Protamine enhances the proliferative activity of hepatocyte
growth factor in rats
Ke-Xin
Liu1,
Yukio
Kato1,
Tai-Ichi
Kaku2,
Kunio
Matsumoto3,
Toshikazu
Nakamura3, and
Yuichi
Sugiyama1
1 Faculty of Pharmaceutical
Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113;
2 Bioproducts Industry Company,
Ltd., Tomigaya, Shibuya-ku, Tokyo 151; and
3 Biomedical Research Center,
Osaka University School of Medicine, Suita, Osaka 565, Japan
 |
ABSTRACT |
The effect of protamine on the proliferative
activity of hepatocyte growth factor (HGF) was examined in
-naphthyl
isothiocyanate-intoxicated rats. Protamine preinjection increased the
hepatocyte labeling index induced by HGF four- to fivefold. A similar
effect was also observed in partially hepatectomized rats. Because a
cell surface heparin-like substance can bind to HGF and protamine has
an affinity for heparin, protamine may affect HGF pharmacokinetics. In
fact, protamine injection caused a transient increase in plasma HGF concentrations after administration of HGF and, in vitro, protamine eluted HGF prebound to heparin-Sepharose. Protamine also reduced the
plasma clearance of HGF and increased 2.5-fold the exposure of
hepatocytes to HGF in vivo. The enhancing effect of protamine on the
mitogenic response of hepatocytes to HGF was also observed in vitro
(~2-fold after protamine pretreatment compared with HGF alone),
suggesting that the enhancing effect of protamine on HGF-induced liver
regeneration results from dual effects exerted by protamine 1) lowering the overall elimination
of HGF and 2) directly stimulating hepatocyte mitosis induced by HGF.
liver regeneration; liver function; drug delivery
system
 |
INTRODUCTION |
HEPATOCYTE GROWTH FACTOR (HGF) is a heterodimer protein
with a molecular mass of 82-85 kDa (18). HGF stimulates
proliferation of a variety of epithelial cells, including hepatocytes
(6, 8, 16, 20). Its gene expression is increased not only when there is
hepatic damage, such as with partial hepatectomy (25, 31) and carbon
tetrachloride poisoning (10), but also after renal (5) and pulmonary
injury (20). In such cases HGF levels in circulating plasma are
increased, and therefore HGF is believed to be a hepatotrophic,
renotrophic, and pulmotrophic factor (5, 20, 31).
HGF is a basic polypeptide and one of the heparin-binding proteins (2,
19). HGF can bind to heparan sulfate expressed on the surface of
ubiquitous cells and in the extracellular matrix (15, 32). Mutational
deletion of its NH2-terminal
hairpin loop or second kringle domain reduces the affinity of HGF for heparin, suggesting that these structures are the heparin-binding domains on the HGF molecule (17). An oligosaccharide moiety in heparan
sulfate required for binding to HGF has also been identified and is
different from that required for binding to basic fibroblast growth
factor, another heparin-binding protein (26). Low concentrations (<0.1-10 µg/ml) of sulfated oligosaccharides of sufficient
length (6 glucose units) induce dimerization of HGF and also increase its mitogenic effect on cultured rat hepatocytes (24). This effect may
result from stabilization of the HGF dimer, which stimulates dimerization of the HGF receptor on the cell surface (24).
HGF markedly accelerates regeneration of damaged organs in animals with
hepatic and renal failure (6). However, a large dose (>100 µg/kg)
is usually required to exert such a pharmacological effect (4). This
may be one of the stumbling blocks for the clinical application of HGF.
We have been trying to identify the way in which HGF is cleared from
the circulation (11-14), and we have previously suggested that
both receptor-mediated endocytosis and another low-affinity uptake
system, probably mediated by a cell surface heparin-like substance in
the liver, are involved in the systemic clearance of HGF (11-14).
When HGF is premixed with heparin and then given intravenously, its
plasma clearance is reduced (8, 14). Thus a heparin-HGF complex such as
this one may be used to increase the plasma residence time of HGF. Such
an inhibitory effect of heparin on HGF clearance possibly results from
occupation of the heparin-binding domain on the HGF molecule by
heparin, which results in a reduction in HGF binding and subsequent
internalization through a cell surface heparin-like substance (8,
11-14). However, high concentrations (>100 µg/ml) of heparin
reduce the mitogenic activity of HGF (8). In addition, heparin has
anticoagulant activity. Therefore, further studies need to be performed
to develop a reliable and efficient drug delivery system (DDS) for HGF.
Protamine is a basic protein with an affinity for heparin, and it has
been used clinically to neutralize any excessive pharmacological effect
exerted by heparin. The molecular mass of protamine is usually ~4
kDa, and more than one-half of its amino acid sequence consists of
arginine. If protamine can bind to a cell surface heparin-like
substance and inhibit the binding of HGF to this substance, it may be
that it can be used as another type of DDS to increase the HGF plasma
residence time. Hence, in the present study, we examined the effect of
protamine on both the proliferative activity and pharmacokinetics of
HGF in rats.
 |
MATERIALS AND METHODS |
Animals.
Male Wistar rats weighing 250 g (Nisseizai, Tokyo, Japan) were used and
treated humanely. Studies were carried out in accordance with the
Declaration of Helsinki and the Guide for the Care and Use of Laboratory Animals [DHEW Publication No.
(NIH) 85-23, Revised 1985, Office of Science and Health Reports,
DRR/NIH, Bethesda, MD 20205] as adopted and promulgated by the
National Institutes of Health.
-Naphthylisothiocyanate (ANIT)
dissolved in olive oil was injected intraperitoneally at a dose of 50 mg/kg body wt. While rats were under ether anesthesia, partial (30%)
hepatectomy was performed by removing the left lateral lobe of the
liver through a subxyphoid incision.
Materials.
Protamine sulfate was obtained from salmon roe from Wako Pure Chemical
(Osaka, Japan), ANIT and 3,3'-diaminobenzidine were from Sigma
(St. Louis, MO), and
125I-deoxyuridine was from New
England Nuclear (Boston, MA). Human recombinant HGF was purified from a
culture medium of C127 cells transfected with plasmid containing human
HGF cDNA (18). Epidermal growth factor (EGF) was supplied by Wakunaga
Pharmaceutical (Hiroshima, Japan).
HGF injection.
HGF dissolved in saline was administered through the penile vein 30 min
before and 8, 22, 32, 46, 56, 70, 80, 94, 104, and 118 h after ANIT
treatment or 8, 22, 32, and 46 h after partial hepatectomy. Rats were
killed 12, 24, 48, 72, 96, and 120 h after ANIT treatment or 48 h after
30% partial hepatectomy.
Protamine injection.
Under light ether anesthesia, protamine dissolved in saline was
administered through the penile vein 10 min before HGF or EGF
injection.
Measurement of labeling index.
One hour before rats were killed, 5-bromo-2'-deoxyuridine
dissolved in normal saline was injected intraperitoneally (100 mg/kg body wt). One hour after injection, the rats were exsanguinated via the
abdominal artery, under light ether anethesia. The liver was then
removed and fixed in 10% buffered Formalin for 24 h. The fixed samples
were embedded in paraffin, and the paraffin sections (4 µm) were
mounted on glass slides. After deparaffinization of the liver sections,
endogenous peroxidase was inactivated in 0.3% hydrogen peroxide in
absolute methanol, and nuclei incorporating 5-bromo-2'-deoxyuridine were stained with the use of a cell
proliferation kit (Amersham, Arlington Heights, IL). The labeling index
of hepatocytes was determined by counting more than 500 nuclei in
photographs of three randomly selected fields under light microscopy.
Determination of bilirubin concentration and activity of liver
cytosolic enzymes in serum.
The total bilirubin concentration and the activity of liver-specific
cytosolic enzymes such as glutamic-pyruvic transaminase (GPT), lactate
dehydrogenase (LAP), alkaline phosphatase (ALP), and
-glutamyltransferase (
-GTP) in rat serum obtained 48 h after ANIT
treatment were determined using the appropriate assay kits (Wako Pure
Chemical).
Pharmacokinetic analysis of HGF in ANIT-treated rats.
Under light ether anesthesia, protamine (0 or 1.6 mg/kg) was
administered to rats through the penile vein 24 h after ANIT treatment.
Ten minutes after the protamine injection, HGF (300 µg/kg body wt)
dissolved in saline was also given through the penile vein. Plasma was
collected from the external jugular vein, and the HGF concentration was
determined using an enzyme-linked immunoassay (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 by a nonlinear iterative least-squares method (11)
|
(1)
|
where
and
are the apparent rate constants,
A and
B are the corresponding
time 0 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 the fitting was the damping Gauss-Newton method.
The area under the plasma concentration-time curve (AUC) and area under
the moment curve (AUMC) were calculated as
|
(2)
|
|
(3)
|
The
plasma clearance (CLplasma),
distribution volume of the central compartment
(V1), and steady-state
distribution volume (Vdss) were
calculated from Eqs.
4, 5,
and 6, respectively
|
(4)
|
|
(5)
|
|
(6)
|
Effect of protamine injection on the plasma elimination of HGF.
Under light ether anesthesia, 1 µg/kg HGF was injected through the
penile vein of normal rats. At indicated times, blood was withdrawn
through the left external jugular vein. At 3.5 min, 250 µl saline
containing protamine (0-20 mg/kg) were also injected through the
penile vein, and blood samples were collected. The plasma concentration
of HGF was determined by EIA.
Assay for DNA synthesis in primary cultured rat hepatocytes.
Parenchymal hepatocytes were plated at a density of 1.25 × 105 cells/1.88
cm2 and cultured for 3 h for HGF
or 24 h for HGF and EGF, as described previously (8). The nonattached
cells were removed by washing, and the culture medium containing
protamine was applied to the monolayer. HGF or EGF was added 10 min
after the addition of protamine. Then, 22 h after HGF addition,
125I-deoxyuridine was added, and
its incorporation for 6 h was assayed as described previously (8).
Cellular protein was determined by the Bradford method, using the
Bio-Rad protein assay kit with bovine serum albumin as standard (9).
Elution of HGF by protamine using heparin-Sepharose column
chromatography.
HGF (1.0 ml; 250 ng/ml) dissolved in phosphate-buffered saline (PBS)
was added to a heparin column (1-ml bed volume, heparin-Sepharose CL6B;
Pharmacia) at a rate of 0.3 ml/min and incubated for 30 min on ice.
Then, 1.0 ml PBS or 1.0 ml protamine (20 mg/ml) was applied to the
column 13 times at the same rate. Finally, 1.0 ml PBS containing 2 M
sodium chloride was added to the column six times. Each eluted solution
was collected to determine HGF by EIA. The ratio of the amount of HGF
eluted in each sample to that added to the column was calculated as the
recovery of HGF.
Statistical analysis.
Statistical analysis was performed by Student's
t-test to identify significant
differences between various treatment groups.
 |
RESULTS |
Effect of protamine on liver regeneration induced by HGF in rats
with liver damage in vivo.
Protamine (1.6 mg/kg body wt) was injected intravenously into
ANIT-intoxicated rats 10 min before administration of HGF (300 µg/kg), and hepatocyte labeling indexes were determined at specified times after ANIT intoxication (Fig. 1). The
labeling indexes after the administration of protamine before HGF
injection were 0.95 ± 0.08, 1.37 ± 0.33, 4.63 ± 1.01, 3.87 ± 0.45, 5.02 ± 1.08, and 1.32 ± 0.32% (means ± SE;
n = 6) at 12, 24, 48, 72, 96, and 120 h after ANIT treatment, respectively (Fig. 1). These values were respectively 1.2, 2.4, 4.9, 3.8, 2.1, and 1.4 times the value after the
injection of HGF alone (Fig. 1). On the other hand, when protamine
alone was injected, the labeling indexes were much lower compared with
those after administration of HGF alone and protamine before the
injection of HGF, at any time after ANIT intoxication (Fig. 1).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 1.
Time profiles of DNA synthesis in hepatocytes of
-naphthylisothiocyanate (ANIT)-intoxicated rats treated with
hepatocyte growth factor (HGF) alone or protamine before HGF injection.
ANIT-intoxicated rats were treated with HGF (300 µg/kg) alone ( ),
protamine (1.6 mg/kg) before injection of HGF ( ), or protamine alone
( ), and the labeling index in hepatocytes was determined at
designated times after ANIT intoxication. Data are means ± SE of
3-6 rats. * P < 0.05, ** P < 0.01, significantly
different from HGF alone.
|
|
To examine the protamine dose dependence, a similar experiment with
various doses of protamine was performed and the labeling indexes were
determined 48 h after ANIT intoxication (Fig.
2A). Protamine alone could not stimulate liver regeneration in
ANIT-intoxicated rats (Fig. 2A).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of protamine on liver regeneration induced by HGF in
ANIT-intoxicated (A) and partially
hepatectomized (B) rats.
ANIT-intoxicated rats were treated with HGF (300 µg/kg) alone (H) or
with various doses of protamine before HGF injection (H+P), or
protamine (1.6 mg/kg) alone (P), and the labeling index in hepatocytes
was determined 48 h after ANIT intoxication
(A). Similar experiments with HGF
were also performed in rats after partial (30%) hepatectomy
(B). C(+) and C( ) represent
ANIT-intoxicated rats treated with saline and nonintoxicated rats
treated with saline, respectively. Numbers in parentheses represent
dose of protamine (mg/kg). Data are means ± SE of 3-5 rats.
* P < 0.05, ** P < 0.01, significantly
different from HGF alone.
|
|
The labeling index increased in a protamine dose-dependent manner when
the dose of protamine was increased from 0 to 1.6 mg/kg, and the peak
value was reached at 1.6 mg/kg protamine (Fig.
2A). When the dose of protamine was
further increased to over 1.6 mg/kg, a dose-dependent reduction in the
labeling index was observed (Fig.
2A). The effect of protamine on
liver regeneration induced by HGF fell to almost the control level when
the dose of protamine was 6.4 mg/kg (Fig.
2A). An enhancing effect of
protamine on liver regeneration induced by HGF was also found in
partially (30%) hepatectomized rats (Fig.
2B). The protamine dose dependence
in the labeling indexes was similar to that in ANIT-intoxicated rats (Fig. 2). When the dose of protamine was increased, the peak value of
the labeling index occurred at a protamine dose of 1.6 mg/kg (Fig.
2B).
Effect of protamine on bilirubin concentration and activity of liver
cytosolic enzymes in ANIT-intoxicated rats.
To examine whether protamine promotes the repair of liver function
induced by HGF in ANIT-intoxicated rats, we determined the change in
total bilirubin concentration and activity of liver cytosolic enzymes
such as GPT, LAP, ALP, and
-GTP in serum from rats after
administration of HGF alone or protamine before HGF injection (Fig.
3). Protamine alone did not reduce the
total bilirubin concentration or the activity of liver cytosolic
enzymes in serum (Fig. 3). The increase in total bilirubin
concentration and activity of liver cytosolic enzymes in serum caused
by ANIT administration was significantly countered by injection of HGF
(300 µg/kg) alone (Fig. 3). When protamine at a dose of 0.8 or 1.6 mg/kg was administered before HGF injection, the serum level of
-GTP
was significantly lower than that after injection of HGF alone (Fig.
3). Protamine slightly enhanced the reduction produced by HGF in
bilirubin concentration, GPT, and LAP, although this effect was not
significant (Fig. 3).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 3.
Change in bilirubin (BIL) concentration and activity of liver cytosolic
enzymes in serum in ANIT-intoxicated rats treated with HGF alone or
protamine before injection of HGF. ANIT-intoxicated rats were treated
with HGF (300 µg/kg) alone (H), various doses of protamine before HGF
injection (H+P), or protamine (1.6 mg/kg) alone (P). Total bilirubin
concentration and activity of liver cytosolic enzymes in serum were
determined 48 h after ANIT intoxication. ALP, alkaline phosphatase;
GPT, glutamic-pyruvic transaminase; -GTP, -glutamyltransferase;
LAP, lactate dehydrogenase. Data are means ± SE of 3 rats.
* P < 0.05, ** P < 0.01, significantly
different from C(+). # P < 0.05, significantly different from HGF alone.
|
|
Effect of protamine on HGF clearance from the circulation in
ANIT-intoxicated rats.
To examine whether protamine reduces the clearance of HGF from the
circulation, plasma concentration-time profiles of HGF in
ANIT-intoxicated rats were determined after intravenous administration of HGF alone or HGF following protamine treatment (Fig.
4). The elimination of HGF from plasma
after injection of HGF following protamine treatment was slower
compared with that after administration of HGF alone (Fig. 4). The AUC
after administration of HGF following protamine injection was 2.48-fold
that after HGF injected alone (Table 1).
CLplasma,
V1, and
Vdss after administration of HGF following protamine injection fell to 39.5%, 34.7%, and 19.1%, respectively, of the values after administration of HGF without protamine treatment (Table 1).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of protamine on pharmacokinetics of HGF in ANIT-intoxicated rats
24 h after ANIT intoxication. HGF (300 µg/kg) alone ( ) or
protamine (1.6 mg/kg) followed by HGF (300 µg/kg) ( ) was
given intravenously and plasma HGF concentrations were
determined using enzyme-linked immunoassay (EIA). Pharmacokinetic
parameters obtained are shown in Table 1. Data are means ± SE of 3 rats.
|
|
View this table:
[in this window]
[in a new window]
|
Table 1.
Comparison of the pharmacokinetic parameters of HGF in ANIT-intoxicated
rats after intravenous administration of HGF alone and protamine before
HGF injection
|
|
To examine whether the stimulant effect of protamine on liver
regeneration induced by HGF can be attributed to the increase in HGF
AUC produced by protamine preinjection, the hepatocyte labeling index
was plotted against AUC (Fig. 5). The
labeling index at 300 µg/kg HGF following 1.6 mg/kg protamine
treatment was 5.23 ± 0.99%, which was much higher than that after
administration of HGF alone at a dose of 500 µg/kg (0.530 ± 0.104%) (Fig. 5), although in both cases the AUC had almost the same
value (Fig. 5).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 5.
Relationship between HGF area under the plasma concentration-time curve
(AUC) and liver regeneration in ANIT-intoxicated rats. Twenty-four
hours after ANIT intoxication, indicated doses of HGF (0, 300, 500 µg/kg) alone or protamine (1.6 mg/kg) followed by HGF (300 µg/kg)
were given intravenously and plasma HGF AUCs were determined.
ANIT-intoxicated rats were treated with same doses of HGF alone or
protamine before injection of HGF, and the labeling index in
hepatocytes was thus obtained 48 h after ANIT intoxication. Labeling
indexes were plotted against AUCs after the corresponding dose. Data
are means ± SE of 3-5 rats.
|
|
Effect of protamine on DNA synthesis rate induced by HGF and EGF in
primary cultured rat hepatocytes.
To examine the direct effect of protamine on hepatocytes, we examined
the effect of protamine on DNA synthesis in primary cultured
hepatocytes in the presence of HGF (Fig. 6,
A and
B). When the protamine concentration
was increased to 12.5 µg/ml, no significant change in the DNA
synthesis rate induced by HGF was observed in hepatocytes cultured for
3 and 24 h (Fig. 6, A and
B). When the protamine concentration
in the medium was increased to 25 µg/ml, DNA synthesis in hepatocytes
cultured for 24 h was increased approximately twofold compared with
that in the presence of HGF alone (Fig.
6B). The DNA synthesis rate in
hepatocytes in the presence of any concentration of HGF was inhibited
almost completely when the protamine concentration in the medium was 200 µg/ml (Fig. 6, A and
B). To examine whether the enhancing effect of protamine is specific to HGF, we performed the same experiment with EGF (Fig. 6C). In
the presence of 6-25 µg/ml protamine, DNA synthesis was
increased approximately two- to threefold compared with DNA synthesis
in the presence of EGF alone (Fig.
6C). When the concentration of
protamine was increased to 50 and 200 µg/ml, DNA synthesis of
hepatocytes was inhibited (Fig. 6C).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of protamine on mitogenic response to HGF and EGF in primary
cultured rat hepatocytes. In rat hepatocytes cultured for 3 (A) or 24 h
(B,
C), protamine was applied to give
final protamine concentrations of 0 ( ), 6 ( ), 12.5 ( ), 25 ( ), 50 ( ), and 200 ( ) µg/ml. Ten minutes later, HGF
(A,
B) or EGF
(C) was applied to give indicated
final concentrations, followed by determination of DNA synthesis. Data
are means ± SE of 3 rats. * P < 0.05, ** P < 0.01, significantly different from protamine
concentration of 0 µg/ml.
|
|
Protamine causes a transient increase in the plasma concentration
profile of HGF after intravenous administration of HGF.
To support the hypothesis that protamine competes with HGF for binding
to a heparin-like substance in vivo, we studied the effect of protamine
injection on the plasma concentration-time profile of HGF in normal
rats (Fig. 7). After intravenous
administration of HGF (1 µg/kg), plasma HGF concentrations fell
rapidly (Fig. 7). After various doses of protamine (0.48-20 mg/kg)
were injected, the plasma concentrations of HGF increased immediately
in a protamine dose-dependent manner (Fig. 7). However, this protamine
dose dependence differed from that for the enhancing effect on the
labeling index (Fig. 2) and reached a maximum at 20 mg/kg protamine
(Fig. 7).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of protamine injection on plasma elimination of HGF. HGF (1 µg/kg) was injected through penile vein of normal rats. At indicated
times, blood was withdrawn through the left external jugular vein. At
3.5 min, 250 µl saline in control rats ( ) or protamine at 0.48 ( ), 1.6 ( ), 5.0 ( ), or 20 mg/kg ( ) dissolved in 250 µl
saline was injected through the penile vein, and blood samples were
collected. Plasma concentrations of HGF were determined by EIA. Data
are means ± SE of 3 rats.
* P < 0.05, ** P < 0.01, significantly
different from control.
|
|
Protamine elutes HGF prebound to heparin-Sepharose in a column
chromatography experiment.
HGF bound to heparin in a heparin affinity column could not
be washed off by PBS but was easily eluted with 2 M
sodium chloride (Fig.
8A). The
recovery of HGF from the heparin column was 85.7% (Fig.
8A). To further support the
hypothesis of competition for the binding of HGF to heparin by
protamine, we added protamine (20 mg/ml) to the heparin affinity column
prebound with HGF (Fig. 8B). The HGF
bound to the column was eluted by addition of protamine, and the
recovery of HGF was 84.4% (Fig.
8B). After elution with protamine,
only a small amount of HGF was further eluted by 2 M sodium chloride
(Fig. 8B). In this analysis we
confirmed that the determination of HGF by EIA was not influenced by 20 mg/ml protamine (data not shown).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 8.
Protamine elutes HGF prebound to a heparin-immobilized column. HGF
(1 ml; 250 ng) dissolved in phosphate-buffered saline (PBS) was applied
to a heparin-immobilized column (1-ml bed volume), which was then
eluted with PBS (A) or protamine (20 mg/ml) (B) and subsequently 2 M
sodium chloride. Amount of HGF in eluate (1 ml per fraction) was
determined by EIA.
|
|
 |
DISCUSSION |
In the present study, we found that, when administered before injection
of HGF, protamine enhances HGF-induced liver regeneration (Fig. 1).
Such an enhancing effect of protamine was found in both ANIT-intoxicated rats and partially hepatectomized rats (Fig. 2), and
the protamine dose dependence in the hepatocyte labeling indexes was
almost identical in both cases (Fig. 2), suggesting that this effect
may be general for a number of liver diseases. Protamine also
significantly further reduces
-GTP (Fig. 3) at 300 µg/kg HGF, and
at 50 µg/kg HGF, bilirubin concentration and the activity of all
cytosolic marker enzymes examined were significantly reduced by
preinjection of protamine, compared with those with HGF alone (data not
shown). Thus the effect of protamine is also observed in the repair of
liver function. The dosage of protamine in clinical situations is
10-15 mg for the neutralization of 1,000 U heparin (29). Because
the regular clinical single dose of heparin is 100 U/kg iv,
1.0-1.5 mg/kg protamine is usually used as an antidote for
heparin. In the present study, we required 1.6 mg/kg protamine to
observe its maximum enhancing effect on liver regeneration (Fig. 2).
Thus this dose of protamine is very similar to the clinical dose and
therefore may also be used in clinical situations. The dose of
protamine should be strictly controlled, since a higher dose of
protamine reduces the mitogenic response to HGF (Figs.
2A and 6), probably because of its
cytotoxic effect.
The CLplasma of HGF was reduced by
preadministration of protamine (Fig. 4 and Table 1). We believe that
the likely mechanism involves inhibition of the nonspecific clearance
of HGF by protamine. HGF has two binding sites on epithelial cell
surfaces: 1) the HGF receptor, a
specific binding site, and 2) a
heparin-like substance, which has a lower affinity for HGF (2, 5). In
previous studies, we suggested that one of the major clearance
mechanisms for HGF is its nonspecific uptake in the liver, which is
probably mediated by a heparin-like substance (16-19). Given that
protamine has a high affinity for heparin (7) and can elute HGF
molecules prebound to heparin-Sepharose (Fig. 8), a transient increase
in plasma HGF after intravenous administration of protamine (Fig. 7)
may reflect the transfer of HGF molecules bound to the heparin-like substance on cell surfaces and/or the extracellular matrix of various tissues into the circulating plasma after protamine injection. Thus protamine and HGF bind to the same region of the heparin-like substance or, at least, to a similar location so that each compound can
affect the binding of the other.
There are two possible mechanisms for the enhancing effect of protamine
on HGF-induced liver regeneration in vivo: one is the increase in HGF
AUC, which results from inhibition of the nonspecific uptake of HGF by
protamine (Fig. 4), and the other is a direct stimulatory effect on the
mitogenic response of hepatocytes to HGF (Fig.
6B). Protamine increases HGF AUC
2.5-fold (Table 1), whereas the increase in liver regeneration,
assessed as the area under the time course of the labeling index after
ANIT intoxication, was approximately fivefold (Fig. 1). Therefore, the
enhancing effect of protamine on HGF-induced liver regeneration can be
partially explained by increased exposure of hepatocytes to HGF. As
shown in Fig. 5, HGF at a dose of 300 µg/kg plus protamine has a
markedly higher labeling index than HGF alone at 500 µg/kg but has an
AUC nearly identical to that of HGF alone at 500 µg/kg. The data
shown in Fig. 5 provide clear evidence against a direct relationship between HGF availability (as expressed by the AUC) and liver
regeneration (as expresssed by the labeling index). Thus the mechanism
of the effect of protamine on HGF-induced liver regeneration is not
principally related to its inhibitory effect on HGF clearance. The
difference in the protamine dose dependence between the labeling index
(Fig. 2) and plasma disappearance of HGF (Fig. 7), where maximum
effect can be observed at 1.6 and 20 mg/kg protamine,
respectively, also supports that the enhanced liver regeneration cannot
be fully explained by such an indirect effect. In fact, the DNA
synthesis in hepatocytes in primary culture induced by HGF was
increased approximately twofold through the direct stimulatory effect
of protamine (Fig. 6B). Therefore,
the direct effect of protamine on hepatocytes may be considered one of
the more rational mechanisms for the beneficial effects of protamine on
the labeling index in vivo.
The effect of protamine on several cytokine receptors has been
investigated (23, 28). Lokeshwar et al. (14a) reported that protamine
induced an increase in the number of EGF receptors by activating
cryptic or inactive receptors to become functionally active in Swiss
3T3 cells and human epidermoid carcinoma A431. Protamine also increases
EGF-induced phosphorylation of the EGF receptor. In the present study,
we also found that protamine enhanced EGF-induced DNA synthesis in
hepatocytes in vitro (Fig. 5C). This indicates that the direct enhancement effect of protamine on hepatocyte DNA synthesis is not specific to HGF. Sacks and McDonald (23) have also
reported that protamine enhanced the insulin-induced autophosphorylation activity of insulin receptors. Like EGF and insulin
receptors, HGF receptors are also transmembrane protein tyrosine kinase
receptors (27). The diverse biological actions of HGF are a result of
signaling through this receptor (22, 30). According to current
thinking, HGF activates its corresponding PTK receptors by inducing
receptor dimerization and autophosphorylation as a first step in an
intracellular signaling cascade (3). Therefore, such an interaction of
protamine with the HGF receptor or its signal transduction cascade may
occur, resulting in the increase in DNA synthesis.
The suppressive effect of protamine on bilirubin concentration and the
activity of cytosolic enzymes, except
-GTP, was not significant
(Fig. 3). Because the suppressive effect of HGF alone on bilirubin
concentration and the activity of cytosolic enzymes could not be
further increased even when the dose of HGF was raised to
710-1,000 µg/kg (data not shown), we believe that 300 µg/kg HGF exerts an almost maximal effect in suppressing bilirubin
concentration and the activity of these cytosolic enzymes in serum in
ANIT-intoxicated rats.
In conclusion, we find that protamine enhances HGF-induced liver
regeneration in vivo. Such an effect of protamine can be explained by
its dual effects, 1) a direct
stimulatory effect on hepatocyte DNA synthesis and
2) an indirect effect on HGF
clearance, which results in increased exposure to HGF.
 |
ACKNOWLEDGEMENTS |
We are very grateful to Drs. Kohji Tanaka in Development Research
Laboratories, Dainippon Pharmaceutical Co., Ltd., and Satoru Inagaki in
Development Research Laboratories, Banyu Pharmaceutical Co., Ltd., for
kindly advising us how to determine the labeling index in hepatocytes
in rats.
 |
FOOTNOTES |
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.
Address for reprint requests: Y. Sugiyama, Faculty of Pharmaceutical
Sciences, Univ. of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
Received 30 December 1996; accepted in final form 9 September
1997.
 |
REFERENCES |
1.
Ando, T.,
M. Yamasaki,
and
K. Suzuki.
Protamines. Molecular Biology, Biochemistry and Biophysics, edited by A. Kleinzeller,
G. F. Springer,
and H. G. Wittmann. Berlin: Springer-Verlag, 1973, vol. 12, p. 1-30.
2.
Gohda, E.,
H. Tsubouchi,
H. Nakayama,
S. Hirono,
O. Sakiyama,
K. Takahashi,
H. Miyazaki,
S. Hashimoto,
and
Y. Daikuhara.
Purification and partial characterization of hepatocyte growth factor from plasma of a patient with fulminant hepatic failure.
J. Clin. Invest.
81:
414-419,
1988[Medline].
3.
Heldin, C. H.
Dimerization of cell surface receptors in signal transduction.
Cell
80:
213-223,
1995[Medline].
4.
Huang, J. S.,
J. Nishimura,
S. S. Huang,
and
T. Deuel.
Protamine inhibits platelet derived growth factor receptor activity but not epidermal growth factor activity.
J. Cell. Biochem.
26:
205-220,
1984[Medline].
5.
Igawa, T.,
K. Matsumoto,
S. Kanda,
Y. Saito,
and
T. Nakamura.
Growth factor may function as a renotropic factor for regeneration in rats with acute renal injury.
Am. J. Physiol.
265 (Renal Fluid Electrolyte Physiol. 34):
F61-F69,
1993[Abstract/Free Full Text].
6.
Ishiki, Y.,
H. Ohnishi,
Y. Muto,
K. Matsumoto,
and
T. Nakamura.
Direct evidence that hepatocyte growth factor is a hepatotrophic factor for liver regeneration and for potent anti-hepatitis action in vivo.
Hepatology
16:
1227-1235,
1992[Medline].
7.
Jaques, L. B.
Protamin-antagonist, to heparin.
J. Can. Dent. Assoc.
108:
1291-1297,
1973.
8.
Kato, Y.,
K. X. Liu,
T. Nakamura,
and
Y. Sugiyama.
Heparin-hepatocyte growth factor complex with low plasma clearance and retained hepatocyte proliferating activity.
Hepatology
20:
417-424,
1994[Medline].
9.
Kato, Y.,
and
Y. Sugiyama.
Binding, internalization, degradation, and mitogenic effect of epidermal growth factor in cultured rat hepatocytes.
Sci. Tech. Pharm. Pharm. Sci.
3:
75-82,
1993.
10.
Lindroos, P. M.,
R. Zarnegar,
and
G. K. Michalopoulos.
Hepatocyte growth factor (Hepatopoietin A) rapidly increases in plasma before DNA synthesis and liver regeneration stimulated by partial hepatectomy and carbon tetrachloride administration.
Hepatology
13:
743-749,
1991[Medline].
11.
Liu, K. X.,
Y. Kato,
M. Narukawa,
D. C. Kim,
M. Hanano,
O. Higuchi,
T. Nakamura,
and
Y. Sugiyama.
The importance of the liver in the plasma clearance of hepatocyte growth factor in rats.
Am. J. Physiol.
263 (Gastrointest. Liver Physiol. 26):
G642-G649,
1992[Abstract/Free Full Text].
12.
Liu, K. X.,
Y. Kato,
T. Terasaki,
S. Aoki,
K. Okumura,
T. Nakamura,
and
Y. Sugiyama.
Contribution of parenchymal and non-parenchymal liver cell to the clearance of hepatocyte growth factor from the circulation in rats.
Pharm. Res.
12:
1737-1740,
1995[Medline].
13.
Liu, K. X.,
Y. Kato,
T. Terasaki,
T. Nakamura,
and
Y. Sugiyama.
Change in hepatic handling of hepatocyte growth factor during liver regeneration in rats.
Am. J. Physiol.
269 (Gastrointest. Liver Physiol. 32):
G745-G753,
1995[Abstract/Free Full Text].
14.
Liu, K. X.,
Y. Kato,
M. Yamazaki,
O. Higuchi,
T. Nakamura,
and
Y. Sugiyama.
Decrease in the hepatic uptake clearance of hepatocyte growth factor (HGF) in CCl4-intoxicated rats.
Hepatology
17:
651-660,
1993[Medline].
14a.
Lokeshwar, V. B.,
S. S. Huang,
and
J. S. Huang.
Protamine enhances epidermal growth factor (EGF)-stimulated mitogenesis by increasing cell surface EGF receptor number.
J. Biol. Chem.
264:
19318-19326,
1989[Abstract/Free Full Text].
15.
Masumoto, A.,
and
N. Yamamoto.
Sequestration of a hepatocyte growth factor in extracellular matrix in normal adult rat liver.
Biochem. Biophys. Res. Commun.
174:
90-95,
1991[Medline].
16.
Matsumoto, K.,
K. Hashimoto,
K. Yoshikawa,
and
T. Nakamura.
Marked stimulation of growth and motility of human keratinocytes by hepatocyte growth factor.
Exp. Cell Res.
196:
114-120,
1991[Medline].
17.
Mizuno, K.,
H. Inoue,
M. Hagiya,
S. Shimizu,
T. Nose,
Y. Shimohigashi,
and
T. Nakamura.
Hairpin loop and second kringle domain are essential sites for heparin binding and biological activity of hepatocyte growth factor.
J. Biol. Chem.
269:
1131-1136,
1994[Abstract/Free Full Text].
18.
Nakamura, T.,
T. Nishizawa,
M. Hagiya,
T. Seki,
M. Shimonishi,
A. Sugimura,
K. Tashiro,
and
S. Shimizu.
Molecular cloning and expression of human hepatocyte growth factor.
Nature
342:
440-443,
1989[Medline].
19.
Nakamura, T.,
H. Teramoto,
and
A. Ichihara.
Purification and characterization of a growth factor from rat platelets for mature parenchymal hepatocytes in primary cultures.
Proc. Natl. Acad. Sci. USA
83:
6489-6493,
1986[Abstract].
20.
Ohmichi, H.,
K. Matsumoto,
and
T. Nakamura.
In vivo mitogenic action of HGF on lung epithelial cells: pulmotrophic role in lung regeneration.
Am. J. Physiol.
270 (Lung Cell. Mol. Physiol. 14):
L1031-L1039,
1996[Abstract/Free Full Text].
21.
O'Reilly, R. A.
Anticoagulant, antithrombotic, and thrombolytic drugs.
In: The Pharmalogical Basis of Therapeutics, edited by A. G. Goodman.,
L. S. Gilman,
and A. Gilman. New York: Macmillan, 1980, p. 1347-1366.
22.
Rubin, J. S.,
D. P. Bottaro,
and
S. A. Aaronson.
Hepatocyte growth factor/scatter factor and its receptor, the c-Met proto-oncogene product.
Biochim. Biophys. Acta
1155:
357-371,
1993[Medline].
23.
Sacks, D. B.,
and
J. M. McDonald.
Insulin-stimulated phosphorylation of calmodulin by rat liver insulin receptor preparations.
J. Biol. Chem.
263:
2377-2383,
1988[Abstract/Free Full Text].
24.
Schwall, R. H.,
L. Y. Chang,
P. J. Godowski,
D. W. Kahn,
K. J. Hillan,
K. D. Bauer,
and
T. F. Zioncheck.
Heparin induces dimerization and confers proliferative activity onto the hepatocyte growth factor antagonists NK1 and NK2.
J. Cell Biol.
133:
709-718,
1996[Abstract].
25.
Shiota, G.,
T. C. Wang,
T. Nakamura,
and
E. V. Schmidt.
Hepatocyte growth factor in transgenic mice: effect on hepatocyte growth, liver regeneration and gene expression.
Hepatology
19:
962-972,
1994[Medline].
26.
Turnbull, J. E.,
D. G. Fering,
Y. Ke,
M. C. Wilkinson,
and
J. T. Gallagher.
Identification of the basic fibroblast growth factor binding sequence in fibroblast heparan sulfate.
J. Biol. Chem.
267:
10287-10293,
1992.
27.
Vigna, E.,
L. Naldini,
L. Tamagnone,
P. Longati,
A. Bardelli,
F. Maina,
C. Ponzetto,
and
P. M. Comoglio.
Hepatocyte growth factor and its receptor, the tyrosine kinase encoded by the c-Met proto-oncogene.
Cell Mol. Biol. (Oxf.)
40:
597-604,
1994.
29.
Wakefield, T. W.,
C. B. Hantler,
S. K. Wrobleski,
B. A. Crider,
and
J. C. Stanley.
Effect of differing rats of protamine reversal of heparin anticoagulation.
Surgery
119:
123-128,
1996[Medline].
30.
Weidner, K. M.,
M. Sachs,
and
W. Birchmeier.
The Met receptor tyrosine kinase transducers motility, proliferation, and morphogenic signals of scatter factor/hepatocyte growth factor in epithelial cells.
J. Cell Biol.
121:
145-154,
1993[Abstract].
31.
Zarnegar, R.,
M. C. DeFrances,
D. P. Kost,
P. M. Lindroos,
and
G. K. Michalopoulos.
Expression of hepatocyte growth factor mRNA in regenerating rat liver after partial hepatectomy.
Biochem. Biophys. Res. Commun.
177:
559-565,
1991[Medline].
32.
Zarnegar, R.,
M. C. DeFrances,
L. Oliver,
and
G. K. Michalopoulos.
Identification and partial characterization of receptor binding sites for HGF on rat hepatocytes.
Biochem. Biophys. Res. Commun.
173:
1179-1185,
1990[Medline].
AJP Gastroint Liver Physiol 274(1):G21-G28
0193-1857/98 $5.00
Copyright © 1998 the American Physiological Society