Skip to main content

Advertisement

SpringerLink
Log in

Activated thrombin-activatable fibrinolysis inhibitor attenuates the angiogenic potential of endothelial cells: potential relevance to the breast tumour microenvironment

  • Research Paper
  • Published:
Clinical & Experimental Metastasis Aims and scope Submit manuscript

Abstract

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a basic carboxypeptidase zymogen present in blood plasma. Proteolytic activation of TAFI by thrombin, thrombin in complex with the endothelial cell cofactor thrombomodulin, or plasmin results in an enzyme (TAFIa) that removes carboxyl-terminal lysine residues from protein and peptide substrates, including cell-surface plasminogen receptors. TAFIa is therefore capable of inhibiting plasminogen activation in the pericellular milieu. Since plasminogen activation has been linked to angiogenesis, TAFIa could therefore have anti-angiogenic properties, and indeed TAFIa has been shown to inhibit endothelial tube formation in a fibrin matrix. In this study, the TAFI pathway was manipulated by providing exogenous TAFI or TAFIa or by adding a potent and specific inhibitor of TAFIa. We found that TAFIa elicited a series of anti-angiogenic responses by endothelial cells, including decreased endothelial cell proliferation, cell invasion, cell migration, tube formation, and collagen degradation. Moreover, TAFIa decreased tube formation and proteolysis in endothelial cell culture grown alone and in co-culture with breast cancer cell lines. In accordance with these findings, inhibition of TAFIa increased secretion of matrix metalloprotease proenzymes by endothelial and breast cancer cells. Finally, treatment of endothelial cells with TAFIa significantly inhibited plasminogen activation. Taken together our results suggest a novel role for TAFI in inhibiting tumour angiogenic behaviors in breast cancer.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Abbreviations

BME:

Basement membrane extract

CM:

Conditioned media

ε-ACA:

ε-aminocaproic acid

ECM:

Extracellular matrix

HUVECs:

Human umbilical vein endothelial cells

MMPs:

Matrix metalloproteinases

PAS:

Plasminogen activation system

PTCI:

Potato tuber carboxypeptidase inhibitor

TAFI:

Thrombin-activatable fibrinolysis inhibitor

TAFIa:

Activated thrombin-activatable fibrinolysis inhibitor

TM:

Thrombomodulin

tPA:

Tissue-type plasminogen activator

uPA:

Urokinase plasminogen activator

uPAR:

Urokinase plasminogen activator receptor

VEGF:

Vascular endothelial growth factor

References

  1. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100(1):57–70

    Article  CAS  PubMed  Google Scholar 

  2. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144(5):646–674. doi:10.1016/j.cell.2011.02.013

    Article  CAS  PubMed  Google Scholar 

  3. Bergers G, Benjamin LE (2003) Tumorigenesis and the angiogenic switch. Nat Rev Cancer 3(6):401–410. doi:10.1038/nrc1093

    Article  CAS  PubMed  Google Scholar 

  4. Folkman J (1995) Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1(1):27–31

    Article  CAS  PubMed  Google Scholar 

  5. Folkman J (1971) Tumor angiogenesis: therapeutic implications. N Engl J Med 285(21):1182–1186. doi:10.1056/NEJM197111182852108

    Article  CAS  PubMed  Google Scholar 

  6. Gimbrone MA Jr, Leapman SB, Cotran RS, Folkman J (1972) Tumor dormancy in vivo by prevention of neovascularization. J Exp Med 136(2):261–276

    Article  PubMed  PubMed Central  Google Scholar 

  7. Talmadge JE, Fidler IJ (2010) AACR centennial series: the biology of cancer metastasis: historical perspective. Cancer Res 70(14):5649–5669. doi:10.1158/0008-5472.CAN-10-1040

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Zetter BR (1998) Angiogenesis and tumor metastasis. Annu Rev Med 49:407–424. doi:10.1146/annurev.med.49.1.407

    Article  CAS  PubMed  Google Scholar 

  9. Oh CW, Hoover-Plow J, Plow EF (2003) The role of plasminogen in angiogenesis in vivo. J Thromb Haemost 1(8):1683–1687

    Article  CAS  PubMed  Google Scholar 

  10. Andreasen PA, Egelund R, Petersen HH (2000) The plasminogen activation system in tumor growth, invasion, and metastasis. Cell Mol Life Sci 57(1):25–40. doi:10.1007/s000180050497

    Article  CAS  PubMed  Google Scholar 

  11. Law RH, Abu-Ssaydeh D, Whisstock JC (2013) New insights into the structure and function of the plasminogen/plasmin system. Curr Opin Struct Biol 23(6):836–841. doi:10.1016/j.sbi.2013.10.006

    Article  CAS  PubMed  Google Scholar 

  12. Ceruti P, Principe M, Capello M, Cappello P, Novelli F (2013) Three are better than one: plasminogen receptors as cancer theranostic targets. Exp Hematol Oncol 2(1):1–11. doi:10.1186/2162-3619-2-12

    Article  Google Scholar 

  13. Pepper MS (2001) Role of the matrix metalloproteinase and plasminogen activator-plasmin systems in angiogenesis. Arterioscler Thromb Vasc Biol 21(7):1104–1117

    Article  CAS  PubMed  Google Scholar 

  14. Rakic JM, Maillard C, Jost M, Bajou K, Masson V, Devy L, Lambert V, Foidart JM, Noel A (2003) Role of plasminogen activator-plasmin system in tumor angiogenesis. Cell Mol Life Sci 60(3):463–473

    Article  CAS  PubMed  Google Scholar 

  15. Boffa MB, Koschinsky ML (2007) Curiouser and curiouser: recent advances in measurement of thrombin-activatable fibrinolysis inhibitor (TAFI) and in understanding its molecular genetics, gene regulation, and biological roles. Clin Biochem 40(7):431–442. doi:10.1016/j.clinbiochem.2006.10.020

    Article  CAS  PubMed  Google Scholar 

  16. Redlitz A, Tan AK, Eaton DL, Plow EF (1995) Plasma carboxypeptidases as regulators of the plasminogen system. J Clin Invest 96(5):2534–2538. doi:10.1172/JCI118315

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Bazzi ZA, Lanoue D, El-Youssef M, Romagnuolo R, Tubman J, Cavallo-Medved D, Porter LA, Boffa MB (2016) Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates breast cancer cell metastatic behaviors through inhibition of plasminogen activation and extracellular proteolysis. BMC Cancer 16(1):328. doi:10.1186/s12885-016-2359-1

    Article  PubMed  PubMed Central  Google Scholar 

  18. Higuchi T, Nakamura T, Kakutani H, Ishi H (2009) Thrombomodulin suppresses invasiveness of HT1080 tumor cells by reducing plasminogen activation on the cell surface through activation of thrombin-activatable fibrinolysis inhibitor. Biol Pharm Bull 32(2):179–185

    Article  CAS  PubMed  Google Scholar 

  19. Reijerkerk A, Meijers JC, Havik SR, Bouma BN, Voest EE, Gebbink MF (2004) Tumor growth and metastasis are not affected in thrombin-activatable fibrinolysis inhibitor-deficient mice. J Thromb Haemost 2(5):769–779. doi:10.1111/j.1538-7836.2004.00682.x

    Article  CAS  PubMed  Google Scholar 

  20. Atkinson JM, Pullen N, Johnson TS (2013) An inhibitor of thrombin activated fibrinolysis inhibitor (TAFI) can reduce extracellular matrix accumulation in an in vitro model of glucose induced ECM expansion. Matrix Biol 32(5):277–287. doi:10.1016/j.matbio.2013.01.006

    Article  CAS  PubMed  Google Scholar 

  21. Guimaraes AHC, Laurens N, Weijers EM, Koolwijk P, van Hinsbergh VWM, Rijken DC (2007) TAFI and pancreatic carboxypeptidase B modulate in vitro capillary tube formation by human microvascular endothelial cells. Arterioscl Throm Vas 27 (10):2157–2162. doi:10.1161/Atvbaha.107.150144

    Article  CAS  Google Scholar 

  22. Chazaud B, Ricoux R, Christov C, Plonquet A, Gherardi RK, Barlovatz-Meimon G (2002) Promigratory effect of plasminogen activator inhibitor-1 on invasive breast cancer cell populations. Am J Pathol 160(1):237–246. doi:10.1016/S0002-9440(10)64367-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Grant DS, Lelkes PL, Fukuda K, Kleinman HK (1991) Intracellular mechanisms involved in basement-membrane induced blood-vessel differentiation invitro. In Vitro Cell Dev B 27 (4):327–336

    Article  Google Scholar 

  24. Kubota Y, Kleinman HK, Martin GR, Lawley TJ (1988) Role of laminin and basement-membrane in the morphological-differentiation of human-endothelial cells into capillary-like structures. J Cell Biol 107(4):1589–1598. DOI:10.1083/jcb.107.4.1589

    Article  CAS  PubMed  Google Scholar 

  25. Sameni M, Dosescu J, Moin K, Sloane BF (2003) Functional imaging of proteolysis: stromal and inflammatory cells increase tumor proteolysis. Mol Imaging 2(3):159–175

    Article  PubMed  Google Scholar 

  26. Sloane BF, Sameni M, Podgorski I, Cavallo-Medved D, Moin K (2006) Functional imaging of tumor proteolysis. Annu Rev Pharmacol Toxicol 46:301–315. doi:10.1146/annurev.pharmtox.45.120403.095853

    Article  CAS  PubMed  Google Scholar 

  27. Sameni M, Cavallo-Medved D, Dosescu J, Jedeszko C, Moin K, Mullins SR, Olive MB, Rudy D, Sloane BF (2009) Imaging and quantifying the dynamics of tumor-associated proteolysis. Clin Exp Metastasis 26(4):299–309. doi:10.1007/s10585-008-9218-7

    Article  CAS  PubMed  Google Scholar 

  28. Romagnuolo R, Marcovina SM, Boffa MB, Koschinsky ML (2014) Inhibition of plasminogen activation by apo(a): role of carboxyl-terminal lysines and identification of inhibitory domains in apo(a). J Lipid Res 55(4):625–634. doi:10.1194/jlr.M036566

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Deutsch DG, Mertz ET (1970) Plasminogen: purification from human plasma by affinity chromatography. Science 170(3962):1095–1096. doi:10.1126/science.170.3962.1095

    Article  CAS  PubMed  Google Scholar 

  30. Mohamed MM, Cavallo-Medved D, Rudy D, Anbalagan A, Moin K, Sloane BF (2010) Interleukin-6 increases expression and secretion of cathepsin B by breast tumor-associated monocytes. Cell Physiol Biochem 25(2–3):315–324. doi:10.1159/000276564

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Hanahan D, Folkman J (1996) Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86(3):353–364

    Article  CAS  PubMed  Google Scholar 

  32. Staton CA, Stribbling SM, Tazzyman S, Hughes R, Brown NJ, Lewis CE (2004) Current methods for assaying angiogenesis in vitro and in vivo. Int J Exp Pathol 85(5):233–248. doi:10.1111/j.0959-9673.2004.00396.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Kessenbrock K, Plaks V, Werb Z (2010) Matrix metalloproteinases: regulators of the tumor microenvironment. Cell 141(1):52–67. doi:10.1016/j.cell.2010.03.015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Ulisse S, Baldini E, Sorrenti S, D’Armiento M (2009) The urokinase plasminogen activator system: a target for anti-cancer therapy. Curr Cancer Drug Targets 9(1):32–71

    Article  CAS  PubMed  Google Scholar 

  35. Pepper MS, Vassalli JD, Montesano R, Orci L (1987) Urokinase-type plasminogen activator is induced in migrating capillary endothelial cells. J Cell Biol 105(6 Pt 1):2535–2541

    Article  CAS  PubMed  Google Scholar 

  36. Pepper MS, Montesano R, Orci L, Vassalli JD (1991) Plasminogen activator inhibitor-1 is induced in microvascular endothelial cells by a chondrocyte-derived transforming growth factor-beta. Biochem Biophys Res Commun 176(2):633–638

    Article  CAS  PubMed  Google Scholar 

  37. Cavallo-Medved D, Rudy D, Blum G, Bogyo M, Caglic D, Sloane BF (2009) Live-cell imaging demonstrates extracellular matrix degradation in association with active cathepsin B in caveolae of endothelial cells during tube formation. Exp Cell Res 315(7):1234–1246. doi:10.1016/j.yexcr.2009.01.021

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Redlitz A, Tan AK, Eaton DL, Plow EF (1995) Plasma carboxypeptidases as regulators of the plasminogen system. J Clin Invest 96(5):2534. doi:10.1172/jci118315

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Swaisgood CM, Schmitt D, Eaton D, Plow EF (2002) In vivo regulation of plasminogen function by plasma carboxypeptidase B. J Clin Invest 110(9):1275–1282. doi:10.1172/jci0215082

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Okumura N, Koh T, Hasebe Y, Seki T, Ariga T (2009) A novel function of thrombin-activatable fibrinolysis inhibitor during rat liver regeneration and in growth-promoted hepatocytes in primary culture. J Biol Chem 284(24):16553–16561. doi:10.1074/jbc.M109.011452

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. LeBleu VS, Macdonald B, Kalluri R (2007) Structure and function of basement membranes. Exp Biol Med 232(9):1121–1129. doi:10.3181/0703-MR-72

    Article  CAS  Google Scholar 

  42. Itoh T, Tanioka M, Yoshida H, Yoshioka T, Nishimoto H, Itohara S (1998) Reduced angiogenesis and tumor progression in gelatinase A-deficient mice. Cancer Res 58(5):1048–1051

    CAS  PubMed  Google Scholar 

  43. Egeblad M, Werb Z (2002) New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2(3):161–174. doi:10.1038/nrc745

    Article  CAS  PubMed  Google Scholar 

  44. Hollborn M, Stathopoulos C, Steffen A, Wiedemann P, Kohen L, Bringmann A (2007) Positive feedback regulation between MMP-9 and VEGF in human RPE cells. Invest Ophthalmol Vis Sci 48(9):4360–4367. doi:10.1167/iovs.06-1234

    Article  PubMed  Google Scholar 

  45. Toth M, Sohail A, Fridman R (2012) Assessment of gelatinases (MMP-2 and MMP-9) by gelatin zymography. Methods Mol Biol 878:121–135. doi:10.1007/978-1-61779-854-2_8

    Article  CAS  PubMed  Google Scholar 

  46. van Hinsbergh VW, Engelse MA, Quax PH (2006) Pericellular proteases in angiogenesis and vasculogenesis. Arterioscler Thromb Vasc Biol 26(4):716–728. doi:10.1161/01.ATV.0000209518.58252.17

    Article  PubMed  Google Scholar 

  47. Lamalice L, Le Boeuf F, Huot J (2007) Endothelial cell migration during angiogenesis. Circ Res 100(6):782–794. doi:10.1161/01.RES.0000259593.07661.1e

    Article  CAS  PubMed  Google Scholar 

  48. van Hinsbergh VW, Koolwijk P (2008) Endothelial sprouting and angiogenesis: matrix metalloproteinases in the lead. Cardiovasc Res 78(2):203–212. doi:10.1093/cvr/cvm102

    Article  PubMed  Google Scholar 

  49. Sameni M, Dosescu J, Moin K, Sloane BF (2003) Functional imaging of proteolysis: stromal and inflammatory cells increase tumor proteolysis. Mol Imaging. doi:10.1162/153535003322556903

    PubMed  Google Scholar 

  50. Sameni M, Moin K, Sloane BF (2000) Imaging proteolysis by living human breast cancer cells. Neoplasia. doi:10.1038/sj.neo.7900116

    PubMed  PubMed Central  Google Scholar 

  51. Sameni M, Dosescu J, Sloane BF (2001) Imaging proteolysis by living human glioma cells. Biol Chem 382(5):785–788. doi:10.1515/bchm.2001.382.5.785

    Article  CAS  PubMed  Google Scholar 

  52. Deryugina EI, Quigley JP (2012) Cell surface remodeling by plasmin: a new function for an old enzyme. J Biomed Biotechnol. doi:10.1155/2012/564259

    PubMed  PubMed Central  Google Scholar 

  53. Everts V, van der Zee E, Creemers L, Beertsen W (1996) Phagocytosis and intracellular digestion of collagen, its role in turnover and remodelling. Histochem J 28(4):229–245

    Article  CAS  PubMed  Google Scholar 

  54. Grove AD, Prabhu VV, Young BL, Lee FC, Kulpa V, Munson PJ, Kohn EC (2002) Both protein activation and gene expression are involved in early vascular tube formation in vitro. Clin Cancer Res 8(9):3019–3026

    CAS  PubMed  Google Scholar 

  55. Schnaper HW, Barnathan ES, Mazar A, Maheshwari S, Ellis S, Cortez SL, Baricos WH, Kleinman HK (1995) Plasminogen activators augment endothelial cell organization in vitro by two distinct pathways. J Cell Physiol 165(1):107–118. doi:10.1002/jcp.1041650114

    Article  CAS  PubMed  Google Scholar 

  56. Franses JW, Edelman ER (2011) The evolution of endothelial regulatory paradigms in cancer biology and vascular repair. Cancer Res 71(24):7339–7344. doi:10.1158/0008-5472.CAN-11-1718

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Wu C, Kim PY, Swystun LL, Liaw PC, Weitz JI (2016) Activation of protein C and thrombin activable fibrinolysis inhibitor on cultured human endothelial cells. J Thromb Haemost 14(2):366–374. doi:10.1111/jth.13222

    Article  CAS  PubMed  Google Scholar 

  58. Kim SJ, Shiba E, Ishii H, Inoue T, Taguchi T, Tanji Y, Kimoto Y, Izukura M, Takai S (1997) Thrombomodulin is a new biological and prognostic marker for breast cancer: an immunohistochemical study. Anticancer Res 17(3c):2319–2323

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was funded by a Seeds4Hope grant from the Windsor Essex Cancer Centre Foundation.

Author information

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Windsor, Windsor, ON, Canada

    Zainab A. Bazzi & Jennifer Balun

  2. Department of Biochemistry, Room 4245A Robarts Research Institute, University of Western Ontario, 1151 Richmond Street North, London, ON, N5B 3P7, Canada

    Zainab A. Bazzi & Michael B. Boffa

  3. Department of Biological Sciences, University of Windsor, Windsor, ON, Canada

    Dora Cavallo-Medved & Lisa A. Porter

Authors
  1. Zainab A. Bazzi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jennifer Balun
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dora Cavallo-Medved
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lisa A. Porter
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael B. Boffa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michael B. Boffa.

Additional information

Zainab A. Bazzi and Jennifer Balun have contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 324 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bazzi, Z.A., Balun, J., Cavallo-Medved, D. et al. Activated thrombin-activatable fibrinolysis inhibitor attenuates the angiogenic potential of endothelial cells: potential relevance to the breast tumour microenvironment. Clin Exp Metastasis 34, 155–169 (2017). https://doi.org/10.1007/s10585-017-9837-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10585-017-9837-y

Keywords

Search

Navigation