 |
INTRODUCTION |
Hemeostasis requires a proper balance between the coagulation and
fibrinolytic systems. In response to vascular injury, a hemostatic plug
is generated by converting fibrinogen to an insoluble fibrin clot
through the action of thrombin, the terminal enzyme of the coagulation
cascade. Fibrinolysis, the breakdown of the fibrin clot, is achieved
primarily by the activation of plasminogen to the serine protease
plasmin, which catalyzes degradation of the insoluble fibrin clot to
soluble fibrin degradation products (FDPs).1 The activation of
plasminogen can be catalyzed by both endogenous activators such as
tissue-type plasminogen activator (t-PA) and urokinase or exogenous
activators such as streptokinase, staphylokinase, and Desmodus
rotundus salivary plasminogen activators (DSPAs). These enzymes
have all been used as thrombolytic agents for the dissolution of
pathological thrombi, which can cause both myocardial infarction and stroke.
The fibrin clot is not only the substrate for plasmin but also a
cofactor for plasmin generation by the various plasminogen activators.
Both t-PA and DSPA
1 are known as fibrin-selective plasminogen
activators, because the rate of plasminogen activation with both
activators is increased several orders of magnitude in the presence of
fibrin, as compared with fibrinogen (1). Extensive plasminogen
activation in the plasma, mediated via the cofactor effect of
fibrinogen, results in systemic, plasmin-mediated fibrinogenolysis and
consumption of
2-antiplasmin, severely compromising the
coagulation potential of the plasma (1, 2). Fibrin selectivity is thus
highly desirable for systemically administered thrombolytic agents.
DSPA
1 is considerably more fibrin-selective than t-PA, as the
catalytic efficiency of DSPA
1 is stimulated 13,000-fold, compared
with only 820-fold for t-PA, when fibrin is the cofactor instead of
fibrinogen (1). Furthermore, DSPA
1 is intrinsically less
fibrinogenolytic than t-PA because the catalytic efficiency of DSPA
1
is 13-fold lower than t-PA when fibrinogen is the cofactor (50 versus 640 M
1
s
1) (1).
The stimulation of plasminogen activation by fibrin is mediated by
interactions of both the activator and plasminogen with fibrin (3).
Structures within t-PA by which it interacts with fibrin are its
fibronectin finger-like domain and its kringle-2 domain. The
interaction of DSPA
1 with fibrin is presumably mediated solely by
its finger domain, since it lacks a kringle-2 domain, although at least
one other low affinity interaction is likely, because DSPA
and
DSPA
, highly homologous relatives of DSPA
1 (89 and 91% identity,
respectively) lacking the finger domain, are also stimulated by fibrin,
albeit to a much lesser extent (1). The interaction of plasminogen with
fibrin occurs by its lysine-binding kringle domains. Two forms of
plasminogen exist. Glu-plasminogen, the full-length form found
circulating in plasma, interacts only weakly with intact fibrin but
strongly with partially degraded fibrin possessing carboxyl-terminal
lysine and/or arginine residues (4). Lys-plasminogen, a truncated
version of plasminogen produced by the plasmin-catalyzed removal of a
77-residue peptide from the amino terminus, binds to both native and
partially degraded fibrin tightly (4). Lys-plasminogen is a
considerably better substrate for t-PA, and its formation during
t-PA-mediated fibrinolysis confers positive feedback on the process (3,
4). Since the cleavage of fibrin by plasmin exposes carboxyl-terminal
lysine and arginine residues, producing a fibrin surface containing
high affinity plasminogen-binding sites, partial degradation of fibrin by plasmin results in the recruitment of plasminogen to the partially degraded fibrin surface (5-7). The partially degraded fibrin is a
superior cofactor than intact fibrin for plasminogen activation (8).
This effect can be eliminated by the basic plasma carboxypeptidase, TAFIa, which removes the carboxyl-terminal lysine and arginine residues
from the fibrin surface (8). Plasmin production, therefore, results in
a positive feedback loop that can be down-regulated through the
generation of TAFIa from its precursor TAFI.
Plasmin-catalyzed digestion of fibrin produces soluble FDPs which,
owing to their structural similarity to fibrin and/or fibrinogen, likely act as cofactors for plasminogen activation. We have recently demonstrated that FDPs released from a perfused clot are composed of
noncovalently associated products whose masses range from 250 kDa (the
mass of DD/E) to ~10,000 kDa (9). Furthermore, our work showed that
the majority of the FDPs compose structures much larger than DD/E.
Since the relationship between the sizes of FDPs and their cofactor
effects has not been studied, we isolated FDPs with different masses to
study the relationship between FDP mass and cofactor activity in
reactions with t-PA and DSPA
1. Furthermore, since the FDPs contain
carboxyl-terminal lysine and arginine residues as a result of plasmin
degradation, we also investigated the effect of the TAFIa-catalyzed
removal of the carboxyl-terminal lysine and arginine residues on the
cofactor activity of FDPs. The work described in this paper represents the first extensive study of the cofactor effect of FDPs on plasminogen activation catalyzed by the fibrin-specific plasminogen activators, t-PA and DSPA
1.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Human fibrinogen, plasminogen (Pgn), prothrombin,
and factor XIII (FXIII), were prepared as described previously (9).
Plasmin (Pn), thrombin (IIa), and FXIIIa were prepared from the
purified zymogens as described (9). Recombinant human
thrombin-activable fibrinolysis inhibitor (TAFI) was produced as
described by Boffa et al. (10). The recombinant, active site
serine to cysteine mutant of human plasminogen, plasminogen(S741C), was
produced as described by Horrevoets et al. (11). The
fluorescein derivative, plasminogen(S741C-fluorescein), hereafter
referred to as 5AF-Pgn, was prepared as described (11). A soluble form
of human thrombomodulin, Solulin, was a kind gift of Dr. John
Morser of Berlex Biosciences (Richmond, CA). The recombinant
plasminogen activator from the vampire bat Desmodus
rotundus, DSPA
1, was a kind gift of Dr. Peter Bringmann at
Schering, AG (Berlin, Germany). The recombinant tissue-type plasminogen
activator, activase, was a kind gift from Dr. Gordon Vehar at Genentech
(South San Francisco, CA).
Preparation of FXIIIa Cross-linked Soluble Fibrin Degradation
Products--
Soluble FDPs were prepared as described previously (9),
except that 1) clots were formed in columns with 9 ml of available volume, 2) the clots were perfused with 0.2 nM plasmin
instead of 0.05 nM plasmin, and 3) the FDPs
from four perfusions were pooled prior to gel filtration. The pooled
FDPs (~25 mg) were subjected to gel filtration on Sephacryl S-1000
and the weight average molecular weight
(
w) of the FDPs in the eluate was
determined on-line using multiangle laser light scattering (9). Samples from the gel filtration column having
w = 0.48 × 106, 1.08 × 106, 1.93 × 106, 3.08 × 106, 3.97 × 106, and 4.94 × 106 g/mol were prepared
by pooling appropriate fractions, and the samples were concentrated to
>6.5 µM by centrifugal concentration as described (9).
The concentration of the FDPs refers to the concentration of fragment
X equivalents present in the sample. Samples were in stored
0.02 M Hepes, 0.5 M NaCl, 0.001% Tween 80, pH
7.4 at 4 °C.
Activation of 5AF-Pgn by t-PA and DSPA
1 in the Presence of
Soluble FDP Cofactors of Varying Sizes--
The activation of 5AF-Pgn
(to 5AF-Pn) by t-PA and DSPA
1 in the presence of soluble FDP
cofactors, both native and TAFIa-treated (see below), was monitored by
fluorescence spectroscopy as described previously (3). Aliquots (90 µl) of 5AF-Pgn, in 0.02 M Hepes, 0.053 M
NaCl, 0.0256% Tween 80, 2.56 mM CaCl2, pH 7.4, were added to the wells of a fluorescence microtiter plate (Microfluor,
Dynatech, Chantilly, VA), and the fluorescence intensities of the
samples were determined over 10-30 min using a PerkinElmer Life
Sciences LS-50B fluorescence spectrophotometer equipped with the plate reader accessory. The samples were excited at 495 nm (5 nm slit), and
the fluorescence was measured at 535 nm (3 nm slit) using a 515 nm
cut-off filter. FDPs (25 µl) were then added to the wells, and the
fluorescence of the 5AF-Pgn/FDP samples was determined over 10-30 min.
The combination of the 5AF-Pgn and FDPs gave a solution containing 0.02 M Hepes, 0.15 M NaCl, 2 mM
CaCl2, 0.02% Tween 80, pH 7.4. Plasminogen activation
reactions containing FDPs (33.3-500 nM final) and 5AF-Pgn
(33.3-500 nM final) were then initiated by the addition of
15 µl of either t-PA (1-4 nM final) or DSPA
1 (4-10
nM final), and the fluorescence of the reactions was
monitored every 80 s for 80 min. Both t-PA and DSPA
1 were in
0.02 M Hepes, 0.15 M NaCl, 2 mM
CaCl2, 0.02% Tween 80, pH 7.4. The fluorescence of 5AF-Pgn
in reactions without FDPs was determined identically to the reactions
described above, except that 0.02 M Hepes, 0.5 M NaCl, 0.001% Tween 80, pH 7.4, was used in place of the
FDPs. All experiments were performed at ambient temperature
(~20 °C).
Binding of 5AF-Pgn to Soluble FDP Cofactors of Varying
Sizes--
The binding of 5AF-Pgn to the FDPs was measured based on
the decrease in fluorescence of the 5AF-Pgn in the presence of FDPs. Binding was determined by measuring the fluorescence of the reactions containing 5AF-Pgn and FDPs prior to the addition of the activator (I)
and subtracting it from the measured fluorescence of the 5AF-Pgn in
each reaction prior to the addition of FDP, corrected for dilution (Io). The dilution factors accounting for the fluorescence change upon addition of FDPs and activator were determined from the
experiments described above in which no FDPs were used. The difference
in fluorescence
I = Io
I was measured for all
concentrations of Pgn and FDP, and the binding was analyzed by
nonlinear regression of the data according to Equation 1,
|
(Eq. 1)
|
where [Pgn-FDP] is the concentration of bound 5AF-Pgn, and
Ibound is the difference in fluorescence coefficients
(fluorescence units/5AF-Pgn) between free and bound 5AF-Pgn. These
coefficients are referred to as ICfree and
ICbound, respectively. The concentration of bound 5AF-Pgn
is found from the quadratic binding Equation 2
|
(Eq. 2)
|
where [Pgn]o and [FDP]o are the total
concentrations of 5AF-Pgn and FDP, respectively, and
Kd is the binding constant for the Pgn-FDP
interaction. The data were fit globally by nonlinear regression to
Equation 3. The independent variables were [Pgn]o and
[FDP]o; the dependent variable was I/Io, and the best
fit parameters were
Ibound and Kd.
ICfree is known as the fluorescence intensity of free
5AF-Pgn divided by its concentration.
|
(Eq. 3)
|
Treatment of FDPs with TAFIa--
TAFI was activated to TAFIa by
thrombin in the presence of Solulin essentially as described
previously (10). Briefly, 1.0 µM TAFI was reacted with 20 nM thrombin, 80 nM Solulin in 0.02 M Hepes, 0.15 M NaCl, 5 mM
CaCl2, 0.001% Tween 80, pH 7.4, for 10 min at ~22 °C.
The TAFIa was then stored on wet ice until used. A TAFIa titration was
performed to determine the amount of TAFIa required to achieve maximal
"deactivation" of the FDPs. The titration was based on the
activation of 5AF-Pgn in the presence of FDPs treated with or without
varying amounts of TAFIa. The 1.93 × 106 g/mol FDP
was used as the cofactor and 5 nM DSPA
1 as the
plasminogen activator. FDPs (2.6 µM) were treated with
varying concentrations of TAFIa (0.1-30 nM) for 60 min at
room temperature. The TAFIa/FDP solutions were diluted in half with
0.02 M Hepes, 0.5 M NaCl, 0.001% Tween 80, pH
7.4, and a 25-µl aliquot was added to 90 µl of 180 nM
5AF-Pgn in the wells of a fluorescence microtiter plate. DSPA
1 (15 µl of 43.3 nM) was added to initiate
cofactor-dependent activation, and the fluorescence of the
reaction was monitored over time (see above). Based on the results of
the TAFIa titration, the FDP samples were treated with 10 nM TAFIa. The FDPs (2.6 µM in 0.02 M Hepes, 0.5 M NaCl, 0.001% Tween 80, pH 7.4)
were treated with 10 nM TAFIa for 60 min at room
temperature followed by a 2-h incubation at 37 °C which served to
both maximally inactivate the FDPs and inactivate the TAFIa. The
TAFIa-treated FDPs were stored at 4 °C.
Data Analysis--
The rates of 5AF-Pgn activation were
determined from the initial slopes of the activation reactions, using
the fact that conversion of 5AF-Pgn to 5AF-Pn results in a 50%
decrease in the fluorescence of the active site fluorescein label (11).
The fluorescence of the 5AF-Pgn, prior to the addition of activator,
was found to decrease as a result of 5AF-Pgn binding to the native but
not TAFIa-treated FDPs. The rate data were corrected for this effect using the fact that the final fluorescence, i.e. the
fluorescence of a reaction taken to completion, was dependent only on
the initial 5AF-Pgn concentration and not on the initial fluorescence
of the 5AF-Pgn/FDP solutions (data not shown (11)). Since the end point fluorescence of the reactions was independent of the FDP concentration, the difference between the fluorescence of the 5AF-Pgn/FDP solutions and the end point fluorescence for the 5AF-Pgn concentration defined the full-scale fluorescence change upon complete conversion of 5AF-Pgn
to 5AF-Pn.
The data from the reactions were modeled according to the steady-state
template model for plasminogen activation as described by Horrevoets
et al. (3), with the terms for fibrin substituted by FDPs.
The rate Equation 4 is given by
|
(Eq. 4)
|
where rate is the velocity of the reaction per nominal activator
concentration; kcat is the turnover number for
the reaction; [Pgn]free is the concentration of free
plasminogen (calculated from the above binding Equation 4);
[FDP]o is the total FDP concentration; Km
is the Michaelis constant for the reaction; KA is
the dissociation constant of the activator for the FDPs, and
K is a constant whose value is equal to the concentration of
FDPs required to give a rate equal to half kcat at saturating Pgn.
The data from the experiments were initially analyzed using direct
plots. The reaction rates at each [FDP]o were plotted against
the [Pgn]free, and the resulting curves were analyzed by nonlinear regression according to the Michaelis-Menten equation for
each set of reaction conditions (see Equation 5),
|
(Eq. 5)
|
where kcat(app) and
Km(app) are the apparent kcat
and Km measured by varying the [Pgn]o at a
given FDP concentration. The ratio
kcat(app)/Km(app) was plotted against [FDP]o and analyzed by nonlinear regression according to Equation 6,
|
(Eq. 6)
|
by using the relationships (Equations 7 and 8)
|
(Eq. 7)
|
|
(Eq. 8)
|
to obtain values for the true
kcat/Km ratio and
KA. Finally, the data from all reactions for each
activator were analyzed globally, according to Equation 4.
The rates of 5AF-Pn formation from the experiments using the
TAFIa-treated FDPs did not show saturation with respect to
[Pgn]o at any FDP concentration for all TAFIa-treated FDPs.
We may assume, therefore, that for all TAFIa-treated FDPs,
Km(app)
[Pgn]free in all
reactions (from Equation 5). Furthermore, since binding of 5AF-Pgn to
TAFIa-treated FDPs was not detected in these experiments,
[Pgn]free can be set equal to [Pgn]o. By using
these assumptions, the rate obtained at each TAFIa-treated FDP
concentration was plotted against [Pgn]o, using Equation 9, a
modified form of Equation 5, to obtain values for the
kcat(app)/Km(app) ratio at
each FDP concentration.
|
(Eq. 9)
|
The kcat(app)/Km(app)
was then plotted against [FDP]o and analyzed by nonlinear
regression according to Equation 10, a modified form of Equation 6.
|
(Eq. 10)
|
The Effect of TAFIa on t-PA- and DSPA
1-mediated Fibrinolysis
in Plasma--
Clots (200 µl), made from 66.6 µl of
TAFIa-deficient human plasma, 56 µl of 0.02 M Hepes, 0.15 M NaCl, 0.02% Tween 80, pH 7.4, 4 µl of 500 nM Solulin, 20 µl of 6 nM t-PA or
DSPA
1, 20 µl of 0-200 nM TAFI, and 33.3 µl of 36 nM thrombin, 60 mM CaCl2 were
formed in the wells of a microtiter plate. The clotting and subsequent
lysis of the clots were monitored by turbidity at 405 nm at 37 °C.
The lysis time, the time at which the turbidity has decreased to
one-half the maximal plateau value, was determined for each sample, and
the results are presented as relative lysis times, which are the lysis
times for each reaction divided by the lysis time for the reaction in
the absence of TAFI.
 |
RESULTS |
Isolation of FDPs with
w Ranging from 0.48 × 106 to 4.94 × 106 g/mol--
FDPs
were made using a perfused clot system and subjected to gel filtration
on Sephacryl S-1000. The
w of the FDPs in
the eluate was determined on-line using multiangle laser light
scattering (9). Fig. 1 shows a plot of
the protein concentration and corresponding
w of the FDPs versus the
volume of the eluate. The fractions that were pooled to give FDP
samples having
w of 0.48 × 106, 1.08 × 106, 1.93 × 106, 3.08 × 106, 3.97 × 106 and 4.94 × 106 g/mol are indicated by
shading.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 1.
Isolation of FDPs with different
w. Pooled FDPs from perfusion
fibrinolysis experiments were concentrated and passed over a
Sephacryl-S1000 gel filtration column. The eluate from the column was
passed through an absorbance monitor and a multiangle laser light
scattering detector arranged in tandem. The figure shows the
concentration of FDPs and the corresponding
w versus the volume of the
eluate. The shaded boxes show the fractions that were pooled
and concentrated to obtain the different samples. The
w (g/mol) values of the different samples
are as follows: I = 4.94 × 106, II = 3.97 × 106, III = 3.08 × 106,
IV = 1.93 × 106, V = 1.08 × 106, and VI = 0.48 × 106.
|
|
Binding of 5AF-Pgn to FDPs--
During the course of the
t-PA/DSPA
1 cofactor activity experiments (see below), it was found
that the fluorescent plasminogen derivative, 5AF-Pgn, displayed a
reduced fluorescence in the presence of the FDPs relative to 5AF-Pgn
alone. We used this property to investigate the binding of the 5AF-Pgn
to the FDPs. The fluorescence changes versus the
concentration of FDP were collected for all FDP sizes at all 5AF-Pgn
concentrations. The data from each FDP sample of a particular
w were analyzed by nonlinear regression. This analysis showed no dependence of the Kd on the
FDP
w. Therefore, the data were fit
globally using a single value for Kd and letting
Ibound, the change in 5AF-Pgn fluorescence upon binding
FDPs, vary for each of the FDP samples. Fig.
2 presents the observed data for the
binding of 500 nM 5AF-Pgn to increasing concentrations of
the different FDP samples (Fig. 2, symbols) as well as the
calculated fit lines for each FDP sample from the global fit of the
data at all concentrations of FDP and 5AF-Pgn. The percent decrease in
fluorescence for the 5AF-Pgn bound to the different FDP samples was
5.8 ± 0.8, 11.6 ± 1.2, 19.4 ± 1.8, 20.6 ± 1.9,
20.5 ± 1.9, and 21.7 ± 2.0% for the FDPs having
w of 0.48 × 106,
1.08 × 106, 1.93 × 106, 3.08 × 106, 3.97 × 106, and 4.94 × 106 g/mol, respectively. Despite the different extents of
quenching of fluorescence, all FDPs bound with the same affinity
(Kd = 225 ± 60 nM). Although the
differences in intensity changes suggest subtle
size-dependent differences in the environment of bound
5AF-Pgn, the relevant parameter for modeling of kinetics is the
Kd. No binding was detected after treating the FDP
samples with TAFIa (data not shown). The Kd value and the apparent dependence of binding on carboxyl-terminal lysines and/or arginines are consistent with results reported by others (5-7)
with partially degraded fibrin.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 2.
Binding of 5AF-Pgn to FDPs of different
w. The binding of 5AF-Pgn to
the FDPs was determined as described under "Experimental
Procedures." The figure shows the fluorescence intensity of the
5AF-Pgn/FDP mixtures (I), relative to free 5AF-Pgn (Io),
versus the FDP concentration at 500 nM 5AF-Pgn.
The FDPs of different w (g/mol) are shown
by open circles 0.48 × 106, open
squares 1.08 × 106, open triangles
1.93 × 106, closed circles 3.08 × 106, closed squares 3.97 × 106, and closed triangles 4.94 × 106. The 5AF-Pgn bound to the FDPs with a global
Kd of 225 ± 60 nM. The
fluorescence decrement of FDP-bound 5AF-Pgn relative to free 5AF-Pgn
( Ibound/Io) was dependent on the
w of the FDP as follows:
Ibound/Io = 5.8 ± 0.8, 11.6 ± 1.2, 19.4 ± 1.8, 20.6 ± 1.9, 20.5 ± 1.9, and 21.7 ± 2.0% for the FDPs having w (g/mol) of
0.48 × 106, 1.08 × 106, 1.93 × 106, 3.08 × 106, 3.97 × 106, and 4.94 × 106, respectively. The
solid lines were obtained by nonlinear regression of the
data to Equation 3. The lines for FDPs of
w (g/mol) 3.08 × 106
and 3.97 × 106 overlap at this scale.
|
|
FDPs of
w Ranging from 0.48 × 106 to 4.94 × 106 g/mol Display
Differential Cofactor Activity with Respect to t-PA and
DSPA
1--
The different FDP samples were tested for their ability
to serve as cofactors in the t-PA and DSPA
1-catalyzed conversion of
5AF-Pgn to 5AF-Pn. The data from both the t-PA- (Fig.
3) and DSPA
1 (Fig.
4)-catalyzed reactions were initially
analyzed by regression of the data to the Michaelis-Menten equation
according to the steady-state template model for Pgn activation as
described by Horrevoets et al. (3).

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 3.
t-PA cofactor activity of the FDPs of
different w. The figure shows
the initial rates of 5AF-Pgn conversion to 5AF-Pn by t-PA
versus 5AF-Pgn concentration in the presence of increasing
concentrations of FDPs. For all panels, [FDP]: 33.3 nM
open circles, 66.6 nM open squares,
100.0 nM open triangles, 175.0 nM
closed circles, 300.0 nM closed
squares, and 500.0 nM closed triangles. The
w (g/mol) values of the FDPs used in each
experiment are as follows: A, 0.48 × 106;
B, 1.08 × 106; C, 1.93 × 106; D, 3.08 × 106;
E, 3.97 × 106; and F, 4.94 × 106. The solid lines are fit lines derived
from regression analysis of the data to the steady-state template
model. The figure shows that all FDPs functioned as effective cofactors
for t-PA and that the cofactor activity of all FDPs displayed saturable
kinetics with respect to both 5AF-Pgn and FDP concentration.
|
|

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 4.
DSPA 1 cofactor
activity of the FDPs of different
w. The figure shows the
initial rate of 5AF-Pgn conversion to 5AF-Pn by DSPA 1
versus 5AF-Pgn concentration in the presence of increasing
concentrations of FDPs. For all panels, [FDP]: 33.3 nM
open circles, 66.6 nM open squares,
100.0 nM open triangles, 175.0 nM
closed circles, 300.0 nM closed
squares, and 500.0 nM closed triangles. The
w (g/mol) values of the FDPs used in each
experiment are as follows: A, 0.48 × 106;
B, 1.08 × 106; C, 1.93 × 106; D, 3.08 × 106;
E, 3.97 × 106; and F, 4.94 × 106. The inset in A is the same
plot on an expanded scale. The solid lines are fit lines
derived from regression analysis of the data to the steady-state
template model. DSPA 1 cofactor activity of the FDPs was dependent on
the FDP mass, with the smallest FDPs (A) being ~10-fold
less active than the large FDPs (C-F) under the conditions
tested. The FDPs with w 1.93 × 106 g/mol displayed saturable kinetics with respect to both
5AF-Pgn and FDP concentration, whereas saturation was not achieved
under the conditions used for the FDP with
w 1.08 × 106 g/mol.
In contrast to the lack of cofactor activity observed with fragment
DD/E (2), the figure shows that large soluble FDPs possess significant
DSPA 1 cofactor activity.
|
|
The kcat(app), Km(app), and
kcat(app)/Km(app) values for
the reactions with t-PA, derived from the direct Michaelis-Menten
plots, are shown in Table I. The data
show that the reactions with t-PA exhibit Michaelis-Menten kinetics at
any fixed FDP concentration for each of the different FDP samples when
the substrate concentration is expressed as free 5AF-Pgn (Equation 5).
The kcat(app) was found to increase with
increasing FDP concentration, as expected in a cofactor-mediated,
template-dependent reaction. No significant differences in
kcat(app) between the different FDP samples, at
any particular FDP concentration, were found. With the exception of the
smallest FDP (
w = 0.48 × 106 g/mol), the Km(app) value for the
reactions was fairly insensitive to the FDP concentration. This is
consistent with reactions where KA ~ K
(Equation 8). For the FDP of
w = 0.48 × 106 g/mol, the Km(app) was
found to decrease with increasing FDP concentration, consistent with
KA > K for this FDP. The data indicate
that the true Km value for reactions with t-PA
(obtained at saturating FDP concentration, Equation 8) is the same for
all FDPs, regardless of
w. The
kcat(app)/Km(app) ratios for
all FDPs increase with increasing FDP concentration, indicative of a
template mechanism.
View this table:
[in this window]
[in a new window]
|
Table I
Kinetic parameters of t-PA-catalyzed 5AF-Pgn activation in the presence
of FDPs of different w
The rate of 5AF-Pn formation at each FDP concentration was plotted
against the 5AF-Pgn concentration, and the kcat(app)
and Km(app) values were determined by regression to
the Michaelis-Menten equation. The data show that all the reactions
with FDPs exhibited Michaelis-Menten kinetics, with the
kcat(app) increasing with FDP concentration as
expected in a cofactor-mediated reaction. The
Km(app) values for the reactions with the FDPs
w = 0.48 × 106 g/mol decreased
with FDP concentration, indicating that KA > K. The Km(app) values for the reactions
with the FDPs of w 1.08 × 106 g/mol was essentially independent of FDP concentration,
indicating that KA ~ K for these
reactions. The kcat(app)/Km(app)
ratio for all FDP samples increased with increasing FDP concentration,
indicative of a template mechanism. All data are shown ± S.E. as
returned by the nonlinear regression algorithm. ND indicates not
determined.
|
|
The kcat(app), Km(app), and
kcat(app)/Km(app) values for
the reactions with DSPA
1, derived from the direct Michaelis-Menten
plots, are shown in Table II. The values
for kcat(app) and Km(app) for
the small FDPs of
w = 0.48 × 106 and 1.08 × 106 g/mol could not be
determined individually, since the plots did not exhibit saturable
kinetics over the concentrations of 5AF-Pgn and FDPs used (Fig. 4). For
the FDPs of higher
w the plots exhibited an approach to saturation with respect to substrate concentration and
the kcat(app) increased with increasing FDP
concentration. The data show that for FDPs with
w
1.93 × 106 g/mol,
the kcat(app) with DSPA
1 was insensitive to
the
w of the FDPs. In contrast to that
seen with t-PA, the Km(app) for the larger FDPs was
found to increase with increasing FDP concentration, indicating that
the KA < K for these reactions (Equation 8). Although the lack of saturation in the Michaelis-Menten plots of
the reactions with the smaller FDPs (0.48 × 106 and
1.08 × 106 g/mol) precluded separate determinations
of the kcat(app) and Km(app),
and therefore any conclusions regarding the relationship between
KA and K, the behavior of the
kcat(app)/Km(app) with
respect to FDP concentration was found from the slopes of the direct
plots. Since the apparent catalytic efficiency was found to be a
function of the FDP concentration, these data indicate that the smaller
FDPs act as cofactors. The larger FDPs were found to influence
plasminogen activation in a manner fairly independent of FDP size, as
indicated by the behavior of the
kcat(app)/Km(app) ratios with
respect to FDP concentration (Table II).
View this table:
[in this window]
[in a new window]
|
Table II
Kinetic parameters of DSPA 1-catalyzed 5AF-Pgn activation in the
presence of FDPs of different w
The rate of 5AF-Pn formation at each FDP concentration was plotted
against the 5AF-Pgn concentration, and the kcat(app)
and Km(app) values were determined by regression to
the Michaelis-Menten equation. The data show that the reactions with
FDPs of w 1.93 × 106 g/mol
exhibit Michaelis-Menten kinetics with kcat(app)
values increasing with FDP concentration, as expected in a
cofactor-mediated reaction. The Km(app) values for
these reactions increased with FDP concentration, indicating that
KA < K. The reactions using FDPs of
w 1.08 × 106 g/mol did not
show saturation over the 5AF-Pgn concentration range used. Thus, the
kcat(app) and Km(app) values for
these reactions could not be determined; however, the
kcat(app)/Km(app) ratio was found
from the slope of the rate versus 5AF-Pgn plot. The
kcat(app)/Km(app) ratio for all
FDP samples increased with increasing FDP concentration, indicative of
a template mechanism. All data are shown ± S.E.
|
|
By using the results from the direct plots as a guide, we fit the
experimental data from each of the FDP samples with t-PA as the
activator to the steady-state template model (Equation 4). When the
data from all 5AF-Pgn and FDP concentrations were regressed together,
we were unable to assign values simultaneously for all four parameters
(kcat, Km,
KA, and K) for each FDP size. We could,
however, determine values for kcat, KA, and K, using a single value of
Km, or for kcat,
Km, and K, using a single value for
KA, for all FDP sizes. From these two fits of the
data, we found that the values for kcat and
K were essentially invariant, with respect to FDP
w, when either the Km
was fixed for all FDPs (kcat = 0.065-0.081
s
1, K = 122-198
nM) or when the KA was fixed for all
FDPs (kcat = 0.076-0.091
s
1, K = 140-300
nM). Since the results from the direct Michaelis-Menten plots (Table I) suggested that the true Km value
(obtained at saturating [FDP]o, Equation 8) is likely the
same for all FDP samples when t-PA was used as the activator, and since kcat and K values were essentially
unaffected by fixing either Km or
KA, we modeled the t-PA cofactor activities for each
FDP size according to differences in KA, using a
single Km for all FDP sizes. The results of the
regression analysis are indicated by the solid lines in Fig.
3. The kinetic parameters obtained from the regression analysis for
t-PA with each of the different FDP samples are shown in Table
III. Consistent with the observations
from the individual Michaelis-Menten plots, the value of
KA is greater than the value of K for
t-PA with 0.48 × 106 g/mol FDPs, whereas
KA ~K with FDPs
1.08 × 106 g/mol. The data show that the FDP cofactors yield a low
Km (45 nM) reaction with t-PA, and the
kcat (0.065-0.081 s
1)
and K (122-198 nM) values of the reactions are
essentially independent of FDP
w. The
difference in cofactor activity between the smallest (
w = 0.48 × 106 g/mol)
and the other (
w
1.08 × 106 g/mol) FDPs is minimal when t-PA is the activator and
is attributable to an approximate 5-fold difference in the value of
KA. The increasing FDP
w coincides with a decrease in the
KA of the reactions, implying that larger FDPs have
either more sites or higher affinity sites for t-PA binding.
View this table:
[in this window]
[in a new window]
|
Table III
Summary of the kinetic parameters of t-PA- and DSPA 1-catalyzed
5AF-Pgn activation in the presence of FDPs of different
w
The parameters from the regression of the data to the steady-state
template model are presented ± S.E. The reactions were modeled
with kcat, KA, and K
being dependent on FDP w and a single
Km for all FDPs. FDP cofactors yielded lower
Km values with t-PA than with DSPA 1.
kcat and K were insensitive to the FDP
w with both activators. The data show that
whereas DSPA 1 has a higher turnover number, reactions with t-PA are
more sensitive to FDP and 5AF-Pgn concentrations because of their
respective lower K and Km values.
DSPA 1 activity displayed a profound size dependence on FDP
w, whereas that seen with t-PA was modest.
These differences are reflected by the ranges of KA
values observed for the two activators.
|
|
The data from the DSPA
1 experiments were also analyzed according to
the steady-state template model based on differences in
KA. When the data were modeled with either a single KA or Km, a fixed
kcat was required for a solution for the small
FDPs of
w = 0.48 × 106
and 1.08 × 106 g/mol, due to the lack of curvature of
the Michaelis-Menten curves for each FDP. For the FDPs of
w
1.93 × 106 g/mol,
the kcat values were essentially invariant
(kcat = 0.327-0.384 s
1) and thus the value for
kcat for the FDPs having
w = 0.48 × 106 g/mol
and 1.08 × 106 g/mol was fixed at the average
kcat for FDPs
w
1.93 × 106 g/mol (0.358 s
1). Repeating the analysis showed that FDP
of
w = 0.48 × 106 g/mol
also required a fixed K for a solution to the rates observed at all FDP concentrations. The value of K for FDP with
w
1.08 × 106 g/mol
was essentially invariant (1660-2450 nM), and thus the value of K for FDP of
w = 0.48 × 106 g/mol was fixed at the average value of
K for the FDPs of
w
1.08 × 106 g/mol (1990 nM). Fixing the
parameters at the average values as an approximation was supported by
the fact that a global fit of the data for all FDP sizes, using single
kcat, Km, and K
for all FDPs, yielded values of 0.35 ± 0.15 s
1, 670 ± 340 and 2090 ± 970 nM for kcat, Km,
and K, respectively. The results of the regression analysis
are indicated by the solid lines in Fig. 4. The kinetic
parameters for DSPA
1 with the different FDP samples are shown in
Table III. Consistent with the individual Michaelis-Menten plots using
DSPA
1 as the activator, the data show that KA < K (239-588 versus 1660-2450 nM) for
FDPs with
w
1.93 × 106 g/mol. Furthermore, the data show that the
kcat and K values are independent of
the FDP
w when DSPA
1 is the activator.
Finally, the data show that the large decrease in the DSPA
1 cofactor
activity with the small FDPs is attributable to a large increase
(~43-fold) in the KA, indicating that the high
affinity sites for DSPA
1 disappear as the
w of the FDP approaches 0.48 × 106 g/mol. This is consistent with the observations of
Stewart et al. (2), who showed that DSPA
1 binds to intact
fibrin with high affinity (KA = 150 ± 40 nM) but not to fragment DD/E (KA
3000 nM), the smallest possible FDP
(
w = 0.25 × 106
g/mol).
TAFIa Treatment of FDPs Eliminates the High Affinity 5AF-Pgn:FDP
Binding, Markedly Reduces Both t-PA and DSPA
1 Cofactor Activity, and
Abrogates the FDP Size Dependence of DSPA
1 Cofactor
Activity--
The rate constant for 5AF-Pgn activation is increased
2.5-fold by Pn-mediated exposure of carboxyl-terminal lysine and/or arginine residues on the fibrin surface during fibrinolysis (8). This
feedback activation is down-regulated by the removal of the carboxyl-terminal lysines and arginines from the degraded fibrin surface by activated TAFIa, resulting in an attenuation of fibrinolysis (8). Since the soluble FDPs contain carboxyl-terminal lysine and
arginine residues, we investigated the effect of TAFIa treatment of the
FDPs on both t-PA- and DSPA
1-catalyzed plasminogen activation. The
FDPs from
w = 0.48 × 106 to 3.08 × 106 g/mol were treated with
TAFIa and then analyzed for their cofactor activity as described for
the native FDPs.
The reaction rates were strictly linear with respect to the 5AF-Pgn
concentrations for all TAFIa-treated FDP samples. Thus, we were unable
to obtain individual kcat(app) and
Km(app) values. The
kcat(app)/Km(app) ratio for
each reaction was found by regressing the data to the Michaelis-Menten
model under conditions in which the substrate does not bind the
cofactor and the substrate concentration is low relative to the
apparent Km (Equation 9). The data obtained with
DSPA
1 are shown in Fig. 5. Although
not shown graphically, the results with t-PA were also linear, and no
FDP
w-dependent differences
in the rates of the reactions were observed.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 5.
DSPA 1 cofactor
activity of TAFIa-treated FDPs of different
w. The figure shows the
initial rate of 5AF-Pgn conversion to 5AF-Pn by DSPA 1
versus 5AF-Pgn concentration in the presence of increasing
concentrations of TAFIa-treated FDPs. For all panels, [TAFIa-treated
FDP]: 33.3 nM open circles, 66.6 nM
open squares, 100.0 nM open
triangles, 175.0 nM closed circles, 300.0 nM closed squares, and 500.0 nM
closed triangles. The w
(g/mol) values of the TAFIa-treated FDPs used in each experiment are as
follows: A, 0.48 × 106; B,
1.08 × 106; C, 1.93 × 106; D, 3.08 × 106. The
solid lines were obtained by regression of the data to the
modified Michaelis-Menten Equation (see Equation 9 under
"Experimental Procedures"). The figure shows that the DSPA 1
cofactor activity of the TAFIa-treated FDPs displayed only a modest
dependence on w. Furthermore, whereas all
of the TAFIa-treated FDPs served as cofactors for DSPA 1, they were
all substantially less effective cofactors than their nontreated FDP
counterparts (see Fig. 4).
|
|
The kcat(app)/Km(app) ratios
as functions of the FDP concentration for each FDP
w were analyzed by regression according to Equation 10 to obtain estimates of the true
kcat/Km and for each
activator with the TAFIa-treated FDPs. Table
IV shows the dependence of the
kcat(app)/Km(app) on FDP
concentration for both t-PA and DSPA
1. All TAFIa-treated FDPs
exhibited cofactor behavior as seen by the increasing
kcat(app)/Km(app) as a
function of FDP concentration. Since these values showed at best a
modest dependence on FDP
w, a global
analysis of the data, using Equation 10 and a single true
kcat/Km value for all
TAFIa-treated FDPs, was performed. These yielded estimates of 0.20 ± 0.01 × 105 and 0.028 ± 0.004 × 105 M
1
s
1 for t-PA and DSPA
1, respectively.
View this table:
[in this window]
[in a new window]
|
Table IV
Catalytic efficiency of t-PA- and DSPA 1-catalyzed 5AF-Pgn activation
in the presence of TAFIa-treated FDPs of different w
The kcat(app)/Km(app) ratios were
determined at each FDP concentration by linear regression of the
reaction rate versus 5AF-Pgn concentration. The
kcat(app)/Km(app) ratio for all
FDPs samples increased with increasing FDP concentration, indicative of
a template mechanism. TAFIa treatment yielded FDPs that were less
active cofactors for both t-PA and DSPA 1 than were their non-treated
counterparts. t-PA was equally responsive to all FDPs, and the size
dependence seen with DSPA 1 was markedly attenuated by treatment of
the FDPs with TAFIa. All data are shown ± S.E.
|
|
A summary of the intrinsic kcat,
Km, and
kcat/Km (catalytic
efficiency) values for t-PA and DSPA
1-stimulated 5AF-Pgn activation
using FDPs as cofactors, before and after treatment of the FDPs with
TAFIa, is presented in Table V. The
kcat and Km values presented
for t-PA and DSPA
1 with native FDPs were determined for each
activator by fitting the data for all FDPs to a global steady-state
template model using single values for kcat,
Km, and K and values that were dependent
on the FDP
w for KA.
For comparison, Table V also shows values obtained by others, using
intact fibrin (1, 3) or fibrinogen (1) as the cofactor and either t-PA
(1, 3) or DSPA
1 (1) as the activator with Glu-plasminogen (1, 3) and
Lys-plasminogen (3) as substrates.
View this table:
[in this window]
[in a new window]
|
Table V
Comparison of the intrinsic kinetic parameters of t-PA- and
DSPA 1-catalyzed plasminogen activation with FDPs, TAFIa-treated
FDPs, fibrin, and fibrinogen as cofactors
The data from the reactions with t-PA and DSPA 1 were globally
regressed to the steady-state template model using single values for
kcat, Km, and K for
all FDPs. The data show that the intrinsic catalytic efficiency of t-PA
is higher than that of DSPA 1 when the cofactor is either FDPs or
TAFIa-treated FDPs. With FDPs as the cofactor, the intrinsic catalytic
efficiency of t-PA is increased by a factor of 3(1) to 10(3) compared
with fibrin and approaches that seen with fibrin when the substrate is
Lys-plasminogen (3). Although the intrinsic catalytic efficiency of
DSPA 1 with FDPs was the same as that found by others (1) with
fibrin, the effect of TAFIa on DSPA 1-mediated fibrinolysis (Fig. 6)
shows that the intrinsic catalytic efficiency of DSPA 1 with FDPs is
higher than that seen with fibrin (see "Discussion"). Although
TAFIa treatment of the FDPs markedly decreases the catalytic
efficiencies of t-PA (90-fold) and DSPA 1 (210-fold), to values below
those for intact fibrin, TAFIa-treated FDPs are still superior
cofactors for both t-PA (30-fold) and DSPA 1 (50-fold) in comparison
to fibrinogen.
|
|
The Effect of TAFIa on t-PA- and DSPA
1-mediated Fibrinolysis in
Plasma--
The fibrinolysis of clots formed in human plasma, induced
by t-PA or DSPA
1, was prolonged by TAFIa in a
concentration-dependent, saturable manner. As shown in Fig.
6, fibrinolysis induced with t-PA and
DSPA
1 was equally prolonged at low (<2 nM)
concentrations of TAFIa, and the extent of prolongation with saturating
TAFIa was marginally higher with t-PA (3-fold) than with DSPA
(2.5-fold). When t-PA is the activator, TAFIa can prolong fibrinolysis
by three separate mechanisms (8). As a basic carboxypeptidase, TAFIa
removes exposed carboxyl-terminal lysine and arginine residues, thus
preventing the plasmin-mediated up-regulation of the fibrin cofactor
activity. In addition, TAFIa suppresses the plasmin-catalyzed conversion of Glu-plasminogen to Lys-plasminogen, a much better substrate for t-PA, thereby eliminating up-regulation through this
mode. Finally, TAFIa can directly inhibit the activity of plasmin,
although high TAFIa concentrations are required to achieve inhibition
(8). The data in Fig. 6 show that fibrinolysis by DSPA
1 is also
down-regulated by TAFIa, most likely by the same mechanisms involved
with t-PA.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of TAFIa on t-PA and
DSPA 1-mediated fibrinolysis in plasma.
Clots were made in TAFI-deficient human plasma by the addition of
purified human thrombin in the presence of varying concentrations of
TAFI. Either t-PA or DSPA 1 was included as the plasminogen
activator. Solulin was included to promote the rapid formation
of TAFIa. Fibrinolysis was followed by monitoring the turbidity of the
clots at 405 nm. The figure shows the effect of TAFIa on the time
required to achieve the Lysis Time, the time required to
reach 50% lysis, expressed relative to the Lysis Time
observed in the absence of TAFIa. The figure shows that TAFIa increases
the lysis time of both t-PA and DSPA 1 mediated reactions in a
saturable manner and that TAFIa maximally prolongs DSPA 1-mediated
reactions marginally less (2.5-fold) than it does reactions with t-PA
(3-fold).
|
|
 |
DISCUSSION |
The Catalytic Properties of t-PA and DSPA
1 with FDPs as
Cofactors--
The intrinsic catalytic efficiency
(kcat/Km) of t-PA exceeds
that of DSPA
1 by ~3-fold when FDPs are used as cofactors and
5AF-Pgn is the substrate. t-PA and DSPA
1 are qualitatively similar,
however, in that their intrinsic kcat and
Km values, and thus their intrinsic catalytic
efficiencies are not dependent on the molecular weight of the FDP
(Table III). The similarity in the kcat values
with the different cofactors shows that the conversion of fibrin into
FDPs and, by analogy, to partially degraded fibrin does not
substantially alter the influence of the cofactor on the turnover of
the ternary complex with either activator. Nonetheless, because three
components are involved in the reactions, the reaction rates with both
activators are sensitive to the FDP size when the reactions are not
saturated with respect to the concentrations of FDP and 5AF-Pgn. Both
activators showed decreased rates with decreasing FDP
w. The dependence is modest with t-PA but
substantial with DSPA
1 (Figs. 3 and 4). With both activators, the
decrease in reaction rate with decreasing FDP
w can be accounted for by a decrease in
the binding affinity of the activator for the smaller FDPs. With t-PA
the decrease in affinity is modest, whereas with DSPA
1 the decrease
is large. For example, the KA values for the binding
of t-PA to the two smallest FDPs (
w = 0.48 and 1.08 × 106 g/mol) are only 4.5- and 1.5-fold
greater, respectively, than the average KA value for
FDPs with
w
1.93 × 106 g/mol (182 nM). In contrast, with DSPA
1,
the corresponding KA values are 43- and 4-fold
higher, respectively, than the average value for the FDPs with
w
1.93 × 106 g/mol
(432 nM).
These differences can be rationalized on the basis of differences in
structure between t-PA and DSPA
1. t-PA interacts with fibrin by both
its finger domain (Kd = 260 nM (4)) and
its kringle-2 domain (Kd = 690 nM, (4)).
DSPA
1, which lacks the kringle-2 domain, interacts with intact
fibrin with high affinity via the finger domain (Kd = 150 nM (1, 2)). Light scattering measurements by Stewart
et al. (2) of the binding of t-PA to fragment DD/E, the
terminal product of fibrin degradation by plasmin, indicated high
affinity binding (Kd = 20 nM). The
authors concluded that the binding was mediated by kringle-2 since the
lysine analogue
-amino caproic acid abolished the binding, and no
binding of DSPA
1 to the fragment was detected (Kd
3000 nM). Thus, we infer that for the smallest FDPs
used in our studies the predominant mode of binding of t-PA involves
the kringle-2 domain and the lack of DSPA
1 binding reflects its lack
of a kringle-2 domain. This inference is supported by the quantitative
agreement between the KA value for the binding of
t-PA to the smallest FDP sample (895 nM), as determined by
kinetics, and the direct measurement of the binding of a fingerless
t-PA mutant to intact fibrin (690 nM (4)). The interactions
between DSPA
1 and the larger FDPs must be mediated, therefore, by
its finger domain, and the modest increase seen in the affinity of t-PA
for the larger FDPs is consistent with the ability of the larger FDPs
to support both finger domain and kringle-2
domain-dependent binding. As a corollary, the smallest FDP
does not contain the elements of structure necessary for
finger-mediated binding, whereas the large FDPs do.
Comparison of Cofactor Activities of FDPs and Fibrin--
The
cofactor activities of FDPs and fibrin with t-PA can be quantitatively
compared because prior measurements with t-PA (3) were made using an
experimental approach identical to that used in the present study.
Under these conditions, with 5AF-Pgn (Glu-plasminogen) as the
substrate, the kcat value with fibrin (0.058 s
1) is very similar to the globally measured
value for the FDPs (0.075 s
1, Table V). The
Km value with Glu-plasminogen and fibrin is much
greater than that observed with FDPs as the cofactor (410 versus 43 nM, Table V). Thus, with
Glu-plasminogen as the substrate, the intrinsic catalytic efficiency of
t-PA with FDPs exceeds that of intact fibrin by a factor of 12.4 (Table
V). With fibrin as the cofactor and Lys-plasminogen as the substrate,
the intrinsic catalytic efficiency of t-PA exceeds that observed when
Glu-plasminogen is the substrate by 19.2-fold, an effect that is also
reflected by a large Km difference between the
substrates. Thus, proteolytic modification of the substrate by plasmin
(the conversion of Glu- to Lys-plasminogen) has the same impact on the
catalytic efficiency as proteolytic modification of the cofactor (the
conversion of intact fibrin to large FDPs).
The catalytic efficiency of DSPA
1 with FDPs from the current data
cannot be compared quantitatively to those obtained by Bringmann
et al. (1) with intact fibrin as the cofactor because the
conditions under which the data were obtained are not identical. In
addition, the model used for the kinetic analysis in the present study
and in the work of Horrevoets et al. (3) involved a template mechanism, whereas that used by Bringmann et al. (1)
involves a bimolecular mechanism. Thus, despite the apparent identity
of the intrinsic catalytic efficiency of DSPA
1 measured here with FDPs (6.0 × 105
M
1 s
1)
and that measured by Bringmann et al. (1) with intact fibrin (6.8 × 105 M
1
s
1), FDPs are undoubtedly more active than
fibrin because TAFIa suppresses the up-regulation of fibrinolysis (Fig.
6), a phenomenon shown to be due to the suppression of the
up-regulation of cofactor activity that occurs upon plasmin-catalyzed
modification of fibrin (8).
The value of the parameter K in the template model indicates
the FDP concentration at which the reaction rate will be half-maximal at saturating plasminogen concentration. With both t-PA and DSPA
1 this value is essentially independent of FDP
w (Table III). The average value for t-PA
(158 nM) is considerably lower than that for DSPA
1 (1990 nM). Since the physiologic concentration of plasminogen (2-3 µM) is near saturation for both activators, the
lower value of K for t-PA indicates that plasminogen
activation would be saturated at much lower levels of FDPs with t-PA
than with DSPA
1. Thus, FDPs of all sizes are superior cofactors for
t-PA compared with DSPA
1, in that at subsaturating concentrations of
FDPs more activity will be elicited with t-PA than with DSPA
1.
The Effect of TAFIa on the Cofactor Activity of FDPs--
In the
presence of TAFIa, the up-regulation of plasminogen activation, through
the formation of modified fibrin by plasmin, is eliminated by the
removal of the lysine and arginine residues from the modified fibrin
(8). FDPs (and degraded fibrin) increase the intrinsic catalytic
efficiency of t-PA and DSPA
1. TAFIa attenuates this up-regulation,
decreasing the intrinsic catalytic efficiency of both t-PA (0.20 × 105 M
1
s
1) and DSPA
1 (0.028 × 105 M
1
s
1) by 90- and 210-fold, respectively. These
catalytic efficiency values nonetheless exceed those seen for t-PA
(0.0064 × 105
M
1 s
1)
and DSPA
1 (0.00053 × 105
M
1 s
1)
when the fibrinogen is the cofactor (1) by ~30- and 50-fold, respectively. Therefore, although TAFIa-treated FDPs are much less
active than intact fibrin, they are considerably more active than
fibrinogen. Interestingly, when the TAFIa-treated FDPs are cofactors,
the size dependence seen with DSPA
1 is markedly attenuated and that
seen with t-PA is absent. Since DSPA
1 has only finger binding, one
would expect the TAFIa-treated FDPs to exhibit the same size dependence
as the native FDPs. In addition, since TAFIa treatment removes
carboxyl-terminal lysines, one would expect the t-PA kringle-2
interaction to be affected and thus the size dependence seen with t-PA
should mirror that seen with DSPA
1. That the expected results did
not occur can be explained by a comparison of the DSPA
1 activity to
DSPA
and DSPA
, highly homologous relatives of DSPA
1 (89 and
91% identity, respectively) which lack either the finger domain
(DSPA
) or the finger and epidermal growth factor domains (DSPA
)
(12). Although neither activator binds to fibrin with high affinity
(1), the intrinsic catalytic efficiencies are increased 1650- (DSPA
)
and 800-fold (DSPA
) in the presence of fibrin (1), indicating the
existence of a finger-independent, low affinity interaction with
fibrin. Given the shared identity among the DSPAs, the same interaction
likely occurs with DSPA
1. The effect of this interaction on
plasminogen activation is not observed with intact fibrin and FDPs not
treated with TAFIa due to the overwhelming contribution of finger-bound DSPA
1 to the kinetics of plasmin generation. However, with
TAFIa-treated FDPs the finger-independent, low affinity interaction of
DSPA
1 contributes a significant fraction of the total plasmin
generating activity. Therefore, the total activity of DSPA
1 is a
composite of the activities of DSPA
1 bound by both
finger-dependent and finger-independent modes. Since the
larger FDPs contain more finger-binding sites, the small observed
dependence of the rate on FDP
w results from the low activity, due to the loss of the high affinity plasminogen binding, of the finger-bound DSPA
1. This interpretation receives support from the similarity of the intrinsic catalytic efficiency of
DSPA
1 with the TAFIa-treated FDPs (0.028 × 105
M
1 s
1)
to the catalytic efficiencies of both DSPA
(0.099 × 105 M
1
s
1) and DSPA
(0.035 × 105 M
1
s
1) (1) with intact fibrin. Similarly, the
lack of size dependence with t-PA and the TAFIa-treated FDPs reflects
catalysis through a mode independent of finger binding of t-PA to FDPs.
Thus, finger-bound activator loses the ability to form a ternary
complex due to the loss of the plasminogen-binding site. That t-PA
interacts with TAFIa-treated FDPs via a finger-independent mode
indicates that t-PA binding is not strictly dependent on the lysine
residues that are the targets of plasmin. The contribution of t-PA
interacting through this mode to the overall rate of plasmin generation
masks that of finger bound t-PA with TAFIa-treated FDPs. This
interpretation receives support from the fibrin-dependent
intrinsic catalytic efficiency of a recombinant t-PA lacking the finger
domain (0.17 × 105
M
1 s
1)
(3). In cleaving the carboxyl-terminal lysines from FDPs, TAFIa does
not simply convert the high affinity sites back to low affinity sites,
it removes the sites altogether.
Implications for the Fibrin Specificity of Plasminogen
Activators--
Recently, Stewart et al. (2) demonstrated
that plasminogen activation catalyzed by t-PA, but not DSPA
1, is
effectively stimulated by the fibrin degradation product DD/E. The
authors speculated that this property may increase the fibrin
selectivity of DSPA
1, relative to t-PA, as the DD/E fragments
released from the clot would continue to stimulate the t-PA-catalyzed
but not the DSPA
1-catalyzed plasminogen activation, and thus, the
release of DD/E into the circulation would promote fibrinogenolysis in the presence of t-PA but not DSPA
1. This speculation rests on the
assumption that DD/E is the primary product of plasmin-catalyzed fibrin
degradation. We, however, recently demonstrated that FDPs produced in a
perfused, cross-linked clot vary in molecular weight from 250 (the mass
of DD/E) to 10,000 kDa, with the majority of the mass contained in
fragments larger than DD/E (9). This suggests that FDPs much larger
than DD/E are released into circulation during fibrinolysis, and indeed
FDP complexes of at least 2.0 × 106 Da have been
observed in the plasma of patients with disseminated intravascular
coagulation (13, 14) and chronic subdural hematoma (15). Whereas a
large difference in activity between t-PA and DSPA
1 would be
expected with DD/E, this difference disappears with larger FDPs.
Therefore, the relative fibrin specificity of DSPA
1 compared with
t-PA would be attenuated in the presence of larger FDPs.
Considerations on the Use of Plasminogen Activation to Measure
Soluble Fibrin and Fibrin Degradation Products in Plasma--
The t-PA
and DSPA
1 cofactor activity of FDPs larger than DD/E can be
attenuated by the action of TAFIa. Our studies have implications
regarding the use of plasminogen activation assays for the measurement
of soluble fibrin and/or FDPs in plasma. The rate of plasminogen
activation in plasma will be determined by the relative concentrations
of soluble fibrin, native FDPs, FDPs exposed to TAFIa, and fibrinogen.
Since the Km of the reactions with the FDPs is
10-fold lower and the intrinsic catalytic efficiency is 10-fold greater
than with fibrin, and therefore presumably with soluble fibrin, small
amounts of soluble FDPs could compromise the measurement of soluble
fibrin. Furthermore, TAFIa treatment attenuates the cofactor activity
of FDPs by ~100-fold. In determinations of FDPs, the rate of plasmin
generation in the assay will essentially be a measure of the
concentration of the native FDPs, which may or may not reflect the
total concentration, the value of which will be underestimated
according to the extent of FDP exposure to TAFIa. Thus, measurements
using plasminogen activation represent the composite cofactor activity
of plasma, which may or may not be a good measure of the species in
question. The complexity of the cofactor mixture and differences in the kinetics associated with each component in the mixture may explain the
differences observed with antibody-based and cofactor activity-based assays in the determination of the concentrations of both soluble fibrin and FDPs (16-18).