(Received for publication, September 11, 1996)
From the Departments of Biochemistry and Medicine,
Queen's University, Kingston, Ontario, Canada, K7L 3N6, and the
§ Department of Biochemistry, Academic Medical Centre,
University of Amsterdam, 1105 AZ Amsterdam, The Netherlands
A variant of recombinant plasminogen with the
plasmin active site serine (S741) replaced by cysteine was produced and
labeled with fluorescein at this residue to provide the derivative
Plg(S741C-fluorescein). Studies of cleavage, conformation, and
fibrin-binding properties of the derivative showed it to be a good
model substrate to study plasminogen activation. Both in solution and
in a fully polymerized fibrin clot, cleavage of the single chain
zymogen to the two-chain "plasmin" molecule was accompanied by a
50% quench of fluorescence intensity. This change allows facile,
continuous monitoring of the kinetics of cleavage. Measurements of
cleavage by single chain t-PA within intact, fully polymerized 3 µM fibrin yielded apparent kcat
and Km values of (0.08 s1, 0.52 µM) and (0.092 s
1, 0.098 µM)
for [Glu1]- and
[Lys78]Plg(S741C-fluorescein), respectively. These values
are similar to those obtained by others with plasma plasminogen. The
approach used here might generally be useful in simplifying the
analysis of zymogen activation kinetics in cases where the product
(protease) has a great influence on its own formation via positive or
negative feedback loops.
The fibrinolytic system leads to the formation of plasmin, which converts fibrin, the major protein constituent of blood clots, to soluble products. Activation of its inactive precursor plasminogen by tissue-type plasminogen activator (t-PA)1 occurs efficiently only when fibrin, but not fibrinogen, is present (1). Thus, fibrin is both a substrate for plasmin and a cofactor for plasmin formation (2). This makes an analysis of the t-PA-mediated activation process complex due to several proposed feedback loops. First, the generation of carboxyl-terminal lysines after limited proteolysis of the fibrin cofactor results in the generation of new, high affinity binding sites for plasminogen (3-5). Second, plasmin readily converts single chain t-PA to the more active two-chain form. Third, plasmin converts native [Glu1]plasminogen to the truncated form [Lys78] plasminogen, which is a superior substrate for t-PA (2, 6). The latter process is stimulated up to 200-fold by partly digested but not intact fibrin (7). A detailed kinetic analysis of the fibrinolytic process therefore necessitates the performance of steady state measurements during the different stages of fibrin degradation. Since this generates active plasmin, however, the structure of the cofactor fibrin, the substrate plasminogen and the enzyme t-PA will be subject to continuous change. Ideally, one would like to perform activation studies without the generation of active plasmin. All systems used so far, however, were based on measuring the generation of plasmin.
A clue to solving this problem came from the work of Drs. P. Bock and J. Shore, and co-workers, who constructed a plasminogen variant that was fluorescently labeled. They treated plasminogen with streptokinase in the presence of a chloromethyl ketone and thereby introduced a fluorescent label into plasminogen, such that subsequent cleavage could be monitored without the concurrent generation of active plasmin (8, 9). Studies with other proteases, inactivated by fluorescent labeling at the active site, also indicated that the properties of these labels thus positioned are sensitive probes to monitor changes in protease conformation (9-11). In this paper we describe the expression of a variant of recombinant human plasminogen, in which the plasmin active site serine has been replaced by cysteine: Plg(S741C). After labeling with a cysteine-specific fluorescent probe, we could quantify the rate of cleavage of this zymogen, Plg(S741C-fluorescein), without generating active plasmin.
The full-length plasminogen cDNA was a generous gift of Dr. L.-O. Heden (KabiGen AB, Stockholm, Sweden). The cysteine-specific fluorescent probe 5-iodoacetamidofluorescein (5-IAF) was obtained from Molecular Probes Inc. (Eugene, Oregon). Dulbecco's modified Eagle's medium:nutrient mixture F-12 (1:1), Opti-MEM I, and newborn bovine serum were from Life Technologies Inc. Methotrexate sodium injection (David Bull Laboratories, Mulgrave, Victoria, Australia) was purchased at the local hospital pharmacy. Chromogenic substrate D-Val-Leu-Lys-para-nitroanilide (S2251) and dansyl-Glu-Gly-Arg-chloromethyl ketone were obtained from Chromogenix (Molndal, Sweden) and Helena laboratories (Mississauga, Ontario, Canada), respectively. All DNA modifying enzymes were obtained from either Life Technologies, Inc. or Promega and were used according to the manufacturers' instructions. Sequenase 2.0 was obtained from U. S. Biochemical Corp.
ProteinsActivase (t-PA) was generously provided by Dr. G. Vehar (Genentech, San Francisco, California). High molecular weight
urokinase (u-PA) (>60,000 units/mg) was from Calbiochem; aprotinin was
Trasylol (Bayer, Leverkusen, Germany). Human -thrombin was produced
as described (12), and human fibrinogen (>98% clottable) was prepared from fresh-frozen, citrated plasma according to published procedures (13, 14). Human [Glu1]plasminogen was isolated from fresh
frozen plasma on lysine-Sepharose (Pharmacia Biotech Inc., Uppsala,
Sweden) as described (15). Lys-plasminogen and plasmin were produced as
described (6). Concentrations of the proteins were determined by
absorbance at 280 nm using the following specific absorbances for 1%
protein solutions: fibrinogen = 16.0 (16), plasminogen = 16.2 (17).
The full-length
plasminogen cDNA (18) was inserted as a
BalI-SphI fragment into the multiple cloning site
(mp-18) of pATA-18 as described (19). Site-directed mutagenesis was
achieved by the polymerase chain reaction overlap-extension technique
(20). We employed the two partly complementary oligonucleotides 2 and 3 (Table I) to change the codon for Ser741
(AGT) to Cys (TGT), whereas amplification of the 760 base pairs 3-end
of the plasminogen cDNA was achieved by oligonucleotides 1 and 4. The mutated fragment was used to substitute the "wild-type" EcoRV-SphI fragment of plasminogen in pATA-18.
The absence of undesired mutations was verified by DNA sequencing of
the entire fragment, using oligonucleotides 1, 3, and 4.
|
Stable cell lines expressing [Glu1]Plg(S741C) were constructed essentially as described for native Plg (19). In brief, cDNA for human [Glu1]Plg(S741C) was removed from the pATA-18 plasmid by digestion with HindIII and blunt ends were generated with T4-DNA polymerase. The expression vector pNUT (21) was digested with SmaI, treated with alkaline phosphatase, and ligated to the [Glu1]Plg(S741C) cDNA. The correct insertion and identity of [Glu1]Plg(S741C) cDNA was evidenced by sequencing, using oligonucleotides 5 and 6. BHK cells were cultured in Dulbecco's modified Eagle's medium/F-12, supplemented with 5% newborn bovine serum from which (bovine) plasminogen was removed by passage over lysine-Sepharose. Baby hamster kidney (BHK-21) cells were transfected with the pNUT -Plg(S741C) plasmid according to the calcium phosphate precipitate method (22). Sixteen hours after transfection, the growth medium was supplemented with 0.44 mM methotrexate to select for pNUT (dihydrofolate reductase). Two weeks after transfection, individual clones were screened for [Glu1]Plg(S741C) production by a sandwich enzyme-linked immunosorbent assay for human plasminogen (Affinity Biologicals, Ancaster, Ontario, Canada). Typical production levels were 10 µg/106 cells/day.
Production, Purification, and Fluorescein Labeling of [Glu1]Plg(S741C)Cell lines were grown in
500-cm2 triple flasks (Nunc) for large scale production. At
confluence, the cells were washed and the selection medium was replaced
by serum-free Opti-MEM I, supplemented with 50 µM
ZnCl2. Conditioned media were collected every other day;
supplemented with 1 µM dansyl-Glu-Gly-Arg-chloromethyl
ketone, 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA;
and loaded onto lysine-Sepharose. After washing the column with PBS (20 mM NaPi, pH 7.4, 150 mM NaCl) until
the A280 of the effluent was <0.002,
[Glu1]Plg(S741C) was eluted with PBS, 10 mM
6-aminohexanoic acid. Fractions containing >0.25 mg/ml
[Glu1]Plg(S741C) based on A280
were pooled. Labeling was performed directly on this pooled fraction
(typically 10-15 ml, 6-10 µM [Glu1]Plg(S741C)) by adding 150 µl of 20 mM
5-IAF in N,N-dimethylformamide. The reaction was continued
for 30 min at room temperature in the dark. Excess label was removed
from this incubation mixture on a 1-ml DEAE-fast flow column
(Pharmacia, Uppsala, Sweden), equilibrated and run in PBS, 0.05% Tween
80, resulting in elution of Plg(S741C-fluorescein) in the flow-through
and binding of 5-IAF to the resin. Subsequently, the protein was
concentrated and freed from remaining traces of 5-IAF and
6-aminohexanoic acid (6-AHA) by applying to a 5-ml DEAE-fast flow
column after a 1:5 dilution with 20 mM Tris-HCl, pH 8.0. The column was washed with 20 mM HEPES-NaOH, pH 7.4, and
the labeled protein eluted with HBST (20 mM HEPES-NaOH, pH
7.4, 150 mM NaCl, 0.02% Tween 80) and stored in aliquots
at
70 °C. All chromatographic steps were performed in the dark,
and control runs were performed with "wild-type" plasminogen to
determine the absence of nonspecific labeling. The amount of
fluorescein incorporation was determined spectrophotometrically using
an extinction coefficient at 495 nm for fluorescein of 84,000 M
1 cm
1 (Molecular Probes). The
concentration of the labeled protein was determined from absorbance at
280 nm, after correction for the contribution of fluorescein
(A280 = 0.19·A495).
Typical incorporation levels were at 0.9 mol/mol.
[Glu1]Plg(S741C-fluorescein) was converted to
[Lys78]Plg(S741C-fluorescein) by adding 5 µl of 188 µM plasmin to 2.5 ml of 15 µM plasminogen
in HBST supplemented with 5 mM 6-aminohexanoic acid. After
90 min, plasmin was removed by binding to 1 ml of aprotinin-agarose.
The flow-through, containing the
[Lys78]Plg(S741C-fluorescein), was treated with 10 µM valylphenylalaninyllysyl chloromethyl ketone for
1 h. The absence of traces of plasmin was verified by incubating
aliquots of the protein at 37 °C with 1 mM S2251 for
2 h, in which period no increase of the absorbance at 405 nm was
observed. Samples of Plg(S741C-fluorescein) (Glu1 and
Lys78 form) were subjected to urea/acetic acid 7.5% PAGE
(23) at 120 V in a minigel system and mobilities were compared to
native plasminogen. Gels were stained with Coomassie Brilliant Blue and destained.
Binding of [Glu1]- or
[Lys78]Plg(S741C-fluorescein) forms to a fibrin clot was
measured as follows. To a series of microcentrifuge tubes containing 2 µl of 30 nM -thrombin in HBST, 0.5 M
CaCl2 equilibrated at 37 °C, were added 98 µl of HBST
with a fixed concentration of Plg(S741C-fluorescein) and various
concentrations of fibrinogen. Clotting of the fibrinogen was complete
within 1 min, after which the incubation was continued for 10 min at
37 °C. Subsequently, the fibrin clot was pelleted by centrifugation
for 1 min at 10,000 × g and the supernatant was
removed immediately. The amount of non-fibrin-bound
Plg(S741C-fluorescein) was determined by quantitation of fluorescence
intensity of the supernatant, as follows. Aliquots of 25 and 75 µl of
each supernatant were added to 75 and 25 µl of HBST in 96-well
fluorescence plates, and fluorescence intensity was measured at
excitation and emission wavelengths of 495 and 535, respectively,
employing a 530-nm emission cut-off filter. Fluorescence intensities
were converted to concentration of plasminogen in the supernatant by
comparison to the supernatant of an otherwise identically treated
sample that did not contain fibrin. The latter was identical to the
intensity of untreated plasminogen, directly diluted from the stock
solution. Fluorescence intensities obtained in this way were linear
with respect to plasminogen concentration as established by measuring,
under identical conditions, the fluorescence intensities in wells that
contained serial dilutions of fluorescent plasminogen. In an
alternative experiment, we determined the binding of various
concentrations of [Lys78]Plg(S741C-fluorescein) to 1 µM fibrin clots. Experimental conditions were as
described above, but fibrin was kept constant at 1 µM and
concentrations of [Lys78]Plg(S741C-fluorescein) ranged
from 0.5 to 4 µM.
Experiments to measure the kinetics of u-PA-catalyzed cleavage of [Glu1]- or [Lys78]Plg(S741C-fluorescein) at Arg561-Val562 were performed in 96-well fluorescence plates (Dynatech) at 20 °C, using a Perkin Elmer LS50B Luminescence Spectrometer equipped with a fluorescence plate reader. Fluorescence intensities were measured at excitation and emission wavelengths of 495 and 535, respectively, employing a 530-nm emission cut-off filter. [Glu1]- or [Lys78]Plg(S741C-fluorescein) (90 µl) was added to wells with HBST and equilibrated until a stable fluorescence signal was obtained. Then, 6-AHA was added at various concentrations and again a stable signal was obtained. Next, 15 units of u-PA were added to give a final volume of 100 µl and fluorescence intensity was followed at 1-min intervals. The time courses of cleavage of fluorescent plasminogen were subjected to nonlinear regression analysis according to a single exponential decay process, from which the rate constants and, therefore, rates of the reaction were determined. This analysis was justified since rates of cleavage activation were linear with respect to both the u-PA and [Glu1]- or [Lys78]Plg(S741C-fluorescein) concentration. Second order rate constants were obtained from the pseudo first order rate constants by assuming 60,000 units/mg u-PA and a molecular weight of 55,000.
Fluorescent Plasminogen Cleavage by t-PAExperiments to
measure the kinetics of t-PA-catalyzed cleavage of
[Glu1]- or
[Lys78]Plg(S741C-fluorescein) at
Arg561-Val562 were performed in 96-well
fluorescence plates (Dynatech) at 20 °C, using a Perkin Elmer LS50B
Luminescence Spectrometer equipped with a fluorescence plate reader.
Fluorescence intensities were measured at excitation and emission
wavelengths of 495 and 535, respectively, employing a 530-nm emission
cut-off filter. Wells were pre-equilibrated with HBST for 1 h to
prevent absorbance of proteins to the plastic. Subsequently, wells were
loaded with 90 µl of HBST containing various concentrations of
[Glu1]- or [Lys78]Plg(S741C-fluorescein)
and 3.3 µM fibrinogen, and were equilibrated at 20 °C.
The stability of the fluorescence intensity was verified for 5 min. The
reaction was initiated by adding 10 µl of HBST, 100 mM
CaCl2 containing 60 nM human -thrombin and
t-PA (1-10 nM final concentration). Data were collected
every 60 s and stored as print files for each individual well
using a data acquisition program written by Dr. W. K. Stevens in our
laboratory. Initial rates of fluorescence decrease were determined by
linear regression analysis and converted to rates of plasminogen
activation according to Equation 1.
![]() |
(Eq. 1) |
Recombinant
[Glu1]Plg(S741C) was expressed in stably transformed BHK
cells at production levels of 10 µg of plasminogen/ml of serum-free
medium (106 cells)/day. [Glu1]Plg(S741C) was
purified from the BHK conditioned medium to apparent homogeneity by
conventional affinity chromatography on lysine-Sepharose. Analysis of
the protein on acid urea gels (Fig. 1) showed the absence of detectable amounts of degraded products. Laser densitometry of Coomassie-stained acid/urea gels showed that the recombinant plasminogen displays a similar ratio of the two glycoforms: 35% plasminogen-I (glycosylated at Asn288 and
Thr345) and 65% plasminogen-II (glycosylated at
Thr345 only) as observed for plasma plasminogen (24) and
recombinant wild-type plasminogen (19).
The production levels in BHK cells and yields after purification on
lysine-Sepharose of [Glu1]Plg(S741C) were identical to
those for wild-type recombinant plasminogen (19). This suggests that no
gross alterations in the structure result from the introduction of a
new, free cysteine replacing serine 741. To further substantiate the
validity of this plasminogen variant as a model, we analyzed by
intrinsic fluorescence one of the most striking properties of
[Glu1]plasminogen, namely its tight, activation-resistant
conformation (25, 26). The increase of this intrinsic fluorescence
(Fig. 2) has a sigmoidal relationship with increasing
concentrations of the lysine analog 6-AHA as shown previously for
plasma derived plasminogen (27) and "wild-type" recombinant
plasminogen (19), indicating the tight (activation-resistant)
conformation of the Glu1 form of this variant.
Plg(S741C) Lacks Detectable Proteolytic Activity
The absence of amidolytic and proteolytic activity in the variant plasminogen molecule (S741C) was substantiated by "activation" with the three plasminogen activators u-PA, t-PA, and streptokinase as follows. [Glu1]Plg(S741C) at 5 µM was incubated with 150 units/ml u-PA in the presence of the chromogenic substrate S2251 (1.0 mM), and the absorbance at 405 nm was followed in time for 20 h. The progress curve did not vary from similar experiments without plasminogen, whereas identical experiments with native plasminogen indicated a lower detection limit of 0.5 nM plasmin. Incubations of 5 µM [Glu1]Plg(S741C) with 5 or 50 units/ml streptokinase, which generates an active site in plasminogen by 1:1 complex formation rather than cleavage at Arg561-Val562, were performed in a similar fashion as described (19), and gave an amidolytic activity (S2251) corresponding to 0.0125% of the input [Glu1]Plg(S741C), when compared to the native plasminogen-streptokinase complex in an identical experiment. Finally, 5 µM [Glu1]Plg(S741C) was included in a clot lysis experiment with 3 µM fibrin and 5 nM t-PA, as described previously (19, 28). The turbidities of these clots were stable for 16 h, whereas in control experiments run simultaneously at little as 0.1 nM native plasminogen can be detected by a decrease in turbidity due to lysis of the fibrin. Based on these assays we conclude that [Glu1]Plg(S741C) when "activated" does not possess sufficient intrinsic amidolytic activity to perturb the experiments that are described in this paper.
Fluorescent Labeling of [Glu1]Plg(S741C) and Characterization of [Glu1]Plg(S741C-fluorescein)Fluorescent labeling of the introduced "active site" cysteine to generate [Glu1]Plg(S741C-fluorescein) could be accomplished nearly quantitatively (0.85 ± 0.1 mol of fluorescein/mol of plasminogen, n = 10) as described in detail under "Experimental Procedures". Upon full conversion of [Glu1]Plg(S741C-fluorescein) to the two-chain "plasmin" form by u-PA, the fluorescein label was bound exclusively to the protease domain of plasminogen, as deduced from SDS-PAGE analysis (see below). Recombinant "wild-type" plasminogen did not incorporate detectable levels of fluorescein when produced and subjected to labeling under identical conditions.
The interaction of [Glu1]Plg(S741C-fluorescein) with
6-AHA was analyzed by quantifying the change in fluorescence intensity of the reporter group (Fig. 3). Contrary to the results
on the intrinsic fluorescence change (Fig. 2), the intensity decreases (21.4% at saturation) and can be described best by a model that embodies binding to a single binding site with Kd of 2.52 mM. The Hill coefficient for the inferred binding
suggested minimal cooperativity (h = 1.1). The decrease
in fluorescence intensity of
[Lys78]Plg(S741C-fluorescein) was < 1.5% at 10 mM 6-AHA. Intrinsic (Trp) fluorescence cannot be studied
for these molecules due to interference by the fluorescein label,
precluding a direct comparison with plasma plasminogen or
[Glu1]Plg(S741C). Comparison of the results of Figs.
2 and 3, however, suggests that the interaction between 6-AHA and
plasminogen measured by intrinsic fluorescence, with a Hill coefficient
of 2.0 and a transition midpoint at 0.52 mM 6-AHA, is
different from the interaction measured by extrinsic fluorescence.
The existence of an activation-resistant conformation has been used to
rationalize the weak fibrin binding of [Glu1]- as
compared to [Lys78]plasminogen. To determine whether the
Glu1 and Lys78 forms of the fluorescent
plasminogen exhibit these differences in affinity for fibrin, we
measured their binding to fully polymerized clots (Fig.
4). Fibrin binding of
[Glu1]Plg(S741C-fluorescein) was weak with an estimated
Kd of 30 µM, whereas that of
[Lys78]Plg(S741C-fluorescein) was stronger
(Kd = 1.2 µM, n = 1.8 sites/fibrin). These trends are identical to those of the "wild-type" recombinant plasminogen species (19), and the values of
the binding parameters are similar to those reported for the plasma
plasminogen forms (Kd = 38 µM and
0.32, respectively) (29).
The Activation Cleavage of Plg(S741C-fluorescein) by u-PA in Solution
[Glu1]Plg(S741C-fluorescein) was treated
with 6-AHA, and the initial decrease in fluorescence was measured. Then
urokinase was added and the progressive decrease over time was
monitored continually. An example is shown in Fig.
5A. Functional homogeneity of the fluorescent
plasminogen is suggested by the coincidence of data and the line
obtained by linear regression to the equation for first order decay. In
this experiment, samples were withdrawn at regular intervals after the
addition of urokinase and subjected to SDS-PAGE. The fluorescent bands
were photographed (Fig. 5A, inset), and the gel
was stained with Coomassie Blue, destained, and scanned with a
densitometer. As the inset of Fig. 5A indicates, the progressive decline in intensity correlates with cleavage and, as
expected, the fluorescence is associated exclusively with either the
zymogen or the light chain, with no visible fluorescence in the heavy
chain. Although the data are not shown, densitometry indicated that the
decline in fluorescence after the addition of urokinase was linear in
the extent of cleavage of [Glu1]Plg(S741C-fluorescein),
and linear regression of intensity values to the extent of the reaction
predicted an overall drop in intensity (including that due to 6-AHA) of
54% at 100% completion of the reaction.
Notably, the maximal decrease in fluorescence intensity of
[Glu1]Plg(S741C-fluorescein) upon activation at different
6-AHA concentrations was inversely proportional to the initial change
upon adding 6-AHA (data not shown), making the combined effects of
6-AHA and u-PA constant at about a 50% decrease. From this observation
we suggest that the [Glu1] plasmin molecule has a
relaxed conformation similar to [Lys78] plasminogen,
and therefore no effect of 6-AHA on the overall intensity change is
observed. This is in agreement with results on the properties described
for active site blocked Glu-plasmin, which are similar to
Lys-plasminogen, with respect to, for example, affinity for fibrin
(30). The influence of 6-AHA on the rate of plasminogen activation by
urokinase in solution has been well documented and resulted in the
proposal of a tight, activation-resistant conformation of
[Glu1]plasminogen in the absence of such lysine analogs
(26, 31). A similar effect of 6-AHA on activation rates is shown for
[Glu1]Plg(S741C-fluorescein) in Fig. 5B. The
decrease at higher concentrations of 6-AHA has been shown to result
from the direct inhibitory effect of lysine analogs on the activity of
u-PA (31). The maximal rate of cleavage in this experiment corresponds
to a second order rate constant of 0.016 µM1 s
1, which is similar to
that determined for recombinant [Glu1]plasminogen (0.02 µM
1 s
1) and that reported for
plasma [Glu1]plasminogen (0.027 µM
1 s
1) by Lenich et
al. (32). For [Lys78]Plg(S741C-fluorescein), no
increase in activation rate was observed upon addition of 6-AHA, with
second order rate constants for u-PA activation of 0.019 µM
1 s
1 in the absence of
6-AHA and 0.017 µM
1 s
1 in the
presence of 5 mM 6-AHA, and total decrease in fluorescence intensity of 40% (data not shown).
The
t-PA-mediated cleavage of the Glu1 and Lys78
forms of Plg(S741C-fluorescein) in the absence of any cofactor proceeds
at a very low rate (Fig. 6). Under these
conditions, the second order rate constant for
[Lys78]Plg(S741C-fluorescein) activation is 4.2-fold
higher than for [Glu1]Plg(S741C-fluorescein):
1.43 × 103 and 3.44 × 10
4
µM
1 s
1, respectively. Rates
did not show any sign of saturation up to [Glu1]- or
[Lys78]Plg(S741C-fluorescein) concentrations of 8 µM, probably reflecting a high Km for
this reaction, as reported for plasma plasminogen (2).
The time courses of fluorescence intensity obtained when solutions of
[Glu1]Plg(S741C-fluorescein) (0.2 µM,
final) and fibrinogen (0.05 µM or 3.0 µM,
final) were added to the wells of microtiter plates, and the reactions
were initiated with a solution of CaCl2 (10 µM, final), thrombin (6.0 nM, final), and
t-PA (25 nM, final) are indicated in Fig.
7A. Upon initialization of the reactions, an
initial decrease in intensity (~10%) occurred because of dilution. Although a small additional change (4.8%) followed the polymerization of fibrin at the high input level of fibrinogen, as evidenced by the
difference in control (minus t-PA) signals of Fig. 7A, the
magnitude of the subsequent decreases when the reactions approached completion were virtually identical at both the high and low fibrin concentrations. The relative lack of influence of fibrin polymerization on the signal, potentially due to, for example, light scattering can
most likely be attributed to the plate reader format whereby both the
excitation and emission optics are above the sample and the sample well
is quite reflective. The lines of Fig. 7A are the regression
lines obtained by fitting the data to the equation for first order
decay, a procedure that is justified because of the relatively low
input concentration of [Glu1]Plg(S741C-fluorescein). The
good fit of the data to the equation implies functional homogeneity.
The regression analysis indicated a 50% decrease in intensity upon
completion of the reaction. The data from similar experiments with
[Lys78]Plg(S741C-fluorescein) did not fit as well to the
first order decay equation (due to low Km), but
monitoring for extended periods indicated a 40% decline in intensity
at completion of the reaction (data not shown), at both low and high
input concentrations of fibrinogen. In the absence of the
fluorescein-labeled protein, the signal was negligible (7.0% or less
than that with the fluorescent protein over the range of protein
concentrations studied). This blank value, however, was subtracted from
all relevant data.
In order to measure initial rates, activator concentrations were
decreased so that the approximately linear portion of the reaction
could be measured. An example is shown in Fig. 7B. In this
case the magnitude of signal change encompassed by the exhibited data
is about 10% of the total signal, which corresponds to about 20%
consumption of the substrate. Over this range the rate was essentially
constant. In typical measurements of initial rates, data such as those
of Fig. 7B were subjected to linear regression to determine
the slope. This approach was employed to obtain the apparent
kcatand Km values for the
t-PA-catalyzed cleavage of [Glu1]- and
[Lys78]Plg(S741C-fluorescein) at a single input
concentration of fibrinogen and thus compare them to values obtained by
others with native plasminogen utilizing soluble substitutes for fibrin
or fibrin films. Initial rates of cleavage versus nominal
concentrations of the substrate fit well to a rectangular hyperbola,
indicating Michaelis-Menten-like kinetics (Fig. 8).
We describe the production of a variant of plasminogen in which the serine of the plasmin catalytic triad has been replaced by cysteine. This enabled the introduction of a fluorescein label at the position of this residue in plasminogen to produce [Glu1]Plg(S741C-fluorescein). Characterization of the activating cleavage, conformation, and fibrin-binding properties of this variant showed it to be a good model substrate to study plasminogen activation. Both in solution and in a fully polymerized fibrin clot, the activation of the single chain zymogen to the two-chain "plasmin" molecule was accompanied by an approximate 50% quench of the fluorescence intensity of the fluorescein reporter group (40% for the Lys78 form), indicating a substantial change in the micro-environment of this probe upon occurrence of the activation cleavage. Results for active proteases, which had been labeled via protein-chemical approaches, had already indicated the sensitivity of this position within the protease domain (9-11). We show that this approach yields a variant of the zymogen, at high levels of expression, which differs from the native zymogen only by a serine to cysteine substitution. In the case of plasminogen, we did not find indications of malfolding as a result of this change, since production levels in BHK cells as well as other properties of this variant were identical to those obtained for "wild-type" plasminogen (19).
Recently Bock and co-workers (9) described the production of a fluorescent plasminogen derivative similar to that reported here. They treated plasma plasminogen with streptokinase and covalently modified the active site with a thioester chloromethyl ketone. The thioester was subsequently hydrolyzed with hydroxylamine, and the thiol group was covalently modified with an anilinonaphthylsulfonate moiety. The fluorescent derivative was then separated from streptokinase. The active site-modified fluorescent derivative yielded readily measured spectral changes upon interaction with lysine analogues, with streptokinase, and upon cleavage to the plasmin derivative. The kinetics of cleavage by urokinase of the derivative and unmodified plasma plasminogen were very similar, indicating that the derivative is a good surrogate for unmodified plasminogen whereby the properties of plasminogen can be monitored. The cleaved form of the derivative yielded plasmin activity of about 1.0% of native plasmin. The presently described derivative Plg(S741C-fluorescein) has many of the properties of the derivative described by Bock et al. (9). Its fluorescence properties are sensitive to lysine analogues and cleavage by plasminogen activators. In addition, it appears to be a good derivative for analyzing the interactions and reactions of plasminogen. Unlike the derivative prepared with streptokinase, however, the presently described derivative exhibits no detectable plasmin activity when cleavaged by plasminogen activators, whether or not cysteine 741 is modified with a fluorescent probe. This lack of activity is undoubtedly the result of the cysteine for serine substitution at position 741. Thus, Plg(S741C) appears ideally suited as a tool to produce plasminogen derivatives that can be labeled with thiol specific probes and that generate no plasmin activity upon cleavage by plasminogen activators.
Plg(S741C-fluorescein) enabled us for the first time to do steady state
determinations of activation rates in a fully polymerized fibrin
clot without solubilization of the clot. Since no active protease was
formed, none of the positive feedback loops of fibrinolysis have taken
place. The stimulation factors (rate fibrin absent/rate fibrin present)
were several hundredfold for both forms of plasminogen. In a very
detailed study in which plasminogen activation rates were deduced from
rates of release of isotope from 125I-labeled fibrin films
due to generated plasmin, kcat and
Km values of 0.1 s1, 0.16 µM and 0.2 s
1, 0.02 µM were
obtained for [Glu1]- and
[Lys78]plasminogen, respectively (2). Our results on the
activation in an intact fully polymerized 3 µM
fibrin clot by single-chain t-PA yield apparent
kcat and Km values of 0.08 s
1, 0.52 µM and 0.092 s
1,
0.098 µM for [Glu1]- and
[Lys78]Plg(S741C-fluorescein), respectively. The values
for [Glu1]Plg(S741C-fluorescein) are similar to those
reported on soluble fibrin monomer and native
[Glu1]plasminogen, where initial rates in the early
phase of plasminogen activation were deduced from extrapolation to time
zero (e.g. before plasmin feedback): 0.17 s
1
and 1 µM (33). It has been suggested that the conversion
of single-chain t-PA to two-chain t-PA, [Glu1]plasminogen
to [Lys78]plasminogen, and intact fibrin to
proteolytically modified fibrin, which are all plasmin-catalyzed, might
result in substantial acceleration of the rate of plasminogen
activation. The kinetic parameters presented in the present study,
however, are very similar to those obtained in assays in which all of
the mentioned feedback loops do occur. Hence, our results indicate that
the effects of the proposed feedback loops in a polymerized fibrin clot
might be limited under non-pathological conditions. Differences are
restricted to decreased Km values for plasminogen
activation, being 0.52 µM
([Glu1]plasminogen, intact fibrin, single chain t-PA;
this study) to minimally 0.02 µM
([Lys78]plasminogen, two chain t-PA, plasmin-modified
fibrin; Ref. 2). At physiological levels of fibrin (9 µM)
and plasminogen (1.5 µM), this will result in a 25%
increase in rate of plasminogen activation. Adverse conditions like
clot retraction, however, might lead to plasminogen depletion, and
therefore the decrease in Km could have a more
dramatic effect.
In conclusion, the approach to label a zymogen at serine of the corresponding active site of the enzyme via mutation to cysteine and incorporation of a fluorescent, cysteine-specific probe might have wide applicability, since it enables steady state activation studies through a readily measured signal. In addition, it simplifies the interpretation of kinetics in cases where the product (protease) has a great influence on its own formation via positive or negative feedback loops.
We thank Drs. P. Bock and J. Shore for making their fluorescent plasminogen species available to us during preliminary stages of the work presented here. The technical and administrative assistance of Tom Abbott and the gift of purified fibrinogen by John Walker are greatly appreciated.