Existence of two nonlinear elimination mechanisms for
hepatocyte growth factor in rats
Ke-Xin
Liu1,
Yukio
Kato1,
Motohiro
Kato1,
Tai-Ichi
Kaku2,
Toshikazu
Nakamura3, and
Yuichi
Sugiyama1
1 Faculty of Pharmaceutical
Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113;
2 Bioproducts Industry Company,
Tomigaya, Shibuya-ku, Tokyo 151; and
3 Biomedical Research Center,
Osaka University School of Medicine, Suita, Osaka 565, Japan
 |
ABSTRACT |
Nonlinearity in the overall elimination of
hepatocyte growth factor (HGF) was examined in rats. After intravenous
administration, the plasma clearance
(CLplasma) of HGF exhibited a
dose-dependent biphasic reduction with high- and low-affinity
components. If we consider our previous finding that both
receptor-mediated endocytosis (RME) and a low-affinity uptake
mechanism, probably mediated by heparan sulfate proteoglycan (HSPG), in
the liver are major HGF clearance mechanisms, it may be that saturation
of CLplasma at lower and higher
doses represents saturation of RME and HSPG-mediated uptake,
respectively. At an HGF dose (1.46 nmol/kg), which completely saturates
the high-affinity component,
CLplasma was almost completely reduced when HGF was premixed with heparin. However,
CLplasma was reduced by heparin
to, at most, one-fifth that after HGF alone in a dose near the linear
range (3.66 pmol/kg). Saturation of CLplasma for HGF premixed with
heparin was monophasic and nonlinear only at the lowest HGF doses. In
vitro, high-affinity binding of
[35S]heparin to HGF was found,
showing that one HGF molecule binds to the penta- or hexasaccharide
unit. Because mitogenic activity of HGF has been reported in the
presence of heparin, these results suggest that heparin mainly inhibits
low-affinity HGF uptake by complexing with HGF, whereas its effect on
RME is relatively minor.
receptor-mediated endocytosis; heparin
 |
INTRODUCTION |
HEPATOCYTE GROWTH FACTOR (HGF) was first identified as
a potent mitogen for mature hepatocytes and is now recognized as a mitogen for a variety of types of epithelial cells (18). The biological
effect of HGF is not restricted to its mitogenic activity, but HGF is
identical to the scatter factor that acts as a motogen stimulating the migration of epithelial cells (31). HGF is also a
morphogen for epithelial cells, and it induces a multicellular architecture (20) as well as being a potent angiogenic factor capable
of inducing endothelial cells to proliferate and migrate (3). The
biological activity of HGF is exerted through its binding to a specific
receptor. The HGF receptor is a protooncogene c-met product (2, 6) and is expressed
on ubiquitous epithelial cells (25, 29). It has been suggested that HGF
binding to the receptor induces receptor dimerization, resulting in
reciprocal trans-phosphorylation of
each receptor and subsequent interaction with the other cytoplasmic
effectors (1, 11). HGF has an affinity for heparin and can bind to the
so-called heparin-like substance on the cell surface and/or
extracellular matrix (17, 32). Lyon et al. (16) demonstrated that the
heparan sulfate proteoglycan (HSPG) derived from the liver binds to
HGF. Thus HSPG is thought to be responsible for the binding to HGF as a heparin-like substance. Both a heparin-binding domain and a
receptor-binding domain on the HGF molecule have been identified at the
NH2-terminal half of the
-chain. The former is located on the
NH2-terminal hairpin loop and the
second Kringle domain, whereas the latter is within the region of the
hairpin loop and the first Kringle domain (19).
HGF also exhibits biological activity in vivo in several types of
animal with experimentally induced liver and kidney disease (9, 27).
However, the dose of HGF needed to produce a pharmacological effect is
usually high (>1.22 nmol/kg) except when HGF is administered through
the portal vein (4, 10), in which 0.61-3.05 pmol/kg HGF stimulates
hepatocyte growth. One of the reasons that such a high dose is needed
may be the short plasma half-life of HGF (~4 min) (12). For the
clinical application of HGF, it is important to clarify the mechanism
of its elimination from the circulating plasma. We have been studying
the clearance mechanism of HGF and suggested that both
receptor-mediated endocytosis (RME) and a low-affinity uptake
mechanism, probably mediated by a cell-surface HSPG, mainly contribute
to the systemic clearance of HGF under tracer conditions (12-14).
However, its nonlinear pharmacokinetic behavior has not been completely
clarified, and so in the present study, we investigated a number of
dose-dependent pharmacokinetic profiles.
To display the pharmacological activity of HGF in vivo, it is important
to develop an efficient drug-delivery system for HGF (8, 27). Because
the HSPG on the cell surface may mediate the uptake of HGF, its plasma
clearance can be reduced when HGF is premixed with heparin to form a
heparin-HGF complex (8). A mitogenic response by hepatocytes to HGF can
be observed in the presence of heparin (8, 32, 33), suggesting that HGF can bind to its receptor even when it forms a complex with heparin. We
previously reported the reduction in HGF clearance after its coinjection with heparin in rats (8). In that study, however, because
trichloroacetic acid precipitation was used to determine the plasma
concentration of HGF, the experiment was carried out within a short
time (<30 min) of its administration (8). In this study, we used
enzyme immunoassay (EIA) to investigate the plasma concentration-time
profile of HGF for a long period (~48 h) to examine the effect of
heparin on HGF disposition.
 |
MATERIALS AND METHODS |
Animals.
Male Wistar rats weighing 250 g (Nisseizai, Tokyo, Japan)
were used. All animals received humane care in compliance with the National Research Council's criteria for humane care as outlined in
"Guide for the Care and Use of Laboratory Animals" prepared by
the National Academy of Sciences and published by the National Institutes of Health (NIH publication no. 86-23, revised 1985).
Materials.
Porcine intestinal mucosa heparin with a molecular mass of
18-23 kDa (185.5 U/mg) was purchased from Sigma (St. Louis, MO); human recombinant HGF was purified from a culture medium of C-127 cells
transfected with plasmid containing human HGF cDNA (23).
Pharmacokinetic analysis of HGF or heparin-HGF complex.
Heparin dissolved in saline was incubated with HGF for
50 min at 25°C (8). With the animals under light ether
anesthesia, HGF alone or the heparin-HGF mixture was administered
through the penile vein, and at specified times, blood samples were
withdrawn through the jugular vein without cannulation. Plasma HGF
concentrations were determined using an EIA kit (Institute of
Immunology, Tochigi, Japan).
The plasma concentration
(Cp)-time profiles of HGF after
intravenous administration were fitted to the following two-exponential equation using a nonlinear iterative least-squares method (12)
|
(1)
|
where
and
are the apparent rate constants.
A and
B are the corresponding zero-time
intercepts, and t is time. The input data were weighted as the reciprocal of the square of the observed values, and the algorithm used for fitting was the damping Gauss Newton
method.
The area under the plasma concentration-time curve from
time 0 to
t
[AUC(0-t)]
was calculated
as
|
(2)
|
The area under the plasma concentration-time curve from
time 0 to infinity
[AUC(0-
)] was
calculated as
|
(3)
|
The
plasma clearance (CLplasma) was
calculated from
|
(4)
|
Ligation of portal vein and hepatic artery.
With the animals under light ether anesthesia, both the portal vein and
hepatic artery were ligated before intravenous injection of HGF. In a
sham operation, rats were anesthetized, and laparatomies were performed
without ligation.
In vitro binding of [35S]heparin to
HGF.
The protamine-affinity fraction of
[35S]heparin (15-25 mCi/g,
Amersham, Arlington Heights, IL) was chromatographed on a Sephadex G-100 column (114 cm × 1.5 cm ID) at a flow rate of 0.18 ml/min using phosphate-buffered saline as elution buffer (30). Blue dextran
(Pharmacia, Uppsala, Sweden), fluorescein-dextran with molecular masses
of 40 and 10 kDa (Cosmo-Bio, Tokyo, Japan),
[14C]inulin (New
England Nuclear, Boston, MA), and
3H2O
(New England Nuclear) were also separately eluted as molecular mass
markers. The volume of each fraction was 2.0 ml. Fractions 44-46
and 56-58 were collected as
[35S]heparin with molecular
masses of 21-23 and 12-13 kDa, respectively. The molecular
mass of the [35S]heparin was
determined from a plot of
kavg vs. the
logarithm of the molecular mass of each marker where
|
(5)
|
The
Ve,
Vo, and
Vt were peak
fraction numbers for each molecular mass marker, Blue dextran, and
3H2O,
respectively. [35S]heparin
dissolved in the buffer containing 120 mM NaCl, 4.8 mM KCl, 1.0 mM
KH2PO4,
1.2 mM MgSO4, 5.0 mM glucose, 2.2 mM CaCl2, and 20 mM
2-(N-morpholino)ethanesulfonic acid (pH 7.4) was incubated with HGF (50 nM) for 50 min at 25°C. Total and unbound
radioactivity was determined by ultrafiltration with a molecular mass
limitation of 30 kDa. The binding parameters were obtained by fitting
the data to the following equation using an iterative nonlinear
least-squares method
|
(6)
|
where
Cb,
Cf,
n,
Pt,
Kd, and
are
the concentration of heparin bound to HGF, unbound heparin
concentration, specific binding capacity, HGF concentration,
dissociation constant, and proportional constant for nonspecific
binding, respectively.
 |
RESULTS |
Nonlinear elimination of HGF.
After administration of various doses of HGF (1.22 pmol/kg-12.2
nmol/kg) as bolus intravenous injections, plasma concentration-time profiles were investigated using EIA (Fig.
1). At HGF doses of <12.2 pmol/kg, HGF in
plasma rapidly disappeared, whereas at >12.2 pmol/kg, such plasma
disappearance was delayed (Fig. 1). From the data shown in Fig. 1,
CLplasma was obtained (Fig.
2).
CLplasma exhibited a
dose-dependent reduction with increasing dose (Fig. 2). This reduction
was biphasic, showing both a high-affinity component saturated at
relatively low doses (1-3 µg/kg, 12.2-36.6 pmol/kg) and a
low-affinity component saturated at relatively higher doses
(300-1,000 µg/kg, 3.66-12.2 nmol/kg) (Fig. 2).

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Fig. 1.
Plasma concentration-time profiles of hepatocyte growth factor
(HGF) after intravenous administration of various doses of HGF. After
intravenous administration of 1.22 ( ), 3.66 ( ), 12.2 ( ), 36.6 ( ), 122 ( ), or 366 ( ) pmol/kg HGF
(A) and 1.46 ( ), 3.66 ( ), 6.10 ( ), 8.66 ( ), or 12.2 ( ) nmol/kg HGF
(B) as a bolus dose, plasma
concentration-time profiles were determined using enzyme immunoassay
(EIA). Each point and vertical bar represent mean ± SE of 3 rats.
Plasma clearance (CLplasma) of
HGF was calculated based on these time profiles and is shown in Fig.
2.
|
|

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Fig. 2.
Two saturable components in
CLplasma of HGF. From plasma
concentration-time profiles of HGF shown in Fig. 1,
CLplasma of HGF in normal rats was
estimated and plotted against dose. Each point and vertical bar
represent mean ± SE of 3 rats. Vertical bar is not shown when SE
value is smaller than symbol.
|
|
Elimination profile of HGF in rats with ligated portal vein and
hepatic artery.
To directly demonstrate the importance of the liver for the plasma
clearance of HGF, the elimination profile of HGF in plasma was examined
in rats after ligation of both the portal vein and hepatic artery (Fig.
3). The plasma disappearance of HGF at all doses examined was delayed in ligated rats compared with the control (sham-operated) rats (Fig. 3).
CLplasma in ligated rats was 14.5 ± 1.1, 9.18 ± 0.90, 0.903 ± 0.349, and 0.538 ± 0.362 ml · min
1 · kg
1
(means ± SE, N = 3) at HGF doses
of 1.22, 3.66, and 36.6 pmol/kg and 1.10 nmol/kg, respectively, also
showing a dose-dependent reduction.

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Fig. 3.
Elimination profile of HGF in rats with ligated portal vein and hepatic
artery. With animals under light ether anesthesia, both portal vein and
hepatic artery were ligated ( ), followed by intravenous
administration of bolus doses of HGF: 1.22 (A), 3.66 (B), or 36.6 pmol/kg
(C) or 1.10 nmol/kg
(D). Plasma concentration-time
profiles were determined using EIA. As a control experiment, plasma
concentration-time profile of HGF was also examined in sham-operated
rats ( ). Each point and vertical bar represent mean ± SE of 3 rats.
|
|
Effect of heparin on nonlinear elimination of HGF.
HGF was first mixed with heparin (0-20 mg/kg) and then given
intravenously (Fig.
4A) at
an HGF dose of 1.46 nmol/kg (120 µg/kg) when the high-affinity
component of CLplasma was almost
completely saturated (Fig. 2). Plasma concentration-time profile of HGF
after injection of a mixture with heparin at 0.004 mg/kg was almost identical to that after injection of HGF alone (Fig. 4). At >0.02 mg/kg heparin, the plasma disappearance of HGF was delayed after injection of heparin-HGF mixture (Fig. 4) compared with that after injection of HGF alone. This effect was heparin dose dependent (Fig. 4)
and reached a maximum when the dose of heparin was 0.4 mg/kg (Fig. 4).
From the data shown in Fig. 4A, a
kinetic parameter, the
AUC(0-180), representing the
exposure of HGF in plasma was determined (Fig.
4B). The
AUC(0-180) was 0.0423 ± 0.0039 nmol · min · ml
1
after injection of HGF (1.46 nmol/kg) alone and increased ~21-fold (0.899 ± 0.079 nmol · min · ml
1)
after injection of a mixture with 0.4 mg/kg heparin (Fig.
4B).

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Fig. 4.
Plasma concentration-time profiles
(A) and area under curve
[AUC(0-180);
B] of HGF after intravenous
administration of HGF alone or HGF premixed with heparin.
A: HGF was mixed with heparin to give
final heparin doses of 0 (large ), 0.004 (small ), 0.02 (large
), 0.04 ( ), 0.2 ( ), 0.3 ( ), 0.4 ( ), or 20 (small )
mg/kg and then injected intravenously. Plasma concentration-time
profiles of HGF were determined using EIA and normalized for injected
dose. B: from data shown in
A,
AUC(0-180) was calculated
using Eq. 2. Each point and vertical
bar represent mean ± SE of 3 rats.
|
|
The plasma concentration-time profiles of HGF after intravenous
injection of HGF alone or a heparin-HGF mixture were determined for a
longer period (~48 h) to accurately estimate
CLplasma (Fig. 5). At an HGF dose of 3.66 pmol/kg (0.3 µg/kg) near the linear dose range for
CLplasma (Fig. 2), HGF was first
mixed with enough heparin (0.4 mg/kg), and the mixture was then given
intravenously (Fig. 5A). The plasma
disappearance of HGF was also delayed compared with that after an
injection of 3.66 pmol/kg HGF alone (Fig.
5A). However, the reduction in
CLplasma at an HGF dose of 3.66 pmol/kg was not so marked (Fig. 5A,
Table 1) compared with the HGF dose of 1.46 nmol/kg (Fig. 5B, Table 1). At an HGF
dose of 3.66 pmol/kg, CLplasma
after administration of HGF with heparin was 21% that after
administration of HGF alone (Table 1). On the other hand, at an HGF
dose of 1.46 nmol/kg, CLplasma of
HGF with heparin was only 2.4% that after administration of HGF alone
(Table 1). The saturable component of
CLplasma was estimated by
subtracting CLplasma at 1.46 nmol/kg from that at 3.66 pmol/kg for both HGF alone and the
heparin-HGF mixture; this was ~10
ml · min
1 · kg
1
for the heparin-HGF mixture, approximately one-half that for HGF alone
(Table 1).

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Fig. 5.
Plasma concentration-time profiles of HGF after intravenous
administration of HGF alone or HGF premixed with heparin. At HGF doses
of 3.66 pmol/kg (A) and 1.46 nmol/kg
(B), HGF alone ( ) or HGF premixed
with sufficient heparin (0.4 mg/kg, ) was injected intravenously.
Plasma concentration-time profiles of HGF were determined by EIA. Each
point and vertical bar represent mean ± SE of 3 rats.
Pharmacokinetic parameters obtained are shown in Table 1.
|
|
Binding of [35S]heparin and HGF.
The binding of the protamine-affinity fraction of
[35S]heparin and HGF was
determined by ultrafiltration (Fig. 6). For
both [35S]heparins with
molecular masses of 21-23 and 12-13 kDa, saturable and
nonsaturable components could be observed for the binding with HGF
(Fig. 6). Kd
values were 0.310 ± 0.130 and 0.487 ± 0.292 nM,
n values were 0.0862 ± 0.0126 and
0.121 ± 0.024 mol of heparin/mol of HGF, and
values were 0.0115 ± 0.0012 and 0.0165 ± 0.0018 (means ± calculated SD) for
the 21- to 23- and 12- to 13-kDa
[35S]heparins, respectively
(Fig. 6).

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Fig. 6.
Scatchard plot representing binding of
[35S]heparin to HGF.
Protamine-affinity fraction of
[35S]heparin with a molecular
weight of 21-23 (A) or
12-13 kDa (B) was incubated
with HGF at 25°C for 50 min, and
[35S]heparin binding to HGF was
determined by ultrafiltration. Lines in figure represent calculated
curves obtained by fitting to Eq. 6.
Cb, heparin bound to HGF;
Cf, unbound heparin concentration;
Pt, HGF concentration.
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|
 |
DISCUSSION |
We have investigated the elimination mechanism of HGF and come to
several conclusions. The major clearance organ for HGF is the liver
(12), and the clearance mechanism for HGF consists of at least two
systems, RME and a low-affinity uptake mechanism probably through a
cell-surface HSPG (13, 14). Because the nonlinear elimination profile
of HGF from the circulation has never been reported previously, we
analyzed the plasma concentration-time profiles of HGF after
intravenous administration of several different doses (Figs. 1 and 3).
The present study supports the liver as the major clearance organ for
HGF at any of the doses of HGF examined, since the disappearance of
plasma HGF was significantly delayed in rats with their portal vein and
hepatic artery ligated (Fig. 3).
When we consider that RME contributes to HGF clearance (12-14), it
may be that its plasma elimination exhibits nonlinearity because of
saturated receptor binding and/or subsequent endocytosis. Actually, CLplasma showed biphasic
saturation with increasing HGF doses (Fig. 2); e.g.,
CLplasma was reduced at
12.2-36.6 pmol/kg and 3.66-12.2 nmol/kg (Fig. 2). This result
suggests that the clearance mechanism consists of at least two systems,
a high-affinity clearance site and a low-affinity one. The saturation
in CLplasma was observed at
12.2-36.6 pmol/kg, at which the plasma concentration ranged from
10 to 100 pM (Fig. 1). Because the equilibrium dissociation constant of
the HGF receptor is 20-40 pM (7), this result suggests that the
saturation at the lower dose range (12.2-36.6 pmol/kg) comes from
saturation of RME. On the other hand,
CLplasma also exhibited saturation
over the dose range 3.66-12.2 nmol/kg (Fig. 2). This can be
explained if we consider that not only RME but also the low-affinity
uptake mechanism, probably mediated by HSPG, can be saturated at this
higher HGF dose range.
If the nonlinearity in CLplasma
observed at the lower dose range (12.2-36.6 pmol/kg) results from
the saturation of RME, the HGF clearance at the much higher dose range
should be almost exclusively governed by the low-affinity uptake
mechanism and not RME. Under such conditions,
CLplasma should be almost
completely stopped when HGF prebound to heparin is injected, since the
heparin-binding site on the HGF molecule is occupied by the heparin so
that the heparin-HGF complex cannot bind to the HSPG (8). HGF has an affinity for heparin (18, 22, 24). In the present study, we showed that
HGF can bind to heparin with high affinity and exhibits an equilibrium
dissociation constant of 0.3-0.5 nM (Fig. 6). When we gave
intravenous HGF (1.46 nmol/kg) prebound to sufficient heparin (0.4 mg/kg), CLplasma was almost
completely reduced to zero compared with that after the injection of
HGF alone (Fig. 5B, Table 1). This
result also suggests that the low-affinity clearance site, which cannot
be saturated at the lower dose range (~3.66 nmol/kg), represents this
HSPG. The details of the mechanism of this low-affinity clearance site
are still unknown. In an earlier study, we found that part of the
125I-HGF internalization is not
saturated even in the presence of unlabeled HGF (135 pM) and is also
insensitive to phenylarsine oxide, an inhibitor of RME, in perfused rat
liver (12). Thus the sensitivity of each clearance site to the RME
inhibitor may be different. However, we cannot deny that the
low-affinity component is also mediated by an RME-like mechanism and
further studies are needed to clarify the mechanism of the low-affinity
component.
On the other hand, when HGF near the linear dose range (3.66 pmol/kg)
was premixed with heparin and administered intravenously, CLplasma was not completely
reduced but was ~20% of that after administration of HGF (3.66 pmol/kg) alone (Fig. 5A, Table 1). This can be explained by considering that HGF prebound to heparin can
still bind to the HGF receptor and be eliminated through RME. Actually,
even when HGF was premixed with a sufficient amount of heparin, a
saturable component in CLplasma of
HGF could still be observed (Table 1). In addition, we and others (8,
32, 33) have reported that the mitogenic response to HGF can be observed even in the presence of heparin in primary cultured rat hepatocytes. These results support our hypothesis that HGF bound to
heparin can still bind to its receptor. However, the saturable component in CLplasma after
administration of heparin-HGF complex was at most one-half that after
administration of HGF alone (Table 1). When we consider that this
saturable portion mainly reflects RME, this result suggests that the
efficiency in RME of HGF prebound to heparin is approximately one-half
that of HGF alone. On the other hand, the half-effective concentration
of the mitogenic effect of HGF in cultured rat hepatocytes increased
two- to threefold after the addition of heparin (8), suggesting that
the affinity of heparin-HGF complex for the HGF receptor is also
one-half that of HGF alone. Thus it can be speculated that the
relatively lower affinity of heparin-HGF complex for the receptor
results in the lower saturable component in
CLplasma. Actually, Naka et al.
(21) analyzed the interaction between HGF and its receptor in the
presence of heparin and found that heparin added during the binding of 125I-HGF to its receptor
significantly reduced the cross-linking of
125I-HGF to the HGF receptor. This
result implies that heparin can inhibit the receptor binding of HGF.
Nevertheless, they also showed in their study that the mitogenic
response to HGF was exhibited in the presence of heparin. Thus,
although it is likely that the affinity of heparin-HGF complex for the
receptor is relatively low, this complex still exhibits the biological
activity of HGF.
It has been reported that HGF exhibits marked pharmacological activity
in several types of experimental animal models of liver and kidney
dysfunction (4, 9, 10, 27). However, a large dose (>1.22 nmol/kg) is
needed to obtain any effect in vivo (9, 27), although biological
activity can be observed at very low (~100 pM) concentrations in
vitro (7, 32). Therefore, for its clinical application, we need to
develop a drug-delivery system so that the pharmacological effects can
be obtained at much lower doses. We suggest that the heparin-HGF
complex may be a candidate for such a drug-delivery system (8). In this
study, CLplasma of HGF can be
reduced to 2% of that of the controls by complex formation with
heparin at an HGF dose (1.46 nmol/kg) within the range where its
pharmacological activity can be observed (Table 1). This effect of
heparin on HGF clearance is heparin dose dependent and reaches a
maximum at a heparin dose of 0.4 mg/kg (Fig. 3), corresponding to 74 U/kg. Because an intravenous clinical dose of heparin is usually 100 U/kg (5), this dose of heparin is within the clinical dose range. Our
present study therefore suggests that clinical doses of heparin can
almost completely block HGF clearance.
CLplasma also showed nonlinearity
in rats with ligated portal vein and hepatic artery (Fig. 3). This
saturation in CLplasma was almost
complete and observed at a dose of <1.22 nmol/kg (15 ml · min
1 · kg
1
at 1.22 pmol/kg and 0.5 ml · min
1 · kg
1
at 1.10 nmol/kg). This result suggests that the clearance mechanism in
extrahepatic organs mainly consists of the high-affinity component mediated by RME. Expression of the HGF receptor is not restricted in
the liver but is also observed in extrahepatic organs such as kidney,
spleen, and lung (25, 29). Therefore, clearance via these organs may be
part of the extrahepatic organ clearance. However, we should be careful
about the absolute value of
CLplasma under such
unphysiological conditions. In order to estimate more accurately the
contribution of the liver, a further study needs to be done.
Our [35S]heparin binding study
with HGF revealed that the binding capacity of HGF (n) was 0.086 mol
heparin/mol HGF for 21- to 23-kDa
[35S]heparin (Fig. 6). This
means that 1 heparin molecule can bind to ~12 HGF molecules, and 1 unit in the heparin molecule, which can bind to 1 HGF molecule, should
have a molecular weight of 1.9 kDa (corresponding to a
hexasaccharide). On the other hand, n was 0.12 mol heparin/mol HGF for 12- to 13-kDa [35S]heparin (Fig. 6),
indicating that one unit in the heparin molecule binding to one HGF
molecule should have a molecular weight of 1.5 kDa (corresponding to a
pentasaccharide). Lyon et al. (16) demonstrated that the minimal size
binding component in heparan sulfate derived from the liver is a
hexasaccharide. Heparan sulfate derived from liver possesses more
heparin-like properties than a heparan sulfate species derived from
other sources (15). Zioncheck et al. (33) reported that low
concentrations of sulfated oligosaccharides of sufficient length (6 glucose units) induce dimerization of HGF and also increase its
mitogenic effect on cultured rat hepatocytes. Both our findings and
their report can be understood if a penta- or hexasaccharide can bind
to an HGF molecule and stimulate its conformational change, resulting
in dimerization of HGF. It is well known that receptor binding and the
subsequent mitogenic effect of fibroblast growth factor (FGF) are
regulated by HSPG on the cell surface (26, 28). One of the proposed
mechanisms for such regulation by HSPG is that binding of FGF with
heparan sulfate may induce a conformational change in FGF, resulting in its receptor binding (26, 28). Such a regulation may also occur in the
interaction between HGF and its receptor.
In conclusion, the plasma clearance of HGF is governed by two
mechanisms, RME and a low-affinity uptake mechanism, probably mediated
by a cell-surface HSPG, and exhibits nonlinearity that stems from the
saturation of these mechanisms as the dose increases. HGF premixed with
a clinical dose of heparin shows a much lower plasma clearance compared
with HGF alone, mainly by preventing binding to HSPG.
 |
ACKNOWLEDGEMENTS |
This study was supported in part by a Grant-in Aid for Scientific
Research provided by the Ministry of Education, Science and Culture of
Japan.
 |
FOOTNOTES |
Address for reprint requests: Y. Sugiyama, Faculty of Pharmaceutical
Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo
113, Japan.
Received 24 February 1997; accepted in final form 7 July 1997.
 |
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