgeUSA

Personalised recommendations