The Proteolytic Profile of Human Cancer Procoagulant Suggests That It Promotes Cancer Metastasis at the Level of Activation Rather Than Degradation

Abstract

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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Abbreviations

ECM:

Extracellular matrix

CP:

Cancer procoagulant

FX:

Factor X

RVV:

Russell’s viper venom

MALDI-TOF MS:

Matrix-assisted laser desorption/ionisation time of flight mass spectrometry

AMC:

7-Amino-4-methylcoumarin

MCA:

7-Methoxycoumarin-4-acetyl

DMSO:

Dimethyl sulfoxide

PMSF:

Phenylmethylsulfonylfluoride

PAR:

Protease-activated receptor

EMT:

Epithelial-mesenchymal transition

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Acknowledgments

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.

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Correspondence to Carminita L. Frost.

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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

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Keywords

  • Factor X
  • Fibronectin
  • CP assay
  • Peptide substrates
  • Kinetics
  • Extracellular matrix