From The Scripps Research Institute, La Jolla, California 92037, ¶ The University of Notre Dame, Notre Dame 46556, Indiana, and the § Institut De Recerca Oncologica, 08907 Barcelona, Spain
Received for publication, February 13, 2001, and in revised form, March 21, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
When Glu-plasminogen is
bound to cells, plasmin (Pm) formation by plasminogen (Pg) activators
is markedly enhanced compared with the reaction in solution. It
is not known whether the direct activation of Glu-Pg by Pg activators
is promoted on the cell surface or whether plasminolytic conversion of
Glu-Pg to the more readily activated Lys-Pg is necessary for enhanced
Pm formation on the cell surface. To distinguish between these
potential mechanisms, we tested whether Pm formation on the cell
surface could be stimulated in the absence of conversion of Glu-Pg to
Lys-Pg. Rates of activation of Glu-Pg, Lys-Pg, and a mutant Glu-Pg,
[D646E]Glu-Pg, by either tissue Pg activator (t-PA) or urokinase
(u-PA) were compared when these Pg forms were either bound to human
umbilical vein endothelial cells (HUVEC) or in solution.
([D646E]Glu-Pg can be cleaved at the
Arg561-Val562 bond by Pg activators but
does not possess Pm activity subsequent to this cleavage because of the
mutation of Asp646 of the serine protease catalytic triad.)
Glu-Pg activation by t-PA was enhanced on HUVEC compared with the
solution phase by 13-fold. In contrast, much less enhancement of Pg
activation was observed with [D646E]Glu-Pg (~2-fold). Although the
extent of activation of Lys-Pg on cells was similar to that of Glu-Pg,
the cells afforded minimal enhancement of Lys-Pg activation compared with the solution phase (1.3-fold). Similar results were obtained when
u-PA was used as activator. When Glu-Pg was bound to the cell in the
presence of either t-PA or u-PA, conversion to Lys-Pg was observed, but
conversion of ([D646E]Glu-Pg to ([D646E]Lys-Pg was not detected,
consistent with the conversion of Glu-Pg to Lys-Pg being necessary for
optimal enhancement of Pg activation on cell surfaces.
Furthermore, we found that conversion of [D646E]Glu-Pg to
[D646E]Lys-Pg by exogenous Pm was markedly enhanced (~20-fold) on
the HUVEC surface, suggesting that the stimulation of the conversion of
Glu-Pg to Lys-Pg is a key mechanism by which cells enhance Pg activation.
When Glu-plasminogen
(Glu-Pg),1 the native
circulating form of the zymogen, is bound to cell surfaces, plasmin
(Pm) generation by plasminogen (Pg) activators is markedly stimulated
compared with the reaction in solution (1-10). This results in arming
cell surfaces with the proteolytic activity of Pm. In the case of
endothelial cells, Pm becomes localized to sites of thrombus formation
and, in the case of leukocytes, the cells become armed with proteolytic activity required for processes in which cells degrade extracellular matrices to migrate. However, a key component of the mechanism of
stimulation of Pg activation on the cell surface is not understood. It
is not known whether: 1) direct activation of Glu-Pg by Pg activators
is promoted on the cell surface or 2) plasminolytic conversion of
Glu-Pg to Lys-Pg is necessary to observe enhanced Pm formation on the
cell surface. (Pm catalyzes cleavage of Glu-Pg at the carboxyl sides of
Lys62, Arg68, Lys77 (11-13) and at
additional minor sites (14) to generate new amino termini of Pg,
resulting in Pg molecular forms that are collectively termed
"Lys-Pg". Lys-Pg is more readily activated by Pg activators (15-17).) In the first mechanism, Glu-Pg remains uncleaved, yet its
direct activation is promoted on the cell surface relative to the
solution phase, perhaps through conformational changes induced in the
molecule upon its interaction with the cell surface. In the second
mechanism, conversion of Glu-Pg by Pm to yield the more readily
activated Lys-Pg is necessary, leading to increased Pm production on
the cell surface. Furthermore, it is not known whether localization of
Glu-Pg on the cell surface enhances its conversion to Lys-Pg by Pm.
This question has been addressed also in the current study.
In previous studies, using kinetic assays (1-10) it was not
possible to distinguish between the two mechanisms listed above to
explain the stimulation of activation of Glu-Pg in the presence of
cells. In these earlier studies, cell-associated Glu-Pg was converted
to Lys-Pg (~50-60% conversion) on the surfaces of both U937
monocytoid cells (5) and HUVEC (18) in the absence of added Pg
activators or Pm. Thus, it was not possible to distinguish whether the
direct activation of Glu-Pg was enhanced on the cell surface compared
with the reaction in solution. In contrast, in our studies, Glu-Pg
remained in its native form, without conversion to Lys-Pg when bound to
HUVEC. This enabled us to address the role of conversion of Glu-Pg to
Lys-Pg in enhancement of activation on the cell surface. In the current
investigation, we employed a Pg recombinant variant, [D646E]Glu-Pg,
to assess the requirement for conversion of Glu-Pg to Lys-Pg on the
cell surface for stimulation of Pg activation. This recombinant Pg/Pm
variant can be converted to the molecular form of Pm, but does not
possess Pm activity because of the absence of the necessary Asp residue
in the serine proteolytic catalytic triad (19). Hence, [D646E]Glu-Pg
is not converted to [D646E]Lys-Pg following its cleavage by Pg
activators. [D646E]Glu-Pg was used as the inactive mutant to be as
conservative as possible in introducing an amino acid substitution into
the active site triad. The rates of activation of Glu-Pg, Lys-Pg, and
[D646E]Glu-Pg by either t-PA or u-PA were compared when these forms
were either bound to HUVEC or in solution. These experiments were
designed to distinguish between two potential mechanisms by which Pm
formation is enhanced on the cell surface compared with the soluble
phase: 1) direct activation of Glu-Pg forms by Pg activators is
promoted on the cell surface compared with the reaction in solution, or
2) plasminolytic cleavage is necessary for stimulation of Glu-Pg
activation on the cell surface. Furthermore, we examined whether
conversion of [D646E]Glu-Pg to [D646E]Lys-Pg by exogenous Pm was
enhanced on the HUVEC surface.
Proteins--
Glu-Pg was purified from fresh human blood
collected into 3 mM benzamidine, 3 mM EDTA, 100 units/ml Trasylol (Pentex Miles, Inc., Kankakee, IL), and 100 µg/ml
soybean trypsin inhibitor (Sigma). The plasma was subjected to affinity
chromatography on lysine-Sepharose (20) in phosphate-buffered saline
(0.01 M sodium phosphate pH 7.3, 0.15 M NaCl)
with 1 mM benzamidine, 0.02% NaN3, and 3 mM EDTA, followed by molecular exclusion chromatography on
Biogel A 1.5 M (Bio-Rad, Hercules, CA). The Pg
concentration was determined spectrophotometrically at 280 nm using an
extinction coefficient of 16.8. Lys-Pg was from the National Institute
for Biological Standards and Control (Holly Hill, Hampstead, London).
The Lys-Pm control was prepared by incubating Lys-Pg with 10 nM high molecular weight (hmw) u-PA. (Calbiochem, San
Diego, CA) for 30 min at 37 °C. Pm was from Amersham Pharmacia
Biotech/Chromogenix (Uppsala, Sweden). [D646E]Glu-Pg was generated by
primer-directed mutagenesis of single-stranded p119/HPg (21) as
previously described (22) and expressed in baculovirus-infected
lepidopteran cells, followed by purification on lysine-Sepharose as
described (20). The characteristics of this mutant have been described
(19). Pg forms were radiolabeled using a modified chloramine T
procedure as described (1). t-PA was from Genentech (South San
Francisco, CA). Low molecular weight (lmw) u-PA was from Calbiochem
(San Diego, CA).
Cells, Cell Culture, and Ligand Binding Analyses--
HUVEC were
purchased from Clonetics/BioWhittaker (Walkersville, MD) and cells of
passage four and below were used in these experiments. HUVEC were grown
to confluence in MCDB 131 medium containing 2% fetal calf serum, 12 µg/ml bovine brain extract, 50 ng/ml amphotericin B, 50 µg/ml
gentamicin, 1 µg/ml hydrocortisone, 1 ng/ml human epidermal growth factor.
The binding of radiolabeled Pg forms to HUVEC was performed as
previously described from our laboratory (23). Briefly, HUVEC, grown to
confluence in 24-well culture dishes (6-8 × 104
cells/cm2), were washed three times with HBSS. The
radiolabeled Pg forms were incubated with the cells in a final total
volume of 200 µl at a final concentration of 25 nM.
Reactions were terminated by aspirating the fluid from the wells and
rapidly washing the cultures twice with HBSS-BSA. The cell-bound
radioactivity was extracted with 100 µl of reduced sample buffer
(31.2 mM TrisHCl, pH 7.2, 2% SDS, 10% sucrose, 0.002%
bromphenol blue, 15 mM dithiothreitol, 10 mM
EDTA, 10 mM benzamidine, 2 mM
phenylmethylsulfonyl fluoride, 10 µg/ml soybean trypsin inhibitor
(Sigma), 0.02% Na azide, 5 units/ml Trasylol (Miles, Inc., Kankakee,
IL)). Greater than 90% of the cell-associated ligand was eluted by
this procedure as assessed by comparing counts bound to the cells prior
to elution, with counts eluted. Samples were subjected to 7% SDS-PAGE
under reducing conditions that distinguish two-chain Pm from the single chain Pg and were exposed to Biomax MR film. The autoradiograms were
scanned on an Alpha ImagerTM 2000.
Role of the Glu-Pg to Lys-Pg Conversion in Pg Activation by t-PA on
the Cell Surface--
To monitor the activation of
125I-Glu-Pg, 125I-[D 646E]Glu-Pg and
125I-Lys-Pg by t-PA, SDS-PAGE was employed because kinetic
assays could not be performed on [D646E]Glu-Pg because its Pm form is proteolytically inactive. We found that in the absence of added t-PA,
>95% of the added 125I-Glu-Pg and
125I-[D646E]Glu-Pg remained in their Glu-Pg forms, and Pg
activation was not detected on these cells (Fig.
1). Activation of the three ligands by 20 nM t-PA was compared on HUVEC with the reaction in the
solution phase, in the absence of cells. The cell-bound ligand was
recovered and subjected to 7% SDS-PAGE under reducing conditions,
which distinguish native Glu-Pg from Lys-Pg and distinguish the heavy
chains of Glu- and Lys-Pm (Fig. 1). Greater than 90% of the cell-bound
ligand was recovered by the elution procedure. The percent Pm formation
was calculated by dividing the sum of the densities of the Glu-Pm
heavy chain and Lys-Pm heavy chain bands by the sum of the densities of
the Glu-Pg and Lys-Pg bands and the Glu-Pm and Lys-Pm heavy chain
bands. (The light chain of Pm does not incorporate 125I in
proportion to the heavy chain and was not used in the calculation of
100% cell-associated ligand). The activation of
125I-Glu-Pg was enhanced ~13-fold at 10 min compared with
the reaction in the solution phase (Fig. 1, panel A and Fig.
2). (Enhancement was observed at later
time points, also, but the extent of enhancement was limited because
the percent Pm formation that could be attained was finite.) The
predominant form of the Pm heavy chain corresponded to the Lys-Pm heavy
chain consistent with production of Pm activity (Fig. 1, panel
A). Both Glu-Pg and Lys-Pg were present on the cell surface
following t-PA activation, although Lys-Pg formation was not detected
when the reaction was carried out in solution. Lys-Pg
accounted for 27% of the total counts in cell-bound Pg at 10 min and
40% of the total counts in cell-bound Pg at 20 min. A similar
distribution of Glu-Pg and Lys-Pg was observed also at 60 min and at
120 min.
From the foregoing experiments, as in previous studies with Pg
activation by u-PA on U937 cells (5), it was not possible to determine
whether Glu-Pg or Lys-Pg was the predominant substrate for t-PA. To
address this issue, cleavage of 125I-[D646E]Glu-Pg by
t-PA was analyzed. Cleavage of cell-associated 125I-[D646E]Glu-Pg by t-PA was only 2-fold greater than
the reaction in the absence of cells at 10 min (Fig. 1, panel
B and Fig. 2). The predominant form of the Pm heavy chain was the
125I-[D646E]Glu-Pm form, and no conversion of
125I-[D646E]Glu-Pg to 125I-[D646E]Lys-Pg
was observed on the cell surface, consistent with the absence of Pm
activity. Notably, the extent of Pm formation when
125I-[D646E]Glu-Pg was bound to the cells was less than
that observed when 125I-Glu-Pg was bound to the cells
(Figs. 1 and 2). The extent of cleavage of cell-associated
125I-Lys-Pg by t-PA was similar to that of
125I-Glu-Pg (Fig. 1, panels A and C
and Fig. 2). However, at 10 min, the HUVEC-stimulated activation of
Lys-Pg was only 1.3-fold greater than the reaction in solution. The
cells afforded only this minimal enhancement of Lys-Pg activation by
t-PA, compared with the solution phase, because Lys-Pg was activated
more rapidly in solution than was Glu-Pg. (At 10 min, 51% of the
Lys-Pg in solution was cleaved to Pm whereas only 3% of the Glu-Pg in
solution was cleaved to Pm).
To ensure that the increase in Pm associated with the cells was not
because of a higher affinity or capacity of the cells for Pm
versus Pg, we compared the number of molecules of
125I-plasmin(ogen) forms bound to the cells in either the
presence or absence of t-PA (Table I).
The molecules of 125I-Glu-Pg bound to the cells did not
increase in the presence compared with the absence of t-PA.
Furthermore, in the presence of t-PA, the molecules of ligand bound to
the cell were not greater when cell-bound 125I-Glu-Pg was
activated by t-PA compared with treatment of cell-bound 125I-[D646E]Glu-Pg with t-PA. These data suggest that the
enhanced formation of Pm versus Pg on the cell surface could
not be accounted for by enhanced binding of Pm to the cells.
Role of the Glu-Pg to Lys-Pg Conversion in Pg Activation by u-PA on
the Cell Surface--
We also examined cleavage of the Pg forms by
another Pg activator, hmw u-PA. Cleavage of Glu-Pg to Pm by hmw u-PA
(10 nM) was stimulated 4-fold at 10 min, when Glu-Pg was
bound to the HUVEC surface compared with the reaction in solution (Fig.
3, panels A and B).
The predominant form of the Pm heavy chain was Lys-Pm (Fig. 3,
panel A). In addition, Lys-Pg accounted for 43% of the
uncleaved Pg on the cell surface. In contrast, cleavage of
cell-associated [D646E]Pg was still markedly less than cleavage of
cell-associated 125I-Glu-Pg and cleavage of cell-associated
[D646E]Pg was not enhanced compared with the reaction in solution
(Fig. 3). The formation of [D646E]Lys-Pg was not detected on the cell
surface. The percentage cleavage of Lys-Pg on the cell surface was
similar to that of Glu-Pg, but cleavage of Lys-Pg in solution was
markedly greater than that of Glu-Pg. (At 10 min, 71% of the Lys-Pg in
solution was cleaved to Pm whereas only 17% of the Glu-Pg in solution
was cleaved to Pm). Hence, cleavage of Lys-Pg was enhanced only
1.1-fold.
The foregoing data showed that cell-associated
125I-[D646E]Glu-Pg was less readily cleaved than either
cell-associated 125I-Glu-Pg or 125I-Lys-Pg,
which is consistent with less direct cleavage of Glu-Pg forms compared
with Lys-Pg. However, the case of 125I-[D646E]Glu-Pg was
also distinct from that of the other ligands because Pm was not
produced following cleavage by the Pg activators. Pm cleaves
single-chain t-PA to two-chain t-PA (24), although the catalytic
efficiency of both forms are virtually identical (24). Pm also cleaves
hmw u-PA to lmw u-PA, which also retains catalytic activity (25). To
exclude other potential effects of cleavage of the Pg activator by Pm
as an explanation for our results, we compared cleavage of
125I-Glu-Pg and 125I-[D646E]Glu-Pg by lmw
u-PA (which is not cleaved by Pm). The cleavage of these Pg forms by
lmw u-PA was compared when the ligands were either bound to the HUVEC
or in solution (Fig. 4, panels A and B). The extent of Pm formation when
cell-associated Glu-Pg was activated with lmw u-PA was similar to that
when cell-associated Glu-Pg was activated with hmw u-PA (compare Figs.
3 and 4). When cell-associated Glu-Pg was activated with lmw u-PA, the
predominant form of the Pm heavy chain was Lys-Pm and Lys-Pg accounted
for 41% of the uncleaved Pg on the cell surface. Under these
conditions, activation of Glu-Pg in solution was not detected. Cleavage
of 125I-[D646E]Pg bound to HUVEC was not detected under
these conditions. These results suggest that the differences in extent
of cleavage of 125I-[D646E]Pg compared with
125I-Glu-Pg could not be ascribed to an effect of Pm
(produced in the latter reaction) on the Pg activator.
Effect of Cells on Conversion of [D646E]Glu-Pg to [D646E]Lys-Pg
by Exogenous Pm--
We examined whether localization of Glu-Pg on the
cell surface could enhance its conversion to Lys-Pg by exogenous Pm,
compared with the solution phase. 125I-[D646E]Glu-Pg was
used in this analysis to avoid any contribution of the added ligand to
the conversion. 125I-[D646E]Glu-Pg was incubated with
either buffer or adherent HUVEC in the presence of increasing
concentrations of Pm for 20 min at 37 °C. The conversion of the
cell-bound and solution phase 125I-[D646E]Glu-Pg to
[D646E]Lys-Pg was monitored by SDS-PAGE. A marked enhancement in
conversion to the Lys-form was observed when the ligand was bound to
the HUVEC, compared with the solution phase (Fig.
5). For example, under conditions where
59% of the cell-bound ligand was in the Lys-form, only 3% of the
ligand in the solution phase was in the Lys-form.
In this study we provide the first demonstration that activation
of cell-bound Glu-Pg is markedly enhanced only when its conversion to
Lys-Pg on the cell surface is permitted. This result allowed us to
distinguish between two potential mechanisms for stimulation of Pm
formation when Glu-Pg is bound to the cell surface (1-10). In the
first mechanism, the direct activation of Glu-Pg is promoted on the
cell surface. In the second mechanism, initial conversion of Glu-Pg to
Lys-Pg is necessary, so that formation of Pm is enhanced because the
more readily activated Lys-Pg becomes the predominant substrate for Pg
activators. These two potential mechanisms for this key step in cell
surface Pg activation have not been distinguished in previous studies.
With both native Glu-Pg and [D646E]Glu-Pg, we detected <5%
conversion to either Lys-Pg or [D646E]Lys-Pg following binding to
HUVEC in the absence of a Pg activator. This finding allowed us to
carry out our study to address the role of the Glu-Pg to Lys-Pg
conversion in stimulation of Pg activation on the HUVEC surface. In
contrast, in an earlier study ~50-60% of Glu-Pg was converted to
Lys-Pg on the HUVEC surface, in the absence of the addition of
exogenous Pm (18). (These studies also suggested that Pm was not the
source of proteolytic activity for conversion of Glu-Pg to Lys-Pg (5,
18)). This difference from the previously published data may be
ascribed to differences in the culture conditions of the HUVEC in
different laboratories or to differences in the added ligand. Thus, the
ability to convert Glu-Pg to Lys-Pg, in the absence of exogenous Pm or
Pg activator is not a consistent property of the HUVEC surface.
We found that when Glu-Pg was bound to the cells and activated with
either t-PA or hmw u-PA (conditions where Lys-Pg and Lys-Pm formation
occurred), the rate of Pm formation was similar to that of cell-bound
Lys-Pg. This resulted in stimulation of Glu-Pg activation 4-13-fold
compared with the reaction in solution. This first observation would be
compatible with either of the two activation mechanisms proposed above.
To distinguish between these mechanisms we studied cleavage by t-PA of
a mutant Glu-Pg, [D646E]Glu-Pg, that does not produce Pm activity
following cleavage by Pg activators, so that a [D646E]Lys-Pg is not
produced. Cleavage of [D646E]Glu-Pg on the HUVEC surface was not
markedly enhanced compared with the reaction in solution. Furthermore,
the rates of cleavage of cell-bound and soluble Lys-Pg were also
similar, suggesting that the interaction of Lys-Pg with the cells also
did not markedly stimulate its activation. Thus, in the absence of the
reaction step in which Glu-Pg is converted to Lys-Pg, Pm formation was
not markedly enhanced on the cell surface, i.e. the direct
cleavage of [D646E]Glu-Pg was not markedly stimulated upon binding to
the cell surface, and the cleavage of Lys-Pg was not markedly
stimulated on the cell surface compared with the reaction in solution.
In contrast, we observed a marked stimulation of activation of
cell-bound Glu-Pg, a reaction in which the Glu-Pg to Lys-Pg conversion
did occur.
In addition to the transition to the Lys-Pg form, the activation of
both Glu-Pg and of Lys-Pg differs from that of [D646E]Glu-Pg because
active Pm is formed in the former cases, but not in the latter case.
Therefore, we considered whether other activities of Pm might account
for the greater activation of Glu-Pg and Lys-Pg compared with the
cleavage of [D646E]Glu-Pg on the cell surface. Firstly, treatment of
U937 cells with Pm has been reported to increase the binding of Pg to
this cell type (26). In the present study we found that the molecules
of 125I-Glu-Pg bound to the cells did not increase in the
presence when compared with the absence of t-PA. Furthermore, in the
presence of t-PA, the molecules of ligand bound to the cell were not
greater when cell-bound 125I-Glu-Pg was activated by t-PA
compared with treatment of cell-bound 125I-[D646E]Glu-Pg
with t-PA. These data suggest that the enhanced formation of Pm
versus Pg on the cell surface could not be accounted for by
enhanced binding of Glu-Pg versus [D646E]Glu-Pg to the cells because of the generation of new binding sites by Pm generated in
the reaction. The differences between our data and the previous report
(26) may be ascribed to differences in responsiveness of HUVEC to Pm
and/or to lower levels of Pm generated in our experiments.
Pm also cleaves single-chain t-PA to two-chain t-PA and hmw u-PA to lmw
u-PA. Although, the catalytic efficiencies of both forms are similar
(24, 25) we excluded other potential effects of cleavage of the Pg
activator by Pm by examining cleavage of cell-bound Pg forms by lmw
u-PA (a Pg activator that is not cleaved by Pm). Glu-Pg activation was
also markedly stimulated on the HUVEC surface by lmw u-PA under
conditions where cleavage of [D646E]Glu-Pg was not detected. Because
similar differences in activation were seen using both lmw and hmw
u-PA, these results suggest that cleavage of the Pg activator by Pm did
not account for the observed differences in Pg activation on the HUVEC
surface with the different Pg forms.
The number of molecules of Lys-Pg bound to the cells compared with both
Glu-Pg and [D646E]Glu-Pg, was higher as reflected by the data in
Table I and consistent with previous reports of a higher affinity of
Lys-Pg versus Glu-Pg for cells (27, 28). However, this
increased binding did not appear to increase stimulation of Pg
activation on the cell surface because the rates of activation of
Glu-Pg and Lys-Pg (when bound to the cell surface) were similar. Although Lys-Pg and Lys-Pm were formed on the cell surface when cell-bound Glu-Pg was activated with t-PA, the number of molecules of
ligand bound/cell was not increased. This suggests that the affinity of
Glu-Pg for the cells remained the same even in the presence of t-PA
when Lys-Pg was formed on the cell surface, most likely because >94%
of the ligand in solution remained as Glu-Pg (data not shown).
Cleavage of the variant Pg, [D646E]Glu-Pg, was not markedly
stimulated when bound to the HUVEC, although [D646E]Glu-Pg was still
susceptible to cleavage by both t-PA and by u-PA when bound to the cell
surface. Thus, cleavage of [D646E]Glu-Pg by Pg activators can occur
(as it does in the solution phase) but direct cleavage of
[D646E]Glu-Pg does not appear to be stimulated upon binding of the
ligand to the cell. Analogously, a small amount of direct activation of
native Glu-Pg on the cell surface, should provide a source of Pm for
conversion of cell-bound Glu-Pg to Lys-Pg, leading to amplification of
Pg activation on these cells.
The rate of cleavage of cell-bound Lys-Pg by t-PA was similar to the
rate of cleavage of cell-bound Glu-Pg. However, binding to the cells
only minimally increased Lys-Pg activation by the Pg activators
compared with the reaction in the solution phase. The small amount of
stimulation of activation of [D646E]Glu-Pg and Lys-Pg when bound to
the cell surface could be because of the effect of concentration of
reactants as previously suggested (10), particularly because the HUVEC
express binding sites for both t-PA (29-36) and for u-PA (23, 32, 37).
Alternatively, or in addition, some common conformational change
induced in both Glu-Pg and Lys-Pg upon binding to cells may contribute
to the enhancement in activation. Furthermore, although these
experiments were performed under conditions where only a small
proportion of t-PA receptors would be saturated and at an excess of hmw
u-PA that would allow cleavage by soluble as well as by cell-bound u-PA, we cannot exclude that the effect of concentration of Pg on the
cell surface, as well as the presence of receptors for the Pg
activators is also absolutely required for stimulation of activation on
the cell surface.
Our results also suggest an additional new profibrinolytic function of
localization of plasmin(ogen) on the cell surface: Conversion of Glu-Pg
to Lys-Pg by Pm was enhanced when the ligand was cell-associated
compared with being in the solution phase. This may be because of
colocalization and concentration of Pm and Pg on the cell surface,
enhanced enzymatic activity of cell-bound Pm as described for U937
cells (26) and/or a more accessible conformation of cell-associated
Glu-Pg compared with the solution phase.
Taken together, our results suggest that the conversion of Glu-Pg to
Lys-Pg by Pm is necessary for maximal enhancement in Glu-Pg activation
on cell surfaces relative to the reaction in solution and that the
conversion of Glu-Pg to Lys-Pg is enhanced when Glu-Pg is bound to
cells. Thus, the enhancement of formation of the more readily activated
Lys-Pg allows cells to promote Pg activation on their surfaces, a key
step in both thrombolysis and in physiologic and pathophysiologic
processes involving cell migration.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (50K):
[in a new window]
Fig. 1.
Role of the Glu-Pg to Lys-Pg conversion in Pg
activation on the cell surface. A concentration of 25 nM of either 125I-Glu-Pg (A),
125I-[D646E]Glu-Pg (B), or
125I-Lys-Pg (C) was incubated with buffer
(HBSS-0.4% BSA) or HUVEC (5 × 104) in the presence
or absence of 20 nM t-PA for the indicated times at
37 °C. Cell-bound ligand was obtained as described under
"Experimental Procedures" and subjected to electrophoresis on
7% SDS-PAGE under reducing conditions.
View larger version (20K):
[in a new window]
Fig. 2.
Comparison of cleavage of Pg forms by t-PA on
HUVEC and in solution. The autoradiograms in Fig. 1 were scanned.
The percent Pm formation was calculated by dividing the sum of the
densities of the Glu-Pm heavy chain and Lys-Pm heavy chain bands by the
sum of the densities of the Glu-Pg and Lys-Pg bands and the Glu-Pm and
Lys-Pm heavy chain bands. The percent Pm in the starting material was
subtracted. Symbols are as follows: , 125I-Glu-Pg + t-PA;
, 125I-Glu-Pg + t-PA + HUVEC;
,
125I-[D646E]Glu-Pg + t-PA;
,
125I-[D646E]Glu-Pg + t-PA + HUVEC;
,
125I-Lys-Pg + t-PA;
, 125I-Lys- Pg + t-PA + HUVEC.
Effect of t-PA on plasminogen binding to HUVEC
View larger version (50K):
[in a new window]
Fig. 3.
Role of the Glu-Pg to Lys-Pg conversion in Pg
activation by hmw u-PA on the cell surface. A, a
concentration of 25 nM of either 125I-Glu-Pg,
125I-[D646E]Glu-Pg, or 125I-Lys-Pg was
incubated with buffer (HBSS-0.4% BSA) or HUVEC (5 × 104) for 10 min at 37 °C in the presence or absence of
10 nM hmw u-PA. Cell-bound ligand was obtained as described
under "Experimental Procedures" and subjected to electrophoresis on
7% SDS-PAGE under reducing conditions. B, the
autoradiograms in A were scanned. The percent Pm formation
was calculated by dividing the sum of the densities of the Glu-Pm heavy
chain and Lys-Pm heavy chain bands by the sum of the densities of the
Glu-Pg and Lys-Pg bands and the Glu-Pm and Lys-Pm heavy chain bands.
The percent Pm in the starting material was subtracted.
View larger version (32K):
[in a new window]
Fig. 4.
Role of the Glu-Pg to Lys-Pg conversion in Pg
activation by lmw u-PA on the cell surface. A, a
concentration of 25 nM of either 125I-Glu-Pg or
125I-[D646E]Glu-Pg was incubated with buffer (HBSS-0.4%
BSA) or HUVEC (5 × 104) for 10 min at 37 °C in the
presence or absence of 15 nM lmw u-PA. Cell-bound ligand
was obtained as described under "Experimental Procedures" and
subjected to electrophoresis on 7% SDS-PAGE under reducing conditions.
B, the autoradiograms in A were scanned. The
percent Pm formation was calculated by dividing the sum of the
densities of the Glu-Pm heavy chain band and Lys-Pm heavy chain band by
the sum of the densities of the Glu-Pg and Lys-Pg bands and the Glu-Pm
and Lys-Pm heavy chain bands. The percent Pm in the starting material
was subtracted.
View larger version (41K):
[in a new window]
Fig. 5.
Effect of HUVEC on conversion of
[D646E]Glu-Pg to [D646E]Lys-Pg by exogenous Pm.
125I-[D646E]Glu-Pg (25 nM) was incubated with
either HUVEC (5 × 104)( ) or buffer (
) in the
presence of the indicated concentration of Pm for 20 min at 37 °C.
The cell-bound ligand was obtained as described under "Experimental
Procedures" and subjected to electrophoresis in parallel with the
solution phase samples on 7% SDS-PAGE under reducing conditions
(A). The autoradiograms were scanned (B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENT |
---|
We thank Holly Hapworth for excellent editorial assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants HL38272, HL45934, and HL31950 (to L. A. M.) and HL13423 (to F. J. C.), American Heart Association Grant-in-aid 9650636N (to L. A. M.), and Marato-TV3/Cardiovascular and F.R. Areces (to J. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present Address: Han Nong Central Research Center, Dongbu
Advanced Research Inst., 103-2 Moonji-dong, Daeduck Science Town, Taejon 305-380 Korea.
To whom correspondence should be addressed: The
Scripps Research Inst., CVN-26 10550 North Torrey Pines Rd.,
La Jolla, CA 92037. Tel.: 858-784-7105; Fax: 858-784-7374; E-mail:
lmiles@scripps.edu.
Published, JBC Papers in Press, March 22, 2001, DOI 10.1074/jbc.M101387200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Glu-Pg, the native form of plasminogen with N-terminal Glu; [D646E]Glu-Pg, a recombinant human plasminogen with Asp646 mutated to Glu; EACA, epsilon aminocaproic acid; BSA, bovine serum albumin; HBSS, Hanks' balanced salt solution; hmw u-PA, high molecular weight urokinase; HUVEC, human umbilical vein endothelial cells; [D646E]Lys-Pg, [D646E]Glu-Pg cleaved by plasmin; lmw u-PA, low molecular weight urokinase; Lys-Pg, a proteolytic derivative of Glu-Pg with N-terminal Met68, Lys77, or Val78; Pm, plasmin; PAGE, polyacrylamide gel electrophoresis; t-PA, tissue plasminogen activator.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Miles, L. A., and Plow, E. F. (1985) J. Biol. Chem. 260, 4303-4311[Abstract] |
2. | Stricker, R. B., Wong, D., Tak Shiu, D., Reyes, P. T., and Shuman, M. A. (1986) Blood 68, 275-280[Abstract] |
3. |
Hajjar, K. A.,
Harpel, P. C.,
Jaffe, E. A.,
and Nachman, R. L.
(1986)
J. Biol. Chem.
261,
11656-11662 |
4. | Loscalzo, J., and Vaughan, D. E. (1987) J. Clin. Invest. 79, 1749-1755[Medline] [Order article via Infotrieve] |
5. |
Ellis, V.,
Behrendt, N.,
and Dano, K.
(1991)
J. Biol. Chem.
266,
12752-12758 |
6. |
Duval-Jobe, C.,
and Parmely, M. J.
(1994)
J. Biol. Chem.
269,
21353-21357 |
7. | Felez, J., Miles, L. A., Fabregas, P., Jardi, M., Plow, E. F., and Lijnen, R. J. (1996) Thromb. Haemost. 76, 577-584[Medline] [Order article via Infotrieve] |
8. | Lopez-Alemany, R., Longstaff, C., Fabregas, P., Jardi, M., Merton, E., and Felez, J. (1996) Fibrinolysis 10, Supp. 3, 5 |
9. |
Longstaff, C.,
Merton, R. E.,
Fabregas, P.,
and Felez, J.
(1999)
Blood
93,
3839-3846 |
10. |
Sinniger, V.,
Merton, R. E.,
Fabregas, P.,
Felez, J.,
and Longstaff, C.
(1999)
J. Biol. Chem.
274,
12414-12422 |
11. | Wiman, B. (1973) Eur. J. Biochem. 39, 1-9[Medline] [Order article via Infotrieve] |
12. | Wiman, B., and Wallen, P. (1973) Eur. J. Biochem. 36, 25-31[Medline] [Order article via Infotrieve] |
13. | Violand, B. N., and Castellino, F. J. (1976) J. Biol. Chem. 251, 3906-3912[Abstract] |
14. |
Horrevoets, A. J. G.,
Smilde, A. E.,
Fredenburgh, J. C.,
Pannekoek, H.,
and Nesheim, M. E.
(1995)
J. Biol. Chem.
270,
15770-15776 |
15. |
Hoylaerts, M.,
Rijken, D. C.,
Lijnen, H. R.,
and Collen, D.
(1982)
J. Biol. Chem.
257,
2912-2919 |
16. | Markus, G., Evers, J. L., and Hobika, G. H. (1978) J. Biol. Chem. 253, 733-739[Medline] [Order article via Infotrieve] |
17. | Markus, G., Priore, R. L., and Wissler, F. C. (1979) J. Biol. Chem. 254, 1211-1216[Medline] [Order article via Infotrieve] |
18. | Hajjar, K. A., and Nachman, R. L. (1988) J. Clin. Invest. 82, 1769-1778[Medline] [Order article via Infotrieve] |
19. |
Grella, D. K.,
and Castellino, F. J.
(1997)
Blood
89,
1585-1589 |
20. | Deutsch, D. G., and Mertz, E. T. (1970) Science 170, 1995-1996 |
21. | Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol. 154, 367-382[Medline] [Order article via Infotrieve] |
22. | Menhart, N., Hoover, G. J., McCance, S. G., and Castellino, F. J. (1995) Biochemistry 34, 1482-1488[Medline] [Order article via Infotrieve] |
23. | Miles, L. A., Levin, E. G., Plescia, J., Collen, D., and Plow, E. F. (1988) Blood 72, 628-635[Abstract] |
24. |
Rijken, D. C.,
Hoylaerts, M.,
and Collen, D.
(1982)
J. Biol. Chem.
257,
2920-2925 |
25. | Saksela, O., and Rifkin, D. B. (1988) Annu. Rev. Cell Biol. 4, 93-126[CrossRef] |
26. | Gonzalez-Gronow, M., Stack, S., and Pizzo, S. V. (1991) Arch. Biochem. Biophys. 286, 625-628[Medline] [Order article via Infotrieve] |
27. | Hajjar, K. A., Hamel, N. M., Harpel, P. C., and Nachman, R. L. (1987) J. Clin. Invest. 80, 1712-1719[Medline] [Order article via Infotrieve] |
28. |
Miles, L. A.,
Dahlberg, C. M.,
and Plow, E. F.
(1988)
J. Biol. Chem.
263,
11928-11934 |
29. | Hajjar, K. A., Harpel, P. C., and Nachman, R. L. (1986) Circulation 74 Suppl. II, 233 |
30. | Beebe, D. P. (1987) Thromb. Res. 46, 241-254[Medline] [Order article via Infotrieve] |
31. | Beebe, D., and Wood, L. (1989) Blood 74, 1116 |
32. |
Hajjar, K. A.,
and Hamel, N. M.
(1990)
J. Biol. Chem.
265,
2908-2916 |
33. |
Ramakrishnan, V.,
Sinicropi, D. V.,
Dere, R.,
Darbonne, W. C.,
Bechtol, K. B.,
and Baker, J. B.
(1990)
J. Biol. Chem.
265,
2755-2761 |
34. |
Russell, M. E.,
Quertermous, T.,
Declerck, P. J.,
Collen, D.,
Haber, E.,
and Homcy, C. J.
(1990)
J. Biol. Chem.
265,
2569-2575 |
35. | Felez, J., Chanquia, C. J., Levin, E. G., Miles, L. A., and Plow, E. F. (1991) Blood 78, 2318-2327[Abstract] |
36. | Fukao, H., Hagiya, Y., Nonaka, T., Okada, K., and Matsuo, O. (1992) Biochem. Biophys. Res. Commun. 187, 956-962[Medline] [Order article via Infotrieve] |
37. |
Barnathan, E. S.,
Kuo, A.,
Rosenfeld, L.,
Karikó, K.,
Leski, M.,
Robbiati, F.,
Nolli, M. L.,
Henkin, J.,
and Cines, D. B.
(1990)
J. Biol. Chem.
265,
2865-2872 |