From the
Differential scanning calorimetry was used to study the domain
structure and intramolecular interactions of tPA/uPA chimeras. A high
temperature transition centered near 90 °C was observed upon
melting of the tPA/uPA chimera (amino acids 1-274 of tPA and
138-411 of uPA) and its variant lacking the finger and epidermal
growth factor-like modules (residues 1-3 and 87-274 of tPA
and 138-411 of uPA). Since neither of the two parent plasminogen
activators display such a stable structure, one may suggest that a new
stabilizing intramolecular interaction occurs in the chimeras. We found
that occupation of the lysine binding site of tPA by a lysine or
arginine side chain from the urokinase moiety is responsible for the
high temperature transition as well as for the failure of the chimeras
to exhibit the expected fibrin binding properties. All uPA species,
single- and two-chain high molecular weight uPA (Pro-Uk and HMW-Uk) and
two-chain low molecular weight uPA (LMW-Uk), interact intermolecularly
with tPA and its kringle-containing derivatives. This intermolecular
interaction was strongly inhibited by
Tissue-type plasminogen activator (tPA)
tPA and uPA are mosaic proteins consisting of similar
homologous modules that are also found in other plasma proteins. These
include an epidermal growth factor-like (E) module, a kringle module
(K), and a serine protease module (SP) which are common for the two
plasminogen activators. The modular composition of uPA is E-K-SP. tPA
has an additional finger module (F) homologous to fibronectin type 1
repeats, and its modular composition can be presented as F-E-K1-K2-SP.
While the SP module is responsible for enzymatic activity, all other
modules in tPA and uPA are involved in interactions with other proteins
and ligands. K2 of tPA carries a so-called lysine binding site (LBS)
which is responsible for the interaction with
tPA and uPA have
attracted much attention because their recombinant forms are used as
thrombolytic agents. A number of attempts have been made to improve the
fibrinolytic properties of these enzymes by constructing chimeric
plasminogen activators that combine the best features of both molecules
to improve the activating properties and fibrin specificity
(2, 14, 15, 16, 17) . But these
attempts were not as successful as hoped since the created chimeric
molecules did not exhibit appreciable affinity for fibrin. Upon module
shuffling, one needs to be aware of possible intramolecular
domain-domain interactions which might be important for the function.
The domain structures and interactions of tPA and uPA have already been
studied by differential scanning calorimetry
(18, 19, 20) . It was shown that in uPA all
domains are independent and noninteracting, whereas in tPA, the serine
protease domain interacts strongly with the amino-terminal F and/or E
domains. In order to explain the failure of the tPA/uPA chimeras to
bind fibrin effectively, one must first be sure that all modules are
folded properly in their new environment and, second, check whether the
interactions between them are preserved. In the present paper, we
report the results of such a study of the tPA/uPA chimera and its
deletion mutant lacking F and E domains. It is shown that uPA interacts
with tPA intermolecularly by means of the LBS of K2 of the latter. This
interaction is preserved in the tPA/uPA chimeric molecule where the LBS
of tPA is occupied intramolecularly by residues within the
Leu
Fluorescence
anisotropy measurements were performed in TBS at 25 °C with an
SLM-8000C fluorometer. Concentrated solutions of uPA species were
automatically added with a motor-driven syringe to a stirred cuvette
containing the FITC-32/35K tPA fragment at 0.1 µ
M while
monitoring the anisotropy at 524 nm with excitation at 493 nm. The
resulting concentration-dependent increase in anisotropy ( A)
was fit to Equation 1:
Differential scanning calorimetry
measurements were made as described previously
(19, 20) with a DASM-4M or updated DASM-1 calorimeter
(27) at a scan rate of 1 °C/min and protein concentrations
between 0.4 and 2.2 mg/ml. Deconvolution analysis was performed
according to a procedure similar to the recurrent procedure of Freire
and Biltonen
(28) using software provided by Dr. V. Filimonov
(Institute of Protein Research, Pouschino, Russia) as described
previously
(19, 20) .
The homogeneity of all proteins
was greater than 95% as judged by SDS-polyacrylamide gel
electrophoresis, size exclusion chromatography on Superdex-75
(Pharmacia), and amino-terminal sequence analysis with an HPG1000A
Sequencer (Hewlett Packard). Protein concentrations were determined
from absorbance at 280 nm using the molar extinction coefficients
(
M
The melting properties of tPA and uPA were well characterized
earlier at low pH where both proteins were soluble and the denaturation
process was highly reversible
(19, 20) . These
conditions were selected for comparison of the melting behavior of
chimeric molecules with that of tPA and uPA. Original endotherms of the
tPA/uPA chimera and its
Which domain in the chimeric molecule is responsible for the high
temperature transition? It is unlikely that the finger or epidermal
growth factor-like domains are involved since the high temperature
transition occurs in the
Numerous attempts have been made during the last several
years to create an improved thrombolytic agent by constructing chimeric
plasminogen activators that consist of domains from the A-chain of tPA
and the serine protease portion of uPA in order to combine the
mechanisms of fibrin selectivity of both molecules
(2, 14, 15, 16, 17) . Some of
them were very promising. For example,
This new type of interaction was quite
surprising since initially it was expected that the poor fibrin binding
of the chimeras might be accounted for by their failure to duplicate
domain interactions that had been identified in tPA
(19) . In
that molecule, the serine protease domain interacts strongly with the
amino-terminal F and/or E domains. If this interaction were important
for fibrin binding, its absence in the tPA/uPA chimera could have
explained the poor fibrin binding. An alternative explanation would be
that the fibrin binding domains in the amino-terminal region of tPA do
not fold autonomously in the chimeric molecules
(24) . However,
calorimetric data presented in Figs. 1 and 2 indicate that the total
melting enthalpies of the two chimeras are close to what is expected
and that the number of transitions revealed by deconvolution analysis
is consistent with the modular composition of these proteins. In this
study we did not attempt to observe the unfolding status of the
superstable finger module in the chimera since in tPA it melts off
scale under the conditions employed. To observe its melting would
require the presence of denaturants and a very large quantity of the
sample
(19) . But the fact that the chimeric molecules exhibited
detectable fibrin affinity
(24, 35) , which could be
mediated by the second fibrin binding site located within the finger
structure, suggests that this module is also properly folded. Thus, one
can conclude that all domains in the chimeric molecules preserve their
autonomous folding status in their new environment. The diminished
fibrin affinity is attributed to the fact that the LBS, instead of
mediating the interaction with fibrin, is utilized for the
intramolecular domain-domain interaction.
This is not the first time
that an interaction between a kringle domain and a serine protease has
been reported. An interaction of similar affinity was observed between
thrombin and the second kringle of prothrombin
(36, 37) . Although we are not aware of any report of
lysine binding by K2 of prothrombin, a recent crystallographic study
revealed that multiple positively charged group(s) on thrombin form
contacts with an enlarged anionic center in the same region of K2 where
lysine analogues bind to other kringles
(38) . A similar
extended interface could also occur between urokinase and K2 of tPA.
This would explain why the interaction persists at low pH where the
high temperature transition was observed since the participating
carboxyl groups of the LBS would tend to be inaccessible to solvent.
Although the interaction definitely occurs in solution, the apparent
affinity was one or two orders higher when one or the other of the
components was adsorbed to a solid phase (). This
discrepancy may arise from several factors. One is that immobilization
of one of the interacting components on the plastic would decrease its
translational and rotational degrees of freedom, lowering the entropic
cost of complex formation. A second factor is that the high local
concentration of one component on the surface will tend to reduce the
off rate compared to that in solution with a corresponding decrease in
the equilibrium dissociation constant. A third possibility is that if
the fluid-phase component has even a weak tendency to self-associate,
the oligomeric forms could bind through multivalent attachment to
clusters of the solid-phase component. It is possible that the
solid-phase model is more relevant to the biology of the system since
binding of uPA to its receptor or of tPA to a fibrin clot could mimic
the solid-phase conditions used here.
Based on the observed
K
Data obtained with synthetic peptides allowed us to
narrow down the tPA binding site on urokinase within the sequence
144-157. It is clear that the carboxyl-terminal lysine 158 is not
important for this interaction. The latter conclusion is deduced not
only from the data with peptides but also from the fact that
single-chain Pro-Uk binds equally well to tPA. Although
carboxyl-terminal lysine is usually considered to be a primary
candidate for binding to the LBS, that is not the case with tPA since
it was shown to have similar affinity for lysine-Sepharose and
aminohexyl-Sepharose
(10) . The question of which of the 4
positively charged residues within the 144-157 sequence is
crucial remains to be answered. Determination of this residue would
allow it to be mutated in the chimera, liberating the lysine binding
site to interact with fibrin.
It is important to stress that the
synthetic peptides are 100-fold more potent then
The enhanced fibrin selectivity of single-chain uPA
versus the two-chain form is well established although the
mechanism is not
(3, 7) . This selectivity was also
diminished in the single-chain chimera
(24) . Conversion of
single-chain uPA to the two-chain form with lower fibrin selectivity
involves cleavage at the Lys
We thank Dr. Jack Henkin of Abbott Laboratories for
provision of uPA species and monoclonal antibodies.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-aminocaproic acid
indicating that the lysine binding site of tPA is involved. The binding
of uPA with the fluorescein-labeled A-chain of tPA, registered by
changes in fluorescence anisotropy, was estimated to have a
K
range of 1-7 µ
M. The
interaction of tPA with uPA determined by solid-phase assays appeared
to be tighter, with a K
range of
50-300 n
M. Two synthetic peptides, with and without
carboxyl-terminal lysine, corresponding to urokinase residues
144-158 and 144-157, were
100-fold more potent than
-aminocaproic acid with respect to inhibition of the tPA-uPA
interaction, indicating that the tPA binding site on urokinase is
located within this sequence, close to the activation site
Lys
-Ile
. The discovered intermolecular
interaction may be related to the reported synergistic effect of
simultaneous administration of these two plasminogen activators.
(
)
and urokinase-type plasminogen activator (uPA) are the two
major physiological activators of plasminogen, the inactive precursor
of the main fibrinolytic enzyme plasmin. Both of these proteins are
serine proteases that cleave a single Arg-Val peptide bond to
convert plasminogen to plasmin. uPA is a 411-amino acid glycoprotein
that exists in several molecular forms
(1, 2) . High
molecular weight single-chain urokinase-type plasminogen activator
(Pro-Uk) is a M
= 54,000 zymogen with
little or no activity
(3) . Specific cleavage of the
Lys
-Ile
peptide bond generates an
active two-chain enzyme (HMW-Uk). An active two-chain low molecular
weight form
(33, 0) starting from Lys
(LMW-Uk) can be isolated after cleavage with plasmin
(4) .
tPA is a 68-kDa glycoprotein that is synthesized as a single chain of
527 amino acids and may be converted by plasmin into a two-chain form
(2) . In contrast to uPA and most other serine proteases, the
single- and two-chain forms of tPA are both enzymatically active. It is
well established that the activation of plasminogen by tPA is
significantly stimulated by fibrin
(5, 6) . uPA displays
fibrin specificity only when its activation peptide bond
Lys
-Ile
is intact
(7) and is
not dependent on the presence of of the amino-terminal 143 amino acids
(4) .
-aminocarboxylic
acids and is the major site which mediates binding of tPA with fibrin
(8, 9, 10) . It has been reported that in
addition to K2, fibrin binding by tPA also involves the finger module
(9) . The uPA kringle and K1 of tPA do not possess lysine
binding properties or affinity to fibrin
(10, 11) .
Although the uPA kringle is able to bind heparin
(12) , no
function for K1 of tPA is known. The E module of uPA is responsible for
the interaction with the uPA receptor
(13) .
-Lys
sequence of the urokinase
moiety. This unnatural filling of the major fibrin binding site
explains the poor fibrin affinity of the chimeras.
Proteins
All urokinase species were kindly
provided by Dr. J. Henkin from Abbott Laboratories. Recombinant high
molecular weight Pro-Uk and HMW-Uk were expressed and purified from
SP2/0 cell culture by Abbott Biotech (Needham, MA)
(21) . Low
molecular weight urokinase (Abbokinase) was from Abbott Laboratories
and was further purified by affinity chromatography on
benzamidine-Sepharose
(22) . Recombinant single-chain tPA was a
Genentech product known under the trade name ``Activase.''
The deletion variant F
E-rtPA
(23) was obtained from
Dr. B. Isaacs of the Genetics Institute (Boston, MA). The 32/35K
subtilisin fragment of tPA consisting of F-E-K1-K2 domains was prepared
and isolated as described elsewhere
(19) . The extended chimeric
tPA/uPA protein consisting of amino acids 1-274 of tPA and
138-411 of uPA was expressed in CHO cells as described
(24) . The
F
E-tPA/uPA deletion mutant (residues
1-3, 87-274 of tPA, and 138-411 of uPA) was obtained
by a similar approach
(25) . The monoclonal antibody directed
against tPA K1 (PAM-2) was purchased from American Diagnostica (Product
No. 372). The monoclonal antibody directed against the urokinase
A-chain was obtained from Dr. J. Henkin, Abbott Laboratories.
-Aminocaproic acid (
-ACA), Tris, glycine, and the proteolytic
enzymes subtilisin, trypsin chymotrypsin were obtained from Sigma. All
other chemicals were of reagent grade or higher.
Fluorescence Binding Measurements
Fluorescent
32/35K tPA fragment was prepared by labeling with fluorescein
isothiocyanate (FITC). Protein was dialyzed against 0.1
M NaHCO, pH 9.5, mixed with a 10
M excess of
FITC (Sigma), and incubated for 2 h at 37 °C. Unreacted dye was
removed by chromatography on a Sephadex P-10 prepacked column with
subsequent dialysis against 0.02
M Tris, 0.15
M NaCl,
pH 7.4 (TBS). The degree of labeling was determined optically as
described elsewhere
(26) . The protein concentration was
measured from the absorbance at 280 nm, correcting for the contribution
from covalently attached fluorescein
(26) .
A =
A
[L]/ K
+ [L] where K
= dissociation constant and [L] =
concentration of free ligand.
A
was treated
as a fitting parameter because the amount of protein added was not
enough to achieve saturation. Since the concentration of labeled
protein was small compared to the K
, the
concentration of free protein was assumed to be equal to the total
concentration. Competitive displacement studies were performed by
premixing 0.1 µ
M FITC-32/35K fragment with a sufficient
amount of uPA to provide
75% saturation and titrating with
-ACA or the synthetic peptides while measuring the decrease in
fluorescence anisotropy.
Solid-phase Binding Assay
Solid-phase binding was
performed in plastic microtiter plates using an enzyme-linked
immunosorbent assay. Microtiter plate wells were coated with proteins
at 3.0 µg/ml in coating buffer (0.1
M NaCO
, pH 9.5) for 2 h at 37 °C or
overnight at 4 °C. Unbound sites were blocked with 3% nonfat milk
in TBS for 1 h at 37 °C. After washing with TBS containing 0.05%
Tween 20 (TBS-Tween), tPA or uPA species were added to the wells at
concentrations from 0-5 µ
M in TBS-Tween. Following a
4-h incubation at 37 °C the wells were washed with TBS-Tween. Bound
proteins were reacted with either PAM-2 or Uk monoclonal antibodies (1
µg/ml IgG in TBS-Tween in the presence of 3% nonfat milk) followed
by goat anti-mouse IgG conjugated to horseradish peroxidase (Bio-Rad).
A 1:1 mixture of 3,3`,5,5`-Tetramethylbenzidine peroxidase substrate
with hydroxyperoxide solution (Kirkegaard & Perry Laboratories
Inc.) was added, and the absorbance at 650 nm was measured. The data
were analyzed using Equation 1.
cm
, 280 nm)
calculated from the amino acid composition
(29) . The values
are: rtPA, 115,600;
F
E-rtPA, 90,000; 32/35K tPA fragment,
65,400; Pro-Uk and HMW-Uk, 70,000; LMW-Uk, 44,500; tPA/uPA, 110,000;
F
E-tPA/uPA, 93,800.
Peptide Synthesis
Peptides corresponding to
urokinase sequences Leu-Lys
and
Leu
-Phe
were synthesized on a
MilliGen 9050 Pep Synthesizer using Fmoc chemistry with
pentafluorophenyl amino acid active esters and a polyethylene glycol
polystyrene support with a
5-(4`-Fmoc-aminomethyl-3`-5`-dimethoxyphenoxy)valeric acid linker.
After synthesis, peptides were purified by reverse-phase high
performance liquid chromatography and lyophilized. Concentration of the
synthetic peptides was determined by amino acid analysis using a Waters
Pico Tag system.
F
E deletion variant are presented in
Fig. 1
. The dotted lines are simulated curves which were
created by the superposition of the melting transitions of the Uk
serine protease domain and domains from the A-chain of tPA, taken from
the deconvolution analyses presented in Fig. 2 B.
Experimental endotherms of the chimeras would be expected to match the
simulated curves if no new domain-domain interactions were introduced.
However, the experimental curves differ from the simulated ones in the
high temperature range. The most prominent difference is the appearance
of a new heat-absorption peak centered near 90 °C in both chimeric
molecules. This becomes especially evident when the deconvoluted excess
heat capacity curves for the chimeras (Fig. 2 A) are
compared with the corresponding curves for tPA and LMW-Uk
(Fig. 2 B). The new transition reflects melting of a
single domain since it is well described by one two-state transition.
Since neither of the two parent molecules display such a stable
structure, one may conclude that one of the domains is stabilized,
i.e. we are dealing with a newly acquired interaction unique
to the chimeric molecule.
Figure 1:
Original differential scanning
calorimetry curves of tPA/uPA ( A) and F
E-tPA/uPA
( B) chimeras ( solid lines) in 50 m
M glycine,
pH 3.4. The dotted lines next to each original curve are
simulated profiles expected for these proteins in the case of the
absence of the domain-domain interactions.
Figure 2:
Deconvolution of excess heat capacity
functions obtained with tPA/uPA and F
E-tPA/uPA chimeras
( A) and tPA and LMW-Uk ( B) in 50 m
M glycine,
pH 3.4. The uneven curves represent the experimental curves,
and the smooth curves represent the best fits, i.e. the sums of the component transitions obtained by deconvolution.
Individual transitions on the tPA curve were assigned as in Ref. 19. A
schematic diagram illustrating the modular composition of each species
is presented in each panel. The tPA domains are shaded, and
the uPA serine protease module is
unshaded.
The total enthalpies of denaturation of
the full-length chimera and its deletion variant were found to be equal
to 299 and 245 kcal/mol. They are in good agreement with the expected
values of 310 and 245 kcal/mol, respectively, obtained by summing up
the enthalpies of the constituent transitions in
Fig. 2B. The number of transitions in both chimeric
molecules revealed by deconvolution analysis correlates with their
modular composition. Recall that the SP module actually comprises two
independently folded domains and that the transition corresponding to
melting of the finger module is not present on these curves since it
melts at very high temperature and requires addition of denaturants to
be detected
(19) . Thus, the calorimetric data strongly suggest
that all domains are folded properly in the chimeric molecules.
F
E derivative as well (Fig.
2 A). Kringle domains are known to be stabilized when their
lysine binding sites (LBS) are occupied with lysine or related ligands
(30, 31, 32) . In particular, the isolated
recombinant K2 of tPA, when loaded with
-ACA, melts at a
temperature of 86.5 °C, close to that of the high temperature
transition found in the chimera molecules
(33) . We therefore
propose the following hypothesis: in the tPA/uPA chimera the lysine
binding site of K2 is occupied intramolecularly by the lysine or
arginine residues coming from the urokinase moiety resulting in a
strong thermal stabilization of its structure. The most direct way
to test this hypothesis is to test directly for binding of LMW-Uk to
the A-chain of tPA in solution. For this purpose, the 32/35K A-chain
fragment of tPA was labeled with fluorescein and titrated with LMW-Uk
while monitoring the change in fluorescence anisotropy. As seen from
Fig. 3 A, LMW-Uk indeed interacts with the tPA fragment judging
by the hyperbolic increase of anisotropy upon titration. A similar
hyperbolic increase in anisotropy of FITC-labeled tPA A-chain was
observed upon addition of Pro-Uk (Fig. 3 B). The solid
lines on these graphs represent the best fits of the experimental data
to Equation 1 to obtain the dissociation constants of 7 µ
M for LMW-Uk and 2 µ
M for Pro-Uk. No changes in
anisotropy of FITC-labeled 32/35K tPA fragment were observed upon
titration with bovine serum albumin and several active site-blocked
serine proteases such as thrombin, trypsin, and chymotrypsin used as
controls. Thus, fluorescence titration data unequivocally indicate that
tPA interacts with uPA in solution.
Figure 3:
Titration of fluorescein-labeled 32/35K
subtilisin fragment of tPA with diisopropyl fluorophosphate-inhibited
LMW-Uk ( A) and Pro-Uk ( B). FITC-32/35K fragment at a
concentration of 0.1 µ
M was titrated with the indicated
proteins in TBS at 25 °C while monitoring the anisotropy at 524 nm.
Curves represent a best fit to Equation 1 to yield the K values indicated on the panels.
To localize the Uk binding site
on tPA, a solid-phase binding assay was utilized. Results are shown on
Fig. 4
, and the obtained dissociation constants are listed in
Table I. Whole tPA, its F
E deletion mutant, and its A-chain
all bind to LMW-Uk coated plates. The continuous best-fit curves in the
figure indicate that in all cases the binding is close to saturation
and consistent with a single class of binding sites. Since the kringle
domains are the only structures common for all three tPA species, they
should be responsible for the interaction with uPA. To prove that the
LBS is involved in this interaction, a competition assay with
-ACA
was performed. In this assay, a constant amount of tPA (1
µ
M) was added to the LMW-Uk coated wells containing
-ACA at varying concentrations. The displacement curve presented
in the inset to Fig. 4reflects the fact that the
described reaction could be inhibited by
-ACA. This strongly
suggests that the LBS of K2 is responsible for the interaction with
urokinase. Solid-phase binding was also demonstrated in the opposite
direction, i.e. with tPA adsorbed on the plate. As seen in
Fig. 5
, both Pro-Uk and HMW-Uk bind to solid-phase tPA, and,
again, this interaction was strongly inhibited by the presence of
-ACA. Data in the inset show that this process is also inhibited
by fluid-phase tPA. Thus, the solid-phase data confirm the fluorescence
results that tPA and uPA interact with each other and allow us to
conclude that the LBS of tPA mediates this intermolecular interaction.
Figure 4:
Enzyme-linked immunosorbent binding assay.
Increasing concentrations of tPA (),
F
E-tPA (
),
and 32/35K subtilisin fragment of tPA (
) were incubated with
microtiter wells coated with LMW-Uk or BSA (
) as a control. Bound
tPA species were detected with monoclonal antibody PAM2. The data are
representative of four experiments, each performed in duplicate. The
inset shows the displacement of tPA from the LMW-Uk coated
plate by
-ACA.
Figure 5:
Enzyme-linked immunosorbent binding assay.
Increasing concentrations of Pro-Uk (), HMW-Uk (
), and
HMW-Uk in the presence of 0.1
M
-ACA (▾) were
incubated with microtiter wells coated with tPA or BSA (
) as a
control. Bound uPA species were detected with monoclonal antibody
directed against urokinase A-chain. The data are representative of two
experiments, each performed in duplicate. The inset shows
competition of the binding by fluid-phase
tPA.
Having established a role for the LBS in binding of tPA with uPA, it
is necessary to address the question of what part of the uPA molecule
is reacting with LBS. A clue can be found in the fact that the isolated
A-chain of the chimera, in contrast to the A-chain of tPA, did not
express fibrin affinity
(24) . In other words, the LBS is not
available for binding even when separated from the bulk of the
urokinase moiety. The only difference between these two molecules is
the presence of a short residual peptide from Uk in the A-chain of the
chimera. This peptide, comprising residues
Leu-Lys
, has a carboxyl-terminal
lysine residue which might interact with the LBS of K2. It was also
recently reported that a similar peptide isolated from reduced LMW-UK
was able to inhibit the fibrin-stimulated activation of plasminogen by
tPA
(34) . Therefore, this peptide was synthesized in two
versions, with and without the carboxyl-terminal lysine, in order to
check its ability to inhibit the tPA-Uk interaction.
Fig. 6
presents competitive displacement data which were obtained
by premixing of Pro-Uk with FITC-labeled 32/35K tPA fragment and
titrating the complex with synthetic peptides or
-ACA. One can see
that both peptides displaced the FITC-labeled A-chain of tPA from its
complex with Pro-Uk. The K
for inhibition
by these peptides agreed within a factor of 2, precluding any
significant role of the carboxyl-terminal lysine residue. In addition,
the synthetic peptides are
100-fold more potent then
-ACA in
disrupting the complex proving that the inhibition is very specific.
This specificity was also demonstrated in a solid-phase assay in which
Leu
-Phe
at a concentration of 1
m
M increased the apparent K
for
binding of tPA to solid-phase uPA more than 100-fold whereas an
irrelevant cationic peptide NVSPPRRARVTDA at 4 m
M had no
significant effect (data not shown). Thus, the results indicate that
the tPA binding site on Uk is located within the sequence
Leu
-Phe
.
Figure 6:
Displacement of fluorescein-labeled 32/35K
subtilisin fragment of tPA from Pro-Uk by -ACA and synthetic
peptide corresponding to Uk sequence 144-158 with
( +K) and without ( -K) carboxyl-terminal
lysine. The FITC-labeled tPA fragment was premixed with 4 µ
M Pro-Uk in TBS, and increasing amounts of competitors were added
continuously while monitoring the anisotropy at 524 nm. Curves represent the best fit of the experimental data to Equation
1.
F
E-tPA/uPA exhibited a
3-16-fold enhanced thrombolytic potency in hamster, rabbit, and
baboon models of thrombolysis due to a 6-20-fold prolonged
circulating half-life in vivo (2) . But in
vitro, none of these chimeras demonstrated either the fibrin
affinity of tPA or the fibrin selectivity of uPA. Results presented in
this paper provide an explanation for this phenomenon: the lysine
binding site on kringle two of the tPA part (which is also the major
fibrin binding site) is occupied intramolecularly by one or more
positively charged side chains coming from that part of the urokinase
molecule that is responsible for its fibrin selectivity. Thus, in a
unique twist of fate, the advantages of both molecules canceled each
other in the chimera.
in fluid phase, the concentrations of
tPA and uPA in blood are seldom high enough for significant complex
formation. However, even the fluid-phase interaction could be important
in certain situations where the two plasminogen activators are
administered simultaneously in high concentration. During such
treatment, a synergistic effect was demonstrated in several animal
models of thrombosis
(39, 40) as well as in studies of
patients with acute myocardial infarction
(41, 42) . The
mechanism of this phenomenon is poorly understood, more so because of
inconsistent observations in vitro (43, 44) .
The interaction observed here may shed new light on this phenomenon
since it would tend to facilitate a local increase of uPA near the
fibrin clot.
-ACA with respect
to inhibition of the interaction between tPA and urokinase
(Fig. 6). This fact is in agreement with Song et al. (34) who showed that the corresponding region of the urokinase
A-chain, when isolated from reduced LMW-Uk, was 3500-fold more
effective than
-ACA as an inhibitor of fibrin-stimulated
activation of plasminogen by tPA. This also suggests that the peptide
binds with much higher affinity than
-ACA to the LBS on tPA.
Better understanding of this interaction, in addition to potentially
improving the chimera, could also lead to an improved antifibrinolytic
agent.
-Ile
bond
immediately adjacent to the tPA binding site identified here. This
suggests that this region is somehow involved in the fibrin
selectivity. Since in the chimera this region is already engaged
intramolecularly with the LBS on K2 of tPA, it might be less available
to confer fibrin selectivity. One may further speculate that this
region mediates interaction not only with tPA but with other proteins
that possess kringle structures with lysine binding properties. Recent
data showing an interaction of uPA with plasminogen
(45) are
consistent with this idea and indicate that possible LBS-mediated
interactions with uPA should always be taken into account.
Table:
Dissociation constants for the interaction
between tPA and uPA species obtained by enzyme-linked immunosorbent
assay or fluorescence anisotropy
F
E-tPA/uPA
chimera, recombinant protein consisting of amino acids 1-3 and
87-274 of tPA and 138-411 of uPA; F, finger module; E,
epidermal growth factor-like module; K, kringle module; SP, serine
protease module;
-ACA,
-aminocaproic acid; FITC, fluorescein
isothiocyanate; LBS, lysine binding site.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.