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.

Fibroblast Activation Protein Cleaves and Inactivates Fibroblast Growth Factor 21

“…At the same time, elevated hepatic FAP expression has been observed in activated stellate cells in livers with cirrhosis (33,47). Plasma FAP is also increased in human subjects with cirrhosis in the liver, but to a significantly lesser extent, suggesting that an increase in circulating FAP is mostly due to increased hepatic expression (33). Therefore, in livers with advanced NASH with fibrosis, FAP may negatively regulate the hepatoprotective activity of FGF21.…”

“…It is believed that FGF21 in these conditions has a protective role via modulation of hepatic lipid metabolism and amelioration of endoplasmic reticulum stress (8,13,14). At the same time, elevated hepatic FAP expression has been observed in activated stellate cells in livers with cirrhosis (33,47). Plasma FAP is also increased in human subjects with cirrhosis in the liver, but to a significantly lesser extent, suggesting that an increase in circulating FAP is mostly due to increased hepatic expression (33).…”

“…FAP is a type II transmembrane serine protease that is also found in human serum as a soluble form lacking the transmembrane domain (30 -32). In adult animals, FAP activity is found in a variety of tissues, including the pancreas, skin, lymph nodes, skeletal muscle, colon, and adipose tissues (33). FAP expression is also induced at sites of wound healing, tissue remodeling, fibrosis, inflammation, and tumorigenesis (32,34).…”

Plasma levels of soluble fibroblast activation protein in arterial thrombosis; determinants and cleavage of its substrate alpha-2-antiplasmin

“…Although this variant does not seem to be expressed in vivo, there might be other circulating sFAP variants with reduced activity. Measurement of sFAP activity [16] instead of antigen level could possibly elucidate this.…”

Heterogeneity of molecular forms of dipeptidyl peptidase-IV and fibroblast activation protein in human glioblastomas

“…DPP-IV and FAP are type II-transmembrane proteins that are enzymatically active as homodimers with a molecular weight (MW) typically of 220-240 kDa and 170 kDa, respectively 14,15 . Enzymatically active soluble forms of both enzymes lacking the transmembrane region are found in blood plasma, and in the case of DPP-IV also in other bodily fluids, but their origin is largely speculative 16,17 . In addition to these forms which are probably the result of shedding from the plasma membrane by an unknown protease, substantial molecular heterogeneity has been reported for both DPP-IV and FAP from various sources, and these molecular forms are thought to have unique pathophysiological functions.…”