title: Quantitation of fibroblast activation protein (FAP)-specific protease activity in mouse, baboon and human fluids and organs.
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.
authors.affiliation: Centenary Institute, Camperdown, NSW, Australia ; Sydney Medical School, University of Sydney, NSW, Australia.
authors.forename: Fiona M
authors.initials: FM
authors.lastname: Keane
authors.affiliation: Centenary Institute, Camperdown, NSW, Australia ; Sydney Medical School, University of Sydney, NSW, Australia.
authors.forename: Tsun-Wen
authors.initials: TW
authors.lastname: Yao
authors.affiliation: Centenary Institute, Camperdown, NSW, Australia.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Seelk
authors.affiliation: Centenary Institute, Camperdown, NSW, Australia ; Sydney Medical School, University of Sydney, NSW, Australia.
authors.forename: Margaret G
authors.initials: MG
authors.lastname: Gall
authors.affiliation: Centenary Institute, Camperdown, NSW, Australia ; Sydney Medical School, University of Sydney, NSW, Australia.
authors.forename: Sumaiya
authors.initials: S
authors.lastname: Chowdhury
authors.affiliation: Sackler School of Biomedical Sciences, Tufts University School of Medicine, Boston, MA, USA.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Poplawski
authors.affiliation: Sackler School of Biomedical Sciences, Tufts University School of Medicine, Boston, MA, USA.
authors.forename: Jack H
authors.initials: JH
authors.lastname: Lai
authors.affiliation: Sackler School of Biomedical Sciences, Tufts University School of Medicine, Boston, MA, USA.
authors.forename: Youhua
authors.initials: Y
authors.lastname: Li
authors.affiliation: Sackler School of Biomedical Sciences, Tufts University School of Medicine, Boston, MA, USA.
authors.forename: Wengen
authors.initials: W
authors.lastname: Wu
authors.affiliation: Department of Renal Medicine, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
authors.forename: Penny
authors.initials: P
authors.lastname: Farrell
authors.affiliation: Centenary Institute, Camperdown, NSW, Australia ; Sydney Medical School, University of Sydney, NSW, Australia.
authors.forename: Ana Julia
authors.initials: AJ
authors.lastname: Vieira de Ribeiro
authors.affiliation: Centenary Institute, Camperdown, NSW, Australia ; Sydney Medical School, University of Sydney, NSW, Australia.
authors.forename: Brenna
authors.initials: B
authors.lastname: Osborne
authors.affiliation: Centenary Institute, Camperdown, NSW, Australia ; Sydney Medical School, University of Sydney, NSW, Australia.
authors.forename: Denise M T
authors.initials: DM
authors.lastname: Yu
authors.affiliation: Centenary Institute, Camperdown, NSW, Australia ; Drug Health Services, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
authors.forename: Devanshi
authors.initials: D
authors.lastname: Seth
authors.affiliation: Drug Health Services, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
authors.forename: Khairunnessa
authors.initials: K
authors.lastname: Rahman
authors.affiliation: Sydney Medical School, University of Sydney, NSW, Australia ; Drug Health Services, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
authors.forename: Paul
authors.initials: P
authors.lastname: Haber
authors.affiliation: Pediatric Endocrinology, Faculty of Medicine, Cukurova University, Adana, Turkey.
authors.forename: A Kemal
authors.initials: AK
authors.lastname: Topaloglu
authors.affiliation: Sydney Medical School, University of Sydney, NSW, Australia ; Collaborative Transplantation Research Group, Bosch Institute, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
authors.forename: Chuanmin
authors.initials: C
authors.lastname: Wang
authors.affiliation: Sydney Medical School, University of Sydney, NSW, Australia ; Department of Renal Medicine, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
authors.forename: Sally
authors.initials: S
authors.lastname: Thomson
authors.affiliation: Department of Renal Medicine, Royal Prince Alfred Hospital, Camperdown, NSW, Australia ; School of Medicine, University of Western Sydney, NSW, Australia.
authors.forename: Annemarie
authors.initials: A
authors.lastname: Hennessy
authors.affiliation: Mater Medical Research Institute, University of Queensland, and Department of Endocrinology, Princess Alexandra Hospital, Brisbane, QLD, Australia.
authors.forename: John
authors.initials: J
authors.lastname: Prins
authors.affiliation: Sydney Medical School, University of Sydney, NSW, Australia ; Department of Endocrinology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Twigg
authors.affiliation: Sydney Medical School, University of Sydney, NSW, Australia ; Department of Endocrinology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
authors.forename: Susan V
authors.initials: SV
authors.lastname: McLennan
authors.affiliation: Centenary Institute, Camperdown, NSW, Australia ; Sydney Medical School, University of Sydney, NSW, Australia.
authors.forename: Geoffrey W
authors.initials: GW
authors.lastname: McCaughan
authors.affiliation: Sackler School of Biomedical Sciences, Tufts University School of Medicine, Boston, MA, USA.
authors.forename: William W
authors.initials: WW
authors.lastname: Bachovchin
authors.affiliation: Centenary Institute, Camperdown, NSW, Australia ; Sydney Medical School, University of Sydney, NSW, Australia.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Gorrell
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