From the Departments of Medicine and Biomolecular
Chemistry, University of Wisconsin, Madison, Wisconsin 53706 and the
¶ Hospital "La Fe", Centro de Investigacion, Avenida Campanar
21, 46009 Valencia, Spain
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Single-chain urokinase-type plasminogen activator
(scu-PA) possesses enzymatic activity that increases by 2-3 orders of
magnitude upon binding to its cellular cofactor, the u-PA receptor
(u-PAR), hence activating an enzymatic cascade initially composed of
zymogens. The present study demonstrates that plasminogen activator
inhibitor type 3 (PAI-3) reversibly inhibits scu-PA in solution,
maintaining the system "off." Because the scu-PA/PAI-3 interaction
is reversible, cellular expression of u-PAR allows partitioning of
scu-PA from PAI-3 to u-PAR with resultant expression of full enzymatic
activity. PAI-3 that was originally complexed to scu-PA remains in
solution, retaining its functional activity. Importantly, the scu-PA on cell surface u-PAR is protected from PAI-3 inhibition, remaining an
effective activator in a PAI-rich environment. Plasmin formed as a
result of scu-PA activity then cleaves scu-PA to the mature protease,
two-chain u-PA (tcu-PA), which is efficiently and irreversibly inhibited by PAI-3 via the standard serpin mechanism, even on u-PAR.
This data generates a new hypothesis which, in contrast to the previous
paradigm, holds that receptor bound scu-PA is the initiating enzyme and
that tcu-PA is generated not to augment enzymatic activity but rather
to allow for inhibition and therefore appropriate regulation of the process.
A number of processes involve proteolytic cascades wherein a given
zymogen is proteolytically activated by the enzyme that precedes it in
the cascade. An example of such a system is
plasmin-dependent cell surface proteolysis, where plasmin
is generated at the cell surface from its inactive precursor,
plasminogen, via the proteolytic activity of urokinase type plasminogen
activator (u-PA)1 (1).
There have been several conundra about the initiation of plasminogen
activation. First, u-PA is synthesized and secreted as a zymogen,
single-chain u-PA (scu-PA), which requires cleavage to the mature
protease, two-chain u-PA (tcu-PA) to manifest full enzymatic activity
in solution (2, 3). Because tcu-PA is required to generate plasmin, and
plasmin is required to generate tcu-PA, how the process is initiated
has been a puzzle (4). However, scu-PA belongs to a select subset of
zymogens that demonstrate a small but measurable amount of enzymatic
activity in solution (5-8). Interestingly, upon binding to the
cellular u-PA receptor (u-PAR), scu-PA increases its
plasminogen-activating efficiency by 2-3 orders of magnitude, while
remaining in the single-chain form (6, 9). Hence it is likely that
scu-PA bound to cellular u-PAR is the initiating enzyme of the cascade.
It remains unexplained why scu-PA in solution within biological fluids
exerts no enzymatic activity, whereas purified scu-PA at identical
concentrations activates plasminogen (3, 5-7, 10).
Another conundrum involves regulation. The generation of plasmin by
tcu-PA and the generation of tcu-PA by plasmin creates a positive
feedback loop that would accelerate proteolysis once initiated.
However, physiologic cell migration requires that cell surface
proteolysis is restricted to the immediate area of the cell membrane
and that it is down-regulated when the cell stops. Hence there must be
a mechanism for damping proteolysis, something that seems inconsistent
with a positive feedback loop (11).
To address these conundra, the present study examined the role of
plasminogen activator inhibitor type 3 (PAI-3, also known as the
protein C inhibitor) in regulating u-PA-mediated plasminogen activation. Although we have previously shown that both PAI-1 and PAI-2
are capable of regulating plasminogen activation (12, 13), PAI-2 is
found in few biological fluids, and PAI-1 is present in concentrations
that may be inadequate to explain the complete masking of scu-PA
enzymatic activity (12, 14). In contrast, PAI-3 is present in high
concentrations in plasma, urine, and seminal fluid (15). It is also the
predominant serpin found complexed to tcu-PA in biological fluids (15).
The experiments in this report demonstrate a novel reversible
interaction between scu-PA (the activating protease) and PAI-3 (the
regulatory serpin). Moreover, rather than the previous paradigm of cell
surface plasminogen activation, the data suggest a hypothesis with
receptor bound scu-PA as the pertinent activating enzyme and with
tcu-PA being generated not to augment activity but to allow the system
to be inhibited.
Proteins and Reagents--
Human scu-PA was generously supplied
by Dr. Jack Henkin of Abbott Laboratories and was sequentially treated
with 5 mM DFP and Glu-Gly-Arg-chloromethylketone as
described previously to inactivate traces of tcu-PA that might be
present (6, 12, 13). Dr. Henkin also generously supplied the
amino-terminal fragment (ATF) of u-PA, which possesses the high
affinity receptor binding determinants of u-PA (16).
Glu158-scu-PA was prepared and treated as described
previously to ensure no plasmin-cleavable form of scu-PA was present
(6, 13). PAI-3 was prepared as described previously (17).
Plasminogen-free fibrinogen was kindly supplied by Dr. Michael
Mosesson, Milwaukee, WI. Plasminogen was prepared as described
previously (12, 18). Polyacrylamide, SDS, and reagents for gels were
from Bio-Rad; ovalbumin was from Sigma; and other reagents were of the
highest grade available.
Methods--
Iodination of proteins, SDS-PAGE, the
125I-fibrin plate assay, and the direct,
plasmin-independent 125I-plasminogen cleavage assay were
carried out exactly as described previously (6, 12, 13, 19).
125I-tcu-PA was generated from 125I-scu-PA by
cleavage with plasmin-Sepharose as described previously (6). In some
preparations, residual 125I-scu-PA remains and is seen as a
55-kDa band on the autoradiograms. Variable amounts of residual
125I-scu-PA did not influence the observed results.
U-PA/PAI-3 Interactions--
Detection of SDS-stable complexes
between 125I-scu-PA and PAI-3 was performed by incubating
75 ng of 125I-scu-PA with buffer or with 75 or 750 ng of
the indicated PAI in a final volume of 30 µl of TBS containing 0.01%
Triton X-100 for 3 h at 37 °C, followed by the addition of gel
buffer containing 2-mercaptoethanol and analysis by 10% SDS-PAGE and
autoradiography. Detecting competition between scu-PA and
125I-tcu-PA for interaction with PAI-3 entailed incubating
the indicated amount of scu-PA (or plasminogen as a control) with 75 ng
of PAI-3 in 10 µl of TBS for 30 min at 37 °C.
125I-tcu-PA (75 ng) was then added in 5 µl of TBS to each
reaction for the indicated time at 37 °C. The reactions were
analyzed by SDS-PAGE under reducing conditions and autoradiography.
Reversibility of u-PA/PAI-3 Interactions--
Preliminary
experiments determined conditions under which all scu-PA in a solution
was complexed to PAI-3 as assessed by inhibition of
125I-plasminogen cleavage. Scu-PA·PAI-3 complexes were
established under such conditions (i.e. all scu-PA in
complex with PAI-3), and increasing numbers of U-937 cells with
unoccupied u-PAR (see below) were added for 30 min at 37 °C to allow
binding of scu-PA to receptor. The cells were centrifuged, washed, and
either counted for radioactivity (to measure binding in experiments
using 125I-scu-PA·PAI-3) or placed into a
125I-fibrin plate assay (to measure enzymatic activity in
experiments using scu-PA·PAI-3).
Alternatively, preliminary experiments determined conditions under
which all PAI-3 was complexed to scu-PA, as assessed by the inability
of PAI-3 to form complexes with 125I-tcu-PA within the time
frame defined in Figs. 1 and 2. Scu-PA·PAI-3 complexes were
established under such conditions (i.e. all PAI-3 in complex
with scu-PA), and increasing numbers of U-937 cells with unoccupied
u-PAR receptors were added for 30 min at 37 °C to allow binding of
scu-PA to the receptors. Such receptor binding of scu-PA would leave
free PAI-3 in the supernatant. The cells were centrifuged, and the
supernatant was recovered and tested for free, functional PAI-3 by the
addition of 125I-tcu-PA for 10 min followed by SDS-PAGE
under reducing conditions and autoradiography. Under these conditions,
125I-tcu-PA will form SDS-stable complexes with free PAI-3
but not with PAI-3 that is in complex with scu-PA (see Fig. 1).
U-PAR was identified as the u-PA binding entity on U-937 cells in the
above experiments by preincubating cells with the indicated concentration of u-PA ATF for 30 min at 37 °C, followed by washing the cells to remove unbound ATF prior to addition of scu-PA-containing solutions. Control incubations included DIP-33-kDa protease domain of
u-PA, which does not contain the high affinity receptor binding determinants.
U-937 Cells--
U-937 cells were grown in RPMI 1640, 10%
bovine calf serum, 25 mM HEPES, pH 7.4. Prior to use, U-937
cells were washed in RPMI 1640, 2 mM EACA, 10 µM cycloheximide, and were then treated exactly as
described for native human monocytes (6, 13). These cells had all
detectable endogenous u-PA removed from their surface u-PAR, and they
synthesized neither u-PA or PAI (6, 13).
Inhibition of Receptor-bound u-PA--
U-937 cells with
unoccupied u-PAR were incubated with 5 µg of the designated form of
u-PA for 30 min at 37 °C, washed, and then added to the designated
concentration of PAI-3 for 30 min at 37 °C, washed, resuspended in
0.1 ml of RPMI 1640, and added to a 125I-fibrin plate assay.
The solution phase enzymatic activity of scu-PA was inhibited in a
concentration-dependent manner by PAI-3 but not by the non-inhibitory serpin, ovalbumin, (Fig.
1A). Parallel experiments using identical conditions, but with scu-PA being the labeled protein,
demonstrated that scu-PA remained in the single-chain form throughout
these reactions (Fig. 1A, legend, and see Refs. 12 and 13).
Also, identical results were obtained using the plasmin-insensitive
mutant Glu158-scu-PA. Hence it was single-chain not
two-chain u-PA that was inhibited by PAI-3.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (45K):
[in a new window]
Fig. 1.
A, PAI-3 inhibits scu-PA enzymatic
activity in solution. 125I-plasminogen (Pg) was
incubated 16 h at 37 °C with scu-PA, scu-PA that had been
preincubated 30 min at 37° with the indicated molar ratios of PAI-3,
or with ovalbumin and analyzed by SDS-PAGE under reducing conditions,
followed by autoradiography. The amount of plasmin (Pn)
light chain (L.C.) formed is an accurate indicator of
plasminogen activation (19). B, PAI-3 inhibits scu-PA
without formation of a covalent complex. 75 ng of
125I-scu-PA and the indicated amounts of individual PAIs
were incubated as under "Experimental Procedures" for 3 h at
37 °C, followed by SDS-PAGE under reducing conditions and
autoradiography. 125I-scu-PA did not form SDS-stable
complexes with PAI-3 under conditions where all scu-PA enzymatic
activity was inhibited. Control reactions with PAI-1 and -2 reveal the
previously described reactivity of scu-PA with these molecules (12,
13). The limited SDS-stable complex formation between
125I-scu-PA and PAI-1 serves as a positive control that
demonstrates the ability of scu-PA to form such complexes with a
cognate serpin under appropriate conditions and indicates that the lack
of such complexes with PAI-3 is accurate.
The inhibition of a mature protease by a serpin-type inhibitor is marked by formation of a 1:1 complex between protease and inhibitor that initially is reversible and then progresses to an acyl-enzyme-containing complex that is stable to denaturants (20-22). Incubation of 125I-scu-PA with PAI-3 under conditions that resulted in inhibition of enzymatic activity did not yield SDS-stable complexes (Fig. 1B), implying that PAI-3 interacts with scu-PA in a manner which results in inhibition of enzymatic activity but not formation of a covalent complex.
To investigate the interaction of scu-PA with PAI-3, we determined
whether scu-PA could specifically interfere with the formation of
covalent complexes between 125I-tcu-PA and PAI-3.
Increasing concentrations of scu-PA were preincubated with PAI-3,
followed by a constant amount of 125I-tcu-PA being added to
each reaction. The reactions were then analyzed by SDS-PAGE and
autoradiography to detect SDS-stable complexes between
125I-tcu-PA and PAI-3. As seen in Fig.
2A, scu-PA demonstrated a concentration-dependent inhibition of complex formation
between 125I-tcu-PA and PAI-3. This inhibition was
specific, as plasminogen, another serine protease zymogen, did not
inhibit the formation of 125I-tcu-PA·PAI-3 complexes
(Fig. 2A). However, the ability of scu-PA to prevent the
formation of covalent complexes between 125I-tcu-PA and
PAI-3 was reversible with time, as the longer 125I-tcu-PA
was allowed to compete with scu-PA for interaction with PAI-3, the more
125I-tcu-PA·PAI-3 complexes were formed (Fig.
2B). Hence, the active zymogen, scu-PA, interacted
specifically with PAI-3 to inhibit the intrinsic activity of scu-PA
without covalent bond formation and in a way that interfered with
complex formation between PAI-3 and the mature protease,
125I-tcu-PA. Moreover, this scu-PA/PAI-3 interaction
appeared to be reversible.
|
A similar inhibitory action by the relatively high plasma concentration of PAI-3 might explain the reversible inhibition of scu-PA added to human plasma (7). In addition, similar reversible interactions between scu-PA and both PAI-1 and PAI-2 (12, 13) suggest that reversible serpin/scu-PA interactions may also explain why endogenous scu-PA in cell culture supernatants does not exhibit enzymatic activity, whereas identical concentrations of purified scu-PA are proteolytically active (3, 5, 6, 10).
These findings imply that PAI-3 may serve to maintain scu-PA in an
inactive state until a signal for cell surface plasminogen activation,
u-PAR, is present. To determine whether the scu-PA·PAI-3 interaction
is consistent with such a role, the following experiments were
performed. Scu-PA·PAI-3 complexes were formed under conditions determined in preliminary experiments to yield all scu-PA complexed to
PAI-3. Increasing numbers of U-937 cells with unoccupied u-PAR (i.e. increasing numbers of available u-PAR) were added, and
the fate of scu-PA initially bound to PAI-3 was determined. As shown in
Fig. 3A,
125I-scu-PA prebound to PAI-3 was as available for binding
to u-PAR as was 125I-scu-PA that had not been precomplexed
to PAI-3. SDS-PAGE and autoradiography demonstrated that
125I-scu-PA bound to U-937 cells remained in the
single-chain form and had neither been cleaved to
125I-tcu-PA nor formed SDS-stable complexes with PAI-3
(data not shown). The scu-PA that was initially complexed to PAI-3 and
then bound to U-937 u-PAR retained enzymatic activity equal to that of
scu-PA that had not been precomplexed with PAI-3 (Fig. 3A). The effect of U-937 cells was indeed because of u-PAR, as preincubating U-937 cells with the ATF of u-PA, which contains the high affinity receptor binding determinants of u-PA (16), resulted in parallel concentration-dependent inhibition of
125I-scu-PA binding, and cell surface PA activity (Fig.
3B). The 33 kDa protease domain of u-PA, which binds poorly to the
receptor, demonstrated little inhibition.
|
Control experiments with 125I-PAI-3, either alone or precomplexed to scu-PA, did not demonstrate concentration-dependent binding to U-937 cells. The times required for binding and washing of the cells did not allow differentiation between scu-PA first dissociating from PAI-3 and binding to u-PAR and the initial binding of scu-PA·PAI-3 complexes with subsequent dissociation. Nevertheless, the outcome of these experiments demonstrated fully functional uninhibited scu-PA on cellular u-PAR.
These experiments demonstrate that scu-PA precomplexed to PAI-3 was able to bind to cellular u-PAR and yield PA activity just as efficiently as if it had not been precomplexed to PAI-3. Hence, scu-PA·PAI-3 complexes were reversible, yielding fully functional protease, scu-PA.
To determine whether reversal of scu-PA·PAI-3 complexes also yielded
fully functional serpin, PAI-3, the same experimental framework was
used except that the starting conditions were those determined to yield
all PAI-3 complexed to scu-PA. Increasing numbers of U-937 cells with
unoccupied u-PAR were added, scu-PA was allowed to bind to U-937 u-PAR,
and the cells were centrifuged. If PAI-3 remained functional, it would
have dissociated from scu-PA and remained in the supernatant, available
to form covalent complexes with subsequently added
125I-tcu-PA, which was the pattern seen. As demonstrated in
Fig. 4A, the more U-937 cells
(i.e. the more u-PAR) were added, the more functional PAI-3
was left behind in the supernatant, able to form complexes with
125I-tcu-PA. At the highest concentration of cells tested,
the PAI-3 "liberated" from complexes with scu-PA was able to form
125I-tcu-PA·PAI-3 complexes of almost equal intensity to
those formed between 125I-tcu-PA and PAI-3 that had not
been precomplexed to scu-PA (three experiments, 82, 93, and 98%
intensity of the 125I-tcu-PA·PAI-3 band by laser
densitometry of autoradiograms; with 100% being the intensity of the
band obtained by adding 125I-tcu-PA to supernatants in
which PAI-3 had not been precomplexed to scu-PA). Moreover, the
dissociation of scu-PA·PAI-3 to yield functional PAI-3 required that
scu-PA interact with cell surface u-PAR, as preincubation of U-937
cells with increasing concentrations of the ATF of u-PA yielded a
concentration-dependent inhibition of PAI-3 available for
complex formation with 125I-tcu-PA (Fig. 4B).
Preincubation of U-937 cells with DIP-33-kDa u-PA heavy chain had no
such effect.
|
Hence, scu-PA·PAI-3 complexes were reversible and yielded functional enzyme and functional inhibitor. This demonstrates a novel reversible interaction between a serpin and a serine protease that upon dissociation yields a serpin with its reactive site loop intact and fully able to subsequently form the well described covalent complexes with a mature serine protease (20-23). The key may be that the "protease" that is able to form the novel reversible complex is a zymogen that has some enzymatic activity.
The scu-PA·PAI-3 interaction differs from the well described interaction between a serpin and a mature protease in several ways that may have important biological ramifications. First, this is a biologically reversible interaction. The reaction between a mature protease, and a serpin is chemically reversible with regard to the protease in that cleavage of the serpin P1-P1' bond eventually goes to completion, yielding free functional protease and cleaved nonfunctional serpin (20, 24). However, such an interaction is biologically irreversible, as formation of the stable covalent complex results in exposure of a cryptic determinant with resultant rapid cellular internalization and degradation of the complexes (24-27). In contrast, the interaction described in the present report between an enzymatically active zymogen and cognate serpin is reversible in that the complex is not internalized by cells (data not shown), and dissociation of the complex yields both enzyme and inhibitor, each of which retain full functional activity.
We therefore hypothesize that a zymogen which expresses a small amount of enzymatic activity has that activity masked in solution by a reversible interaction with an inhibitor to prevent inappropriate proteolytic activity. The active zymogen would then dissociate from the serpin and bind to an appropriate cofactor (in the case of scu-PA, it would be u-PAR) to express full proteolytic activity (6, 9).
The hypothesis also holds that the receptor-bound zymogen would not
bind serpin; otherwise, the serpin liberated from the reversible
protease·inhibitor complex would interfere with the receptor-bound
zymogen which would then not express enzymatic activity. To directly
test this, the indicated forms of u-PA (Fig. 5) were bound to U-937 u-PAR, then
exposed to increasing concentrations of PAI-3. Following incubation to
allow the potential interaction of PAI-3 with receptor bound u-PA, the
cells were tested for u-PA activity. As demonstrated in Fig. 5, neither
receptor-bound scu-PA or Glu158-scu-PA were inhibited by
PAI-3, whereas receptor-bound tcu-PA (either added directly to cells or
generated in situ by binding scu-PA then treating with
plasmin) was readily inhibited by PAI-3 in a
concentration-dependent manner. The U-937 cell surface
plasminogen-activating activity which was not PAI-3 inhibitable was
because of u-PAR-bound scu-PA as demonstrated by (i) strict plasminogen
dependence, (ii) inhibition by neutralizing antibodies to u-PA but not
by nonimmune IgG, and (iii) prevention by pretreating U-937 cells with
ATF of u-PA but not by pretreatment with the 33-kDa proteolytic
domain.
|
These experiments could not differentiate between u-PAR· scu-PA not interacting with PAI-3 and interacting in a readily reversible manner. However, the activities of receptor-bound scu-PA were virtually identical whether it had been exposed to PAI-3 or not. Therefore, if PAI-3 does interact with u-PAR·scu-PA, the dissociation is likely to be relatively rapid. The end result of this fleeting interaction would be expression of effective cell surface proteolytic activity even in the presence of PAI-3.
It is also worth noting that heparin did not augment scu-PA inhibition by PAI-3 (data not shown). Therefore, PAI-3 is capable of reversibly inhibiting scu-PA to maintain the system in the "off" mode in the appropriate physiologic compartment which lacks heparin-like molecules, the solution phase. In contrast, the inhibition of tcu-PA by PAI-3 is augmented by heparin (28). Because heparin-like molecules and u-PAR are both found on cell surfaces, u-PAR-bound tcu-PA is localized for optimum inhibition by PAI-3 under physiologic conditions.
We therefore present the following hypothesis (represented in Fig.
6). It has been thought that the zymogen
of u-PA, the initiating enzyme of this pathway, has zero activity or
very close to it, and therefore by definition the enzymatic pathway is
"off." However, data presented here suggests that achieving zero
activity requires that the biological system exert some energy to bring
the small amount of basal proenzyme activity to an unmeasurably low
level. This is achieved by the reversible interaction of scu-PA with a
serpin, PAI-3. This novel serpin activity is distinct from the well
described covalent interaction with a mature protease. When enzymatic
activity is required, cells express u-PAR, allowing for partitioning of
scu-PA from PAI-3 to u-PAR. Upon binding to u-PAR, scu-PA increases its
plasminogen-activating efficiency by 100-1,000-fold (6, 9) and does
not interact efficiently with PAI-3; this results in efficient and
targeted PA, even in a PAI-rich environment.
|
Next in the hypothesis is an important regulatory step that differs significantly from the currently held paradigm which states that tcu-PA is generated to yield augmented plasminogen activation, resulting in a positive feedback loop. Under physiologic conditions, there is instead a built-in negative feedback mechanism in that plasmin cleaves scu-PA to tcu-PA. We hypothesize that the more important physiological role of generating the mature protease tcu-PA is that it (tcu-PA) can be efficiently and irreversibly inhibited, even on the cell surface, as demonstrated in Fig. 5, and as previously shown with PAI-1 and -2 (13, 27, 29). Covalent tcu-PA·PAI complexes thus formed on u-PAR are rapidly internalized via an LRP-dependent mechanism (25, 26), clearing the enzyme from the cell surface and necessitating expression of new receptors on the cell (either recycled or synthesized) for further plasminogen activation. Hence, under this hypothesis, scu-PA bound to u-PAR is the enzyme, and tcu-PA is generated to allow inhibition of the process.
In summary, the data in this report lead us to hypothesize that the effective initiating enzyme is the active zymogen (scu-PA) bound to the activating cofactor (u-PAR). The cleavage of the zymogen (scu-PA) to the mature protease (tcu-PA) serves not to mediate an increase in proteolytic activity but rather to create a species of protease that can be efficiently and irreversibly inhibited, even when bound to the initiating cofactor (u-PAR).
Therefore, in serine protease cascades, the molecularly complex
serpin-type inhibitors could serve two distinct functions: (i) to
effect the reversible inhibition that keeps the system off until the
appropriate initiating cofactor (i.e. u-PAR) is expressed,
and (ii) the biologically irreversible inhibition that limits the
process once initiated. We would hypothesize that the reversible
interaction with the active zymogen might be especially dependent on
secondary sites of contact between serpin and protease (20, 30). These
molecular determinants may therefore have importance for regulating the
initiation of proteolysis as well as facilitating its inhibition.
![]() |
ACKNOWLEDGEMENTS |
---|
The authors would like to thank Ellen Martin, for superb technical assistance, and Marcy Salmon, for expert and patient secretarial help.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant HL43506 (to B. S. S.), and by Fond De Investigaciones Sanitarius Grant 96/1129 (to F. E.).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.
§ To whom correspondence should be addressed. Tel.: 608-262-4982; Fax: 608-263-4969; E-mail: schwartz{at}medicine.wisc.edu.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: u-PA, urokinase-type plasminogen activator; scu-PA, single-chain u-PA; tcu-PA, two-chain u-PA; u-PAR, u-PA receptor; PAI-3, plasminogen activator inhibitor type 3; ATF, amino-terminal fragment of u-PA; TBS, Tris-buffered saline; PAGE, polyacrylamide gel electrophoresis; PA, plasminogen activation; DFP, diisopropyl fluorophosphate; DIP, diisopropyl.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|