MINIREVIEW
Hepatocyte growth factor activator (HGFA): a serine
protease that links tissue injury to activation of
hepatocyte growth factor
Keiji Miyazawa
Department of Biochemistry, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Japan
Introduction
In tissues of multicellular organisms, different types of
cells are intricately and precisely arranged to perform
specific functions. Once such tissue architecture is
destroyed, the regeneration system works to restore the
structure as well as the function of the damaged tissue.
Liver regeneration has long been a subject of active
research, because it displays a dramatic form of organ
regeneration. It also represents a good in vivo model
for understanding the regulation of cell growth:
hepatocytes are usually in a quiescent state, but most
of them enter the cell cycle during liver regeneration.
Humoral factors that trigger liver cell growth have
been detected in the blood circulation of liver-injured
animals, and many researchers have tried to isolate
these factors.
Hepatocyte growth factor (HGF) was originally
identified as a potent mitogen for hepatocytes in pri-
mary culture during studies of liver regeneration [1].
Studies showed that HGF was induced in the blood
plasma and liver in response to liver injury. Therefore,
Keywords
hepatocyte growth factor; plasminogen;
proteolytic activation; tissue injury
Correspondence

coagulation ⁄ fibrinolytic system.
Abbreviations
FXII, coagulation factor II; HGF, hepatocyte growth factor; HGFA, hepatocyte growth factor activator; tPA, tissue-type plasminogen activator.
2208 FEBS Journal 277 (2010) 2208–2214 ª 2010 The Author Journal compilation ª 2010 FEBS
HGF was regarded as the humoral factor that triggers
liver regeneration. HGF was subsequently shown to
have mitogenic, motogenic and morphogenic activities
on various target cells, including epithelial and endo-
thelial cells. HGF is now thought to play a major role
in the repair and regeneration of various tissues,
including the liver, kidney, lung, and stomach [1].
A new question then arose. Why is the liver specifi-
cally targeted to grow after liver injury? HGF and
its receptor, MET tyrosine kinase, are widely expressed
and distributed among normal as well as injured tis-
sues. After liver injury, HGF is induced not only in
the liver but also in blood plasma and other uninjured
tissues, such as spleen [2]. Although HGF is not exclu-
sively induced in the liver, its action is limited to the
injured liver. Apparently, the amount of HGF is not
correlated with the activity of HGF, suggesting that
HGF is latent in normal states, and is activated specifi-
cally at the site of tissue injury. The mechanism of
localized activation of HGF, however, had remained
unclear. In this minireview, I describe the discovery of
a novel serine protease, HGF activator (HGFA),
which has revealed the link between tissue injury and
localized activation of HGF.
Active and inactive forms of HGF
The first step in solving this puzzle was to understand

Plasminogen is synthesized as a single-chain form
that consists of five tandemly repeated kringle domains
and a serine protease domain [1]. It is activated upon
cleavage at a specific site between the fifth kringle
Heavy chain (α-chain)
Light chain (β-chain)
62 kDa
32–34 kDa
Signal
1
I II III IV
2
(K)(N)
(N) (K)
(N)
NK1
NK2
I
I
II
(K)
A
B
Hairpin region
Kringle domain
Serine protease-
like domain
Fig. 1. Schematic structure of HGF (A) and
its variants (B). Circles denote amino acids,
and lines denote disulfide bonds. Arrow-

Proteolytic activation of HGF in
response to tissue injury
In order to elucidate the in vivo roles of proteolytic
processing of HGF, we examined the molecular forms
of HGF by immunoblotting using a heavy chain-
specific monoclonal antibody [11]. The antibody reacts
with both the single-chain and two-chain forms of
HGF, giving positive bands at 92 and 62 kDa, respec-
tively. By scanning of the 92 and 62 kDa bands,
the ratio of the single-chain form (inactive) to the
two-chain form (active) can be quantified.
We first found that HGF from various normal rat
tissues (liver, kidney, lung, and spleen) was present
exclusively in the inactive single-chain form. We next
administered hepatotoxin or nephrotoxin to rats to
induce tissue injury. HGF was extracted from injured
and uninjured tissues and then analyzed. After intraga-
stric administration of carbon tetrachloride, liver tissue
was severely injured and the other tissues were mini-
mally affected. The amount of HGF was dramatically
increased in the liver and spleen tissue, but not in the
kidney or lung tissue. HGF was converted to the
active form only in the liver, which was injured in this
experimental model. Similar results were obtained
when d-galactosamine was used to induce liver injury
through a different mechanism. When we injected
mercuric chloride to induce renal injury in rats, HGF
in the kidney increased in quantity and was activated.
In contrast, HGF in the liver and spleen increased in
quantity but was not activated. These findings

region, a kringle domain, and a catalytic domain
(Fig. 2). These domains are homologous to those
observed in blood coagulation factor XII (FXII), with
an overall amino acid similarity of 39%. Analysis of
genomic DNA coding for HGFA indicated a relation-
ship between HGFA and FXII as well as urokinase
and tPA, activators of plasminogen. These four pro-
teins therefore constitute a family (the PA–FXII–
HGFA family) [13].
Other proteases, such as urokinase, tPA, FXII,
factor XI, plasma kallikrein, matriptase, and hepsin,
were subsequently reported to activate HGF in vitro
[14–18]. The first five of these are of blood plasma ori-
gin, whereas the latter two are transmembrane serine
proteases and may be involved in pericellular activa-
tion of HGF. Among these proteases, matriptase and
hepsin activate HGF with comparable efficiency to
that of HGFA in vitro [17,18]. Although the activities
Hepatocyte growth factor activator K. Miyazawa
2210 FEBS Journal 277 (2010) 2208–2214 ª 2010 The Author Journal compilation ª 2010 FEBS
of the other proteases on HGF are very weak in vitro,
they may be stimulated by a cofactor(s) or by a certain
microenvironment in vivo. These proteases, as well as
HGFA, are thus candidates for the HGF-converting
enzyme in injured tissues.
Recently, Itoh and Kataoka generated mice defi-
cient in hgfa, and directly demonstrated that HGFA
is the serum-derived protease responsible for activa-
tion of HGF [19,20]. HGFA was also shown to be
required for tissue repair in experimental colitis mod-

tion ⁄ migration of vascular endothelial cells in vitro
[24,25]. HGF thus appears to be linked to the blood
coagulation and fibrinolytic system, not only structur-
ally but also functionally (Fig. 3). The blood coagula-
tion system is activated upon injury of blood vessels,
leading to conversion of prothrombin to thrombin.
Thrombin processes fibrinogen and coagulation fac-
tor XIII (plasma transglutaminase) to form stable
blood clots and prevent further hemorrhage from the
injured sites. Concomitantly, thrombin induces activa-
tion of HGF via HGFA. HGF then stimulates prolif-
eration and migration of endothelial cells to repair
blood vessels. It appears rational that the blood coagu-
lation system triggers activation of a growth factor
that promotes angiogenesis. Therefore, the prototypic
function of HGF may be to maintain the integrity of
blood vessels. Thrombin appears to be a bifurcation
point for clotting and endothelial cell migration ⁄ prolif-
eration, and HGFA represents the link between tissue
injury and activation of HGF.
Perspectives
In 1995, Uehara et al. and Bladt and coworkers [26,27]
reported that the embryonic lethality of HGF-knock-
Kringle
Type I
Type II
EGF EGF
1
(2)
Serine protease

epithelial cells to repair the tissue architecture. FVa, factor Va; FXa,
factor Xa; FXIIIa, activated FXIII.
K. Miyazawa Hepatocyte growth factor activator
FEBS Journal 277 (2010) 2208–2214 ª 2010 The Author Journal compilation ª 2010 FEBS 2211
out mice was caused by dysfunction of the placenta
and liver, indicating that HGF plays important roles
during embryonic development. We can explain the
activation of HGF in injured tissues in adults by the
scheme illustrated in Fig. 3, but it remains unclear
how HGF is activated during embryonic development
in which there is no apparent tissue injury. In the
Drosophila embryo, the signal transduction pathway
that establishes the dorsal–ventral pattern is temporally
and spatially regulated through the proteolytic cascade
to activate Spa
¨
tzle, the Toll receptor ligand [28]. It is
likely that a proteolytic cascade also plays an impor-
tant role in the regulation of HGF activity during
mammalian development. Known proteases that acti-
vate HGF in vitro do not appear to be the key prote-
ases activating HGF during embryonic development,
because mice lacking these proteases are not embry-
onic lethal. Alternatively, truncated variant forms of
HGF, NK1, and NK2 (Fig. 1B), which are abundantly
expressed in embryo-derived cells, might contribute to
HGF activity during embryonic development. These
variants exhibit weak agonistic activity, although they
lack the serine protease-like domain [29,30], and do
not appear to require proteolytic activation. Further

HGFA activation in vivo, as well as regulation of
HGFA activity by endogenous protease inhibitors,
including hepatocyte growth factor activator inhibitor-1
(HAI-1) and protein C inhibitor [35–37], will be
important for understanding the pathophysiological
processes regulated by the HGF–HGFA system.
Acknowledgements
I apologize to colleagues in the field for not citing
many important papers, because of the limitation of
the length of this review article. I would like to thank
T. Shimomura, D. Naka and T. Kawaguchi of Mitsu-
bishi Chemical Corp., A. Okajima and A. Kitamura of
Kansai Medical University and N. Kitamura of Tokyo
Institute of Technology for their contributions to the
study of HGFA.
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