(Received for publication, April 24, 1995; and in revised form, June 5, 1995)
From the
Unlike most proteases, tissue-type plasminogen activator (t-PA)
is secreted from cells as an active, single chain
``proenzyme'' whose catalytic efficiency is comparable with
that of the corresponding mature, two-chain enzyme. We have previously
suggested that the absence of the ``zymogen triad''
(Asp
A wide variety of critical biological
processes(1, 2, 3) depend on specific
cleavage of individual target proteins by serine proteases. Important
examples include both the formation and dissolution of blood clots that
are catalyzed, respectively, by the blood coagulation cascade and the
endogenous fibrinolytic system(4, 5) . While blood
coagulation can be initiated by the activity of either Factor VIIa (the
extrinsic pathway) or Factor XIIa (the intrinsic pathway), the
initiating, and rate-limiting, step of the fibrinolytic system is
activation of the circulating zymogen plasminogen by the tissue-type
plasminogen activator (t-PA) ( Unlike typical
chymotrypsin-like enzymes, the single chain or ``proenzyme''
form of t-PA possesses high catalytic
activity(6, 7, 8, 9, 10, 11, 12) .
In the absence of the co-factor fibrin, single chain t-PA is
approximately 8% as active as two-chain t-PA. In the presence of
fibrin, however, single chain and two-chain t-PA display equivalent
enzymatic activity. ``Zymogen activation'' of single chain
t-PA, therefore, can be accomplished either by activation cleavage or
by binding to a specific co-factor. We have previously demonstrated
that introduction of the zymogen triad into t-PA substantially
increases the enzyme's ``zymogenicity,'' or ratio of
activity of the mature, two-chain enzyme to that of the single chain
form, by specifically suppressing the activity of single chain
t-PA(12) . These earlier measurements of the zymogenicity of
the mutated enzyme t-PA/A292S,F305H (
Following mutagenesis,
single-stranded DNA corresponding to the entire 472-base pair EcoRI fragment was fully sequenced to assure the presence of
the desired mutation and the absence of any additional mutations.
Replicative form DNA was prepared for appropriate phage, and the
mutated 472-base pair EcoRI fragments were recovered after
digestion of replicative form DNA with EcoRI and
electrophoresis of the digestion products on an agarose gel. The
isolated EcoRI fragments were used to reconstruct full-length
cDNAs encoding t-PA/R275E,t-PA/R275E,F305H and t-PA/R275E,A292S,F305H.
We have previously reported that the enzymes t-PA/F305H and
t-PA/A292S,F305H display zymogen-like properties when assayed with
synthetic substrates(12) . Kinetic constants for activation of
plasminogen by the single chain form of these variants, however, have
not been reported. To facilitate accurate measurement of these kinetic
constants, we utilized oligonucleotide-directed site-specific
mutagenesis to construct the enzymes t-PA/R275E, t-PA/R275E,F305H and
t-PA/R275E,A292S,F305H. The results of a kinetic analysis of the
activation of plasminogen, in the absence of fibrin, by t-PA,
t-PA/R275E, t-PA/R275E,F305H, and t-PA/R275E,A292S,F305H are depicted
in Table 1. Even before significant hydrolysis of the chromogenic
substrate Spectrozyme PL occurs in this assay, single chain wild type
t-PA is converted into a two-chain enzyme ((9) , data not
shown). By contrast, enzymes containing the R275E mutation remain in
the single chain form for the duration of the assay ((9) , data
not shown). Comparison of k
Table 2presents the results of a kinetic
analysis of plasminogen activation, in the presence of the fibrin
monomer DESAFIB, by the same four enzymes described above. In striking
contrast to their reduced activity in the absence of a co-factor, all
three mutated enzymes exhibit high catalytic activity in this assay. In
the presence of DESAFIB the k
By contrast to the
other human plasminogen activator u-PA, t-PA both binds to and is
substantially stimulated by
fibrin(20, 21, 22) . Table 3lists the
fibrin stimulation factor, defined as the ratio of k
With a molecular mass of almost 400 kDa,
fibrin is a very large protein whose interaction with t-PA appears to
be complex and to involve multiple points of contact with the enzyme
(for review, see (20, 21, 22) ). It is
therefore noteworthy, and perhaps surprising, that the introduction of
only three mutations into t-PA, R275E, A292S, and F305H, can enhance
fibrin stimulation of the enzyme to the extent that it becomes
comparable with that of Desmodusrotundis plasminogen
activator, by far the most fibrin-responsive plasminogen activator that
has been reported (23, 24, 25) . Discounting
the absence of a kringle 2 domain in the Desmodus enzyme, the
primary structures of human t-PA and Desmodusrotundis plasminogen activator still differ at 123 positions. Moreover, the
primary structures of the variant of human t-PA and the Desmodus enzyme differ at all three sites of mutation in the human enzyme
although both enzymes do contain mutations that prevent normal
maturation into a mature, two-chain enzyme. Data in Table 1Table 2, and Table 3establish that the
variants are more fibrin-stimulated than wild type t-PA; data in Fig. 1, on the other hand, indicate that the variants are also
more ``fibrin-selective'' than the wild type enzyme. While
the activity of t-PA is almost equally responsive to fibrin monomers,
cyanogen bromide fragments of fibrinogen, or a stimulatory 13-amino
acid peptide, the three variants show a striking preference for the
fibrin monomers over either of the nonphysiological co-factors. In
fact, again reminiscent of the Desmodus plasminogen activator,
both t-PA/R275E,F305H and t-PA/R275E,A292S,F305H maintain high activity
in the presence of fibrin monomers but are virtually nonresponsive to
the nonphysiological stimulators.
Figure 1:
Standard indirect chromogenic assay of
plasminogen activation in the presence of buffer (
We have previously reported that
the single chain form of the zymogen-like variants of t-PA, t-PA/F305H,
and t-PA/A292S,F305H exhibit resistance to inhibition by
PAI-1(12) . Compared with that of single chain t-PA, the second
order rate constant for inhibition by PAI-1 of single chain t-PA/F305H
or t-PA/A292S,F305H is reduced by a factor of approximately 5 or 25,
respectively. Second order rate constants for inhibition of single
chain t-PA, t-PA/R275E, t-PA/R275E,F305H and t-PA/R275E,A292S,F305H are
listed in Table 4. These data indicate that t-PA/R275E,F305H and
t-PA/R275E,A292S,F305H also exhibit resistance to inhibition by PAI-1;
the second order rate constant for inhibition of these enzymes by PAI-1
is reduced by a factor of approximately 9 or 69, respectively, compared
with t-PA/R275E.
The major findings of this study are that variants
of t-PA containing the zymogen triad exhibit zymogen-like properties
when assayed with the normal substrate plasminogen and that zymogen
activation of these variants can be accomplished by binding to the
co-factor fibrin. While details of the mechanism by which a zymogen is
activated upon binding to a specific co-factor remain completely
unclear, it is clear that this property is not unique to t-PA. Other
examples of this phenomenon include the activation of plasminogen by
streptokinase(26, 27, 28, 29, 30, 31) and
the activation of prothrombin upon binding to
staphylocoagulase(32, 33) . Since several
chymotrypsin-like enzymes have this property, it is tempting to
speculate that this mechanism of activation of zymogens might provide
the initiation event in (a) protease cascade(s) in vivo.
Although there is no current evidence to support the idea, this
strategy has the attractive feature that it avoids the difficulty of
generating proteolytic activity to activate the first zymogen in a
cascade. The mutated enzymes t-PA/R275E,A292S,F305H and
t-PA/R275E,F305H are substantially more fibrin-stimulated, and also
more fibrin-selective, than wild type t-PA. In vivo,
therefore, it is possible that, compared with wild type t-PA, these
enzymes would display significantly enhanced ``clot
selectivity'' due to reduced activity in the circulation but full
activity at a site of fibrin deposition. Whether enhanced clot
selectivity would improve the enzyme as a thrombolytic agent remains
extremely controversial, although this property may assume increased
importance as variants of t-PA with a prolonged half-life are
administered as a single bolus.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-His
-Ser
; chymotrypsin
numbering) contributes to this unusually high enzymatic activity of
single chain t-PA. Consistent with this prediction, the single chain
form of a variant of t-PA containing the zymogen triad displayed
dramatically reduced activity toward synthetic substrates. Activation
cleavage of this variant, however, resulted in a mature, two-chain
enzyme with full catalytic activity. To further examine the functional
significance of the zymogen triad, we used site-specific mutagenesis to
construct a variant of t-PA, t-PA/R275E,A292S,F305H, that contained
this triad but could not be converted into its two-chain form by
plasmin. Characterization of this variant demonstrated that the
presence of the zymogen triad specifically suppressed plasminogen
activation by single chain t-PA in the absence of fibrin. In addition,
these studies indicated that, like wild type t-PA, zymogen activation
of this variant could be accomplished by binding to the co-factor
fibrin. The combination of full activity in the presence of fibrin and
reduced activity in its absence resulted in novel variants of t-PA that
displayed dramatically enhanced stimulation by fibrin. While the
presence of fibrin increased the catalytic efficiency of t-PA toward
plasminogen by a factor of approximately 520, this stimulation factor
increased to 130,000 for t-PA/R275E,A292S,F305H. Plasmin-resistant,
zymogen-like variants of t-PA, therefore, may represent thrombolytic
enzymes with enhanced ``clot selectivity.''
)or urokinase-type plasminogen
activator (u-PA)(4, 5) .
)relied upon activity
assays that utilized small, synthetic substrates. Measuring the
activity of the single chain form of t-PA/A292S,F305H toward the normal
substrate plasminogen proved technically difficult because plasmin
generated during this assay very rapidly and efficiently converted
single chain t-PAs into the corresponding two-chain enzymes by cleaving
the Arg
-Ile
bond of single chain t-PA. To
overcome this technical difficulty, we have now added the mutation
R275E to the zymogen-like variants of t-PA. This strategy rendered the
resulting variants resistant to activation cleavage by plasmin and
therefore allowed accurate measurement of the kinetic constants for
plasminogen activation by the single chain form of the enzymes. These
measurements demonstrated that variants of t-PA that contain the
zymogen triad exhibited zymogen-like properties toward not only
synthetic substrates but also the normal protein substrate plasminogen
and that, as with the wild type enzyme, zymogen activation of these
variants could be accomplished by binding to the co-factor fibrin as
well as by activation cleavage. The combination of these properties
dramatically enhanced the extent of fibrin stimulation of the variants
compared with wild type t-PA. While fibrin enhanced the activity of
t-PA by a factor of approximately 520, the activity of the variant
t-PA/R275E,A292S,F305H was stimulated by fibrin by a factor of
approximately 130,000.
Site-directed Mutagenesis and Construction of
Expression Vectors Encoding Variants of
t-PA
Oligonucleotide-directed site-specific mutagenesis was
performed by the method of Zoller and Smith (13) as modified by
Kunkel(14) . Mutations were introduced into the 472-base pair EcoRI fragment of cDNAs encoding t-PA, t-PA/F305H, and
t-PA/A292S,F305H that had been previously subcloned into bacteriophage
M13 mp18. The mutagenic primer had the following nucleotide sequence:
5`-CAGCCTCAGTTTGAGATCAAAGGAGGG-3`.Expression of Enzymes by Transient Transfection of COS
Cells
cDNAs encoding t-PA, t-PA/R275E, t-PA/R275E,F305H and
t-PA/R275E,A292S,F305H were ligated into the transient expression
vector pSVT7 (15) and then introduced into COS 1 cells by
electroporation using a Bio-Rad Gene Pulser. 20 µg of cDNA, 100
µg of carrier DNA, and approximately 10 COS cells were
placed into a 0.4-cm cuvette, and electroporation was performed at 320
V, 960 microfarads, and
=
. Following
electroporation, cells were incubated overnight at 37 °C in
Dulbecco's modified Eagle's medium containing 10% fetal
calf serum and 5 mM sodium butyrate. Cells were then washed
with serum free medium and incubated in Dulbecco's modified
Eagle's medium for 48 h at 37 °C. After the incubation with
serum-free media, conditioned media were collected, and enzyme
concentrations were determined by enzyme-linked immunosorbent assay.
Purification and Quantitation of
Enzymes
Conditioned media were dialyzed against 20 mM sodium phosphate (pH 7.0), 100 mM NaCl, 20 mM
arginine, and 0.05% Tween 80 and loaded onto a lysine-Sepharose
(Pharmacia Biotech Inc.) column. The column was washed with 20 volumes
of loading buffer, and t-PA was eluted with buffer containing 20 mM sodium phosphate (pH 7.0), 100 mM NaCl, 200 mM arginine, and 0.05% Tween 80. Enzyme concentrations were measured
by enzyme-linked immunosorbent assay.Kinetic Analysis of Plasminogen Activation Using Indirect
Chromogenic Assays
Indirect chromogenic assays of t-PA utilized
the substrates Lys-plasminogen (American Diagnostica) and Spectrozyme
PL (American Diagnostica) and were performed as described previously (15, 16, 17) . Assays were performed both in
the presence and absence of the co-factor DESAFIB (American
Diagnostica). DESAFIB, a preparation of soluble fibrin monomers, was
produced by digesting highly purified human fibrinogen with the
protease batroxobin. Batroxobin cleaved the Arg-Gly
bond in the A
-chain of fibrinogen and consequently released
fibrinopeptide A. The resulting des-AA-fibrinogen or fibrin I monomers
are soluble in the presence of the peptide Gly-Pro-Arg-Pro. The
concentration of Lys-plasminogen was varied from 0.0125 to 0.2
µM in the presence of DESAFIB and from 0.9 to 15
µM in the absence of the co-factor.
Indirect Chromogenic Assays in the Presence of Various
Stimulators
Standard indirect chromogenic assays were performed
as described previously(15, 16, 17) .
Briefly, 0.25-1 ng of enzyme, 0.2 µM Lys-plasminogen, and 0.62 mM Spectrozyme PL were present
in a total volume of 100 µl. Assays were performed either in the
presence of buffer, 25 µg/ml DESAFIB, 100 µg/ml cyanogen
bromide fragments of fibrinogen (American Diagnostica), or 100
µg/ml of the stimulatory, 13-amino acid peptide P368. P368 was
kindly provided by Marshall Runge (University of Texas Medical Center,
Galveston, TX). Assays were performed in microtiter plates, and the
optical density at 405 nm was read every 30 s for 1 h in a Molecular
Devices Thermomax. Reactions were performed at 37 °C.Measurement of Second Order Rate Constants for Inhibition
by PAI-1
Second order rate constants for the inhibition of wild
type and mutated t-PA were measured under pseudo-first order conditions
as described
previously(16, 17, 18, 19) .
Briefly, enzyme and inhibitor were preincubated at 23 °C for
periods of time varying from 0 to 30 min. Following preincubation, the
mixtures were diluted, and the residual enzymatic activity was measured
in a standard indirect chromogenic assay. For each enzyme, the
concentrations of enzyme and inhibitor and the times of preincubation
were chosen to yield several data points for which the residual
enzymatic activity varied between 20 and 80% of the initial activity.
Data were analyzed by plotting ln (residual activity/initial activity) versus time of preincubation and measuring the resulting
slopes. Division of this slope by -[I] yielded the second
order rate constants shown.
/K
values for wild type t-PA and t-PA/R275E, therefore,
indicate that, in accord with previous
reports(6, 7, 8, 9, 10, 11, 12) ,
single chain t-PA has approximately 8% of the activity of two-chain
t-PA in this assay. Compared with that of t-PA/R275E, the catalytic
efficiency of t-PA/R275E,F305H is reduced by a factor of 4.4, while
that of t-PA/R275E,A292S,F305H is reduced 20-fold. Compared with the
activity of wild type t-PA, the activity of t-PA/R275E,F305H is reduced
approximately 58-fold, while that of t-PA/R275E,A292S,F305H is reduced
by a factor of approximately 264. These data demonstrate that the
mutation F305H suppresses the activity of the single chain form of t-PA
toward not only small, synthetic substrates but also the natural
protein substrate plasminogen. Moreover, the addition of a second
mutation, A292S, further suppresses the activity of single chain t-PA
toward plasminogen.
/K
value for
plasminogen activation by all three mutated enzymes varies by less than
6% from that of wild type t-PA. These data demonstrate that, as with
single chain wild type t-PA, zymogen activation of the variants
t-PA/R275E, t-PA/R275E,F305H, and t-PA/R275E,A292S,F305H can be
accomplished by binding to the co-factor fibrin.
/K
in the
presence of fibrin to that in the absence of fibrin, for t-PA,
t-PA/R275E, t-PA/R275E,F305H, and t-PA/R275E,A292S,F305H. As indicated
in Table 3, fibrin enhances the activity of the wild type enzyme
by a factor of approximately 520. Fibrin stimulation of all three
variants, however, is dramatically enhanced compared with the wild type
enzyme. The fibrin stimulation factor for t-PA/R275E, t-PA/R275E,F305H,
and t-PA/R275E,A292S,F305H is, respectively, 6900, 30,000, and 130,000.
Enhanced fibrin stimulation of all three variants results from the
combination of diminished activity in the absence of co-factor and high
activity in its presence.
) DESAFIB
(
), cyanogen bromide fragments of fibrinogen (
), or the
stimulatory peptide P368 (&cjs2134;).
We thank Guy Salvesen, Betsy Goldsmith, Jeff Smith,
Martin Schwartz and Dave Loskutoff for critical review of this
manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.