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Thromb Haemost 1998; 79(03): 602-608
DOI: 10.1055/s-0037-1614953
Review Articles
Schattauer GmbH

Platelets and Endothelial Cells Act in Concert to Delay Thrombolysis – Evidence from an In Vitro Model of the Human Occlusive Thrombus[*]

R. R. Hantgan
1   Departments of Biochemistry
,
W. G. Jerome
2   Pathology, Wake Forest University School of Medicine, Winston-Salem, NC, USA
,
S. Handt
3   Department of Pathology, Aachen University of Technology, Aachen, Germany
› Author Affiliations
Further Information

Publication History

Received 06 March 1997

Accepted after resubmission 28 November 1997

Publication Date:
27 December 2017 (online)

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Summary

The molecular and cellular mechanisms that over a period of hours render a human thrombus progressively resistant to fibrinolysis have been probed with a novel in vitro model. The kinetics of clot formation and fibrinolysis were monitored by laser light scattering with platelet-rich model thrombi contained in cylindrical flow chambers. In selected experiments, human umbilical vein endothelial cells were also cultured to confluence on the inner walls of these “glass blood vessels”. Following an “aging” period (0.5, 2 or 4 h), each thrombus was gently perfused with a bolus of plasminogen/recombinant tissue plasminogen activator to induce fibrinolysis. Platelets delayed lysis of 2 h-aged thrombi by ~70% and (non-stimulated) endothelial cells by ~30%, compared to cell-free control clots. However, even greater lytic delays (~260%) resulted when both vascular cells were present in the same 2 h-aged thrombus. In contrast, rapid lysis was consistently achieved with R298E,R299E t-PA, a genetically engineered plasminogen activator that is insensitive to inhibition by plasminogen activator inhibitor type 1. These observations suggest platelets and endothelial cells act in concert to enrich the fibrin scaffold of an aging human thrombus in plasminogen activator inhibitor. We propose that the presence of both platelets and endothelial cells may contribute to progressive thrombolytic resistance.

* Supported by Grant # 90-IN-5 from the NC Board of Science and Technology, Research Grant # CA12197 from the National Institutes of Health (Bethesda, MD), the Wake Forest University School of Medicine Interim Funding Program, NATO Grant for International Collaboration in Research # CRG 950213, and Grant NC-96-GS-72 from the American Heart Association, North Carolina Affiliate.


 

  • References

  • 1 Collen D, Lijnen HR. On the future of thrombolytic therapy for acute myocardial infarction. Am J Cardiol 1993; 72: 46.
    CrossrefPubMedSearch in Google Scholar
  • 2 Martin GV, Kennedy JW, Marder VJ. Thrombolytic therapy for coronary artery disease. In: Hemostasis and Thrombosis: Basic Principles and Clinical Practice. Colman RW, Hirsh J, Marder VJ, Salzman EW. (eds). Philadelphia: J. B. Lippincott Company; 1994. p 1409.
    Search in Google Scholar
  • 3 Hoylaerts M, Rijken DC, Lijnen HR, Collen D. Kinetics of the activation of plasminogen by human tissue plasminogen activator. Role of fibrin. J Biol Chem 1982; 257: 2912.
    PubMedSearch in Google Scholar
  • 4 Collen D, Lijnen HR. The fibrinolytic system in man. CRC Crit Rev in Onc/Hemat 1985; 4: 248.
    PubMedSearch in Google Scholar
  • 5 de Vries C, Veerman H, Pannekoek H. Identification of the domains of tissue type plasminogen activator inhibitor involved in the augmented binding of fibrin after limited digestion with plasmin. J Biol Chem 1989; 264: 12604.
    PubMedSearch in Google Scholar
  • 6 Tanswell P, Tebbe U, Neuhaus KL. et al. Pharmacokinetics and fibrin specificity of alteplase during accelerated infusions in acute myocardial infarction. J Am Coll 1992; Cardiol (19) 1071.
    PubMedSearch in Google Scholar
  • 7 Chesebro JH, Knatterud G, Roberts R, Borer J, Cohen LS, Dalen J, Dodge HT, Francis CK, Hillis D, Ludbrook P, Markis JE, Mueller H, Passamani ER, Powers ER, Rao AK, Robertson T, Ross A, Ryan TJ, Sobel BE, Willerson J, Williams DO, Zaret BL, Braunwald E. Thrombolysis in myocardial infarction (TIMI) trial, phase 1: a comparison between intravenous tissue plasminogen activator and intravenous streptokinase. Circulation 1987; 76: 142.
    CrossrefPubMedSearch in Google Scholar
  • 8 The GUSTO Investigators.. An international randomized trial comparing four thrombolytic strategies for acute myocardial infarction. New Eng J Med 1993; 329: 673.
    CrossrefPubMedSearch in Google Scholar
  • 9 GUSTO IIa Investigators.. Randomized trial of intravenous heparin versus recombinant hirudin for acute coronary syndromes. Circulation 1994; 90: 1631.
    CrossrefPubMedSearch in Google Scholar
  • 10 Anderson HV, Willerson JT. Thrombolysis in Acute Myocardial Infarction. New Eng J Med 1993; 329: 703.
    CrossrefPubMedSearch in Google Scholar
  • 11 Marder VJ, Hirsh J, Bell WR. Rationale and Practical Basis of Thrombolytic Therapy. In: Hemostasis and Thrombosis: Basic Principles and Clinical Practice. Colman RW, Hirsh J, Marder VJ, Salzman EW. eds. Philadelphia: J.B. Lippincott; 1994. 1514
    Search in Google Scholar
  • 12 Bode C, Nordt TK, Runge MS. Update in thrombolysis 1994 – International Symposium. Ann Hematol 1994; 69: S65.
    CrossrefPubMedSearch in Google Scholar
  • 13 Cannon CP, Antman EM, Walls R, Braunwald E. Time as an Adjunctive agent to thrombolytic therapy. J Thromb Thrombolys 1994; 1: 27.
    CrossrefPubMedSearch in Google Scholar
  • 14 Neuhaus K-L, Feuerer W, Jeep-Tebbe S, Niederer W, Vogt A, Tebbe U. Improved thrombolysis with a modified dose regimen of recombinant tissue-type plasminogen activator. J Am Cell Cardiol 1989; 14: 1566.
    CrossrefPubMedSearch in Google Scholar
  • 15 Carney RJ, Murphy GA, Brandt TR, Daley PJ, Pickering E, White HJ, McDonough TJ, Vermilya SK, Teichman SL. Randomized angiographic trial of recombinant tissue-type plasminogen activator (Alteplase) in myocardial infarction. J Am Coll Cardiol 1992; 20: 17.
    CrossrefPubMedSearch in Google Scholar
  • 16 Neuhaus K-L, Von Essen R, Tebbe U, Vogt A, Roth M, Reiss M, Niederer W, Forycki F, Wirtzfeld A, Maeurer W, Limbourg P, Merx W, Haerten K. Improved thrombolysis in acute myocardial infarction with front-loaded administration of alteplase: results of the rt-PA-APSAC patency study (TAPS). J Am Coll Cardiol 1992; 19: 885.
    CrossrefPubMedSearch in Google Scholar
  • 17 Granger CB, White HD, Bates ER, Ohman EM, Califf RM. A pooled analysis of coronary arterial patency and left ventricular function after intravenous thrombolysis for acute myocardial infarction. Am J Cardiol 1994; 74: 1220.
    CrossrefPubMedSearch in Google Scholar
  • 18 Rogers WJ, Bowlby LJ, Chandra NC, French WJ, Gore JL, Lambrew CT, Rubison RM, Tiefenbrunn AJ, Weaver WD. Treatment of Myocardial Infarction in the United States (1990-1993). Observations from the National Registry for Myocardial Infarction. Circulation 1994; 90: 2103.
    CrossrefPubMedSearch in Google Scholar
  • 19 Levi M, Biemond BJ, Van Zonneveld A-J, Wouter ten Cate J, Pannekoek H. Inhibition of plasminogen activator inhibitor-1 activity results in promotion of endogenous thrombolysis and inhibition of thrombus extension in models of experimental thrombosis. Circulation 1992; 85: 305.
    CrossrefPubMedSearch in Google Scholar
  • 20 Kunitada S, FitzGerald GA, Fitzgerald DJ. Inhibition of clot lysis and decreased binding of tissue-type plasminogen activator as a consequence of clot retraction. Blood 1992; 79: 1420.
    PubMedSearch in Google Scholar
  • 21 Krishnamurti C, Alving BM. Plasminogen activator inhibitor type 1: biochemistry and evidence for modulation of fibrinolysis in vivo. Sem Thromb Hemost 1992; 18: 67.
    Thieme ConnectPubMedSearch in Google Scholar
  • 22 Colucci M, Paramo JA, Collen D. Inhibition of one-chain and two-chain forms of human tissue-type plasminogen activator by the fast-acting inhibitor of plasminogen activator in vitro and in vivo. J Clin Lab Med 1986; 108: 53.
    PubMedSearch in Google Scholar
  • 23 Lindahl TL, Ohlsson P-I, Wiman B. The mechanism of the reaction between human plasminogen-activator inhibitor 1 and tissue plasminogen activator. Biochem J 1990; 265: 109.
    CrossrefPubMedSearch in Google Scholar
  • 24 Lijnen HR, Van Hoef B, Collen D. On the reversible interaction of plasminogen activator inhibitor-1 with tissue-type plasminogen activator and with urokinase-type plasminogen activator. J Biol Chem 1991; 266: 4041.
    PubMedSearch in Google Scholar
  • 25 Fujii S, Sobel BE. Induction of plasminogen activator inhibitor by products released from platelets. Circulation 1990; 82: 1485.
    CrossrefPubMedSearch in Google Scholar
  • 26 Booth NA, Simpson AJ, Croll A, Bennett B, MacGregor IR. Plasminogen activator inhibitor (PAI-1) in plasma and platelets. Brit J Haemotol 1988; 70: 327.
    PubMedSearch in Google Scholar
  • 27 Loskutoff DJ, Curriden SA. The fibrinolytic system of the vessel wall and its role in the control of thrombosis. Ann NY Acad Sci 1990; 598: 238.
    CrossrefPubMedSearch in Google Scholar
  • 28 Slivka SR, Loskutoff DJ. Platelets stimulate endothelial cells to synthesize type 1 plasminogen activator inhibitor. Evaluation of the role of transforming growth factor. Blood 1991; 77: 1013-19.
    PubMedSearch in Google Scholar
  • 29 Handt S, Jerome WG, Braaten JV, Lewis JC, Kirkpatrick CJ, Hantgan RR. PAI-1 released from cultured human umbilical vein endothelial cells binds to fibrin and protects it from fibrinolysis. Fibrinolysis 1994; 8: 104.
    PubMedSearch in Google Scholar
  • 30 Wagner OF, De Vries C, Hohmann C, Veerman H, Pannekoek H. Interaction between plasminogen activator inhibitor type 1 (PAI-1) bound to fibrin and either tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). Binding of t-PA/PAI-1 complexes to fibrin mediated by both the finger and the kringle-2 domain of t-PA. J Clin Invest 1989; 84: 647.
    CrossrefPubMedSearch in Google Scholar
  • 31 Reilly CF, Hutzelmann JE. Plasminogen activator inhibitor-1 binds to fibrin and inhibits tissue-type plasminogen activator-mediated fibrin dissolution. J Biol Chem 1992; 267: 17128.
    PubMedSearch in Google Scholar
  • 32 Keijer J, Linders M, Van Zonneveld A-J, Ehrlich HJ, De Boer J-P, Pannekoek H. The interaction of plasminogen activator inhibitor 1 with plasminogen activators (tissue-type and urokinase-type) and fibrin: Localization of interaction sites and physiologic relevance. Blood 1991; 78: 401.
    PubMedSearch in Google Scholar
  • 33 Braaten JV, Handt S, Jerome WG, Kirkpatrick J, Lewis JC, Hantgan RR. Regulation of fibrinolysis by platelet-released plasminogen activator inhibitor 1 (PAI-1): Light scattering and ultrastructural examination of a model platelet-fibrin thrombus. Blood 1993; 81: 1290.
    PubMedSearch in Google Scholar
  • 34 Kruithof EKO, Nicolosa G, Bachmann F. Plasminogen activator inhibitor 1: development of a radioimmunoassay and observations on its plasma concentration during venous occlusion and after platelet aggregation. Blood 1987; 70: 1645.
    PubMedSearch in Google Scholar
  • 35 Hantgan RR, Hermans J. Assembly of fibrin: A light scattering study. J Biol Chem 1979; 254: 11272.
    PubMedSearch in Google Scholar
  • 36 Handt S, Jerome WG, Tietze L, Hantgan RR. PAI-1 secretion by endothelial cells increases fibrinolytic resistance of an in vitro fibrin clot: Evidence for a key role of endothelial cells in thrombolytic resistance. Blood 1996; 87: 4204.
    PubMedSearch in Google Scholar
  • 37 Bennett WF, Paoni NF, Keyt BA, Botstein D, Jones AJS, Presta L, Wurm FM, Zoller MJ. High resolution analysis of functional determinants on human tissue-type plasminogen activator. J Biol Chem 1991; 266: 5191.
    PubMedSearch in Google Scholar
  • 38 Hantgan RR, Endenburg SC, Cavero I, Marguerie G, Uzan A, Sixma JJ, De Groot PG. Inhibition of platelet adhesion to fibrin(ogen) in flowing whole blood by Arg-Gly-Asp and fibrinogen gamma-chain carboxy terminal peptides. Thromb Haemost 1992; 68: 694.
    Thieme ConnectPubMedSearch in Google Scholar
  • 39 Hantgan RR. A study of the kinetics of ADP-triggered platelet shape change. Blood 1984; 64: 896.
    PubMedSearch in Google Scholar
  • 40 Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 1973; 52: 2745.
    CrossrefPubMedSearch in Google Scholar
  • 41 Carr M, She LL, Hermans J. Mass-length ratio of fibrin fibers from gel permeation and light scattering. Biopolymers 1977; 16: 1.
    CrossrefPubMedSearch in Google Scholar
  • 42 Berkenpas MB, Lawrence DA, Ginsburg D. Molecular evolution of plasminogen activator inhibitor-1 functional stability. EMBO J 1995; 14: 2969.
    PubMedSearch in Google Scholar
  • 43 Braaten JV, Jerome WG, Hantgan RR. Uncoupling fibrin from integrin receptors hastens fibrinolysis at the platelet-fibrin interface. Blood 1994; 83: 982.
    PubMedSearch in Google Scholar
  • 44 Weisel JW. Fibrin assembly. Lateral aggregation and the role of the two pairs of fibrinopeptides. Biophysical Journal 1986; 50: 1079-93.
    CrossrefPubMedSearch in Google Scholar
  • 45 Hantgan RR, McDonagh J, Hermans J. Fibrin assembly. Ann N Y Acad Sci 1983; 408: 344.
    CrossrefPubMedSearch in Google Scholar
  • 46 Erickson LA, Ginsberg MH, Loskeutoff DJ. Detection and partial characterization of an inhibitor of plasminogen activator in human platelets. J Clin Invest 1984; 74: 1465.
    CrossrefPubMedSearch in Google Scholar
  • 47 Lang IM, Schleef RR. Calcium-dependent stabilization of type I plasminogen activator inhibitor within platelet α-granules. J Biol Chem 1996; 271: 2754.
    CrossrefPubMedSearch in Google Scholar
  • 48 Nachman RL, Hajjar KA. Endothelial Cell Fibrinolytic Assembly. Ann N Y Acad Sci 1991; 614: 240.
    CrossrefPubMedSearch in Google Scholar
  • 49 Erickson LA, Lawrence DA, Loskutoff DJ. Reverse fibrin autography: a method to detect and partially characterize protease inhibitors after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal Biochem 1984; 137: 454.
    CrossrefPubMedSearch in Google Scholar
  • 50 Levin EG, Loskutoff DJ. Comparative studies of the fibrinolytic activity of cultured vascular cells. Thromb Res 1979; 15: 869.
    CrossrefPubMedSearch in Google Scholar
  • 51 Kassis J, Hirsh J, Podor TJ. Evidence that postoperative fibrinolytic shutdown is mediated by plasma factors that stimulate endothelial cell type I plasminogen activator inhibitor biosynthesis. Blood 1992; 80: 1758.
    CrossrefPubMedSearch in Google Scholar
  • 52 Bennett WF. Structure-function studies of the t-PA molecule. Fibrinolysis 7 Suppl 1993; 1: 38.
    PubMedSearch in Google Scholar
  • 53 Booth NA, Robbie LA, Croll AM, Bennett B. Lysis of platelet-rich thrombi: The role of PAI-1. Ann NY Acad Sci 1992; 667: 70.
    CrossrefPubMedSearch in Google Scholar
  • 54 Robbie LA, Booth NA, Croll AM, Bennett B. The roles of α2-antiplasmin and plasminogen activator inhibitor 1 (PAI-1) in the inhibition of clot lysis. Thromb Haemost 1993; 70: 301.
    Thieme ConnectPubMedSearch in Google Scholar
  • 55 Stringer HAR, Pannekock H. The significance of fibrin binding by plasminogen activator inhibitor 1 for the mechanism of tissue-type plasminogen activator-mediated fibrinolysis. J Biol Chem 1995; 270: 11205-8.
    CrossrefPubMedSearch in Google Scholar
  • 56 Stringer HAR, Swieten PV, Heijnen HFG, Sixma JJ, Pannekock 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.
    CrossrefPubMedSearch in Google Scholar
  • 57 Wu JH, Siddiqui K, Diamond SL. Transport phenomena and clot dissolving therapy: An experimental investigation of diffusion controlled and permeation-enhanced fibrinolysis. Thromb Haemost 1994; 72: 105.
    Thieme ConnectPubMedSearch in Google Scholar
  • 58 Blinc A, Kennedy SD, Bryant RG, Marder VJ, Francis CW. Flow through clots determines the rate and pattern of fibrinolysis. Thromb Haemost 1993; 71: 230.
    PubMedSearch in Google Scholar
  • 59 Berenbaum MC. The expected effect of a combination of agents: the general solution. J Theor Biol 1985; 114: 413.
    CrossrefPubMedSearch in Google Scholar
  • 60 Tallarida RJ. Statistical analysis of drug combinations for synergism. Pain 1992; 49: 93-7.
    CrossrefPubMedSearch in Google Scholar
  • 61 Berebaum MC. What is synergy?. Pharmacol Rev 1989; 41: 93-141.
    PubMedSearch in Google Scholar
  • 62 Horrevoets AJG, Smilde A, de Vries C, Pannekoek H. The specific roles of finger and kringle 2 domains of tissue-type plasminogen activator during in vitro fibrinolysis. J Biol Chem 1994; 269: 12639-44.
    PubMedSearch in Google Scholar
  • 63 Hantgan RR, Nichols WL, Ruggeri ZM. von Willebrand factor competes with fibrin for occupancy of GPIIb:IIIa on thrombin-stimulated platelets. Blood 1990; 75: 889.
    PubMedSearch in Google Scholar
  • 64 Cheresh DA, Berliner SA, Vicente V, Ruggeri ZM. Recognition of distinct adhesive sites on fibrinogen by related integrins on platelets and endothelial cells. Cell 1989; 58: 945.
    CrossrefPubMedSearch in Google Scholar
  • 65 Cheresh DA. Integrins in thrombosis, wound healing and cancer. Biochem Soc Trans 1991; 19: 835.
    CrossrefPubMedSearch in Google Scholar