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In Vitro Screening of Synthetic Fluorogenic Substrates for Detection of Cancer Procoagulant Activity

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Abstract

Cancer procoagulant (CP), a direct activator of coagulation factor X, is among one of the tumour cell products or activities which may promote fibrin formation and has been suggested to be selectively associated with the malignant phenotype. At present, the most reliable assay for the quantification of CP activity is the three-stage chromogenic assay which utilises the ability of CP to activate factor X. In this assay, the activation of factor X leads to the formation of activated thrombin from prothrombin and the eventual hydrolyses of a thrombin chromogenic substrate which contains a p-nitroaniline leaving group. The complexity of the three-stage chromogenic assay suggests a need for a direct method of assaying CP activity. This study focuses on the design of a fluorogenic substrate that would enable the direct quantification of CP activity. The results of the study show two promising substrates for the determination of CP activity: Boc-PQVR-AMC and PQVR-AMC. Further analysis showed that Boc-PQVR-AMC could be excluded as a potential substrate for CP since it was also cleaved by collagenase.

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Abbreviations

AMC:

7-Amino-4-methylcoumarin

MCA:

7-Methoxycoumarin-4-acetyl

DMSO:

Dimethyl sulfoxide

ECM:

Extracellular matrix

FX:

Factor X

RVV:

Russell’s viper venom

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Acknowledgements

The authors would like to thank the anonymous reviewers as well as the editor for their valuable comments to improve the quality of the final version of the paper. They would also like to thank the nursing staff of the maternity unit of Greenacres Hospital (Port Elizabeth, South Africa) for the collection of tissue samples. This research was funded by the National Research Foundation of South Africa.

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Authors and Affiliations

  1. Department of Biochemistry and Microbiology, Nelson Mandela Metropolitan University, P.O. Box 77000, Port Elizabeth, 6031, South Africa

    Jason Krause & Carminita L. Frost

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  1. Jason Krause
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  2. Carminita L. Frost
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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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Krause, J., Frost, C.L. In Vitro Screening of Synthetic Fluorogenic Substrates for Detection of Cancer Procoagulant Activity. Protein J 37, 151–163 (2018). https://doi.org/10.1007/s10930-018-9758-x

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  • DOI: https://doi.org/10.1007/s10930-018-9758-x

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