Proteases are essential for tumour progression and many are over-expressed during this time. The main focus of research was the role of these proteases in degradation of the basement membrane and extracellular matrix (ECM), thereby enabling metastasis to occur. Cancer procoagulant (CP), a protease present in malignant tumours, but not normal tissue, is a known activator of coagulation factor X (FX). The present study investigated the function of CP in cancer progression by focussing on its enzymatic specificity. FX cleavage was confirmed using SDS-PAGE and MALDI-TOF MS and compared to the proteolytic action of CP on ECM proteins, including collagen type IV, laminin and fibronectin. Contrary to previous reports, CP cleaved FX at the conventional activation site (between Arg-52 and Ile-53). Additionally, degradation of FX by CP occurred at a much slower rate than degradation by conventional activators. Complete degradation of the heavy chain of FX was only visible after 24 h, while degradation by RVV was complete after 30 min, supporting postulations that the procoagulant function of CP may be of secondary importance to its role in cancer progression. Of the ECM proteins tested, only fibronectin was cleaved. The substrate specificity of CP was further investigated by screening synthetic peptide substrates using a novel direct CP assay. The results indicate that CP is not essential for either cancer-associated blood coagulation or the degradation of ECM proteins. Rather, they suggest that this protease may be required for the proteolytic activation of membrane receptors.
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Russell’s viper venom
- MALDI-TOF MS:
Matrix-assisted laser desorption/ionisation time of flight mass spectrometry
Chin D, Boyle GM, Kane AJ, Theile DR, Hayward NK et al (2005) Invasion and metastasis markers in cancer. Br J Plast Surg 58:466–474
David L, Nesland JM, Holm R, Sobrinho-Simões M (1994) Expression of laminin, collagen IV, fibronectin, and type IV collagenase in gastric carcinoma: an immunohistochemical study of 87 patients. Cancer 73:518–527
Armstrong PB, Armstrong MT (2000) Intercellular invasion and the organizational stability of tissues: a role for fibronectin. Biochim Biophys Acta 1470:O9–O20
Gordon SG, Mourad AM (1991) The site of activation of factor X by cancer procoagulant. Blood Coagul Fibrinolysis 2:735–739
Gordon SG (1994) Cancer procoagulant. Methods Enzymol 244:568–583
Gordon SG, Mielicki WP (1997) Cancer procoagulant: a factor X activator, tumor marker and growth factor from malignant tissue. Blood Coagul Fibrinolysis 8:73–86
Kee NLA, Naudé RJ, Blatch GL, Frost CL (2012) The effect of cancer procoagulant on expression of metastatic and angiogenic markers in breast cancer and embryonic stem cell lines. Biol Chem 393:113–121
Buck MR, Karustis DG, Day NA, Honn KV, Sloane BF (1992) Degradation of extracellular-matrix proteins by human cathepsin B from normal and tumour tissues. Biochem J 282:273–278
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Kabuto S, Ogawa T, Muramoto K, Oosthuizen V, Naude RJ (2000) The amino acid sequence of pancreatic α-amylase from the ostrich, Struthio camelus. Comp Biochem Physiol Part B 127:481–490
Frerker N, Wagner L, Wolf R, Heiser U, Hoffmann T et al (2007) Neuropeptide Y (NPY) cleaving enzymes: structural and functional homologues of dipeptidyl peptidase 4. Peptides 28:257–268
Siigur E, Tõnismägi K, Trummal K, Samel M, Vija H et al (2001) Factor X activator from Vipera lebetina snake venom, molecular characterization and substrate specificity. Biochim Biophys Acta 1568:90–98
Dixon M, Webb EC (1979) Enzymes, 3rd edn. Longman, London, pp 344–353
Mielicki WP, Gordon SG (1993) Three-stage chromogenic assay for the analysis of activation properties of factor X by cancer procoagulant. Blood Coagul Fibrinolysis 4:441–446
Tans G, Rosing J (2001) Snake venom activators of factor X: an overview. Haemostasis 31:225–233
Kaplinska K, Mielicki WP (2009) Direct analysis reveals an absence of γ-carboxyglutamic acid in cancer procoagulant from human tissues. Blood Coagul Fibrinolysis 20:315–320
Ramani VC, Haun RS (2008) The extracellular matrix protein fibronectin is a substrate for kallikrein 7. Biochem Biophys Res Commun 369:1169–1173
Pankov R, Yamada KM (2002) Fibronectin at a glance. J Cell Sci 115:3861–3863
Franco E, Vázquez-Prado J, Meza I (1997) Fibronectin-derived fragments as inducers of adhesion and chemotaxis of Entamoeba histolytica trophozoites. J Infect Dis 176:1597–1602
Gebbink MFBG, Bouma B, Maas C, Bouma BN (2009) Physiological responses to protein aggregates: fibrinolysis, coagulation and inflammation (new roles for old factors). FEBS Lett 583:2691–2699
Tsuruo T, Fujita N (2008) Platelet aggregation in the formation of tumor metastasis. Proc Jpn Acad Ser B 84:189–198
Olas B, Mielicki WP, Wachowicz B, Krajewski T (1999) Cancer procoagulant stimulates platelet adhesion. Thromb Res 94:199–203
Olas B, Wachowicz B, Mielicki WP, Buczyński A (2000) Free radicals are involved in cancer procoagulant-induced platelet activation. Thromb Res 97:169–175
Olas B, Wachowicz B, Mielicki WP (2001) Role of phosphoinositide 3-kinase in adhesion of platelets to fibrinogen stimulated by cancer procoagulant. Platelets 12:431–435
Olas B, Wachowicz B, Mielicki WP (2001) Cancer procoagulant and blood platelet activation. Cancer Lett 169:165–171
Kim S, Foster C, Lecchi A, Quinton TM, Prosser DM et al (2002) Protease-activated receptors 1 and 4 do not stimulate Gi signaling pathways in the absence of secreted ADP and cause human platelet aggregation independently of Gi signalling. Blood 99:3629–3636
Coughlin SR (2000) Thrombin signalling and protease-activated receptors. Nature 407:258–264
Arora P, Ricks TK, Trejo J (2007) Protease-activated receptor signalling, endocytic sorting and dysregulation in cancer. J Cell Sci 120:921–928
Hansen KK, Oikonomopoulou K, Li Y, Hollenberg MD (2008) Proteinases, proteinase-activated receptors (PARs) and the pathophysiology of cancer and diseases of the cardiovascular, musculoskeletal, nervous and gastrointestinal systems. Naunyn-Schmiedeberg’s Arch Pharmacol 377:377–392
Naldini A, Filippi I, Ardinghi C, Silini A, Giavazzi R et al (2009) Identification of a functional role for the protease-activated receptor-1 in hypoxic breast cancer cells. Eur J Cancer 45:454–460
Yin Y-J, Salah Z, Maoz M, Cohen Even-Ram S, Ochayon S et al (2003) Oncogenic transformation induces tumor angiogenesis: a role for PAR1 activation. FASEB J 17:163–174
Camerer E, Qazi AA, Duong DN, Cornelissen I, Advincula R et al (2004) Platelets, protease-activated receptors, and fibrinogen in hematogenous metastasis. Blood 15:397–401
Gratio V, Walker F, Lehy T, Laburthe M, Darmoul D (2009) Aberrant expression of proteinase-activated receptor 4 promotes colon cancer cell proliferation through a persistent signaling that involves Src and ErbB-2 kinase. Int J Cancer 124:1517–1525
Ando S, Otani H, Yagi Y, Kawai K, Araki H et al (2007) Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells. Respir Res 8:31. doi:10.1186/1465-9921-8-31
Yin Y-J, Salah Z, Grisaru-Granovsky S, Cohen I, Cohen E-RS et al (2003) Human protease-activated receptor 1 expression in malignant epithelia. Arterioscler Thromb Vasc Biol 23:940–944
Griffin CT, Srinivasan Y, Zheng Y-W, Huang W, Coughlin SR (2001) A role for thrombin receptor signaling in endothelial cells during embryonic development. Science 293:1666–1670
Mackman N (2004) Role of tissue factor in hemostasis, thrombosis, and vascular development. Arterioscler Thromb Vasc Biol 24:1015–1022
Mielicki WP, Mielicka E, Gordon SG (1997) Cancer procoagulant activity studies using synthetic peptidyl substrates. Thromb Res 87:251–256
Jacques SL, Kuliopulos A (2003) Protease-activated receptor-4 uses dual prolines and an anionic retention motif for thrombin recognition and cleavage. Biochem J 376:733–740
The authors wish to thank: Prof. W. Mielicki (Medical University of Łódź, Poland) for help with the purification of CP; nursing staff of the maternity unit of Greenacres Hospital (Port Elizabeth, South Africa) for the collection of tissue samples; Prof. K. Sakka and Dr M. Sakka (Mie University, Tsu, Japan) for N-terminal sequence analysis of FX fragments; and Prof. T. Coetzer (University of KwaZulu-Natal, Pietermaritzburg, South Africa) for MCA standard. This research was funded by the South African Medical Research Council and the National Research Foundation Research Niche Area programme of South Africa. The authors would like to thank the reviewers for their valuable input.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
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Kee, N.L.A., Krause, J., Blatch, G.L. et al. The Proteolytic Profile of Human Cancer Procoagulant Suggests That It Promotes Cancer Metastasis at the Level of Activation Rather Than Degradation. Protein J 34, 338–348 (2015). https://doi.org/10.1007/s10930-015-9628-8
- Factor X
- CP assay
- Peptide substrates
- Extracellular matrix