Skip to main content

Advertisement

SpringerLink
Log in

Localization and Expression of Prothrombin in Rodent Osteoclasts and Long Bones

  • Original Research
  • Published:
Calcified Tissue International Aims and scope Submit manuscript

Abstract

The serum protein prothrombin (PT) is proteolytically converted to thrombin during the coagulation cascade by the cell-associated prothrombinase complex. In vitro, RANKL-differentiated osteoclasts express tissue factor and coagulation factor Xa, which convert PT to thrombin (Karlstrom et al. Biochem Biophys Res Commun 394:593–599, 2010). The present study investigated the localization of PT in bone as well as the expression of PT mRNA in bone and osteoclasts. Herein, immunoblot analysis detected PT and smaller proteolytically cleaved fragments with sizes consistent with the action of prothrombinase in a protein fraction extracted with guanidine-HCl EDTA from mouse tibia. Light microscopic and ultrastructural immunohistochemistry demonstrated the presence of PT in the newly formed bone matrix of the metaphysis. Furthermore, fluorescent immunohistochemistry on metaphyseal trabecular bone showed that PT colocalized with MMP-9-expressing subepiphyseal osteoclasts, whereas cathepsin K-expressing osteoclasts were closely associated with PT of the bone matrix. RT-qPCR analysis revealed that PT mRNA was detected in tibia. Expression of PT mRNA in the tibia was 0.2% of the level in the liver. In addition, PT mRNA expression was increased by RANKL-induced differentiation of bone marrow macrophages to osteoclasts. The results demonstrate that PT is synthesized and proteolytically processed in bone. Furthermore, PT is present mainly in the newly formed bone matrix of the metaphyseal trabecular bone compartment in close association to osteoclasts. In addition, MMP-9-positive osteoclasts contain PT, and PT expression is increased during osteoclastogenesis.

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

Similar content being viewed by others

References

  1. Mann KG (1976) Prothrombin. Methods Enzymol 45:123–156

    Article  PubMed  CAS  Google Scholar 

  2. Barnhart MI (1960) Cellular site for prothrombin synthesis. Am J Physiol 199:360–366

    PubMed  CAS  Google Scholar 

  3. Jamison CS, Degen SJ (1991) Prenatal and postnatal expression of mRNA coding for rat prothrombin. Biochim Biophys Acta 1088:208–216

    PubMed  CAS  Google Scholar 

  4. Norris LA (2003) Blood coagulation. Best Pract Res Clin Obstet Gynaecol 17:369–383

    Article  PubMed  Google Scholar 

  5. Lecrone V, Li W, Devoll RE, Logothetis C, Farach-Carson MC (2000) Calcium signals in prostate cancer cells: specific activation by bone-matrix proteins. Cell Calcium 27:35–42

    Article  PubMed  CAS  Google Scholar 

  6. Zoubine MN, Ma JY, Smirnova IV, Citron BA, Festoff BW (1996) A molecular mechanism for synapse elimination: novel inhibition of locally generated thrombin delays synapse loss in neonatal mouse muscle. Dev Biol 179:447–457

    Article  PubMed  CAS  Google Scholar 

  7. Glazner GW, Yadav K, Fitzgerald S, Coven E, Brenneman DE, Nelson PG (1997) Cholinergic stimulation increases thrombin activity and gene expression in cultured mouse muscle. Brain Res Dev Brain Res 99:148–154

    Article  PubMed  CAS  Google Scholar 

  8. Flynn PD, Byrne CD, Baglin TP, Weissberg PL, Bennett MR (1997) Thrombin generation by apoptotic vascular smooth muscle cells. Blood 89:4378–4384

    PubMed  CAS  Google Scholar 

  9. Festoff BW, Smirnova IV, Ma J, Citron BA (1996) Thrombin, its receptor and protease nexin I, its potent serpin, in the nervous system. Semin Thromb Hemost 22:267–271

    Article  PubMed  CAS  Google Scholar 

  10. Suzuki K, Tanaka T, Miyazawa K, Nakajima C, Moriyama M, Suga K, Murai M, Yano J (1999) Gene expression of prothrombin in human and rat kidneys: basic and clinical approach. J Am Soc Nephrol 10(Suppl 14):S408–S411

    PubMed  CAS  Google Scholar 

  11. Trudel G, Uhthoff HK, Laneuville O (2005) Prothrombin gene expression in articular cartilage with a putative role in cartilage degeneration secondary to joint immobility. J Rheumatol 32:1547–1555

    PubMed  CAS  Google Scholar 

  12. Osterud B, Lindahl U, Seljelid R (1980) Macrophages produce blood coagulation factors. FEBS Lett 120:41–43

    Article  PubMed  CAS  Google Scholar 

  13. Shim K, Zhu H, Westfield LA, Sadler JE (2004) A recombinant murine meizothrombin precursor, prothrombin R157A/R268A, inhibits thrombosis in a model of acute carotid artery injury. Blood 104:415–419

    Article  PubMed  CAS  Google Scholar 

  14. Butenas S, Mann KG (2002) Blood coagulation. Biochemistry (Mosc) 67:3–12

    Article  CAS  Google Scholar 

  15. Ahmad SS, London FS, Walsh PN (2003) The assembly of the factor X-activating complex on activated human platelets. J Thromb Haemost 1:48–59

    Article  PubMed  CAS  Google Scholar 

  16. Krishnaswamy S, Mann KG, Nesheim ME (1986) The prothrombinase-catalyzed activation of prothrombin proceeds through the intermediate meizothrombin in an ordered, sequential reaction. J Biol Chem 261:8977–8984

    PubMed  CAS  Google Scholar 

  17. Mann KG, Nesheim ME, Church WR, Haley P, Krishnaswamy S (1990) Surface-dependent reactions of the vitamin K-dependent enzyme complexes. Blood 76:1–16

    PubMed  CAS  Google Scholar 

  18. Pagel CN, Sivagurunathan S, Loh LH, Tudor EM, Pike RN, Mackie EJ (2006) Functional responses of bone cells to thrombin. Biol Chem 387:1037–1041

    Article  PubMed  CAS  Google Scholar 

  19. Gabazza EC, Taguchi O, Kamada H, Hayashi T, Adachi Y, Suzuki K (2004) Progress in the understanding of protease-activated receptors. Int J Hematol 79:117–122

    Article  PubMed  CAS  Google Scholar 

  20. Pagel CN, de Niese MR, Abraham LA, Chinni C, Song SJ, Pike RN, Mackie EJ (2003) Inhibition of osteoblast apoptosis by thrombin. Bone 33:733–743

    Article  PubMed  CAS  Google Scholar 

  21. Jenkins AL, Bootman MD, Taylor CW, Mackie EJ, Stone SR (1993) Characterization of the receptor responsible for thrombin-induced intracellular calcium responses in osteoblast-like cells. J Biol Chem 268:21432–21437

    PubMed  CAS  Google Scholar 

  22. Abraham LA, Jenkins AL, Stone SR, Mackie EJ (1998) Expression of the thrombin receptor in developing bone and associated tissues. J Bone Miner Res 13:818–827

    Article  PubMed  CAS  Google Scholar 

  23. Frost A, Jonsson KB, Ridefelt P, Nilsson O, Ljunghall S, Ljunggren O (1999) Thrombin, but not bradykinin, stimulates proliferation in isolated human osteoblasts, via a mechanism not dependent on endogenous prostaglandin formation. Acta Orthop Scand 70:497–503

    Article  PubMed  CAS  Google Scholar 

  24. Song SJ, Pagel CN, Campbell TM, Pike RN, Mackie EJ (2005) The role of protease-activated receptor-1 in bone healing. Am J Pathol 166:857–868

    Article  PubMed  CAS  Google Scholar 

  25. Ljunggren O, Johansson H, Ljunghall S, Lerner UH (1991) Thrombin increases cytoplasmic Ca2+ and stimulates formation of prostaglandin E2 in the osteoblastic cell line MC3T3-El. Bone Miner 12:81–90

    Article  PubMed  CAS  Google Scholar 

  26. Kozawa O, Tokuda H, Kaida T, Matsuno H, Uematsu T (1997) Thrombin regulates interleukin-6 synthesis through phosphatidylcholine hydrolysis by phospholipase D in osteoblasts. Arch Biochem Biophys 345:10–15

    Article  PubMed  CAS  Google Scholar 

  27. Shinohara M, Takayanagi H (2007) Novel osteoclast signaling mechanisms. Curr Osteoporos Rep 5:67–72

    Article  PubMed  Google Scholar 

  28. Hu Y, Ek-Rylander B, Karlstrom E, Wendel M, Andersson G (2008) Osteoclast size heterogeneity in rat long bones is associated with differences in adhesive ligand specificity. Exp Cell Res 314:638–650

    Article  PubMed  CAS  Google Scholar 

  29. Karlstrom E, Ek-Rylander B, Wendel M, Andersson G (2010) RANKL induces components of the extrinsic coagulation pathway in osteoclasts. Biochem Biophys Res Commun 394:593–599

    Article  PubMed  Google Scholar 

  30. Hultenby K, Reinholt FP, Oldberg A, Heinegard D (1991) Ultrastructural immunolocalization of osteopontin in metaphyseal and cortical bone. Matrix 11:206–213

    PubMed  CAS  Google Scholar 

  31. Van Noorden S (1986) Immunocytochemistry, modern methods and applications. Wright, Bristol

    Google Scholar 

  32. Zenger S, Hollberg K, Ljusberg J, Norgard M, Ek-Rylander B, Kiviranta R, Andersson G (2007) Proteolytic processing and polarized secretion of tartrate-resistant acid phosphatase is altered in a subpopulation of metaphyseal osteoclasts in cathepsin K-deficient mice. Bone 41:820–832

    Article  PubMed  CAS  Google Scholar 

  33. Takeshita S, Kaji K, Kudo A (2000) Identification and characterization of the new osteoclast progenitor with macrophage phenotypes being able to differentiate into mature osteoclasts. J Bone Miner Res 15:1477–1488

    Article  PubMed  CAS  Google Scholar 

  34. Fischer BE, Schlokat U, Mitterer A, Grillberger L, Reiter M, Mundt W, Dorner F, Eibl J (1996) Differentiation between proteolytic activation and autocatalytic conversion of human prothrombin. Activation of recombinant human prothrombin and recombinant D419N-prothrombin by snake venoms from Echis carinatus and Oxyuranus scutellatus. Protein Eng 9:921–926

    Article  PubMed  CAS  Google Scholar 

  35. Jemtland R, Lee K, Segre GV (1998) Heterogeneity among cells that express osteoclast-associated genes in developing bone. Endocrinology 139:340–349

    Article  PubMed  CAS  Google Scholar 

  36. Vu TH, Shipley JM, Bergers G, Berger JE, Helms JA, Hanahan D, Shapiro SD, Senior RM, Werb Z (1998) MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93:411–422

    Article  PubMed  CAS  Google Scholar 

  37. Saftig P, Hunziker E, Wehmeyer O, Jones S, Boyde A, Rommerskirch W, Moritz JD, Schu P, von Figura K (1998) Impaired osteoclastic bone resorption leads to osteopetrosis in cathepsin-K-deficient mice. Proc Natl Acad Sci USA 95:13453–13458

    Article  PubMed  CAS  Google Scholar 

  38. Tezuka K, Tezuka Y, Maejima A, Sato T, Nemoto K, Kamioka H, Hakeda Y, Kumegawa M (1994) Molecular cloning of a possible cysteine proteinase predominantly expressed in osteoclasts. J Biol Chem 269:1106–1109

    PubMed  CAS  Google Scholar 

  39. Everts V, de Vries TJ, Helfrich MH (2009) Osteoclast heterogeneity: lessons from osteopetrosis and inflammatory conditions. Biochim Biophys Acta 1792:757–765

    PubMed  CAS  Google Scholar 

  40. Nordahl J, Andersson G, Reinholt FP (1998) Chondroclasts and osteoclasts in bones of young rats: comparison of ultrastructural and functional features. Calcif Tissue Int 63:401–408

    Article  PubMed  CAS  Google Scholar 

  41. Robey PG (2002) Bone matrix proteoglycans and glycoproteins. In: Bilezikian JP, Raisz LA, Rodan GA (eds) Principles of bone biology. Academic Press, San Diego, CA, pp 225–237

    Chapter  Google Scholar 

  42. Lian SG, Canalis E, Robey PG, Boskey AL (1999) Bone formation: osteoblast lineage cells, growth factors, matrix proteins and the mineralization process. Lippincott, Williams & Williams, Philadelphia

    Google Scholar 

  43. van den Bos T, Speijer D, Bank RA, Bromme D, Everts V (2008) Differences in matrix composition between calvaria and long bone in mice suggest differences in biomechanical properties and resorption: special emphasis on collagen. Bone 43:459–468

    Article  PubMed  Google Scholar 

  44. Senger DR, Perruzzi CA, Papadopoulos-Sergiou A, Van de Water L (1994) Adhesive properties of osteopontin: regulation by a naturally occurring thrombin-cleavage in close proximity to the GRGDS cell-binding domain. Mol Biol Cell 5:565–574

    PubMed  CAS  Google Scholar 

  45. Hasegawa M, Nakoshi Y, Iino T, Sudo A, Segawa T, Maeda M, Yoshida T, Uchida A (2009) Thrombin-cleaved osteopontin in synovial fluid of subjects with rheumatoid arthritis. J Rheumatol 36:240–245

    Article  PubMed  CAS  Google Scholar 

  46. Yokasaki Y, Sheppard D (2000) Mapping of the cryptic integrin-binding site in osteopontin suggests a new mechanism by which thrombin can regulate inflammation and tissue repair. Trends Cardiovasc Med 10:155–159

    Article  PubMed  CAS  Google Scholar 

  47. Yokosaki Y, Matsuura N, Sasaki T, Murakami I, Schneider H, Higashiyama S, Saitoh Y, Yamakido M, Taooka Y, Sheppard D (1999) The integrin alpha9beta1 binds to a novel recognition sequence (SVVYGLR) in the thrombin-cleaved amino-terminal fragment of osteopontin. J Biol Chem 274:36328–36334

    Article  PubMed  CAS  Google Scholar 

  48. Rao H, Lu G, Kajiya H, Garcia-Palacios V, Kurihara N, Anderson J, Patrene K, Sheppard D, Blair HC, Windle JJ, Choi SJ, Roodman GD (2006) Alpha9beta1: a novel osteoclast integrin that regulates osteoclast formation and function. J Bone Miner Res 21:1657–1665

    Article  PubMed  CAS  Google Scholar 

  49. Yamamoto N, Sakai F, Kon S, Morimoto J, Kimura C, Yamazaki H, Okazaki I, Seki N, Fujii T, Uede T (2003) Essential role of the cryptic epitope SLAYGLR within osteopontin in a murine model of rheumatoid arthritis. J Clin Invest 112:181–188

    PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the Swedish Research Council and the Stockholm County Council.

Author information

Authors and Affiliations

  1. Division of Oral Biology, Department of Dental Medicine, Karolinska Institutet, P.O. Box 4064, 141 04, Stockholm, Sweden

    Erik Karlström, Eszter Somogyi-Ganss, Rachael Sugars & Mikael Wendel

  2. Division of Pathology, Department of Laboratory Medicine, Karolinska Institutet, 141 86, Stockholm, Sweden

    Erik Karlström, Maria Norgård & Göran Andersson

  3. Unit for Electron Microscopy, Division of Clinical Research Centre, Karolinska Institutet, 141 86, Stockholm, Sweden

    Kjell Hultenby

Authors
  1. Erik Karlström
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maria Norgård
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kjell Hultenby
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Eszter Somogyi-Ganss
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Rachael Sugars
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Göran Andersson
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mikael Wendel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Göran Andersson.

Additional information

The authors have stated that they have no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Karlström, E., Norgård, M., Hultenby, K. et al. Localization and Expression of Prothrombin in Rodent Osteoclasts and Long Bones. Calcif Tissue Int 88, 179–188 (2011). https://doi.org/10.1007/s00223-010-9443-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00223-010-9443-3

Keywords

Search

Navigation