(Received for publication, February 28, 1994; and in revised form, September 27, 1994)
From the
Previous studies have shown that hepsin is a putative
membrane-associated serine protease that is required for cell growth
(Torres-Rosado, A., O'Shea, K. S., Tsuji, A., Chou, S.-H., and
Kurachi, K.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7181
7185). In the present study, we have transfected baby hamster kidney
(BHK) cells with a plasmid containing the cDNA for human hepsin and
examined these cells for their ability to activate several blood
coagulation factors including factors X, IX, VII, prothrombin, and
protein C. Little, if any, proteolytic activation of factors X, IX,
prothrombin, or protein C was observed when these clotting factors were
incubated with hepsin-transfected cells. On the other hand,
hepsin-transfected cells proteolytically activated significant
concentrations of human factor VII in a time- and calcium-dependent
manner, whereas essentially no activation of factor VII was observed in
BHK cells transfected with plasmid lacking the cDNA for hepsin. The
factor VII activating activity in the hepsin-transfected BHK cell line
was confined exclusively to the total membrane fraction and was
inhibited >95% by antibody raised against a fusion protein
consisting of maltose-binding protein and the extracellular domain of
human hepsin. An active site factor VII mutant, S344A factor VII, was
cleaved as readily as plasma-derived factor VII by hepsin-transfected
cells, indicating that factor VII was not converted to factor VIIa
autocatalytically on the cell surface. In contrast, an activation
cleavage site factor VII mutant, R152E factor VII, was not cleaved by
hepsin-transfected cells, suggesting that factor VII and S344A factor
VII were activated on these cells by cleavage of the
Arg-Ile
peptide bond. In the copresence of
factor VII and factor X, hepsin-transfected BHK cells supported the
formation of factor Xa. In addition, in the copresence of factor VII,
factor X, and prothrombin, hepsin-transfected BHK cells supported the
formation of thrombin. These results strongly suggest that
membrane-associated hepsin converts zymogen factor VII to factor VIIa,
which in turn, is capable of initiating a coagulation pathway on the
cell surface that ultimately leads to thrombin formation.
Considerable clinical and experimental evidence support the view that a variety of neoplastic cells activate the blood coagulation system, leading to hypercoagulability and intravascular thrombosis(1) . Although the mechanism whereby tumor cells promote blood coagulation is poorly understood, direct activation of the extrinsic pathway of blood coagulation is widely believed to be instrumental in fibrin formation around neoplastic cells. In this regard, several tumor cells synthesize and express cell-surface tissue factor(2) , which serves as a cell-surface receptor and cofactor for factor VIIa. The factor VIIa-tissue factor complex then rapidly activates factors IX and X by limited proteolysis(3, 4, 5, 6) . Precisely how factor VII is activated in the tumor microenvironment is unclear. In addition, several tumor cells generate thrombin and fibrin without detectable cell-surface tissue factor, and precisely how this happens is unknown although cancer procoagulant A synthesis by these cells has been implicated in this mechanism(7) .
Cell-surface
proteases of normal and malignant cells are generally thought to play
roles in cell growth, chemotaxis, endocytosis, exocytosis, blood
coagulation, fibrinolysis, and tissue invasion during
metastasis(8) . Several plasma membrane-associated proteases
have been purified and include endopeptidases, metalloproteases,
neutral serine proteases, carboxypeptidases, aminopeptidases, and
dipeptidases(8) . A novel zymogen of a trypsin-like serine
protease, designated as hepsin, was recently identified by screening a
human liver cDNA library with a synthetic oligonucleotide probe coding
for a highly conserved amino acid sequence found in the serine protease
family(9) . Hepsin (51 kDa) is composed of an
amino-terminal transmembrane domain followed by a potential catalytic
domain featuring the active site triad residues of His, Asp, and Ser
that participate in enzyme catalysis. It is currently assumed that the
transmembrane domain anchors hepsin to the cell membrane and that the
carboxyl-terminal catalytic domain is extracellular. Hepsin is
expressed in high levels in liver tissue but is also expressed at lower
levels in other tissues including kidney, pancreas, lung, thyroid,
pituitary gland, and testis(10) . Western blot analyses of
solubilized fractions of a human hepatocellular carcinoma cell line
(HepG2) indicated that hepsin is located on the membrane but not in the
cytosol or conditioned media of these cells(10) . Although the
precise biological role of hepsin is not clear, recent studies by
Torres-Rosado and co-workers (11) indicate that hepsin may play
a significant role in cell growth and maintenance of cell morphology.
In the present study, we have transfected BHK (
)cells with a
plasmid containing the cDNA for human hepsin. We demonstrate herein
that the hepsin-transfected BHK cells activate human factor VII by
limited proteolytic cleavage of the Arg
-Ile
peptide bond. Furthermore, in the presence of plasma levels of
factor X and prothrombin, newly formed factor VIIa can initiate a
pathway that culminates in thrombin formation. Our results suggest that
hepsin may be involved in the formation of thrombin on tumor cells that
lack demonstrable immunoreactive tissue factor.
Figure 1: Agarose electrophoresis of polymerase cahin reaction-amplified cDNA derived from untransfected BHK cells and hepsin-transfected BHK cells. First strand cDNA from untransfected BHK cells (lane 1) and hepsin-transfected BHK cells (lane 2) was amplified with polymerase chain reaction primers directed against the human Hepsin cDNA as described under ``Experimental Procedures.'' A negative control amplification (lane 3) was included with no cDNA template to assure that reaction products were not due to nonspecific sample contamination with hepsin cDNA sequences. The reaction products were fractionated on 1% agarose and stained with ethidium bromide.
Figure 2:
Activation of factor VII by Hepsin-1/BHK
cells and BHK cells transfected with Zem 229R. Hepsin-1/BHK cell
monolayers were preincubated with either rabbit anti-MBP/hepsin IgG
() or preimmune rabbit IgG (
) for 2 h at 37 °C.
Following aspiration of the IgG solutions, cells were treated with 10
nM factor VII in buffer A
, incubated at 37
°C, and the supernatants assayed for the temporal production of
factor VIIa as described under ``Experimental Procedures.''
Zem 229R-transfected BHK cells (
) were incubated at 37 °C with
10 nM factor VII in buffer A
, and at selected
intervals factor VIIa formed was measured as described under
``Experimental Procedures.'' B, immunoblots of
factor VII following incubation with Hepsin-1/BHK. Aliquots (20 µl)
of the supernatants from Hepsin-1/BHK monolayers pretreated with
preimmune rabbit IgG described in A were reduced,
electrophorized, and reacted with affinity-purified anti-human factor
VII. Lanes 1-5 represent reaction mixtures after 0, 15,
30, 45, and 60 min of incubation, while lane 6 is 20 µl of
350 pM authentic recombinant factor
VIIa.
Figure 3:
Activation of factor VII by cell
components of Hepsin-1/BHK cells. Total membrane fraction (),
cytosol (
), or 10
intact cells (
) were
incubated with 10 nM factor VII in a total volume of 500
µl buffer A
. At selected times, factor VIIa was
determined as described under ``Experimental
Procedures.''
Figure 4:
Effect of trypsin on the ability of
cell-surface hepsin to activate factor VII. A Hepsin-1/BHK cell
suspension (2
10
cells) was incubated with
TPCK-treated trypsin at a final concentration of 5 (
), 50
(
), and 500 (
) µg/ml. At selected times, soybean
trypsin inhibitor (10 mg/ml) was added to the cell suspension to stop
the reaction. The cells were then centrifuged, washed six times, and
subsequently assessed for their ability to activate factor VII. The
results are expressed as percent factor VIIa formed in each system
relative to the amount of factor VIIa formed in an untreated cell
suspension under identical conditions.
Figure 5:
Activation of factor X by Hepsin-1/BHK
cells and untransfected BHK cells in the presence of either factor VII
or S344A factor VII. Hepsin-1/BHK cells (,
) and
untransfected cells (
) were incubated with either 160 nM factor X plus 10 nM factor VII (
,
) or 160
nM factor X plus 10 nM S344A factor VII (
). At
selected times, factor Xa formation was assessed in the supernatants as
described under ``Experimental
Procedures.''
In subsequent studies, we addressed the possibility that factor VIIa
generated on Hepsin-1/BHK forms a complex with potential cell-surface
hamster tissue factor and subsequently activates factor X in part or
totally by a tissue factor-dependent reaction. In these studies,
Hepsin-1/BHK cell monolayers were incubated for 2 h at 37 °C with
either buffer A, preimmune goat IgG (6 mg/ml in buffer A) or purified
goat anti-rabbit tissue factor IgG (6 mg/ml in buffer A) previously
shown to cross-react with hamster tissue factor and inhibit the
prothrombin time of hamster plasma and hamster brain thromboplastin in
a concentration-dependent manner. In the latter studies, goat
anti-rabbit tissue factor IgG prolonged the hamster prothrombin time
15 s at 3 mg/ml and
25 s at 6 mg/ml final concentration. In
this system, a 25-s prolongation of the clotting time was equivalent to
the clotting time observed when the hamster thromboplastin was diluted
5-fold. Thus, the goat anti-rabbit tissue factor IgG, at 6 mg/ml final
concentration, inhibited roughly 80% of the hamster tissue factor in
the thromboplastin preparation. In contrast, rabbit anti-human tissue
factor IgG, at 0.2-6 mg/ml final concentration, had no effect on
the hamster prothrombin time. Following incubation with either buffer A
or the above IgG preparations dissolved in buffer A, the supernatant of
each system was supplemented with CaCl
(5 mM),
factor VIIa (10 nM), and factor X (180 nM) and
allowed to incubate at 37 °C for 1 additional h. Aliquots (200
µl) of each reaction mixture were removed at 15, 30, 45, and 60 min
and assayed for factor Xa using S-2222. The results of these studies
indicated that the rate of factor Xa formation in all three systems was
essentially identical, providing strong evidence that cell-surface
hamster tissue factor was not participating in the factor VIIa-mediated
activation of factor X on these cells.
Having demonstrated that the
Hepsin-1/BHK cells support the activations of factors VII and X in
presumably a tissue factor-independent reaction, we next examined
whether Hepsin-1/BHK cells support the conversion of prothrombin to
thrombin in the presence of plasma levels of factor VII, factor X and
prothrombin. In the presence of prothrombin alone, Hepsin-1/BHK cells
generated small amounts of thrombin (10 pM in 60 min),
while no thrombin formation was observed in this time period on Zem
229R-transfected BHK cells (data not shown). With the addition of
factors VII and X, significant amounts of thrombin (
150 pM in 60 min) were generated on Hepsin-1/BHK cell monolayers (Fig. 6), while Zem-transfected BHK cells under these conditions
generated approximately 10 pM thrombin in 60 min. When factor
VII was deleted from the Hepsin-1/BHK cell system containing factor X
and prothrombin, or replaced with an equal concentration of S344A
factor VII, thrombin formation still occurred, but only 20% of that
seen in the presence of functional factor VII (Fig. 6). Thus,
while hepsin apparently can generate low levels of factor Xa and
thrombin in the absence of factor VII, our findings indicate that the
activation of factor VII by cell-surface hepsin markedly enhances
subsequent factor Xa and thrombin formation on these cells.
Figure 6:
Activation of prothrombin by Hepsin-1/BHK
cells and untransfected BHK cells in the presence of various
combinations of prothrombin, factor X, factor VII, and S344A factor
VII. Hepsin-1/BHK cells (,
,
) and untransfected BHK
cells (
) were incubated with either mixtures of 160 nM factor X, 10 nM factor VII, and 1 µM prothrombin (
,
), 160 nM factor X and 1
µM prothrombin (
), or 160 nM factor X, 10
nM S344A factor VII, and 1 µM prothrombin
(
). At selected times, thrombin formed in the supernatant was
determined as described under ``Experimental
Procedures.''
In the present study, we have transfected BHK cells with a
plasmid containing the cDNA of human hepsin and investigated the
ability of these Hepsin-1/BHK cells to initiate and support coagulation
reactions. Evidence is presented herein that cell-surface-expressed
hepsin converts single-chain zymogen factor VII to two-chain factor
VIIa as a result of proteolytic cleavage of the
Arg-Ile
peptide bond in factor VII. In
addition to the activation of factor VII, we provide evidence that
factor VIIa generated on the Hepsin-1/BHK cell-surface activates factor
X in what appears to be a tissue factor-independent reaction. Factor Xa
formed subsequently converts prothrombin to thrombin in the absence of
exogenous factor V/Va and phospholipids. Thus, in addition to its
ability to regulate cell growth and maintain cell morphology (11) , cell-surface hepsin may be involved physiologically in
initiating the extrinsic pathway of blood coagulation that ultimately
leads to thrombin formation on these cells in the absence of
demonstrable tissue factor.
The activation of factor VII by
cell-surface hepsin was found to be calcium dependent with maximal
activation occurring at 2-5 mM CaCl.
Preliminary data strongly suggest that this calcium requirement, in all
likelihood, involves the calcium-dependent binding of factor VII to
anionic phospholipids on the cell-surface in close proximity to the
extracellular serine protease domain of hepsin. In this regard,
Hepsin-1/BHK cells failed to cleave and activate a gla-domainless
derivative of factor VII under conditions of optimal factor VII
activation (data not shown). In addition, human prothrombin fragment 1,
as well as the isolated human factor VII gla-peptide, inhibited the
activation of factor VII by Hepsin-1/BHK cells in a dose-dependent
manner. In these latter studies, 10 µM factor VII
gla-peptide and 10 µM prothrombin fragment 1 inhibited
factor VII activation by Hepsin-1/BHK cells 40 and 70%, respectively.
Accordingly, these results suggest that factor VII associates with the
cell-surface in a calcium-dependent reaction to kinetically increase
its rate of activation by cell-surface hepsin.
Our data indicating
that factor VII activating activity was observed exclusively in the
total membrane fraction of Hepsin-1/BHK cells confirms the previous
finding of Tsuji et al.(10) who demonstrated that
hepsin in an integral membrane protease in a molecular orientation of
type II membrane-associated proteins. These investigators also showed
that immunoreactive hepsin was virtually all removed from the HepG2
cell-surface after a brief incubation of the cells with 100 µg/ml
trypsin at 0 °C. In contrast, our data indicate that hepsin factor
VII activating activity on the Hepsin-1/BHK cell line was surprisingly
resistant to trypsin digestion, as 20% of hepsin activity still
remained after incubation of these cells with 500 µg/ml trypsin for
30 min at 37 °C. The reason(s) for this discrepancy is not readily
apparent, but may relate to the characteristics of the peptide-specific
antibody used by Tsuji et al.(10) for immunoblot
analysis. In this regard, the antibody used by Tsuji et al.(10) for this experiment was raised against a synthetic
peptide comprising the carboxyl-terminal region of human hepsin
(Glu
-Leu
), and it is conceivable that
trypsin readily cleaves the Lys
-Th
peptide
bond in cell-surface hepsin resulting in loss of immunoreactivity
without appreciably decreasing its proteolytic activity. Alternatively,
our Hepsin-1/BHK cells may be expressing severalfold higher levels of
hepsin in comparison to the HepG2 cells, and this difference may
account for the relatively high levels of residual cell-surface hepsin
activity following trypsinization observed in this study.
Preliminary immunoblot analyses of solubilized Hepsin-1/BHK cell
total membrane fraction using rabbit anti-MBP/hepsin IgG indicated the
presence of thee immunoreactive bands with apparent molecular weight
values of 55,000, 48,000, and 33,000. These three immunoreactive bands
were also observed in solubilized total membrane fractions derived from
an equivalent number of Zem 229R-transfected BHK cells but at an
intensity of 10% of that seen for the Hepsin-1/BHK cell membranes.
Presumably, the latter bands represent cross-reactivity of
anti-MBP/human hepsin IgG with hamster hepsin as was originally
reported by Tsuji et al.(10) using peptide-specific
antibodies based on the human hepsin sequence. The presence of
endogenous hepsin on BHK cells naturally raises the question as to why
these cells did not support factor VII activation in proportion to
their hepsin content. While the answer to this question will require
further experimentation, it is conceivable that the activation of
factor VII by hepsin may be a species-specific reaction.
One
interesting finding of our studies is the fact that newly formed factor
VIIa converts appreciable amounts of factor X to factor Xa on the
Hepsin-1/BHK cell-surface in the absence of tissue factor. While
several studies have demonstrated the slow activation of factor X by
factor VIIa in the presence of calcium and
phospholipids(4, 5, 41, 42) , our
data corroborate a recent report that certain cell types can support
the tissue factor-independent activation of factor X by factor
VIIa(43) . In the latter study, human monocytes, but not an
endothelial cell line, accelerated the activation of factor X by factor
VIIa in a reaction that was not inhibited by either anti-tissue factor
antibody or a 100-fold molar excess of prothrombin fragment
1(43) . The inability of prothrombin fragment 1 to inhibit this
reaction suggests that monocytes express a specific factor VIIa-binding
protein that augments factor X activation by factor VIIa, rather than
binding of factor VIIa to anionic cell-surface phospholipid through its
-carboxyglutamic acid domain. With respect to our results, it is
conceivable that Hepsin-1/BHK cells synthesize and express cell-surface
hamster tissue factor that weakly interacts with nascent human factor
VIIa and augments the activation of factor X by factor VIIa. Our
evidence, although indirect, suggests that this is not the case as
neither Zem-transfected BHK cells nor factor VII-transfected BHK cell
total membranes supported factor VII activation, assuming that
cell-surface hamster tissue factor would support autoactivation of
factor VII as well as it would support factor X activation by factor
VIIa. In addition, pretreatment of Hepsin-1/BHK cells with high levels
of goat anti-rabbit tissue factor apoprotein IgG, an antibody shown to
strongly inhibit the prothrombin time of hamster plasma and hamster
brain thromboplastin, had no measureable effect on the ability of these
cells to generate factor Xa in the presence of factors VIIa and X (data
not shown).
Our studies do not shed light on either the mechanism of
hepsin activation or the number of functional, activated hepsin
molecules on the surface of the Hepsin-1/BHK cells. The findings of
Leytus et al.(9) predict that hepsin is synthesized
as an inactive zymogen that is converted to an active serine protease
by cleavage of the Arg-Ile
peptide bond in
the extracellular domain of hepsin. The identity or the origin of the
protease responsible for this cleavage on our Hepsin-1/BHK cells is not
known. One possibility includes an exogenous source such as proteases
present in the fetal calf serum used in culturing these cells.
Alternatively, hepsin may be synthesized as a single-chain zymogen but
undergoes intracellular cleavage and activation prior to insertion into
the membrane. In light of our results demonstrating inhibition of
factor VII activating activity on Hepsin-1/BHK cells by antithrombin
III, it is also uncertain as to what extent the cell-surface-activated
hepsin is inactivated by antithrombin III (or other serpins) present in
the 10% fetal calf serum. It is perhaps worthwhile to mention that our
Hepsin-1/BHK cells were initially cultured in T-75 flasks and dislodged
from this flask for subculturing by gentle bumping in EDTA-containing
buffer rather than trypsinization. Subsequent treatment of these cells
with trypsin, however, failed to result in a measurable transient
increase in factor VII activating activity that preceded the
time-dependent decline in the ability of the cell to activate factor
VII. This result suggests that either all cell-surface hepsin was fully
activated or that simultaneous degradation and activation of hepsin
occurred precluding any measurable increase in activated hepsin
activity under these conditions. Accordingly, the efficacy of factor
VII activation by cell-surface hepsin is unknown at this point and will
presumably require the isolation of hepsin to homogeneity in order to
address this important question. In preliminary studies, however, the
ability of Hepsin-1/BHK cells to activate factor VII was equivalent to
that seen for factor VII activation on untransfected BHK cells in the
presence of either 20 pM factor Xa or 10 nM factor
IXa (data not shown).
Our finding that cell-surface-expressed hepsin activates factor VII leading to significant thrombin formation on these cells may be relevant to coagulation activation and fibrin deposition in situ on certain tumor tissues and thereby contribute to the progression of malignancy. While tissue factor synthesis and expression by several tumor types has been implicated as the major contributor to fibrin deposition on these tumor cells, there are specific instances of tumor tissues that support fibrin formation yet lack immunoreactive tissue factor(7) . In some of these tumor types, such as malignant melanoma, functional factor Xa has been observed without detectable tissue factor and factor VII antigen(44) , suggesting that these cells synthesize cancer procoagulant, a cysteinyl protease(45) , that activates factor X directly. In other tumor types, such as renal cell carcinoma, significant fibrin was observed in tumor stroma despite the absence of demonstrable tissue factor antigen on these tumor cells (46) . It is tempting to speculate that hepsin synthesis may be dramatically up-regulated in these, and other, tissue factor-deficient tumor cells and contribute to coagulation activation on these cells. In this regard, efforts to address this possibility using hepsin-specific antibodies and immunohistochemical techniques are currently ongoing in our laboratory.