(Received for publication, June 6, 1995; and in revised form, June 15, 1995 )
From the
Urokinase (u-PA) is synthesized and secreted as a single-chain
polypeptide (single-chain u-PA, scu-PA), which has such little
enzymatic activity in solution that it has been considered essentially
enzymatically inert. We found that plasminogen activator inhibitor type
1 (PAI-1), the major PAI in plasma, demonstrated
concentration-dependent inhibition of this solution-phase scu-PA
enzymatic activity. I-scu-PA formed complexes with PAI-1
in a concentration- and time-dependent manner, as detected by
SDS-polyacrylamide gel electrophoresis under reducing conditions. Among
a given population of scu-PA molecules, all measurable enzymatic
activity was inhibited by a 10-fold molar excess of PAI-1. However, at
this stoichiometry, only a minority of
I-scu-PA molecules
formed SDS-stable complexes with PAI-1 (i.e. complexes that
formed a covalent bond upon denaturation), even though the uncomplexed
PAI-1 molecules remained competent to inhibit u-PA enzymatic activity.
Neither the extent nor the time course of complex formation was altered
by using PAI-1 that had been pre-incubated with native human
vitronectin, compared with native PAI-1 alone.
I-scu-PA
PAI-1 complexes that would form a covalent
bond if denatured were reversible and existed in equilibrium with
either non-complexed or loosely complexed reactants. These data suggest
that scu-PA has more enzyme-like properties than previously appreciated
and raises the possibility that it resembles single-chain tissue
type-plasminogen activator in lacking a complete zymogen conformation.
Urokinase-type plasminogen activator (u-PA) ()is
involved in a wide variety of biological
processes(1, 2, 3) . It is synthesized and
secreted as a single-chain molecule (scu-PA), which has little activity
in solution (4, 5, 6, 7) but
exhibits appreciable plasminogen-activating activity when bound to its
specific cell surface receptor (6, 7) or when
plasminogen is bound to an appropriate lysine on partially degraded
fibrin(8) . The plasmin thus generated cleaves scu-PA to
two-chain u-PA (tcu-PA), which has three to four logs more enzymatic
activity than soluble scu-PA (4, 7, 8, 9) and is efficiently
inhibited by plasminogen activator inhibitors (PAIs), members of the
serpin family of protease inhibitors(10) .
There has been debate regarding the enzymatic activity of scu-PA, with the question of whether scu-PA must first be activated to tcu-PA to exhibit meaningful enzymatic activity(5, 6, 7, 11, 12) . Recent data from our laboratory described a reversible noncovalent interaction between scu-PA and plasminogen activator inhibitor type 2 (PAI-2), with resultant complete inhibition of scu-PA enzymatic activity(13) . Such an interaction suggested sufficient enough enzyme-like properties for scu-PA that specific interactions with inhibitors might be important.
Plasminogen activator inhibitor type
1 (PAI-1) is the primary PAI of human plasma, is found in platelets,
and is synthesized and secreted by a variety of cells in
culture(14) . PAI-1 circulates in plasma and exists in
extracellular matrix bound to vitronectin
(VN)(15, 16) , in which state its time-dependent loss
of inhibitory activity is attenuated as compared with isolated
PAI-1(15, 17) . PAI-1 has a high affinity for both
tcu-PA and tissue-type plasminogen activator (t-PA), effectively
limiting the enzymatic activity of these proteases (18) . The
interaction between PAI-1 and tcu-PA follows the reaction paradigm
described for serine proteases and their inhibitors. There is an
initial reversible interaction between the molecules, followed by the
formation of a ``stable intermediate'' complex wherein the
active site serine is complexed to the P residue of the
inhibitor to yield a complex that is stable to denaturing
agents(19) .
In this report, we describe interactions between scu-PA and PAI-1, present findings that support the concept that scu-PA is not an inert proenzyme, and suggest a previously undescribed level of regulation of plasminogen activation.
The following proteins were kindly provided by the individuals noted: recombinant native scu-PA by Dr. Jack Henkin (Abbott Laboratories) and recombinant human PAI-1 by Drs. David Ginsburg and Daniel Lawrence (Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI). Three batches of PAI-1 were used in these studies, exhibiting 47, 53, and >95% active PAI-1 as assessed by inhibition of tcu-PA-mediated hydrolysis of a chromogenic substrate. Human plasma VN was isolated under nondenaturing conditions by Dr. Deane Mosher (University of Wisconsin, Madison, WI), fibrinogen by Dr. Michael Mossesson (University of Wisconsin, Milwaukee, WI), and tcu-PA by Dr. Gene Murano (National Bureau of Standards, Wash., D. C.). Plasminogen was isolated from human plasma as previously described(7, 21) . Chemicals for SDS-polyacrylamide gel electrophoresis (PAGE) (22) were from Bio-Rad. All other chemicals were obtained from Sigma. Proteins were iodinated using the Iodogen method as previously described(7, 13) .
Plasminogen-activating activity was measured using the
plasmin-independent I-plasminogen cleavage assays as
previously described(13, 23) . Inhibition of scu-PA
activity was determined by comparing the generation of
I-plasmin light chain, as assessed by SDS-PAGE under
reducing conditions and autoradiography, of reactions wherein scu-PA
had been preincubated with PAI-1 to scu-PA that had been preincubated
with either ovalbumin or buffer alone.
To detect SDS-stable
complexes, I-scu-PA was incubated with PAI-1 under
conditions specified in the text. The reaction mixture was then boiled
in SDS-PAGE buffer with
-mercaptoethanol and analyzed by SDS-PAGE
followed by autoradiography. Alternatively, unlabeled proteins were
combined as above and analyzed by SDS-PAGE and silver staining. To
determine whether scu-PA and tcu-PA competed with one another for
interaction with PAI-1, varying concentrations of scu-PA were incubated
with PAI-1 in 10 µl of Tris-buffered saline (0.15 M NaCl,
0.01 M Tris, pH 7.4) for 10-15 min at room temperature.
I-tcu-PA in 5 µl of Tris-buffered saline was then
added to each reaction for either a further 10 s or 5 min at 4 °C,
followed by SDS-PAGE under reducing conditions and autoradiography.
Alternatively, to determine whether the association of scu-PA with
PAI-1 was reversible,
I-scu-PA was incubated with PAI-1
at the indicated concentrations for 2 h at 37 °C, followed by the
addition of increasing concentrations of tcu-PA for 3 h at 37 °C.
The samples were then boiled in reducing SDS-gel buffer, which
contained extra tcu-PA such that all samples had the same quantity of
all reactants when analyzed by SDS-PAGE and autoradiography.
To
determine the influence of VN on PAI-1 interactions with scu-PA, native
human VN was incubated with PAI-1 at room temperature for 2 h; this
PAI-1VN mixture was then combined with
I-scu-PA as
described in the text. Preliminary experiments showed the PAI-1:VN
mixture inhibited thrombin activity in a fluorogenic substrate-based
assay(24) , whereas PAI-1 alone did not. Hence, PAI-1
VN
complexes were present in the mixture used in these experiments.
We employed an I-plasminogen cleavage assay
carried out in the presence of high concentrations of trasylol to
monitor the generation of
I-plasmin (13, 23) to demonstrate that scu-PA was inhibited in a
concentration-dependent manner by PAI-1 but not by ovalbumin (Fig. 1). We have previously shown that
I-scu-PA
remains in the single-chain form in this assay(13) . As
demonstrated in Fig. 2A,
I-scu-PA formed
SDS-stable complexes with PAI-1 of molecular mass 98 K
. Identical SDS-stable complexes were
formed between unlabeled scu-PA and PAI-1 as determined by silver
staining of gels (Fig. 2B), indicating that such
complex formation was a property of native scu-PA and not an artifact
of iodination. tcu-PA on the other hand, formed complexes with a
molecular mass of 75 K
as expected (see Fig. 3). Hence, the scu-PA
PAI-1 complexes were not the
result of tcu-PA being generated, then complexing with PAI-1.
Figure 1:
PAI-1 inhibits the enzymatic activity
of native scu-PA. Scu-PA (1 µg) was incubated for 30 min at 37
°C with the indicated amount of PAI-1 or ovalbumin and then
combined with I-plasminogen (Pg) and trasylol
for 16 h at 37 °C as described under ``Materials and
Methods.'' Reaction mixtures were analyzed by SDS-PAGE under
reducing conditions (12% gel), followed by autoradiography to detect
the formation of
I-plasmin. Indicated on the left of the gel are
I-plasminogen, plasmin heavy chain (PnHC), and plasmin light chain (LC), the latter
being the best indicator of plasmin generation. The data are
representative of three similar
experiments.
Figure 2:
The formation of complexes between scu-PA
and PAI-1 is concentration-dependent and is a property of native as
well as iodinated scu-PA. A, a fixed amount of I-scu-PA (75 ng) was incubated with the indicated amounts
of PAI-1 for 3 h at 37 °C. Mixtures were analyzed by SDS-PAGE under
reducing conditions and autoradiography. The data are representative of
five similar experiments. B, a fixed amount of unlabeled
scu-PA was incubated with the indicated amounts of PAI-1 as in A, and analyzed by SDS-PAGE under reducing conditions,
followed by silver staining.
Figure 3:
PAI-1 that does not complex with scu-PA
remains active. I-scu-PA (75 ng) was incubated with PAI-1
(750 ng) for 2 h at 37 °C, at which point additional
I-scu-PA (75 ng) or
I-tcu-PA (75 ng) was
added to the mixtures as noted and incubated for 30 min at 37 °C,
followed by SDS-PAGE under reducing conditions and autoradiography. The
lower molecular weight bands in lanes containing
I-scu-PA are the result of degradation of the labeled
protein during storage and do not represent
I-tcu-PA
(note lack of 75-kDa complexes with PAI-1 unless exogenous
I-tcu-PA was added). The data are representative of two
similar experiments.
The presence of SDS-stable complexes between a serine protease and serpin probably reflects reaction of the tetrahedral protease-serpin intermediate upon denaturation such that a covalent ester bond between the two molecules is formed(19) . Scu-PA apparently forms such an intermediate with PAI-1, which results in covalent bond formation upon denaturation; no such intermediate seems to form between scu-PA and PAI-2, even though scu-PA enzymatic activity is inhibited by PAI-2(13) .
Interestingly, the I-scu-PA was
not quantitatively complexed to PAI-1 in an SDS-stable manner. Even a
100-fold molar excess of PAI-1 over
I-scu-PA did not
yield SDS-stable complexes with all scu-PA molecules (Fig. 2),
despite complete inhibition of enzymatic activity. This is in contrast
to the interaction of PAI-1 with tcu-PA, wherein a 10-fold excess of
PAI-1 results in all tcu-PA becoming complexed with the inhibitor in an
SDS-stable manner (data not shown). However, incubation of
I-scu-PA with a 10-fold excess of PAI-1, followed by
immunoprecipitation of the PAI-1, resulted in coprecipitation of more
than 96% of the
I-scu-PA. Therefore, all of the scu-PA
seems to complex with PAI-1, but only a fraction of the interactions
results in the formation of a stable intermediate.
To determine
whether a given population of PAI-1 molecules was unable to form more
complexes than we observed, I-scu-PA was incubated for 3
h at 37 °C with 10-fold molar excess PAI-1.
I-scu-PA,
I-tcu-PA, or buffer were then added to the incubation
mixtures for a further 30 min, followed by SDS-PAGE under reducing
conditions and autoradiography. As seen in Fig. 3, addition of
excess
I-scu-PA did not result in the formation of more
detectable complexes with PAI-1 compared with the reaction wherein
buffer was added for the second incubation. However, addition of
I-tcu-PA for the second incubation resulted in the
formation of
I-tcu-PA
PAI-1 complexes, with no
decrement in the preformed
I-scu-PA
PAI-1 complexes (i.e. the data do not suggest a single population of active
PAI-1 molecules that complexed to either scu-PA or tcu-PA). Hence, the
previously uncomplexed PAI-1 molecules were competent to form complexes
if the reaction conditions were suitable.
To determine whether
prolonged incubation of I-scu-PA with PAI-1 would alter
SDS-stable complex formation,
I-scu-PA was incubated with
buffer or PAI-1 at either 4 or 37 °C for increasing times, followed
by SDS-PAGE and autoradiography. As seen in Fig. 4, the
formation of SDS-stable complexes was both time- and
temperature-dependent, but once a given proportion of
I-scu-PA molecules was complexed with PAI-1, no further
complex formation could be detected. The use of a 10-fold molar excess
of PAI-1
VN did not result in more rapid, or quantitatively
greater complex formation with
I-scu-PA than found with
the same molar quantity of PAI-1 alone.
Figure 4:
Effects of time, temperature, and presence
of vitronectin on formation of scu-PAPAI-1 complexes.
I-scu-PA (150 ng) was incubated at 4 °C (panelA) or 37 °C (panelB) with PAI-1
(1.5 µg) alone or with PAI-1, which had been preincubated with
vitronectin (2.5 µg) for 30 min at 37 °C (the presence of
PAI-1
VN complexes in these pre-incubations was documented by the
presence of thrombin inhibitory activity as outlined under
``Materials and Methods''(24) ). Aliquots were
removed at the indicated times (0-20 h) and analyzed by SDS-PAGE
under reducing conditions and autoradiography. The data are
representative of three similar
experiments.
The above data suggested
that scu-PAPAI-1 complexes existed in equilibrium between a state
that yielded a covalent bond upon denaturation and a state that did
not. Implicit in this was the reversibility of scu-PA-PAI-1
interactions, which was tested by incubating increasing concentrations
of scu-PA with a constant concentration of PAI-1 for 15 min at 22
°C to allow the formation of scu-PA
PAI-1 complexes. A
constant concentration of
I-tcu-PA was then added to each
reaction for another 10 s at 4 °C, followed by SDS-PAGE and
autoradiography. As demonstrated in Fig. 5A, the
interaction of scu-PA with PAI-1 prevented, in a
concentration-dependent manner, subsequent complex formation between
I-tcu-PA and PAI-1. Plasminogen (also a serine protease
zymogen) demonstrated no such inhibition. However, if the above
experiment were repeated, but with the
I-tcu-PA added for
5 min instead of 10 s prior to SDS-PAGE (to allow a longer time to
compete off the scu-PA), no inhibition of
I-tcu-PA
PAI-1 complex formation by scu-PA could be
demonstrated (Fig. 5B). Hence, the interaction of
scu-PA with PAI-1 seems to be reversible, as PAI-1 that had been
complexed with scu-PA (Fig. 5A) could dissociate from
that scu-PA and subsequently interact with
I-tcu-PA (Fig. 5B).
Figure 5:
Scu-PA competes with I-tcu-PA for binding to PAI-1. A, PAI-1 (75 ng)
was incubated with either scu-PA or plasminogen (Pg) at the
indicated molar ratios (PAI-1:scu-PA or PAI-1:Pg) for 15 min at room
temperature. Mixtures were cooled for 2 min in an ice slurry, 75 ng
I-tcu-PA was added to the mixtures for 10 s, and samples
were analyzed by SDS-PAGE under reducing conditions and
autoradiography. B, the experiment was performed exactly as in A, except
I-tcu-PA was added for 5 min to allow
competition for PAI-1 previously bound to scu-PA. The data are
representative of three similar
experiments.
It is possible that the above reversible
scu-PA-PAI-1 interaction involved only those complexes that did not form a covalent bond upon denaturation, with the more stable
complexes not dissociating. To determine specifically whether the
scu-PAPAI-1 complex, which yields a covalent bond upon
denaturation, is reversible,
I-scu-PA was incubated 2 h
at 37 °C with buffer alone or with a 10-fold molar excess of PAI-1.
Increasing concentrations of unlabeled tcu-PA were then added to
parallel incubations for 3 h at 37 °C, followed by SDS-PAGE and
autoradiography. Fig. 6shows that the more tcu-PA was added to
pre-formed
I-scu-PA
PAI-1 complexes, the fewer of
those complexes remained. This implies that
I-scu-PA
PAI-1 complexes, which would form a
covalent bond upon denaturation, do indeed dissociate. If there is only
I-scu-PA present to interact with the now free PAI-1, new
I-scu-PA
PAI-1 complexes form, and the detected
level of these complexes does not change, resulting in the observed
equilibrium. However, the more tcu-PA is present, the more free PAI-1
becomes complexed to it (tcu-PA), the less PAI-1 is available for
complex formation with
I-scu-PA, and the fewer
I-scu-PA
PAI-1 complexes are detected. Hence,
I-scu-PA
PAI-1 complexes that would form a covalent
bond upon denaturation are reversible, albeit slowly (note in Fig. 3,
I-tcu-PA was added to
I-scu-PA
PAI-1 for only 30 min, compared to 3 h in Fig. 6; the shorter time did not yield an observable decrease in
I-scu-PA
PAI-1 complexes). This is consistent with
scu-PA and PAI-1 existing in a tetrahedral intermediate as described
for
-antitrypsin and elastase(25) .
Figure 6:
Reversible interaction between I-scu-PA and PAI-1.
I-scu-PA (75 ng) was
incubated 2 h at 37 °C with PAI-1 (750 ng). Increasing
concentrations of tcu-PA, as noted in the figure, were then added to
the pre-formed
I-scu-PA
PAI-1 complexes in parallel
incubations for 3 h at 37 °C, followed by SDS-PAGE under reducing
conditions and autoradiography. The relative quantities of
I-scu-PA
PAI-1 complexes remaining were determined
by densitometry and are plotted on the ordinate versus the
amount of tcu-PA added (abscissa). The inset shows
the autoradiogram. This experiment is representative of five
performed.
These data suggest that scu-PA is either more enzyme-like or less zymogen-like than was previously appreciated and that scu-PA interacts with PAI-1 much like a ``conventional'' active serine protease interacts with its cognate serpin. The initial interaction is rapidly reversible and depends on non-active site determinants. The second part of the interaction can involve a ``stable intermediate'' state, which presumably involves the active site serine of scu-PA(19, 25) . Furthermore, it suggests that scu-PA may be more similar to single-chain t-PA than previously appreciated(26) . This would make it more likely that scu-PA is competent to activate plasminogen in the appropriate setting without cleavage to tcu-PA, much as single-chain t-PA can activate plasminogen without cleavage to two-chain t-PA(26, 27) . The mechanisms for initiating plasminogen activation on the surface of either cells or fibrin clots may therefore be mechanistically similar. Each plasminogen activator may lack true zymogen-like properties, as described for t-PA(26) , yet be a poor plasminogen activator in solution. Binding of either enzyme to the appropriate site (cell surface receptor for scu-PA, fibrin for t-PA) in proximity to appropriately bound plasminogen would be the initiating event for the generation of plasmin(7, 8) .
The data presented in
this report may also explain some interesting findings in the
literature. Lijnen et al.(28) described evidence for
reversible inhibition of scu-PA added to plasma; the interaction of
scu-PA with PAI-1 may explain some of this inhibition. We would
hypothesize that because the protein C inhibitor inhibits
tcu-PA(29) , this serpin may also interact in a reversible way
with scu-PA and explain much of the other plasma-derived scu-PA
inhibition (28) . In addition, Ciambrone and McKeown-Longo (30) observed a 98-kDa complex on reduced SDS-PAGE analysis of
supernatants from HT1080 cells that had been cultured with
[S]methionine. These 98-kDa complexes were
immunoprecipitated with antibodies to either u-PA or PAI-1.
We therefore hypothesize that the reversible interaction of scu-PA with u-PA-specific inhibitors of the serpin class may be found in a wide range of situations and may play a role in controlling the initiation of urokinase-mediated plasminogen activation (consistent with the hypothesis presented in (13) ). The observation that scu-PA and PAI-1 can form stable intermediates implies that a molecular conformation of scu-PA possesses appreciable enzyme-like qualities. A more complete description of this must await appropriate studies.