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Thromb Haemost 2002; 88(06): 996-1002
DOI: 10.1055/s-0037-1613346
Involvement of Thrombin Receptors in the Subject-dependent Variability in Ca2+ Signal Generation
Schattauer GmbH

Localization and Identification of Thrombin and Plasminogen Activator Activities in Model Human Thrombi by in situ Zymography

Nicola J. Mutch   *
1   Department of Medicine and Therapeutics, Institute of Medical Science, Aberdeen, UK
2   Department of Molecular and Cell Biology, University of Aberdeen, Institute of Medical Science, Aberdeen, UK
,
Elaine Moir   *
1   Department of Medicine and Therapeutics, Institute of Medical Science, Aberdeen, UK
2   Department of Molecular and Cell Biology, University of Aberdeen, Institute of Medical Science, Aberdeen, UK
,
Linda A. Robbie
1   Department of Medicine and Therapeutics, Institute of Medical Science, Aberdeen, UK
2   Department of Molecular and Cell Biology, University of Aberdeen, Institute of Medical Science, Aberdeen, UK
,
Susan H. Berry
1   Department of Medicine and Therapeutics, Institute of Medical Science, Aberdeen, UK
,
Bruce Bennett
1   Department of Medicine and Therapeutics, Institute of Medical Science, Aberdeen, UK
,
Nuala A. Booth
2   Department of Molecular and Cell Biology, University of Aberdeen, Institute of Medical Science, Aberdeen, UK
› Author Affiliations
Further Information

Publication History

Received 20 May 2002

Accepted after resubmission 05 August 2002

Publication Date:
09 December 2017 (online)

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Summary

Human thrombi vary in their susceptibility to lysis and this is clinically important. Several potential contributory factors were examined in this study by using model thrombi, created under flow; these provide a robust, reproducible and easily-manipulated system. Here we identify the plasminogen activators (PA) active in model thrombi of known age and define the cellular and plasma contribution to activity in different areas. The cell-rich head of model thrombi had strong thrombin and PA activity, with coagulant activity also at the tail. Thrombin activity decreased as model thrombi were aged. PA activity in the thrombus head also decreased on ageing of thrombi but activity emerged around the thrombi, including the tail. Activity in the head of fresh model thrombi was primarily due to uPA, with some contribution from tPA. Experiments with thrombi prepared from platelet-rich plasma and added leucocytes showed that uPA activity at the head of fresh thrombi was derived from PMN. Older thrombi had tPA activity around the tail of the thrombus; this activity occurred in the absence of cells. This study highlights the importance of PMN-derived uPA activity in the lysis of fresh thrombi, with activity originating in the leucocyte-rich head. It also shows that thrombi are dynamic structures in which fibrin can be repeatedly laid down and lysed, observations that are relevant to therapeutic lysis and potential rethrombosis.

Keywords

tPA - uPA - thrombi - fibrinolysis - leucocytes - thrombin

* These authors contributed equally to the research


 

  • References

  • 1 Falk E. Dynamics in thrombus formation. Ann N Y Acad Sci 1992; 667: 204-23.
    CrossrefPubMedGoogle Scholar
  • 2 Mann KG, Jenny RJ, Krishnaswamy S. Cofactor proteins in the assembly and expression of blood clotting enzyme complexes. Ann Rev Biochem 1988; 266: 915-20.
    PubMedGoogle Scholar
  • 3 Carrell RW, Huntington JA, Mushunje A, Zhou A. The conformational basis of thrombosis. Thromb Haemost 2001; 86: 14-22.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 4 Liu CY, Nossel HL, Kaplan KL. The binding of thrombin by fibrin. J Biol Chem 1979; 254: 10421-25.
    PubMedGoogle Scholar
  • 5 Hogg PJ, Jackson CM. Fibrin monomer protects from inactivation by heparin-antithrombin III: Implications for heparin efficacy. Proc Natl Acad Sci 1989; 86: 3619-23.
    CrossrefPubMedGoogle Scholar
  • 6 Weitz JI, Hudoba M, Massel D, Maraganore J, Hirsh J. Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors. J Clin Invest 1990; 86: 385-91.
    CrossrefPubMedGoogle Scholar
  • 7 Mutch NJ, Robbie LA, Booth NA. Human thrombi contain an abundance of thrombin. Thromb Haemost 2001; 86: 1028-34.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 8 Francis CW, Markham Jr RE, Barlow GH, Florack TM, Dobrzynski DM, Marder VJ. Thrombin activity of fibrin thrombi and soluble plasmic derivatives. J Lab Clin Med 1983; 102: 220-30.
    PubMedGoogle Scholar
  • 9 Collen D, Lijnen HR. Basic and clinical aspects of fibrinolysis and thrombolysis. Blood 1991; 78: 3114-3124.
    PubMedGoogle Scholar
  • 10 Booth NA, Simpson AJ, Croll AM, Bennett B, MacGregor IR. Plasminogen activator inhibitor (PAI-1) in plasma and platelets. Brit J Haem 1988; 70: 327-33.
    CrossrefPubMedGoogle Scholar
  • 11 Carmeliet P, Schoonjans L, Kieckens L, Ream B, Degen JL, Bronson R, de Vos R, van den Oord JJ, Collen D, Mulligan RC. Physiological consequences of loss of plasminogen activator gene function in mice. Nature 1994; 368: 419-24.
    CrossrefPubMedGoogle Scholar
  • 12 Bdeir K, Murciano J, Tomaszewski J, Koniaris L, Martinez J, Cines DB, Muzykantov VR, Higazi AAR. Urokinase mediates fibrinolysis in the pulmonary microvasculature. Blood 2000; 96: 1820-6.
    PubMedGoogle Scholar
  • 13 Robbie LA, Bennett B, Croll AM, Brown PAJ, Booth NA. Proteins of the fibrinolytic system in human thrombi. Thromb Haemost 1996; 75: 127-33.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 14 Brommer EJP, van Bockel JH. Composition and susceptibility to thrombolysis of human arterial thrombi and the influence of their age. Blood Coag Fib 1992; 03: 717-25.
    PubMedGoogle Scholar
  • 15 Potter BJvan Loon, Rijken DC, Brommer EJP, van der Mass APC. The amount of plasminogen activator inhibitor type 1 in human thrombi and the relation to ex-vivo lysibility. Thromb Haemost 1992; 67: 101-5.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 16 van Giezen JJJ, Nerme V, Abrahamsson T. PAI-1 inhibition enhances the lysis of the platelet-rich part of arterial-like thrombi formed in vitro. A comparative study using thrombi prepared from rat and human tissue. Blood Coag Fib 1998; 09: 11-8.
    PubMedGoogle Scholar
  • 17 Stringer HAR, van Swieten P, Heijnen FG, Sixma JJ, Pannekoek H. Plasminogen activator inhibitor-1 released from activated platelets plays a key role in thrombolysis resistance: studies with thrombi generated in the Chandler loop. Arterioscler Thromb 1994; 14: 1452-8.
    CrossrefPubMedGoogle Scholar
  • 18 Robbie LA, Young SP, Bennett B, Booth NA. Thrombi formed in a Chandler loop mimic human arterial thrombi in structure and PAI-1 content and distribution. Thromb Haemost 1997; 77: 510-5.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 19 Chandler AB. In vitro thrombotic coagulation of the blood. Lab Invest 1958; 07: 110-4.
    PubMedGoogle Scholar
  • 20 Robbie LA, Robbie LA, Keyt BA, Bennett B, Booth NA. Effective lysis of model thrombi by a t-PA mutant (A473S) that is resistant to α2-antiplasmin. Br J Haematol 2000; 111: 517-23.
    CrossrefPubMedGoogle Scholar
  • 21 Moir E, Booth NA, Bennett B, Robbie LA. Polymorphonuclear leucocytes mediate endogenous thrombus lysis via a u-PA-dependent mechanism. Brit J Haematol 2001; 113: 72-80.
    CrossrefPubMedGoogle Scholar
  • 22 Ritchie H, Robbie LA, Kinghorn S, Exley R, Booth NA. Monocyte plasminogen activator inhibitor 2 (PAI-2) inhibits u-PA mediated fibrinolysis and is cross-linked to fibrin. Thromb Haemost 1999; 81: 96-103.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 23 Naski MC, Shafer JA. α-Thrombin within fibrin clots: Inactivation of thrombin by antithrombin-III. Thromb Res 1993; 69: 453-65.
    CrossrefPubMedGoogle Scholar
  • 24 Northeast ADR, Soo KS, Bobrow LG, Gaffney PJ, Burnand KG. The tissue-plasminogen activator and urokinase response in vivo during natural resolution of venous thrombi. J Vasc Surg 1995; 22: 573-9.
    CrossrefPubMedGoogle Scholar
  • 25 Lang IM, Marsh JJ, Konopka RG, Olman MA, Binder BR, Moser KM, Schleef RR. Factors contributing to increased vascular fibrinolytic activity in mongrel dogs. Circulation 1993; 87: 1990-2000.
    CrossrefPubMedGoogle Scholar
  • 26 Yamamoto K, Loskutoff DJ. Fibrin deposition in tissues from endotoxintreated mice correlates with decreases in the expression of urokinase-type but not tissue-type plasminogen activator. J Clin Invest 1996; 97: 2440-51.
    CrossrefPubMedGoogle Scholar
  • 27 Ichinose A, Fujikawa K, Suyuma T. The activation of pro-urokinase by plasma kallikrein and its inactivation by thrombin. J Biol Chem 1986; 261: 2486-9.
    PubMedGoogle Scholar
  • 28 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: 769-72.
    PubMedGoogle Scholar
  • 29 Moir E, Robbie LA, Bennett B, Booth NA. Polymorphonuclear leucocytes have two opposing roles in fibrinolysis. Thromb Haemost 2002; 87: 1006-10.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 30 Lenich C, Liu J-N, Gurewich V. Thrombin stimulation of platelets induces plasminogen activation mediated by endogenous urokinase-type plasminogen activator. Blood 1997; 90: 3579-86.
    PubMedGoogle Scholar
  • 31 Sinniger V, Merton E, Fabregas P, Felez J, Longstaff C. Regulation of tissue plasminogen activator activity by cells. J Biol Chem 1999; 274: 12414-22.
    CrossrefPubMedGoogle Scholar
  • 32 Plesner T, Ploug M, Ellis V, Rønne E, Høyer-Hansen G, Wittrup M, Pedersen TL, Tscherning T, Danø K, Hansen NE. The receptor for urokinase-type plasminogen activator and urokinase is translocated from two distinct intracellular compartments to the plasma membrane on stimulation of human neutrophils. Blood 1994; 83: 808-15.
    PubMedGoogle Scholar
  • 33 Heiple JM, Ossowski L. Human neutrophil plasminogen activator is localized in specific granules and is translocated to the cell surface by exocytosis. J Exp Med 1986; 164: 826-40.
    CrossrefPubMedGoogle Scholar
  • 34 Higazi AAR, Barghouti I, Abu-Much R. Identification of an inhibitor of tissue-type plasminogen activator-mediated fibrinolysis in human neutrophils. J Biol Chem 1995; 270: 9472-7.
    CrossrefPubMedGoogle Scholar
  • 35 Sakharov DV, Rijken DC. Superficial accumulation of plasminogen during plasma clot lysis. Circulation 1995; 92: 1883-90.
    CrossrefPubMedGoogle Scholar
  • 36 Torr SRBrown, Sobel BE. Attenuation of thrombolysis by release of plasminogen-activator inhibitor type-1 from platelets. Thromb Res 1993; 72: 413-21.
    CrossrefPubMedGoogle Scholar
  • 37 van Giezen JJ, Nerme V, Abrahamson T. PAI-1 inhibition enhances the lysis of the platelet-rich part of arterial-like thrombi formed in vitro. A comparative study using thrombi prepared from rat and human blood. Blood Coag Fibrinol 1998; 09: 11-8.
    PubMedGoogle Scholar
  • 38 Carmeliet P, Stassen JM, Schoonjans L, Ream B, Vandernoord JJ, DeMol M, Mulligan RC, Collen D. Plasminogen-activator inhibitor-1 gene deficient mice 2. Effects on haemostasis, thrombosis and thrombolysis. J Clin Invest 1993; 92: 2756-60.
    CrossrefPubMedGoogle Scholar
  • 39 Farrehi PM, Ozaki CK, Carmeliet P, Fay WP. Regulation of arterial thrombolysis by plasminogen activator inhibitor-1 in mice. Circulation 1998; 97: 1002-8.
    CrossrefPubMedGoogle Scholar
  • 40 Zhu YH, Carmeliet P, Fay WP. Plasminogen activator inhibitor-1 is a major determinant of arterial thrombolysis resistance. Circulation 1999; 99: 3050-5.
    CrossrefPubMedGoogle Scholar