Stabilization Versus Inhibition of TAFIa by
Competitive Inhibitors in Vitro*
John B.
Walker
§,
Bernadette
Hughes¶,
Ian
James¶,
Peter
Haddock¶,
Cornelis
Kluft
, and
Laszlo
Bajzar
**
From the
Henderson Research Centre and
McMaster University, Hamilton, Ontario L8V 1C3, Canada,
¶ Pfizer Global Research and Development, Sandwich Laboratories,
Sandwich CT13 9NJ, United Kingdom, and the
Gaubius
Laboratory, TNO-PG, Leiden 2333 CK, The Netherlands
Received for publication, May 21, 2002, and in revised form, December 2, 2002
 |
ABSTRACT |
Two competitive inhibitors of TAFIa
(activated thrombin-activable fibrinolysis inhibitor),
2-guanidinoethylmercaptosuccinic acid and potato tuber carboxypeptidase
inhibitor, variably affect fibrinolysis of clotted human plasma.
Depending on their concentration, the inhibitors shortened, prolonged,
or had no effect on lysis in vitro. The inhibitor-induced
effects were both tissue-type plasminogen activator (tPA) and TAFIa
concentration-dependent. Inhibitor-dependent
prolongation was favored at lower tPA concentrations. The magnitude of
the prolongation increased with TAFIa concentration, and the maximal
prolongation observed at each TAFIa concentration increased saturably
with respect to TAFIa. A theoretical maximal prolongation of 20-fold
was derived from a plot of the maximum prolongation versus
TAFIa. This represents, for the first time, a measurement of the
maximal antifibrinolytic potential of TAFIa in
vitro.
Because TAFIa spontaneously decays, the stabilization of TAFIa was
investigated as a mechanism explaining the
inhibitor-dependent prolongation of lysis. Both inhibitors
stabilized TAFIa in a concentration-dependent, non-saturable manner. Although their KI values
differed by three orders of magnitude, TAFIa was identically stabilized when the fraction of inhibitor-bound TAFIa was the same. The data fit a
model whereby only free TAFIa decays. Therefore, the variable effects
of competitive inhibitors of TAFIa on fibrinolysis can be rationalized
in terms of free TAFIa and lysis time relative to the half-life of
TAFIa.
 |
INTRODUCTION |
Thrombin-activable fibrinolysis inhibitor
(TAFI)1 is the plasma
zymogen of a carboxypeptidase-B like enzyme, TAFIa (1, 2). TAFIa is
generated from TAFI by thrombin produced during coagulation, in complex
with thrombomodulin, present both on the endothelial cell surface and
in plasma (3-5). TAFIa attenuates fibrinolysis by catalyzing the
removal of C-terminal lysine and arginine residues present in
plasmin-degraded fibrin and fibrin degradation products (6, 7). Because
C-terminal lysine residues facilitate tPA-mediated plasminogen
activation, TAFIa likely exerts its antifibrinolytic effect by
down-regulating tPA-mediated plasminogen activation (6, 7).
Consequently, the inhibition of TAFIa may potentiate thrombolytic
therapy by preventing the TAFIa-mediated attenuation of plasminogen
activation (8-11).
Although there is no known inhibitor of TAFIa present in plasma,
several competitive inhibitors of carboxypeptidase B-like enzymes have
been described, including the small molecule inhibitor GEMSA (12), and
the peptide inhibitor from the potato tuber, PTCI (13).
Physiologically, the only known mechanism for down-regulating TAFIa
activity is an autoregulatory event in which TAFIa activity is lost
spontaneously (1, 2, 14-17). Because inhibition of TAFIa may
prove to be an effective strategy for potentiating thrombolytic therapy
(8), we investigated the effectiveness of GEMSA and PTCI as
profibrinolytic agents in the lysis of clots formed in normal human
plasma. We found that the two inhibitors exhibited complex behavior.
High concentrations of the inhibitors shortened lysis times.
Unexpectedly, low concentrations of the inhibitors prolonged
fibrinolysis by 3-fold (coincidently, an abstract describing this
paradoxical behavior was recently presented by Schneider (18)).
Therefore, the work described in this report was undertaken to
investigate the relationships between competitive TAFIa inhibitors, TAFIa, tPA, and fibrinolysis in an effort to understand the complex behavior exhibited by the competitive inhibitors of TAFIa in an in vitro system.
 |
EXPERIMENTAL PROCEDURES |
Materials--
L-
-Phosphatidylcholine (PC),
L-
-phosphatidylethanolamine (PE), and
L-
-phosphatidylserine (PS) were obtained in chloroform from Avanti Polar Lipids (Alabaster, AL). The TAFIa substrates 3-(2-furyl)acryloyl-Ala-Arg-OH (FAAR) and
3-(2-Furyl)acryloyl-Ala-Lys-OH (FAAK) were obtained from Bachem
Bioscience (King of Prussia, PA). The TAFIa inhibitor
2-guanidinoethylmercaptosuccinic acid (GEMSA) and the thrombin
inhibitor Phe-Pro-Arg-chloromethyl ketone (PPAck) were obtained from
Calbiochem (San Diego, CA). The potato tuber carboxypeptidase inhibitor
(PTCI) was obtained from Sigma-Aldrich (St. Louis, MO). Rabbit lung
thrombomodulin (TM) was obtained from American Diagnostica (Greenwich,
CT). Lipid reconstituted, recombinant tissue factor (TF),
RecombiPlasTin, was from Instrumentation Laboratory (Lexington, MA).
Human thrombin was a generous gift from Dr. M. E. Nesheim
(Queen's University, Kingston, Ontario, Canada). TAFI and TAFIa were
prepared as previously described (7, 17). The recombinant tissue-type
plasminogen activator (tPA), Activase, was a kind gift from Dr. Gordon
Vehar at Genentech (South San Francisco, CA). Pooled normal human
plasma (NHP) was produced from anticoagulated (1/10
acid/citrate/dextrose) whole blood from nine donors. TAFI-deficient
plasma (TdP) was prepared from NHP by immunodepletion of TAFI as
previously described (17). PC:PE:PS (60:20:20, v/v) phospholipid
vesicles were made using the procedure of Barenholz et al.
(19).
The Effect of Competitive Inhibitors of TAFIa on the tPA-induced
Lysis of Clots Formed in NHP or TdP Supplemented with TAFIa--
All
lysis assays were carried out at 37 °C in Dynex Immulon 1B
Removawell strips (Thermo-Labsystems, Franklin, MA). Assays were
initiated by the addition of a 60-µl aliquot, consisting of 25 µl
of 0.02 M Hepes/0.15 M NaCl/0.01% Tween 80 (HBST) plus 35 µl of either NHP or TdP, to a 40-µl aliquot
containing the variable constituents (CaCl2, tPA, thrombin,
TM, PC:PE:PS vesicles, TF, TAFI, TAFIa, GEMSA, or PTCI) of the assay.
The effect of the competitive inhibitors GEMSA and PTCI on the
tPA-induced lysis of clots made in NHP was assessed as follows. NHP
containing either PTCI (0-500 nM) or GEMSA (0-1
mM) was clotted with 3 nM thrombin in the
presence of 5 mM CaCl2, 5 nM TM,
1/800 dilution of TF, 15 µM PC:PE:PS vesicles, and
0.02-0.08 µg/ml tPA. The TM, TF, and PC:PE:PS vesicles were included
in these assays to promote the rapid and complete activation of TAFI to
TAFIa in situ. The effect of GEMSA and PTCI on the
tPA-induced lysis of clots made in TdP supplemented with TAFIa was
assessed as follows. TdP containing GEMSA (0-3 mM) or PTCI
(0-1 µM) was clotted with 3 nM thrombin in
the presence of 5 mM CaCl2, 5 nM
TM, 0.02 µg/ml tPA, and 0-100 nM TAFIa. For all
experiments, the plates were sealed with clear tape and the turbidity
of the clots at 405 nm was monitored at 37 °C using a SpectraMax
Platereader (Molecular Devices, Sunnyvale, CA). The lysis time, the
time at which the turbidity equals one-half of the full-scale value, of
each clot was determined.
The Effect of GEMSA on the Decay of TAFIa--
TAFIa (20 nM) in HBST/5 mM CaCl2 was
incubated at 37 °C in the presence of GEMSA (0-200
µM). Samples (220 µl) were taken at 0, 10, 25, 60, and
120 min and placed on wet ice to prevent further decay. The TAFIa
activity of each sample was determined at the end of the experiment
using the chromolytic TAFIa substrate FAAR. The TAFIa samples (100 µl) were mixed with 100 µl of FAAR (2 mM, in HBST) in
the wells of a microtiter plate, and the absorbance (340 nM) of duplicate reactions was monitored continuously over 3 h at 26 °C. The initial rate of absorbance decrease for each reaction was measured, and the data obtained at each concentration of
GEMSA were fit to the decay equation,
|
(Eq. 1)
|
where ratet is the rate of decrease of
absorbance at 340 nm (FAAR hydrolysis) after incubation at 37 °C for
t minutes, ratet = 0 is the
rate of FAAR hydrolysis prior to incubation at 37 °C, k
is the decay constant, t is the time in minutes, and
C is the intrinsic rate of FAAR hydrolysis. The half-life
(t1/2) of TAFIa at each concentration of GEMSA was
obtained from Equation 1 using the following relationship.
|
(Eq. 2)
|
Modeling the Decay of TAFIa in the Presence of a Large Excess of
Competitive Inhibitor--
Of the models that could be used to fit the
data for the spontaneous decay process or inactivation of TAFIa, the
one that fit our data the best was one where the decay of TAFIa
requires an empty active site.
In this model, the parameters I, TAFIai,
and k represent the inhibitor, the inactive decayed TAFIa,
and the decay constant, respectively. Because the decay of TAFIa is
spontaneous, the rate of decay is directly proportional to the
concentration of TAFIa where k is the decay constant.
|
(Eq. 3)
|
When only unoccupied TAFIa can decay, the rate of decay of TAFIa
in the presence of a competitive inhibitor is therefore proportional to
[TAFIa]free,
|
(Eq. 4)
|
or
|
(Eq. 5)
|
where fTAFIa is the fraction of TAFIa
free. Solving for [TAFIa] yields,
|
(Eq. 6)
|
where C is a constant of integration. When
t = 0, [TAFIa] = [TAFIa]o, and
therefore,
|
(Eq. 7)
|
The half-life, t1/2, of TAFIa in the presence
of a competitive inhibitor is, therefore,
|
(Eq. 8)
|
For a given concentration of TAFIa and inhibitor (I),
[TAFIa]free is related to
[I]free and KI through the
following,
|
(Eq. 9)
|
or
|
(Eq. 10)
|
Because
|
(Eq. 11)
|
then
|
(Eq. 12)
|
When [I]total
[TAFIa]total (i.e. in the case of GEMSA), then
[I]free
[I]total.
Thus,
|
(Eq. 13)
|
Solving for [TAFIa]free yields,
|
(Eq. 14)
|
and dividing out by [TAFIa]total, we obtain the
fraction of TAFIa free (fTAFIa),
|
(Eq. 15)
|
Substitution of Equation 15 into Equation 7 yields the
exponential decay (Equation 16) and the half-life (Equation 17) of
TAFIa in the presence of a competitive inhibitor when [I]
[TAFIa],
|
(Eq. 16)
|
|
(Eq. 17)
|
where t
is the half-life of TAFIa
in the absence of inhibitor. A plot of t1/2 versus the concentration of inhibitor yields a straight line
whose intercept equals t
and whose slope
equals t
/KI. The data
from the experiments using GEMSA were fit globally to Equation 16 using
the Systat program (SPSS Inc., Chicago, IL).
The Effect of PTCI on the Decay of TAFIa--
TAFIa (20 nM) in HBST/5 mM CaCl2 was
incubated at 37 °C in the presence of PTCI (0-70 nM).
Samples (400 µl) were taken at 0, 5, 10, 25, and 60 min and placed on
wet ice to prevent further decay. The TAFIa activity of each sample was
determined at the end of the experiment, using the chromolytic TAFIa
substrate FAAK, as follows. Duplicate samples (180 µl) were mixed
with 20 µl of FAAK (10 mM, in HBST) in the wells of a
microtiter plate, and the absorbance of the reactions at 340 nm was
monitored continuously over 3 h at 26 °C. The initial rate of
absorbance decrease for each reaction was determined.
Modeling the Decay of TAFIa When the Competitive Inhibitor Is Not
in a Large Excess--
According to Reaction 1, the decay of
TAFIa is dependent on [TAFIa]free, which is determined
from the equilibrium binding equation (Equation 10). In the case of the
experiments using PTCI, however, the assumption that
[I]free
[I]total
is not valid, and therefore, unlike the case where GEMSA was used, the
fraction of TAFIa that is TAFIafree changes over time.
Thus, our experiments using PTCI were not modeled using Equations
15-17. However, from Equation 4, the rate of decay is simply
proportional to [TAFIa]free. Using the mass action
relationship in Equation 11, Equation 10 can be rewritten as follows.
|
(Eq. 18)
|
Solving for [TAFIa]bound yields a quadratic whose
solution is,
|
(Eq. 19)
|
substitution into Equation 4 (using Equation 11) yields the
generalized rate of TAFIa decay in the presence of a competitive inhibitor.
|
(Eq. 20)
|
The data from the experiments using PTCI were fit iteratively to
Equation 20. Briefly, [TAFIa]free was determined at
time = t from [TAFIa]total,
[I]total, and KI;
[TAFIa]free then decayed exponentially for dt
seconds with decay constant k; [TAFIa]total at
time t + dt was determined by subtracting the decayed TAFIa; [TAFIa]free was determined at time = t + dt. This cycle was repeated for a total time of 60 min
using a time interval (dt) of 0.1 s. The decay constant
k (and thus t
for TAFIa) and
the KI value for PTCI were determined by non-linear
regression of the observed data to the iteratively calculated data
(varying k and KI) using the Solver tool in Microsoft Excel (Redmond, WA).
Intrinsic Fluorescence of TAFIa--
The intrinsic fluorescence
of TAFIa was monitored over time at 30 °C or 37 °C in the
presence and absence of both GEMSA (20, 40, 50, and 100 µM) or PTCI (120, 180, and 240 nM). In some
experiments, pre-activated TAFIa (4 µM) was added to
HSBT/5 mM CaCl2 in a stirred, thermostatted,
acrylic cuvette in the presence and absence of GEMSA and PTCI to give a
final TAFIa concentration of 100 nM. In other experiments,
100 nM TAFI in HBST/5 mM CaCl2 was
activated to TAFIa by 50 nM thrombin, 50 nM TM
in the presence and absence of GEMSA and PTCI in a stirred,
thermostatted, acrylic cuvette. The activation was initiated by the
addition of thrombin. Activation of TAFI to TAFIa under these
conditions was essentially complete by 60 s. The thrombin in the
reaction was quenched with PPAck (275 nM final) at 90 s. The fluorescence intensity of the samples was monitored in a
PerkinElmer Life Sciences LS-50B fluorescence spectrophotometer using
ex = 280 nm, slit = 5 nm,
em = 340 nm, slit = 4.5 nm with a 290-nm emission filter in place. Samples (110 µl) were removed at various times from the reactions, added to
110 µl of ice-cold HBST/5 mM CaCl2, placed on
ice until the end of the experiment, and then assayed for FAAK activity
as described above.
Determination of the KI for GEMSA and PTCI with
TAFIa--
TAFIa (10 nM) was mixed with GEMSA (0, 5, 10, 20, 50, and 100 µM) or PTCI (0, 10, 20, 30, 40, and 50 nM), and the initial rate of hydrolysis of FAAR (0.5, 1, 1.5, 2, 2.5, and 3 mM) in the presence of the various
concentrations of inhibitor was determined as above. The data was
non-linearly regressed to the Michaelis-Menten equation describing
competitive inhibition,
|
(Eq. 21)
|
where I is the concentration of the inhibitor, using
the Systat program.
 |
RESULTS |
The Effect of the Competitive Inhibitors GEMSA and PTCI on the
tPA-induced Lysis of Clots Formed from NHP--
The lysis of clots
formed from NHP in the presence of various concentrations of tPA was
studied in the presence and absence of various concentrations of GEMSA
and PTCI, two competitive inhibitors of TAFIa. Fig.
1 shows the variable effect of GEMSA
(panel A) and PTCI (panel B) on the lysis of
clots in NHP with 0.08 µg/ml tPA. Compared with the lysis profiles
seen in the absence of inhibitors (Fig. 1, circles), both
inhibitors could either prolong (squares) or shorten
(triangles) lysis, depending on the concentration of the
inhibitor. The concentration dependence of tPA on inhibitor-mediated prolongation of lysis was investigated. Fig.
2 shows the effect of GEMSA (panel
A) and PTCI (panel B) on the lysis time of clots formed
from NHP using 0.02-0.08 µg/ml tPA. In addition, the figure shows
the lysis time in the presence of GEMSA (panel C) and PTCI (panel D) relative to the lysis time in the absence of
inhibitor. The figure also shows that both competitive inhibitors
exhibit a complex, bell-shaped behavior with respect to plasma clot
lysis. Over the concentration ranges of tPA and inhibitor used, both GEMSA (panel C) and PTCI (panel D) can prolong,
shorten, or display no effect on the lysis time. For example, at a PTCI
concentration of 100 nM, the lysis times were prolonged
3.1-fold, 2.0-fold, and 1.4-fold at 0.02, 0.04, and 0.08 µg/ml tPA,
respectively. However, at 500 nM PTCI, the lysis times were
longer at 0.02 µg/ml tPA (1.5-fold) but shorter at 0.04 and 0.08 µg/ml tPA (0.78-fold and 0.48-fold, respectively). The data show that
the effect (prolongation or shortening) of a competitive inhibitor on
the lysis time of clots formed from NHP is dependent on the
concentration of both the inhibitor and tPA, with lower concentrations
of tPA promoting the prolongation of lysis at any given inhibitor
concentration.

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Fig. 1.
Competitive inhibitors of TAFIa can prolong
or shorten fibrinolysis. Representative absorbance profiles of
clots formed from NHP in the absence ( ) or presence of either GEMSA
(A, 100 µM ( ), 1000 µM ( ))
or PTCI (B, 50 nM ( ), 500 nM
( )) at 0.08 µg/ml tPA. The figure shows that, depending on the
concentration of inhibitor, both GEMSA and PTCI can prolong or shorten
tPA-induced fibrinolysis.
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Fig. 2.
The effect of competitive inhibitors on lysis
is dependent on both the inhibitor and tPA concentrations. Clots
were formed from NHP in the presence of various concentrations of
either GEMSA (A and C) or PTCI (B and
D) at 0.02 ( ), 0.04 ( ), and 0.08 ( ) µg/ml tPA.
The data (average of duplicates) are presented as the absolute
(A and B) and relative (C and
D) lysis times at each concentration of tPA and inhibitor.
The figure shows that both inhibitors display complex behavior over the
range of tPA and inhibitor concentrations used and that, for any given
inhibitor concentration, decreasing tPA promotes the
inhibitor-dependent prolongation of fibrinolysis.
|
|
The Inhibitor-induced Prolongation Observed in Plasma Is
TAFIa-dependent--
In order to verify that the
inhibitor-induced prolongation was both specific for TAFIa and not due
to differences in the rates of TAFIa formation by thrombin-TM, the
lysis times of clots formed from TdP supplemented with 0.02 µg/ml tPA
and various concentrations of TAFIa were determined in the absence and
presence of either GEMSA or PTCI. Fig. 3
shows that in the absence of GEMSA (panel A) and PTCI
(panel B), the TAFIa-dependent prolongation is
saturable, with the half-maximal effect observed below 5 nM. The dependence of lysis time on the inhibitor
concentration displayed similar behavior for each TAFIa concentration;
as the inhibitor concentration is increased from zero the lysis time
increases to some maximum value, the magnitude of which increases with
increasing TAFIa concentration. Once the maximum lysis time is
attained, any further increase in the inhibitor concentration decreases
the lysis time. To understand the behavior of the system with respect
to TAFIa concentration, we plotted the maximum prolongation observed,
relative to the lysis time seen in the absence of TAFIa,
versus the input TAFIa concentration. As shown in Fig.
4, the maximum prolongation observed at
each TAFIa concentration in the presence of both GEMSA and PTCI appears
saturable with respect to TAFIa concentration. Thus, the data from each
inhibitor was non-linearly regressed, using the Systat program, to the
rectangular hyperbola,

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Fig. 3.
The inhibitor-induced prolongation of
fibrinolysis observed with both GEMSA and PTCI is
TAFIa-dependent. Clots were formed from TdP
supplemented with 0.02 µg/ml tPA and various concentrations of TAFIa
(0 nM ( ), 5 nM ( ) 10 nM
( ), 20 nM ( ), 30 nM ( ), 50 nM ( ), 70 nM ( ), and 100 nM
( )), in the presence and absence of various concentrations of either
GEMSA (A) or PTCI (B). The data show that the
prolongation of the lysis times for both inhibitors is
TAFIa-dependent and that the maximum prolongation is
dependent on the TAFIa concentration.
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Fig. 4.
The prolongation effect is saturable with
respect to TAFIa. The maximum relative prolongation (the largest
-fold increase in lysis time observed in the presence of an inhibitor)
at each TAFIa concentration in Fig. 3 was plotted against the input
TAFIa concentration for the experiments using GEMSA ( ) and PTCI
( ). The figure shows that the maximum relative prolongation is a
function of TAFIa concentration and that this effect is saturable. The
lines show the fit of the data to a rectangular hyperbola
(Equation 22) and indicate that the maximal relative prolongation for
TAFIa (i.e. -fold prolongation at infinite TAFIa) is
~20-fold.
|
|
|
(Eq. 22)
|
where P[TAFIa] is the maximum relative
prolongation observed at a given TAFIa concentration,
Pmax is the maximal relative prolongation
(i.e. the lysis time expected at infinite TAFIa
concentration relative to the lysis time seen in the absence of TAFIa),
and THalfMax is the TAFIa concentration required
to yield a prolongation equal to one-half of
Pmax. The regression yielded
Pmax values of 23 ± 3-fold and 21 ± 3-fold and THalfMax values of 85 ± 17 and
110 ± 24 nM for GEMSA and PTCI, respectively. It
should be noted that the maximum prolongation observed at any given
TAFIa concentration is not dependent on the inhibitor
KI but on the concentration of inhibitor present
relative to KI, i.e. the maximum
prolongation occurs when the inhibitor is present at some -fold over
KI. Thus, the values of Pmax
and THalfMax are also independent of
KI. The regression curves are shown in Fig. 4.
The Effect of GEMSA and PTCI on the Activity and Spontaneous Decay
of TAFIa--
TAFIa was incubated with various concentrations of
either GEMSA or PTCI at 37 °C and timed samples were taken and
assayed for TAFIa activity. Fig. 5 shows
the TAFIa activity (panels A and B) and the
fraction of initial TAFIa activity (panels C and D) versus the time of incubation at 37 °C in
the presence of various concentrations of GEMSA (panels A
and C) and PTCI (panels B and D). The
figure shows the data points (symbols) as well as the fits
of the data (lines) to Equation 16 for GEMSA and,
iteratively, to Equation 20 for PTCI. That the iterative approach
provided reasonable estimates of t
and
KI for the experiments using PTCI is supported by
the agreement between the estimates obtained for GEMSA using Equation 16 (t
= 9.2 min, KI = 36 µM) and those obtained, iteratively, using Equation 20
(t
= 9.1 min, KI = 41 µM). The figure shows that the inhibition of TAFIa
activity by GEMSA and PTCI (panels A and B) is
accompanied by a stabilization of TAFIa (panels C and
D), resulting in a decreased rate of TAFIa decay. The values
for t
and KI derived
from the decay experiments, as well as the measured
KI values for both inhibitors, are shown in Table
I. To determine the relationship between
the inhibitor concentration, the inhibitor KI, and
the decay of TAFIa for both GEMSA and PTCI, we determined the apparent
half-life of TAFIa (t
) at each
inhibitor concentration by fitting each decay curve (Fig. 5,
C and D) to an exponential decay (Equation 1) and
plotted t
versus the
fraction of TAFIa bound at each inhibitor concentration. Although the
decay of TAFIa activity with PTCI was not truly exponential (see
"Experimental Procedures"), the fit curves (Fig. 5D,
dashed lines) show that a single exponential closely
approximates the decay, and, therefore, the derivation of
t
by this analysis is reasonable. Fig.
6 shows the effect of GEMSA (circles) and PTCI (triangles) on the apparent
half-life of TAFIa versus the fraction of TAFIa bound at
each of the inhibitor concentrations. The fraction of TAFIa bound was
calculated from Equation 19 using KI values derived
from the models (closed symbols) or from
KI values determined independently (open
symbols). The figure shows that, regardless of the method used to
determine the KI, both inhibitors exert the same
effect on the decay of TAFIa when the inhibitors are used at
concentrations that yield the same fraction of TAFIa bound. Therefore,
the extent of stabilization of TAFIa by any given concentration of a
competitive inhibitor is directly related to KI.

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Fig. 5.
The inhibition of TAFIa activity by GEMSA and
PTCI is concomitant with a decrease in the rate of TAFIa decay.
TAFIa (20 nM) was incubated with various concentrations of
either GEMSA (A and C; 0 µM ( ),
3.1 µM ( ), 6.3 µM ( ), 12.5 µM ( ), 25 µM ( ), 50 µM
( ), 100 µM ( ), and 200 µM ( )) or
PTCI (B and D; 0 nM ( ), 10 nM ( ), 20 nM ( ), 30 nM ( ),
40 nM ( ), 50 nM ( ), 60 nM
( ), and 70 nM ( )) at 37 °C for various periods of
time and then assayed, in duplicate, for TAFIa activity. The
solid lines in A and B show the fit of
the data to Equation 16 (GEMSA, A) or Equation 20 (PTCI,
B). The solid lines in C and
D show the decay of TAFIa in the presence of GEMSA
(C) and PTCI (D) calculated from the fit curves
in A and B. Panel D also shows the
deviation of the curves derived using Equation 20 (solid
lines) from a true single exponential decay (dashed
lines). The figure shows that the
concentration-dependent inhibition of TAFIa activity by
GEMSA and PTCI is accompanied by a concentration-dependent
stabilization of TAFIa.
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Table I
The half-life of TAFIa and the KI for GEMSA and PTCI
The table shows the half-life of TAFIa and the KI
value for each inhibitor determined from modeling the data from the
experiments to Equation 16 (GEMSA) using the Systat program (±S.E.) or
Equation 20 (PTCI) using the Solver program (Microsoft Excel). The
values determined experimentally according to Equation 21 using the
Systat program (±S.E.) are shown for comparison.
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Fig. 6.
The effect of GEMSA and PTCI on the apparent
half-life of TAFIa. The apparent half-life of TAFIa, calculated
for each concentration of GEMSA ( , ) and PTCI ( , ), from
the decay curves in Fig. 5 (C and D), was plotted
against the fraction of TAFIa bound, calculated from Equation 19 using
the KI values derived from the models ( , ) or
determined independently ( , ). The figure shows that both GEMSA
and PTCI have equivalent effects on the stability of TAFIa when the
enzyme is expressed as the fraction bound to the inhibitor.
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 |
DISCUSSION |
It is well established that the inhibition of TAFIa, an attenuator
of fibrinolysis, results in the potentiation of tPA-induced clot lysis
in vitro (17, 20, 21). We have observed, however, that two
competitive inhibitors of TAFIa, GEMSA and PTCI, can be
antifibrinolytic, profibrinolytic, or show no effect on fibrinolysis. Furthermore, for a given TAFIa concentration, the effect observed at
any given concentration of the inhibitor is dependent on the tPA
concentration, with lower tPA concentrations favoring the antifibrinolytic effect. Of particular note is the observation that the
maximal prolongation in lysis time is dependent on the TAFIa
concentration for both GEMSA and PTCI. An analysis of the TAFIa
concentration dependence on the prolongation of lysis time showed that
1) the concentration of inhibitor at which the prolongation is greatest
increases as TAFIa concentration increases and 2) the prolongation of
lysis is saturable with respect to TAFIa. By fitting the maximum
prolongation observed at each concentration of TAFIa to a rectangular
hyperbola, we determined the maximal effect of TAFIa, i.e.
the prolongation expected at infinite TAFIa, to be a 20-fold
prolongation of the lysis time seen in the absence of TAFIa. To our
knowledge, this represents the first quantitative estimate of the
maximal antifibrinolytic activity of TAFIa in an in vitro
static system.
Because the phrase "inhibitor-mediated potentiation of enzyme
activity" appears contradictory, we sought a mechanism that could
account for the paradoxical effects of competitive inhibitors of TAFIa
on tPA-induced fibrinolysis. TAFIa decays spontaneously, an intrinsic
property not observed with pancreatic carboxypeptidase B or plasma
carboxypeptidase N, and previous work has shown that GEMSA (14, 22) and
-aminocaproic acid (22-24), both competitive inhibitors of TAFIa,
stabilize the activity of TAFIa toward chromogenic substrates when used
at saturating concentrations. In addition, natural isoforms of TAFI,
which, upon activation, vary in their intrinsic stability have been
characterized and show antifibrinolytic effects correlating with their
intrinsic stability (25). Therefore, we investigated the possibility
that the prolongation of lysis by inhibitors of TAFIa directly results
from stabilization of TAFIa activity. Both GEMSA and PTCI decreased the
activity (Fig. 5, A and B) and concurrently
increased the stability (Fig. 5, C and D) of
TAFIa activity in a concentration-dependent manner. Because
both GEMSA and PTCI inhibit TAFIa activity by occupying the active site
(i.e. competitively), we therefore attempted to rationalize
the stabilization of TAFIa activity according to a model where
inhibitor-bound TAFIa is protected from spontaneous decay.
Two models were proposed to account for the stabilization of TAFIa
activity. In the first model, only free TAFIa is allowed to decay, and
therefore decay is active site-dependent. In the second
model, both free TAFIa and inhibitor-bound TAFIa are allowed to decay
independently, and therefore decay is both active
site-dependent (TAFIa) and active site-independent
(inhibitor-bound TAFIa). Applying a model with a single decay event, in
which only the free TAFIa can decay, we obtained a good global fit to
the data for both GEMSA and PTCI (Fig. 5). The model describes well the
observed decrease in TAFIa activity (Fig. 5, A and
B) and the associated stabilization of the enzyme by each
inhibitor (Fig. 5, C and D). The model returned
calculated t
values for TAFIa at
37 °C of 9.2 (GEMSA) and 9.1 (PTCI) min, in good agreement with
those reported by others (8.7 (14), 9.1 (22), and 8.0 (25) min), as
well as reasonable estimates of the KI values for
both inhibitors (GEMSA = 36 µM, PTCI = 10 nM) when compared with their measured values (GEMSA = 14 µM, PTCI = 4 nM). When the data from
the experiments with both inhibitors were analyzed according to the
second model, the best fit was obtained when the rate constant for the
decay of inhibitor-bound TAFIa was zero, i.e. interpretation
of the results using this model suggests that the effect, if any, of
the decay of inhibitor-bound TAFIa was inconsequential under our
experimental conditions (data not shown). Although our analysis was not
exhaustive, we interpreted our data according to the simpler of the two
models, because no improvement was found when inhibitor-bound TAFIa was
assumed to decay.
Although the derived KI value for each of the
inhibitors studied differs by greater than three orders of magnitude, an analysis of the apparent half-life of TAFIa versus
inhibitor concentration (Fig. 6) showed that GEMSA and PTCI prolonged
the apparent half-life of TAFIa to the same extent when TAFIa was expressed as the fraction bound to the inhibitor. Thus, the degree of
stabilization conferred on TAFIa by any given concentration of GEMSA
and PTCI is directly related to the KI of the inhibitor. Furthermore, the stabilization effect seen with both inhibitors is not saturable (Fig. 6), suggesting that inhibitor-bound TAFIa is protected from spontaneous decay as opposed to simply decaying
at a slower rate than free TAFIa. Thus, the spontaneous decay of TAFIa
in the presence of competitive inhibitors is consistent with a model
whereby the conformational change underlying the thermolability of
TAFIa requires a free active site and, as a corollary, that active-site
bound TAFIa does not decay. Whether or not TAFIa is similarly
stabilized in the presence of its primary substrates, plasmin-degraded
fibrin and fibrin-degradation products, remains to be investigated.
That TAFIa bound to a competitive inhibitor is protected from decay is
consistent with previous findings that showed the decay of the
intrinsic fluorescence of TAFIa to be dose-dependently stabilized by GEMSA (14). The model described in the previous study
employed a two-step, sequential first-order exponential decay process
in which both decay events are stabilized, if not eliminated, by GEMSA
(14). In contrast to the fluorescence decay, the authors found that the
TAFIa activity decayed as a single exponential and proposed that the
activity decayed according to the second, slower transition (14). We
performed similar fluorescence decay experiments in the absence and
presence of GEMSA and PTCI. In agreement with the previous study, both
inhibitors stabilized the intrinsic fluorescence of TAFIa in a
dose-dependent manner with an accompanying stabilization of
TAFIa activity (data not shown). However, no one single mathematical
relationship was found that directly correlated either single or
sequential exponential decay of fluorescence with the TAFIa activity
stabilization data. In addition, we observed several differences
between the two studies. First, we consistently observed only a
10-15% total decrease in intrinsic fluorescence upon activity decay,
significantly less than what has been reported (38% (22), 51% (14)).
Second, we did not consistently observe the presence of two exponential fluorescence decay events. Because TAFIa activity has consistently been
shown to decay according to a single exponential (14, 22, 25), we have
described the stabilization of TAFIa activity according to a model with
a single decay event. Parenthetically, even though we cannot rule out
the two-step model of decay with certainty, due to the apparent rapid
rate of the first decay and the relatively small fluorescence change
associated with it, following a two-step model would not significantly
alter the interpretation of our data. The magnitude of the difference
between the rates of the first (0.50 min
1) and second
(0.064 min
1) transitions in concert with the greater
affinity of the inhibitor (GEMSA) for the second form
(KD = 6 µM) compared with the first
form (KD = 14 µM) of TAFIa would
result in the nearly quantitative accumulation of the second form in
both the presence and absence of the inhibitor (14). As a consequence, the loss of activity and the stabilization of TAFIa in such a two-step
model can effectively be described by a single exponential decay event
of the free TAFIa.
It could be argued that the competitive inhibitors stabilize TAFIa by
binding at sites distinct from the active site. This scenario is
unlikely, because it would be consistent with our data only if 1) the
ratio of the KD values for the exosite(s) for both
inhibitors was, fortuitously, the same as the ratio of the
KI values for the active site and 2) the
KD values for the exosite(s) were essentially
indistinguishable from the KI values, because both
inhibitors express their effects at concentrations approximating their
respective KI values (Fig. 6). Alternatively, one
could envisage a scenario in which the competitive inhibitors stabilize
TAFIa by attenuating TAFIa degradation by proteolysis. For example, a
recent report (26) suggests that plasmin, in contrast to thrombin (14,
24), directly inactivates TAFIa. However, we found little, if any, significant reduction in the activity of TAFIa incubated in the absence
and presence of 100 nM plasmin at either 25 °C or
37 °C: samples taken during a 1-h time course yielded a half-life of 22 versus 19 min at 25 °C and 6.9 versus 6.1 min at 37 °C in the absence and presence of 100 nM
plasmin, respectively. It is unlikely that the steady-state
concentration of plasmin would approach even 10 nM during
fibrinolysis under the conditions used in our study, due to the low tPA
concentrations used and the presence of
2-antiplasmin in
plasma. Furthermore, were plasmin capable of significant proteolytic
inactivation of TAFIa, the generation of significant levels of plasmin
is not temporally coincident with the period of TAFIa activity (14).
Finally, protection from proteolytic inactivation, by itself, would not
extend the half-life of TAFIa beyond that imposed by thermal
instability. For these reasons, proteolytic inactivation of TAFIa
likely represents a minor pathway for inactivation in comparison to
thermal instability, consistent with the conclusions of others (14, 24,
25). As a result, we conclude that the inhibitor-mediated stabilization of TAFIa observed in functional assays, both chromogenic and clot lysis
experiments, occurs via prevention of the spontaneous decay of TAFIa
resulting from intrinsic thermal instability.
Marx et al. (27) have recently shown that the catalytic
Zn2+ ion remains associated with the decayed
TAFIai, indicating that loss of Zn2+ is not
involved in the conformational change underlying the thermal instability of TAFIa. Because proteolytic inactivation of TAFIa is also
not required (14, 24), the protective effect of competitive inhibitors
is likely to involve direct interactions with residues in the active
site. The crystal structures of bovine carboxypeptidase A (CPA) bound
to PTCI (28) and of duck carboxypeptidase D (CPD) domain II (CPD-II) in
complex with GEMSA (29) show that both inhibitors form extensive
contacts with residues at the active site. Both inhibitors directly
coordinate the catalytic Zn2+ ion and form numerous
contacts with residues involved in coordinating the peptide substrate
(28, 29). GEMSA remains intact in CPD-II and forms contacts with active
site residues involved in substrate binding, both proximal and distal
to the scissile bond (29). In contrast, PTCI is cleaved at its C
terminus (Val38-Gly39) by CPA, and the free
Gly39 remains in the active site, at least in the crystal
structure (28). Although nearly all residues in the tail region of PTCI are involved in complex formation, the new C-terminal Val38
contributes half of the binding energy to the PTCI·CPA complex and directly coordinates the Zn2+ ion (28, 30). Thus,
whereas GEMSA inhibits CPD-II as a non-hydrolyzable substrate, PTCI
inhibits CPA as a tight-binding product. Although TAFIa differs
significantly from both CPA and CPD-II, not only in sequence but also
in stability, and it is not known whether PTCI is C-terminally
processed by TAFIa, it is likely that TAFIa interacts with both PTCI
and GEMSA in a manner similar to CPA and CPD-II, respectively. This
assertion stems from the comparison of metallocarboxypeptidases from
different species with different substrate specificities (CPD (duck and
human), carboxypeptidase E (human), CPA (bovine), and carboxypeptidase
B (bovine)) showing a highly conserved active site (29), with the
differences in substrate specificity primarily attributed to the
insertion/deletion of residues in the funnel which leads to the active
site (29, 31). The binding energy involved in the inhibitor-TAFIa
interactions may exert a stabilizing effect by maintaining the
intrinsically unstable positions of critical residues in the active
site. It is also possible, however, that stability is conferred by a
simple steric effect: spontaneous decay may require the "space"
occupied by the inhibitors. In either case, a detailed, structure-based mechanism describing TAFIa stabilization is likely to be provided only
when crystal structure of TAFI, TAFIa, and/or TAFIai have been solved.
The tPA dependence of the inhibitor-induced effect can be rationalized
on the basis that the inhibition of TAFIa is concomitant with the
stabilization of TAFIa. This is best illustrated by considering fibrinolysis in clotted plasma, in the absence of an inhibitor, when
the lysis time is very long relative to the half-life of TAFIa,
e.g. low [tPA], and TAFI activation is rapid and
quantitative. Because the concentration of plasminogen activator is
low, the intrinsic rate of plasmin generation on intact fibrin is low. Thus, the concentration of TAFIa required to maximally inhibit plasminogen activation (TAFIamax), by removing all
C-terminal lysines and arginines before they can be used as cofactors,
represents a small fraction of the initial TAFIa concentration.
However, because the lysis time is very long relative to the half-life of TAFIa, the TAFIa will decay to a concentration less than
TAFIamax long before lysis is complete, even if TAFIa is
initially present at high concentrations. In other words, TAFIa is not
present at a concentration sufficient for complete attenuation of
plasminogen activation for the duration of fibrinolysis.
In the presence of a competitive inhibitor the active concentration of
TAFIa (i.e. [TAFIa]free) is decreased but the
half-life is increased. Because TAFIamax is only a small
fraction of the initial TAFIa concentration, the inhibitor can be
present at high concentrations (relative to KI) and
still leave the initial [TAFIa]free at a concentration
above TAFIamax. Thus, TAFIa activity can be present at an
effective level for a longer period of time than is the case when no
inhibitor is present. Therefore, for any given TAFIa concentration, the
peak of the curve in Fig. 3 represents the concentration of inhibitor
at which both the initial [TAFIa]free and the half-life
of TAFIa are balanced such that effective TAFIa activity is present for
the maximal time. At concentrations of inhibitor less than the peak
amount, the TAFIa decays more quickly, reducing the
inhibitor-dependent prolongation of lysis, whereas at
greater concentrations of the inhibitor, the initial [TAFIa]free is reduced, thereby diminishing the
prolongation effect.
Now consider the two effects that occur as the tPA concentration
increases, again in the absence of an inhibitor. First, the intrinsic
rate of plasmin generation on intact fibrin increases and, therefore,
the concentration of TAFIa required to maximally attenuate plasminogen
activation also increases, i.e. TAFIamax increases. Second, the lysis time is reduced relative to the half-life of TAFIa. Therefore, TAFIa will decay through fewer half-lives before
the lysis time is reached. Consequently, the effect of TAFIa becomes
more dependent on the initial TAFIa concentration and less dependent on
the half-life of TAFIa. In other words, as tPA concentration increases,
more TAFIa is required, but for a shorter period of time. This effect
can be seen in the curves shown in Fig. 2. At any fixed concentration
of inhibitor, the relative lysis time (panels C and
D) decreases as the concentration of tPA increases. This
results from the stabilization of TAFIa being dependent on the
inhibitor concentration but independent of the tPA concentration
(i.e. the fraction of TAFIa bound, and therefore
[TAFIa]free is the same regardless of the tPA
concentration), whereas the intrinsic rate of plasmin generation, and
therefore the intrinsic rate of C-terminal lysine formation on fibrin,
increases as tPA concentration increases. Thus, as tPA concentration
increases, more TAFIa activity is required to maintain the fibrin
surface in a non-stimulatory state (i.e.
TAFIamax increases). Therefore, at any given inhibitor
concentration, [TAFIa]free becomes a smaller fraction of
TAFIamax as the concentration of tPA increases.
The work described in this report provides a rationale for the complex
behavior of TAFIa observed in the presence of competitive inhibitors.
These inhibitors stabilize TAFIa to an extent that is proportional to
the fraction of TAFIa bound. The results can be described according to
a model where TAFIa can spontaneously decay only if it has a free
active site, and thus the stabilization of TAFIa is directly related to
the concentration of an inhibitor through KI. The
tPA dependence of the pleiotropic behavior of the inhibitors can be
rationalized by considering two quantities: 1) TAFIamax, or
the amount of TAFIa required to maximally attenuate plasminogen
activation, and 2) the lysis time in relation the half-life of TAFIa.
The in vitro experiments described here indicate that the
prolongation of lysis is related to the concentration of inhibitor
relative to its KI and is favored under conditions
in which both the concentration of TAFIa is high and that of tPA is
low. Caution should be exercised in extending these results to the
in vivo situation, because the local concentrations of
TAFIa, tPA, and inhibitor at the site of a thrombus in the complex,
non-static in vivo milieu are currently unknown. Although the ramifications of the pleiotropic behavior of TAFIa inhibitors on
their potential clinical utility should be addressed using appropriate
animal models, no antifibrinolytic effects have been reported using
PTCI in vivo in animal models to date (8-11). With this
caveat in mind, however, it is likely that choosing an inhibitor with a
low KI will minimize any prolongation of
fibrinolysis. Finally, we have provided, for the first time, an
estimate of the maximal antifibrinolytic potential of TAFIa in
vitro.
 |
FOOTNOTES |
*
This work was supported in part by a grant (T 4627) from the
Heart and Stroke Foundation of Ontario and funding from Pfizer Global
Research and Development, UK.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Recipient of a Post-doctoral Fellowship from the Heart and Stroke
Foundation of Canada.
**
A Research Scholar of the Heart and Stroke Foundation of Canada. To
whom correspondence should be addressed: Henderson Research Centre, 711 Concession St., Hamilton, Ontario L8V 1C3, Canada. Tel.:
905-527-2299 (ext. 43722); Fax: 905-575-2646; E-mail:
lbajzar@thrombosis.hhscr.org.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M205006200
 |
ABBREVIATIONS |
The abbreviations used are:
TAFI, thrombin-activable fibrinolysis inhibitor;
TAFIa, activated TAFI;
TAFIai, decayed, inactivated TAFIa;
tPA, tissue-type
plasminogen activator;
GEMSA, 2-guanidinoethylmercaptosuccinic acid;
PPAck, Phe-Pro-Arg-chloromethyl ketone;
PTCI, potato tuber
carboxypeptidase inhibitor;
NHP, normal human plasma;
TdP, TAFI-deficient plasma;
PC, L-
-phosphatidylcholine;
PE, L-
-phosphatidylethanolamine;
PS, L-
-phosphatidylserine;
FAAR, 3-(2- furyl)acryloyl-Ala-Arg-OH;
FAAK, 3-(2-furyl)acryloyl-Ala-Lys-OH;
TM, rabbit lung thrombomodulin;
TF, recombinant tissue factor;
CPA, carboxypeptidase A;
CPD, carboxypeptidase D;
CPD-II, carboxypeptidase D domain II.
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