(Received for publication, August 7, 1995)
From the
The relative contribution of the finger/growth factor domains of
tissue-type plasminogen activator (t-PA) and of the other t-PA domains
to the clearance of t-PA by hepatocytes was investigated. A recombinant
finger/growth factor construct inhibited t-PA and t-PA/plasminogen
activator inhibitor type-1 degradation with an IC of 1800
nM, whereas a t-PA mutant lacking the finger and growth factor
domains inhibited degradation with an estimated IC
of 1200
nM. In comparison the IC
of t-PA was found to be
approximately 10 nM. Clearance of t-PA by human or rat
hepatoma cells was not inhibited by high concentrations of fucose (50
mM), which suggests that the fucose on Thr-61 is not involved
in clearance by these cells.
These results suggest that the binding of t-PA involves several low affinity binding sites located on distinct domains of the t-PA molecule.
Tissue-type plasminogen activator (t-PA) ()is
responsible for the degradation of intravascular fibrin deposits. Its
plasma activity is regulated by the rate of its release from the
vascular endothelium, its inhibition by plasminogen activator inhibitor
type-1, and by its rapid hepatic clearance. The latter is mediated to a
large extent by the low density lipoprotein receptor related protein
(LRP)(1, 2, 3, 4) . Free and
PAI-1-complexed t-PA bind to LRP at or near the second cluster of eight
complement-type cysteine-rich repeats(5) . The region of t-PA
mediating the interaction with LRP has not yet been identified with
certainty. Deletion of the finger and growth factor domains leads to a
reduced rate of plasma clearance, suggesting a role for one or both of
these domains(6, 7, 8) . Also, mutation of
Tyr-67 in the growth factor domain (9) affects t-PA clearance.
However, some mutations or deletions in other parts of the t-PA
molecule also lead to a diminished rate of clearance(10) ,
suggesting that t-PA contains more than one receptor binding site or
that the loss of binding to the clearance receptor after deletion of
one domain is due to conformational modifications. A more precise
approach to identify the receptor binding domain(s) would be to
determine the binding affinity of individual domains or domain
clusters.
The aim of the present work was to determine the relative
contribution of the finger/growth factor domains and of the kringle
1/kringle 2/protease domains to the interaction of t-PA with its
clearance receptor. To this end, the inhibitory effects of a t-PA
mutant lacking the finger and growth factor domains (t-PAFG) and
of a recombinant finger/growth factor construct (FG) on the binding
and/or degradation of free and PAI-1 complexed t-PA by hepatocytes were
investigated. The results show that the finger and/or the growth factor
domains interact with the clearance receptor and that additional
binding sites may be located elsewhere on the t-PA molecule.
Previously we observed that t-PAFG at 100 nM had little or no effect on the binding to and degradation by rat
hepatoma cells of free and PAI-1 complexed
I-t-PA(2) . This suggested binding via the finger
or growth factor domain. We therefore investigated the effect of a
recombinant finger/growth factor construct on the degradation of
I-t-PA and
I-t-PA/PAI-1 by Novikoff cells.
Results indicated that FG is able to completely block degradation of
free
I-t-PA (Fig. 1A), but its IC
was 2 orders of magnitude higher than that for recombinant t-PA
(apparent IC
of 1800 nMversus 10.9
nM). The degradation of
I-t-PA/PAI-1 was fully
inhibited by t-PA, with an apparent IC
of 21.4
nM, and partially by FG with an apparent IC
of
1500 nM and an uninhibitable part of
I-t-PA/PAI-1 degradation of 26% (Fig. 1B).
Figure 1:
Effect
of FG on the degradation of I-t-PA and
I-t-PA/PAI-1 by Novikoff cells. 1 million cells in 200
µl of Krebs/BSA were incubated with 10 µl of buffer containing
10,000 cpm (0.1 nM) of
I-t-PA (A) or
I-t-PA/PAI-1 (B) and 40 µl of different
concentrations of t-PA (closed circles) or FG (open
squares) in the appropriate dilution buffers. After 2-h incubation
at 37 °C the cell supernatant was isolated by centrifugation and
analyzed for 10% trichloroacetic acid-soluble material. The results
represent the mean ± S.E. of at least three independent
triplicate experiments. The solid lines represent theoretical
inhibition curves calculated using IC
values for t-PA
(10.9 and 21.4 nM for inhibition of degradation of t-PA and
t-PA/PAI-1, respectively) or FG (1800 and 1500 nM,
respectively) that were obtained using the Ultrafit
program.
FG inhibited the binding of I-t-PA to liver membranes prepared from normal human
liver, with an apparent IC
of 2400 nM and an
uninhibitable part of binding of 15%, whereas t-PA inhibited binding by
50% at a concentration of 23 nM (Fig. 2).
Receptor-associated protein (RAP), an inhibitor of the binding of t-PA
to its hepatic clearance receptor, LRP(2, 4) , also
fully inhibited t-PA binding to human liver membranes (IC
= 6 nM).
Figure 2:
Effect of FG on the binding of I-t-PA to human liver membranes. Human liver membranes
(10 µl; 300 µg/ml) were incubated with 10 µl of
I-t-PA (40 fmol) and 10 µl of buffer containing
various concentrations of unlabeled t-PA (closed circles), of
unlabeled FG (open squares), or of unlabeled RAP (open
triangles). After 15 min at 37 °C, membranes were centrifuged
and membrane-associated radioactivity determined. Results are expressed
as percentage of specific binding (which represents 85% of total
binding). The solid lines represent theoretical inhibition
curves calculated using IC
values for t-PA (23
nM) or FG (2400 nM) that were obtained using the
Ultrafit program.
The low affinity of the FG construct
led us to re-evaluate the effect on t-PA degradation by Novikoff cells
of t-PAFG concentrations up to 1 µM, the maximal
concentration at which t-PA
FG was soluble at the experimental
conditions. At 1 µM a partial inhibition of t-PA
degradation was observed. Curve fitting suggested an IC
of
1200 nM (Fig. 3).
Figure 3:
Effect of t-PAFG on the degradation
of
I-t-PA by Novikoff cells. 1
10
cells in 250 µl of Krebs/BSA were incubated at 37 °C with
0.1 nM
I-t-PA in the presence of various
concentrations of unlabeled t-PA
FG. After 2 h the cell supernatant
was isolated by centrifugation and analyzed for 10% trichloroacetic
acid-soluble material. The results represent the mean ± S.E. of
at least three independent triplicate experiments. The solid line represents the theoretical inhibition curve calculated using the
IC
for t-PA
FG (1200 nM) that was obtained
using the Ultrafit program.
To determine whether the
finger/growth factor part of t-PA could cooperate with the remainder of
the t-PA molecule (t-PAFG) in binding to the clearance receptor,
we performed clearance inhibition experiments in the presence of both
molecules. At concentrations of 350 nM, the inhibitory effect
of the combination of t-PA
FG and FG was only slightly greater than
that of either competitor alone (Table 1).
One study observed that 50 mM fucose blocked the binding and degradation of t-PA by HepG2 cells in suspension(14) , suggesting that the fucose on Thr-61 is involved. As the FG construct used in the present study lacks this fucose, we studied the effect of fucose (up to 50 mM) on t-PA binding and degradation by rat hepatoma cells; no effect was observed (Fig. 4). We also studied the effect of different concentrations of D-fucose on t-PA degradation by adherent HepG2 cells and by HepG2 cells in suspension. At 50 mMD-fucose, the highest concentration which was not cytotoxic, we observed only a modest decrease in t-PA degradation: 88% of control values for HepG2 cells in suspension and 89% for adherent HepG2 cells at 4-h incubation.
Figure 4:
Effect of D-fucose on the binding
and degradation of I-t-PA by Novikoff cells. 1
10
cells in 250 µl of Krebs/BSA were incubated at 37
°C with 0.1 nM of
I-t-PA in the presence or
absence of 100 nM unlabeled ligands. At timed intervals,
cell-associated radioactivity was determined and the cell supernatant
analyzed for 10% trichloroacetic acid-soluble material. A,
percentage of
I-t-PA binding; B, trichloroacetic
soluble radioactivity in the supernatant in the absence (open
circles) or presence of 100 nM unlabeled t-PA (closed
circles) or 50 mMD-fucose (closed
squares). The results represent the mean ± S.E. of at least
three independent triplicate experiments.
The present study was undertaken to identify the domains on the t-PA molecule that interact with its clearance receptor on rat hepatoma cells and on human liver membranes. The strong inhibitory effect of RAP on t-PA binding to human liver membranes suggests that LRP is the principal specific binding site of t-PA on liver membranes. The hepatoma cell model and the human liver membrane model thus appear to address the same receptor system.
For the competition experiments
two complementary constructs were used. The FG construct was chosen
because its secondary structure is known (12) . The t-PAFG
construct represents the remainder of the t-PA molecule. The FG
construct completely inhibited t-PA degradation by Novikoff cells and
binding to human liver membranes, but at concentrations 2 orders of
magnitude higher than for t-PA (degradation by Novikoff cells:
IC
of 1800 for FG versus 10.9 nM for
t-PA; binding to liver membranes: 2400 nMversus 23
nM for t-PA). Similarly most of t-PA/PAI-1 degradation by
Novikoff cells could be inhibited by FG (IC
of 1500
nMversus 21.4 nM for t-PA), but part of
t-PA/PAI-1 degradation was not inhibitable by FG, which suggests the
presence of binding sites on the t-PA/PAI-1 complex not involving the
FG domains. Taken together, these results, as well as our previous
observation that monoclonal antibodies to the growth factor domain
inhibit t-PA clearance by hepatoma cells(13) , provide clear
evidence that the finger and/or the growth factor domains interact with
the t-PA clearance receptor. However, the poor affinity of the
finger/growth factor construct suggests that: 1) other domains of t-PA
contribute to binding (see below), 2) the interaction of finger and
growth factor domains with other t-PA domains (15) is important
for binding of these domains to the clearance receptor, or 3) essential
posttranslational modifications were not made in yeast.
The
consistent inhibitory effect of high concentrations of t-PAFG on
t-PA degradation suggests that this part of the molecule contains a low
affinity binding site. The results, however, should be interpreted with
caution. For a precise determination of IC
it is essential
to employ t-PA
FG concentrations well above the estimated IC
of 1200 nM. However, under the conditions of in
vitro degradation of t-PA by hepatoma cells, precipitation of
t-PA
FG was observed above 1 µM. Thus, the IC
of 1200 nM should be considered a preliminary estimate
rather than a definitive value. Our previous observation that
monoclonal antibodies to the kringle 2 domain interfered with t-PA
clearance (13) is in agreement with the hypothesis that other
domains contribute to the binding to the clearance receptor. We
observed no cooperative interaction between FG and t-PA
FG, which
suggests that these t-PA fragments do not interact with each other
under the experimental conditions.
Recently Hajjar and Reynolds (14) reported that the fucose group on the growth factor domain mediates binding of t-PA to human HepG2 hepatoma cells. However, we observed no inhibitory effect of fucose, even at high concentrations. This suggests that the low affinity of the nonfucosylated FG domain construct is not due to the lack of fucose on Thr-61. These results are in agreement with the observation that t-PA mutants, in which Thr-61 was mutated, efficiently inhibited the degradation of t-PA by human smooth muscle cells(16) .
In conclusion, our results provide clear evidence in favor of a role of the finger and/or the growth factor domains in the interaction of t-PA with its clearance receptor. Results also suggest the presence of other binding sites located on the remainder of the t-PA molecule. The low affinity of the different binding sites may complicate their precise identification.