2. Academic Validation
  3. Quantitation of fibroblast activation protein (FAP)-specific protease activity in mouse, baboon and human fluids and organs

Quantitation of fibroblast activation protein (FAP)-specific protease activity in mouse, baboon and human fluids and organs

  • FEBS Open Bio. 2013 Dec 8;4:43-54. doi: 10.1016/j.fob.2013.12.001.
Fiona M Keane 1,  Tsun-Wen Yao 1,  Stefanie Seelk 2,  Margaret G Gall 1,  Sumaiya Chowdhury 1,  Sarah E Poplawski 3,  Jack H Lai 3,  Youhua Li 3,  Wengen Wu 3,  Penny Farrell 4,  Ana Julia Vieira de Ribeiro 1,  Brenna Osborne 1,  Denise M T Yu 1,  Devanshi Seth 5,  Khairunnessa Rahman 6,  Paul Haber 7,  A Kemal Topaloglu 8,  Chuanmin Wang 9,  Sally Thomson 10,  Annemarie Hennessy 11,  John Prins 12,  Stephen M Twigg 13,  Susan V McLennan 13,  Geoffrey W McCaughan 1,  William W Bachovchin 3,  Mark D Gorrell 1
Affiliations

Affiliations

  • 1 Centenary Institute, Camperdown, NSW, Australia ; Sydney Medical School, University of Sydney, NSW, Australia.
  • 2 Centenary Institute, Camperdown, NSW, Australia.
  • 3 Sackler School of Biomedical Sciences, Tufts University School of Medicine, Boston, MA, USA.
  • 4 Department of Renal Medicine, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
  • 5 Centenary Institute, Camperdown, NSW, Australia ; Drug Health Services, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
  • 6 Drug Health Services, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
  • 7 Sydney Medical School, University of Sydney, NSW, Australia ; Drug Health Services, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
  • 8 Pediatric Endocrinology, Faculty of Medicine, Cukurova University, Adana, Turkey.
  • 9 Sydney Medical School, University of Sydney, NSW, Australia ; Collaborative Transplantation Research Group, Bosch Institute, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
  • 10 Sydney Medical School, University of Sydney, NSW, Australia ; Department of Renal Medicine, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
  • 11 Department of Renal Medicine, Royal Prince Alfred Hospital, Camperdown, NSW, Australia ; School of Medicine, University of Western Sydney, NSW, Australia.
  • 12 Mater Medical Research Institute, University of Queensland, and Department of Endocrinology, Princess Alexandra Hospital, Brisbane, QLD, Australia.
  • 13 Sydney Medical School, University of Sydney, NSW, Australia ; Department of Endocrinology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
PMID: 24371721 DOI: 10.1016/j.fob.2013.12.001
Abstract

The protease fibroblast activation protein (FAP) is a specific marker of activated mesenchymal cells in tumour stroma and fibrotic liver. A specific, reliable FAP Enzyme assay has been lacking. FAP's unique and restricted cleavage of the post proline bond was exploited to generate a new specific substrate to quantify FAP Enzyme activity. This sensitive assay detected no FAP activity in any tissue or fluid of FAP gene knockout mice, thus confirming assay specificity. Circulating FAP activity was ∼20- and 1.3-fold less in baboon than in mouse and human plasma, respectively. Serum and plasma contained comparable FAP activity. In mice, the highest levels of FAP activity were in uterus, pancreas, submaxillary gland and skin, whereas the lowest levels were in brain, prostate, leukocytes and testis. Baboon organs high in FAP activity included skin, epididymis, bladder, colon, adipose tissue, nerve and tongue. FAP activity was greatly elevated in tumours and associated lymph nodes and in fungal-infected skin of unhealthy baboons. FAP activity was 14- to 18-fold greater in cirrhotic than in non-diseased human liver, and circulating FAP activity was almost doubled in alcoholic cirrhosis. Parallel DPP4 measurements concorded with the literature, except for the novel finding of high DPP4 activity in bile. The new FAP Enzyme assay is the first to be thoroughly characterised and shows that FAP activity is measurable in most organs and at high levels in some. This new assay is a robust tool for specific quantitation of FAP Enzyme activity in both preclinical and clinical samples, particularly liver fibrosis.

Keywords

ALD, alcoholic liver disease; AMC, amino-4-methylcoumarin; Biomarker; DMSO, dimethyl sulfoxide; DPP4, dipeptidyl peptidase 4; Dipeptidyl peptidase; EDTA, ethylene diamine tetra acetic acid; FAP, fibroblast activation protein-α; Fibroblast; Fibrosis; HCV, hepatitis C virus; LDS, lithium dodecyl sulphate; LN, lymph node; Liver disease; ND, non-diseased; PBC, primary biliary cirrhosis; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PEP, prolyl endopeptidase; PVDF, polyvinylidene fluoride; Protease activity; Protease substrates; STLV, simian T-cell lymphotrophic virus; gko, gene knock out; het, heterozygous; mAb, monoclonal antibody; wt, wild type; yrs, years.

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