HGFA is a blood coagulation factor XII-like serine protease, responsible for the activation of HGF in tumours and injured tissues. Shimomura et al. (99) first reported the purification of a HGF-converting enzyme present in fetal bovine serum. Subsequently, a protease was purified from human serum with the ability to convert pro-HGF into the active form of HGF in vitro, and was thus termed HGF Activator (HGFA) (87). HGFA has since been shown to be the key mediator of the localised activation of HGF in injured tissue (94).

Many serine proteases are generated from their precursors, via limited proteolysis, upon the initiation of blood coagulation. HGFA appears to follow this trend, as HGFA also exists as a precursor form in the plasma. The HGFA precursor is made up of a single polypeptide chain, consisting of 655 amino acids, has a molecular weight of around 96kDa, and has no HGF converting ability (100). The cDNA sequence for this novel serine protease revealed that the active form of HGFA is derived from the COOH-terminal region of a precursor protein, and is composed of multiple domains. The chromosomal location of the HGFA gene has been determined as 4p16 (101).

The HGFA precursor is inactive in plasma and requires activation to fulfill the function of HGF activator. It was observed that human serum revealed a high degree of HGF converting activity, and Shimomura et al. (100), examined the ability of various serine proteases, from the blood coagulation and fibrinolysis mechanisms, to act as activators of the HGFA precursor. They identified thrombin as the most effective protease for cleavage of the precursor. This cleavage occurred, via limited proteolysis, at the bond between Arg407 and Ile408, in vitro. Thrombin therefore, links HGFA to the blood coagulation cascade, as upon initiation of blood coagulation, the serine protease thrombin is generated from its precursor, pro-thrombin. The HGFA precursor circulates in the plasma in this inactive form and does not have the ability to bind to heparin. However, in the active form HGFA does possess the ability to bind to heparin-like molecules, thereby associating with the cell surface to ensure localised HGFA action. This binding enables a more efficient pro-HGF conversion as the pro-HGF molecule also binds to heparin-like molecules on the cell surface awaiting activation (12, 36, 94). Cleavage of the HGFA precursor results in the generation of two major fragments of 66kDa and 34kDa in size. The 66kDa fragment represents the inactive NH2-terminal region of the precursor, which may have been involved in the binding of the precursor to the cell surface for activation by thrombin (102). Whereas, the 34kDa fragment represents the active form of HGFA and is composed of the COOH-terminal region (Figure 4).

HGF activation by HGFA occurs mainly in the extracellular environment and is the limiting step in the HGF signalling pathway. HGFA was initially detected in the liver, through northern blot analysis, and it has since been established as the main source of HGF in the body (87). HGFA has also been detected in white matter astrocytes of brain tissue, glioma cells, and in colorectal carcinoma (103-105). Our studies have demonstrated in recent years that HGFA is expressed by a wide variety of cancer cell lines, and is also overexpressed in human breast cancer tissues (26, 106).

4.1.2 Matriptase

More recently, a member of the transmembrane type II serine protease family, matriptase, has demonstrated pro-HGF converting properties (88). Matriptase was initially discovered and purified as a matrix degrading protease from breast cancer cell lines and human breast milk (107-109). Interestingly, matriptase has been separately identified by four different groups, subsequent cloning revealed matriptase to be identical to MT-SP1, TADG-15, epithin, and ST14 (110-113).

Matriptase is an 80kDa - 90kDa protease that consists of multiple domains, including a short cytoplasmic domain at the NH2 terminus followed by a putative transmembrane domain; a sperm protein, enteroki-

nase, and agrin domain; two tandem C1r/C1s, urchin embryonic growth factor, and bone morphogenetic protein-1 (CUB) domains; four tandem low-density lipoprotein (LDL) receptor class A domains; and a trypsin-like serine protease domain at its COOH terminus (109) (Figure 4).

Similarly to HGFA, matriptase requires activation via cleavage at its canonical activation motif to convert the single-chain zymogen to a two-chain active protease. However, the activation process of the matriptase zymogen is extraordinarily complex, unique among all serine proteases studied to date, and incompletely understood (114). Matriptase requires proteolytic processing at Gly-149 in the SEA domain of the protease, glycosylation of the first CUB domain and the first serine protease domain, and intact LDL receptor class A domains. It was suggested that activation of matriptase required the presence of its cognate inhibitor, HAI-1 (115). A recent study reports that after its activation, matriptase is rapidly bound to HAI-1. Subsequently, the matriptase-HAI-1 complex is shed into the extracellular milieu (116). These observations indicate that activation and HAI-1-mediated inhibition of matriptase are well organised and controlled in human mammary epithelial cells.

In addition to generating active HGF, matriptase can also create active forms of urokinase-type plasminogen activator and protease-activated receptor 2 (PAR2) (88, 113). Furthermore, purified matriptase was also found to activate one of the important matrix metalloproteases, stromely-sin (MMP-3) (117). The normal physiological role of matriptase may be in epithelial biology, as matriptase is reported to be an essential component of the profilagrin-processing pathway in keratinocytes, a crucial regulator of epidermal terminal differentiation, and also critical for hair follicle growth (116, 118). In addition, transgenic knockout studies have shown that matriptase elimination results in a malfunction in epidermal barrier formation, the cellular immune system, and reduces post-natal survival in mice (119). Matriptase also demonstrates the ability to degrade extracellular matrix proteins, such as gelatin, fibronectin and laminin (109, 120). Therefore, matriptase may contribute to the remodeling of the ECM and aid tumour cell invasion (121). The fact that matriptase is synthesised as a transmembrane form may also prove to aid the pericellular activation of HGF.

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