The Use of Prothrombin(S525C) Labeled with Fluorescein to
Directly Study the Inhibition of Prothrombinase by Antithrombin during
Prothrombin Activation*
Nicole
Brufatto and
Michael E.
Nesheim
From the Department of Biochemistry, Queen's University, Kingston,
Ontario K7L 3N6, Canada
Received for publication, December 21, 2000, and in revised form, February 22, 2001
 |
ABSTRACT |
Serine 525 of human prothrombin was
mutated to cysteine and covalently labeled with fluorescein to make
II(S525C)-fluorescein. Kinetics of cleavage of this derivative by
prothrombinase are identical to those of wild-type prothrombin.
Cleavage is coincident with a 50% increase in fluorescence intensity
and the product is catalytically inactive. Thus, it allows convenient
monitoring of prothrombin activation without generating active
thrombin. The kinetics of inhibition of factor Xa (FXa) by antithrombin (AT) and AT-heparin were measured by monitoring activation of II(S525C)-fluorescein and the hydrolysis of the chromogenic
substrate S2222 in the presence of AT. With S2222 as the substrate
the rate constant for inhibition of FXa, Ca2+, and
unilamellar vesicles of phosphatidylcholine and phosphatidylserine (75:25) (PCPS) vesicles by AT was 3.51 × 103
M
1 s
1; when factor Va (FVa) was
included the rate constant was 1.55 × 103
M
1 s
1. In the absence of FVa,
II(S525C)-fluorescein had no effect on inhibition. When
II(S525C)-fluorescein was the substrate, however, FVa at saturating
concentrations profoundly protected FXa from inhibition by AT,
increasing the half-life from 3 min with FXa, Ca2+,
PCPS, and II(S525C)-fluorescein, to greater than 69 min when FVa
was included. Thus, both FVa and prothrombin are necessary for this
level of protection. In the absence of prothrombin, FVa decreased the
second order rate constant for inhibition by the AT-heparin complex
from 1.58 × 107 M
1
s
1, for FXa, Ca2+, and PCPS, to 7.72 × 106 M
1 s
1.
II(S525C)-fluorescein and factor Va together reduced the rate constant
to less than 1% of that for FXa, Ca2+, and PCPS. At a
heparin concentration of 0.2 unit/ml, this corresponds to a half-life
increase from 1 s to 136 s.
 |
INTRODUCTION |
One of the key steps of coagulation involves activation of
prothrombin to its active form thrombin by the multicomponent complex, prothrombinase. Prothrombinase comprises the serine protease factor Xa,
the activated protein cofactor factor Va, calcium ions, and an
appropriate cell membrane or phospholipid surface (1-5). Although factor Xa alone can slowly generate thrombin by an extremely
inefficient reaction, the rate of thrombin generation is enhanced by
several orders of magnitude by incorporation of the cofactor protein
factor Va and the procoagulant surface (5).
The activities of the clotting serine proteases, such as
thrombin, are regulated, in part, by plasma protease inhibitors of the
serpin superfamily. Of these, antithrombin appears to be the most
important. Antithrombin targets both the product of prothrombin activation, thrombin, and the enzyme responsible for the reaction, factor Xa. Inhibition of serine proteases by serpins occurs through the
formation of a stable complex between the serpin and the active site
of the serine protease. Inhibition by antithrombin is markedly enhanced
by the glycosaminoglycan cofactor, heparin. Heparin accelerates rates
of inhibition of both thrombin and factor Xa several thousand fold (6).
Upon binding, heparin induces a conformational change in
antithrombin (7-9), which is sufficient to accelerate the inhibition
of factor Xa through an allosteric mechanism. The acceleration of
thrombin inhibition, however, is dependent on the formation of a
ternary complex between heparin, antithrombin, and thrombin (10-12).
Several studies demonstrated that incorporation of factor Xa into the
prothrombinase complex changes the ability of the antithrombin-heparin complex to inhibit it. Calcium ion accelerates the heparin catalyzed inactivation of factor Xa by antithrombin through a template mechanism (13). Phospholipids and factor Va play protective roles and decrease
the ability of the heparin-antithrombin complex to inhibit factor Xa
both on synthetic surfaces (14-19) and in whole blood clots (20).
Herault et al. (21), however, indicated that the antithrombin-heparin complex is an effective inhibitor for
phospholipid/factor Va-bound factor Xa in solution and on blood cells.
The substrate prothrombin also plays an important role in the
protection observed when factor Xa is incorporated into the
prothrombinase complex (14).
Prothrombinase activity is usually inferred through measurements of
thrombin activity over time. Therefore, studying
heparin-dependent antithrombin inhibition of prothrombinase
is challenging because the inhibitor recognizes both the product of the
reaction, thrombin, and the enzyme component of prothrombinase, factor
Xa. To resolve this problem in this study, a mutant form of prothrombin
(II(S525C)), in which serine 525 (the active site serine of thrombin)
is replaced with cysteine, was expressed in a mammalian expression
system, isolated, and covalently labeled at Cys525
with fluorescein. II(S525C)-fluorescein exhibits an increase in
fluorescence intensity upon activation to thrombin but is catalytically inactive to both small and macromolecular substrates. Thus, it allows
convenient monitoring of prothrombin activation in the presence of
antithrombin without generating active thrombin. In this study
inhibition of free factor Xa or factor Xa bound to vesicles and factor
Va, by antithrombin or the antithrombin heparin complex was determined
by monitoring over time the hydrolysis of the small factor Xa substrate
S2222 or the activation of II(S525C)-fluorescein.
 |
EXPERIMENTAL PROCEDURES |
Materials
DNA restriction and modification enzymes were obtained
from New England BioLabs (Mississauga, Ontario) or Life Technologies, Inc. (Burlington, Ontario) and Pyrococcus furiosus
(Pfu) DNA polymerase was obtained from Stratagene (La Jolla,
CA). Baby hamster kidney cells and the mammalian expression vector pNUT
were graciously provided by Dr. Ross MacGillivray (University of
British Columbia). Newborn calf serum, Dulbecco's modified Eagle's
medium/F-12 nutrient mixture (1:1), Opti-MEM,
penicillin/streptomycin/Fungizone mixture, and reduced glutathione were
obtained from Life Technologies, Inc. Methotrexate (David Bull
Laboratories, Vaudreuil, Quebec), vitamin K1 (Sabex,
Boucherville, Quebec), and unfractionated heparin (Abbot Laboratories)
were purchased at the local hospital pharmacy. For enzyme-linked
immunosorbent assay, horseradish peroxidase-conjugated sheep anti-human
prothrombin was from Affinity Biologicals (Ancaster, Ontario). XAD-2
resin was from Sigma Chemical Co., and Q-Sepharose Fast Flow anion
exchange resin was from Amersham Pharmacia Biotech (Upsalla, Sweden).
S2222 and S2238 were from Helena Laboratories (Mississauga, Ontario).
5-Iodoacetamidofluorescein
(5-IAF)1 was purchased from
Molecular Probes (Eugene, OR). Phospholipid vesicles (75% PC, 25% PS)
were prepared as described by Bloom et al. (22). Human
prothrombin (23), thrombin (24), Factor Xa (23), Factor Va (25), and
antithrombin (10) were prepared as described previously.
Methods
DNA Construction and Mutagenesis--
pBluescript(SK+)
containing full-length human prothrombin cDNA (26) was digested
with PstI and XbaI restriction endonucleases. The
resulting 393-bp fragment, encompassing nucleotides 1580-1973, was
subcloned into pBluescript(SK+). Oligonucleotides 1 and 2 (Table
I) were constructed to facilitate
mutation of A1714
T resulting in a serine to
cysteine substitution at amino acid Ser525 in the expressed
protein product. PCR using Pfu DNA Polymerase with
oligonucleotide 1 and the T3 promoter primer (Stratagene) produced a
450-bp product (PCRI), whereas that with oligonucleotides 2 and the T7
promoter primer (Stratagene) produced a 217-bp product (PCRII). Each
PCR product was ligated into the EcoRV site of
pBluescript(SK+). PCRI and PCRII in pBluescript were digested with
BspHI and XbaI, and BspHI and
PstI, respectively. The resulting fragments were ligated at
their corresponding BspHI sites and subcloned into pBluescript(SK+) digested with PstI and XbaI. The
presence of mutations and correct PCR amplifications were verified by
DNA sequence analysis using the T3 and T7 promoter primers
(Stratagene). The mutated fragment was ligated back into full-length
prothrombin cDNA to make II(S525C)-cDNA. For expression in
mammalian cells, the II(S525C)-cDNA was excised from
pBluescript(SK+) with XbaI, followed by incubation with T4
DNA Polymerase, and ligated into the pNUT vector (27) at the
SmaI site. This site is downstream of the zinc-inducible
mouse metallothionein I promoter and upstream of the human growth
hormone polyadenylation signal. Proper orientation of the construct was
determined using restriction digest analysis and DNA sequence analysis
using oligonucleotides 3 and 4. The pNUT vector encodes a modified
dihydrofolate reductase gene, which allows for selection under high
methotrexate concentrations.
Cell Culture, Transfection, and Selection--
BHK cells were
cultured in Dulbecco's modified Eagle's medium/F-12 nutrient mixture
(1:1) supplemented with 5% newborn calf serum during transfection and
selection. Transfection of II(S525C)-cDNA was carried out using the
calcium phosphate co-precipitation technique (28). Sixteen hours after
transfection, the medium was supplemented with 0.44 mM
methotrexate to select for pNUT. Fifteen days after transfection,
individual colonies were screened for II(S525C) production by
enzyme-linked immunosorbent assay with horseradish peroxidase-conjugated sheep anti-human prothrombin antibody. High expressing clones were seeded into 500-cm2 triple flasks
for large scale production. At confluence, the growth medium was
replaced by serum-free Opti-MEM I, supplemented with 50 µM ZnCl2, 10 µg/ml vitamin K1,
and penicillin/streptomycin/Fungizone mixture. The medium was collected
at 24-h intervals, supplemented with glutathione (1 mM),
and stored at
20 °C.
Recombinant Protein Purification--
Stored medium was thawed
at 4 °C and loaded onto XAD-2 (2.5 × 15 cm) and Q-Sepharose
(1.4 × 8 cm) columns in tandem at either 4 °C or 21 °C. The
Q-Sepharose column was then washed with 5 column volumes of 0.02 M Tris-HCl, 0.15 M NaCl, pH 7.4 (TBS), followed by elution of II(S525C) with 0.02 M Tris-HCl, 0.5 M NaCl. Protein containing fractions were identified using
a Bio-Rad Assay and pooled. Fractions were collected until all protein
was eluted from the column. Fluorescent labeling was performed directly
on this pooled fraction by adding a 30 M excess of 5-IAF
from a 20 mM stock solution of 5-IAF in
N,N-dimethyl formamide and incubating the sample
for 2 h at room temperature in the dark. The resulting II(S525C)-fluorescein was separated from the excess label by addition of sodium citrate to a final concentration of 0.025 M,
followed by addition of a 1.0 M BaCl2 solution
to a final concentration of 0.08 M. The solution was
stirred at 4 °C for 1 h and centrifuged. The resulting pellet
was washed with the supernatant from a parallel precipitation carried
out in 0.02 M Tris-HCl, 0.5 M NaCl. The adsorbed protein was eluted by dissolving the barium citrate pellet in
0.2 M EDTA, pH 8.0 (1/6 of the original volume). The sample was then dialyzed against TBS, at 4 °C, and subjected to
anion-exchange chromatography on a Amersham Pharmacia Biotech
fast-protein liquid chromatography Mono-Q HR 5/5 column at 4 °C.
This step was carried out to resolve fully
-carboxylated species
from partially
-carboxylated species of II(S525C)-fluorescein (26,
29). The protein was eluted with a 0-30 mM
CaCl2 gradient in TBS (30-ml total volume, flow rate 0.5 ml/min). The first peak containing the fully
-carboxylated II(S525C)-fluorescein eluted at 15.0 mM CaCl2,
whereas the second eluted between 18 and 25 mM
CaCl2. Fractions from the first peak were pooled, and
protein concentrations and labeling efficiency values were determined
by absorbance readings at 280 and 495 nm, respectively.
SDS-PAGE Analysis of Cleavage of
II(S525C)-fluorescein--
II(S525C)-fluorescein (800 nM)
was activated with 50 µM PCPS, 5 nM FVa, 0.1 nM FXa, and 5 mM CaCl2 in TBS.
Fluorescence intensity was monitored at room temperature with an
excitation wavelength of 495 nm and an emission wavelength of 535 nm,
slit widths of 5 nm and 10 nm, respectively, and a 515-nm cut-off
emission filter in place. Aliquots were removed from the reaction and
added to an equal volume of 0.2 M acetic acid. Samples were
concentrated and reduced and subjected to SDS-PAGE on a 5-15%
minigel, which was subsequently photographed over UV light.
SDS-PAGE Analysis of Activation of II(S525C)-fluorescein
Versus II (Wild-type) in the Presence and Absence of
FVa--
II(S525C)-fluorescein or plasma prothrombin (800 nM) was activated in the presence 5 nM FVa with
50 µM PCPS, 0.1 nM FXa, and 5 mM
CaCl2 in TBS, 0.01% Tween 80. The same reactions were
carried out in the absence of FVa, with 10 nM FXa. Aliquots
were removed from the reactions and subjected to SDS-PAGE, under
reducing conditions, on 5-15% minigels, which were subsequently
stained with Coomassie Blue. Densitometry was carried out to quantify
the amount of prothrombin remaining at each time point.
S2222 Subsampling Assays to Determine the Second Order Rate
Constant for Inhibition of Factor Xa by Antithrombin
(k2)--
Reactions (200 µl) were set up containing 1.0 µM AT with 20 nM FXa alone, 20 nM
FXa + 5 mM CaCl2, 20 nM FXa + 5 mM CaCl2 + 50 µM PCPS, 10 nM FXa + 5 mM CaCl2 + 50 µM PCPS + 50 nM FVa, or 20 nM FXa + 5 mM CaCl2 + 50 µM PCPS + 1 µM II(S525C)-fluorescein in TBS, 0.01% Tween 80. Reactions were started by the addition of FXa, and 10-µl aliquots
were removed and diluted in a 96-well plate, which had been pretreated
with TBS, 1% Tween 80. The wells contained TBS, 0.01% Tween 80, with
S2222, and CaCl2 in sufficient amounts to reach final
diluted concentrations of 400 µM and 5 mM,
respectively. Absorbance at 405 nm was read over time in a Spectromax
Plus spectrophotometer (Molecular Devices, Sunnyvale, CA) at room
temperature. Initial rates were calculated. The natural logarithms of
these were then plotted against time and the corresponding slope was
calculated to determine the second order rate constants of inhibition.
Inhibition by S2222 Product--
Reactions containing S2222 at
various concentrations (0-1000 µM), 2 nM
FXa, and 5 mM CaCl2, in TBS, 0.01% Tween 80, were monitored at 405 nm to completion in a 96-well plate. Additional
S2222 (250 µM) was then added, and the resulting
reactions were monitored at 450 nm. Initial rates were determined and
plotted versus [S2222 product], and a
KP value for the inhibition of FXa by the
product of S2222 hydrolysis was determined according to the following equation, where [P] is the concentration of S2222
product:
|
(Eq. 1)
|
In Equation 1, r is the rate of hydrolysis of S2222,
k is kcat,
[E]T is the FXa concentration,
[S] is the concentration of S2222 (250 µM),
Km is the Michaelis-Menten constant for hydrolysis
of S2222, KP is the Michaelis-Menten constant
for the S2222 product, and [P] is the concentration of the
S2222 product. The Km value for hydrolysis of S2222
was determined by measurements of initial reaction rates in the absence
of product. Reactions containing S2222 at various concentrations, 0.1 nM FXa and 5 mM CaCl2 in TBS,
0.01% Tween-80, were monitored at 405 nm at room temperature. Initial
rates were determined and plotted versus [S2222] to
determine Km. Similar analyses were carried out for
0.1 nM FXa alone, 0.1 nM FXa + 5 mM
CaCl2 + 50 µM PCPS, and 0.1 nM
FXa + 5 mM CaCl2 + 50 µM PCPS + 20 nM FVa. The Km values were very
similar for all conditions. In addition they were very similar to the
Kp value determined for one condition. Thus for
all conditions the KP value was assumed to be
identical to the Km value.
End Point Assays to Determine k2 Using
S2222--
Reactions containing 400 µM S2222 with 2 nM FXa alone, 2 nM FXa + 5 mM
CaCl2, 2 nM FXa + 5 mM
CaCl2 + 50 µM PCPS, 2 nM FXa + 5 mM CaCl2 + 50 µM PCPS + 25 nM FVa, or 2 nM FXa + 5 mM
CaCl2 + 50 µM PCPS + 1 µM
II(S525C)-fluorescein, in the presence and absence of 1.0 µM AT, were set up in a 96-well plate, which had been
pretreated with TBS, 1% Tween 80. Reactions were started by addition
of FXa and monitored at 405 nm to completion. End point data were
analyzed to determine the pseudo first order rate constant. The
following rationale was used, taking into account that the product of
S2222 hydrolysis as well as AT inhibit the enzyme. In this analysis,
E is the enzyme, S is the substrate, P
is the product, I is the inhibitor (AT), and
is the
pseudo first order rate constant for inhibition of FXa by AT.
is
equal to the second order rate constant (k2)
multiplied by the inhibitor concentration.
For inhibition,
|
(Eq. 2)
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|
(Eq. 3)
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|
(Eq. 4)
|
For product formation,
|
(Eq. 5)
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|
(Eq. 6)
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|
(Eq. 7)
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Equating Equations 4 and 7 yields Equation 8,
|
(Eq. 8)
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Integration of Equation 8 yields Equation 9,
|
(Eq. 9)
|
When the reaction with AT stops, [E · I] = [E]T and [S] = [S]f. Therefore,
|
(Eq. 10)
|
Consequently,
can be calculated from
k[E]T/Km and
ln([S]o/[S]f), and
k[E]T/Km can be determined from control
experiments with no AT. With the assumption that Km = KP,
|
(Eq. 11)
|
Integration of Equation 11 over time gives Equation 12,
|
(Eq. 12)
|
Equation 12 is rewritten as Equation 13,
|
(Eq. 13)
|
where
= k[E]T/(Km + [S]o).
Raw data were subjected to nonlinear regression to Eq. 13 to find
.
Elimination of k[E]T between
Equations 10 and 13, yields Equation 14, from which the value of
was calculated.
|
(Eq. 14)
|
The second order rate constant (k2) was
then calculated using
|
(Eq. 15)
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End Point Assays to Determine k2 Using
II(S525C)-fluorescein--
End point reactions containing
II(S525C)-fluorescein, and FVa at various concentrations (0-2
nM), were carried out in a 96-well plate, which had been
pretreated with TBS 1%, Tween 80 and were monitored in a Spectra Max
Gemini fluorescence plate reader (Molecular Devices, Sunnyvale, CA) at
excitation and emission wavelengths of 490 nm and 538 nm, respectively,
with a 515-nm emission cut-off filter in place. Reactions were started
by the addition of FXa. Raw data for control experiments, containing no
AT, were fit to the integrated Michaelis-Menten equation:
(Km/vmax)·(ln([S]o/[S]))/[P] + 1/vmax = t/[P], to
determine Km and vmax values
for each condition. Pseudo first order rate constants were then
calculated using,
= k[E]T/Km/ln([S]o/[S]f),
and the second order rate constants using
k2=
/[I].
 |
RESULTS |
Production and Characterization of II(S525C)-fluorescein--
To
obtain II(S525C), stable BHK cell lines expressing II(S525C) were grown
in serum-free medium (Opti-MEM) in triple flasks. Typically 4 liters of
pooled conditioned media were used as the starting material for
purification as outlined under "Methods." II(S525C) behaved
identically to wild-type recombinant prothrombin in all of the
purification steps. II(S525C) was found to be present in conditioned
media at a concentration of 5-10 mg/liter. Only 10% of it, however,
was fully
-carboxylated. II(S525C)-fluorescein was produced with a
typical labeling efficiency of ~50%. Purified II(S525C)-fluorescein
comigrated with plasma prothrombin when subjected to SDS-PAGE (data not
shown). Upon activation of II(S525C)-fluorescein, an increase in
fluorescence intensity of ~50% was observed, which was found to
correlate well with cleavage (Fig. 1).
The fluorescence intensity profile exhibited a transient maximum
in intensity, which is indicative of the expected meizothrombin
intermediate (26, 30). Additionally, upon cleavage, the band that
retains the fluorescent properties corresponds to the B-chain of
thrombin, which contains the active site serine. This confirms that the fluorescein moiety attaches specifically to Cys525 (Fig.
1). When compared with plasma prothrombin, II(S525C)-fluorescein activated at similar rates in both the presence and absence of FVa
(Fig. 2). The thrombin generated from
activation of II(S525C)-fluorescein was catalytically inactive toward
small substrates, as determined by S2238 hydrolysis, in which
II(S525C)-fluorescein was observed to have at least 130,000-fold less
activity (data not shown). II(S525C)-fluorescein was also catalytically
inactive toward macromolecular substrates such as Factor V, fibrinogen,
and itself (data not shown).

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Fig. 1.
Increase in fluorescence intensity of
II(S525C)-fluorescein upon activation correlates well with
cleavage. 800 nM II(S525C)-fluorescein was activated
with 50 µM PCPS, 5 nM FVa, 0.1 nM
FXa, and 5 mM CaCl2 in TBS, 0.01% Tween 80. Fluorescence intensity was monitored as outlined under "Experimental
Procedures." In this figure the blank fluorescence has not been
subtracted. Inset, aliquots were removed at the times
indicated above, and concentrated. Reduced samples were run on a
5-15% gradient gel as outlined under "Experimental Procedures."
Bands were visualized by fluorescence.
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Fig. 2.
Activation of II(S525C)-fluorescein compared
with plasma prothrombin in the presence and absence of FVa.
A, 800 nM II(S525C)-fluorescein (open
circles) or plasma prothrombin (closed circles) were
activated with 50 µM PCPS, 5 nM FVa, 0.1 nM FXa, and 5 mM CaCl2 in TBS,
0.01% Tween 80. Aliquots were removed from the reactions and
concentrated. Reduced samples were subjected to SDS-PAGE on 5-15%
polyacrylamide gels. Densitometry was carried out to determine the
amount of prothrombin remaining at each time point. B,
II(S525C)-fluorescein (open circles) or plasma prothrombin
(closed circles) were activated with 50 µM
PCPS, 10 nM FXa, and 5 mM CaCl2 in
TBS, 0.01% Tween 80. Aliquots were removed and treated as in
A.
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Determination of the Second Order Rate Constant Values
(k2) by Both Subsampling and End Point Analysis Using S2222
Hydrolysis--
The second order rate constants for inhibition of FXa
by antithrombin in the presence and absence of Ca2+, PCPS
vesicles, and FVa were determined from the time courses of hydrolysis
of the FXa substrate S2222. Two methods were employed. One involved
subsampling at various intervals and measuring residual FXa activity
with the substrate. The other involved monitoring the reactions
containing the substrate to completion and calculating the value of the
inhibition constants from the level of unconsumed substrate at the end
of the reaction carried out in the presence of inhibitor. In the first
method the time course of decay of enzymatic activity is directly
measured; thus the results are unambiguous. In the second, the validity
of the inferred value depends on the validity of the model used to
interpret the data. The second method was used with this substrate in
anticipation of the experiments with II(S525C)-fluorescein, in which
the direct method would not be feasible because of the low levels of
FXa used. Thus, the two types of measurements with S2222 were done to
test the validity of the end point method. The analyses involving subsampling are shown in Fig. 3. Two time
courses of the reactions, along with their controls without AT, carried
out with the substrate included (end point analysis) are shown in Fig.
4. In the subsampling experiments, plots
of the natural logarithm of the residual activity versus
time were linear and pseudo first order rate constants were measured
from the slopes. In the end point analyses, reactions containing AT did
not go to completion because the enzyme was consumed, which allowed
determination of the pseudo first order rate constant for inhibition
from the residual substrate concentration and the
vmax/Km ratio for the
control. Initial efforts to find the
vmax/Km ratio by application of
the integrated Michaelis-Menten equation were unsuccessful, because the
time courses of product formation were first order, regardless of the initial substrate concentration, which is indicative of equal affinity
binding of both the substrate and product to the enzyme. Thus, the
Km was found by standard initial rate measurements and the KP for inhibition by product was found
by measuring initial rates of substrate hydrolysis in the presence of
the product at various concentrations (Fig.
5). Km was determined
to be the same regardless of the presence or absence of
Ca2+, PCPS, and FVa. The value inferred by non-linear
regression of the data from Fig. 5 to the Michaelis-Menten equation was
234 ± 40 µM. The KP value
for product inhibition obtained for the data inset in Fig. 5 was
338 ± 14 µM.

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Fig. 3.
S2222 subsampling assays to determine
k2. 200-µl reactions were set up
containing 1.0 µM AT with 20 nM FXa alone
(closed circle), 20 nM FXa + 5 mM
CaCl2 (open circle), 20 nM FXa + 5 mM CaCl2 + 50 µM PCPS
(closed inverted triangle), 10 nM FXa + 5 mM CaCl2 + 50 µM PCPS + 50 nM FVa (open inverted triangle), or 20 nM FXa + 5 mM CaCl2 + 50 µM PCPS + 1 µM II(S525C)-fluorescein
(closed square), in TBS, 0.01% Tween 80. Aliquots were
removed and diluted in a 96-well plate. Absorbance at 405 nm was
monitored, and initial rates were determined and plotted
versus time. Corresponding slopes were used to calculate
k2 values.
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Fig. 4.
S2222 hydrolysis over time. Reactions
containing 400 µM S2222 and 2 nM FXa + 5 mM CaCl2, or 2 nM FXa + 5 mM CaCl2 + 50 µM PCPS + 25 nM FVa, were set up in a 96-well plate in both the absence
(solid line) and presence (dashed line) of 1.0 µM AT. Reactions were started by the addition of FXa and
monitored at 405 nm until completion. End point data were analyzed to
determine the second order rate constants.
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Fig. 5.
Determination of Km
and KP values associated with S2222
hydrolysis. Reactions containing various amounts of S2222, 0.1 nM FXa, and 5 mM CaCl2, in TBS,
0.01% Tween 80 were monitored at 405 nm. Initial rates were determined
and plotted versus [S2222] to determine a
Km value. Inset, reactions containing
various amounts of S2222, 2 nM FXa, and 5 mM
CaCl2 in TBS, 0.01% Tween 80 were monitored at 405 nm in a
96-well plate, until completion. Additional S2222 (250 µM) was added, and the resulting reaction was monitored
at 450 nm. Initial rates were determined and plotted versus
[S2222 product] and a KP value was determined
as outlined under "Experimental Procedures."
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The k2 values for the inhibition of FXa by
antithrombin were determined for FXa alone, FXa + Ca2+, FXa + Ca2+ + PCPS, FXa + Ca2+ + PCPS + FVa, and FXa + Ca2+ + PCPS + II(S525C)-fluorescein. Table
II summarizes the
k2 values determined using both methods. The
values determined were in good agreement with each other. Thus, end
point analysis was determined to be a valid method for measuring
k2 and was used in subsequent experiments with
II(S525C)-fluorescein. Upon addition of Ca2+, FXa was more
susceptible to inhibition by AT, which was expected (13). Addition of
PCPS or PCPS + II(S525C)-fluorescein to the system had little to no
effect when compared with FXa + Ca2+. Finally, addition of
FVa decreased the second order rate constant by ~50% when compared
with that for FXa + Ca2+ + PCPS. In the absence of FVa,
II(S525C)-fluorescein had no effect on inhibition of FXa by AT.
Effects of FVa on AT Inhibition of Prothrombinase in the Absence
and Presence of Prothrombin--
II(S525C)-fluorescein is a useful
substrate for monitoring AT-dependent inhibition of
prothrombinase, because activation of the mutant can be easily
monitored without the generation of active thrombin. For each
experiment, a control experiment, containing no AT, was carried out,
and the raw data were fit to the integrated Michaelis-Menten equation
to determine Km and vmax
values. Unlike S2222 cleavage, prothrombin activation did not appear to exhibit strong product inhibition. Fig. 6
illustrates control and AT-containing experiments for reactions
containing FXa, Ca2+, PCPS, FVa at various concentrations,
and II(S525C)-fluorescein. The end points for the experiments
containing AT increase with increasing FVa, suggesting increasing
protection. Fig. 7A
illustrates that, at saturating levels of FVa, prothrombinase becomes
completely protected and the k2 values for
inhibition by AT fall to undetectable levels. The half-life at a FVa
concentration of 0.75 nM is 69 min, which can be compared
with the half-life in the absence of FVa of 2.9 min. Because this
latter value is very similar to the half-life predicted from the FXa + Ca2+ + PCPS + II(S525C)-fluorescein subsampling
experiments, prothrombin alone does not mediate protection of FXa from
inhibition by AT. When a similar titration of FVa was carried out using
S2222 as the substrate, the k2 values were
observed to fall by only ~50% at saturating levels of FVa (Fig.
7B). Therefore, in the presence of Ca2+ and
PCPS, FXa was observed to have a half-life of 2.0 min, which then
increased to 3.7 min at a FVa concentration of 50 nM,
suggesting that FVa alone does not mediate complete protection of FXa
from inhibition by antithrombin. Thus, FVa and prothrombin must both be
present to observe complete protection.

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Fig. 6.
End point analysis of the effects of FVa on
AT inhibition of prothrombinase. Reactions containing 0.025 nM FVa (A), 0.1 nM FVa
(B), 1 nM FVa (C), and 1 µM II(S525C)-fluorescein, 5 mM
CaCl2, 50 µM PCPS, 10 pM FXa, in
the presence and absence of 2.0 µM AT, were monitored at
excitation and emission wavelengths of 490 and 538 nm, respectively,
with a 515-nm emission filter in place. Reactions were started by
addition of FXa.
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Fig. 7.
Effects of FVa on
k2 values. A, reactions
containing FVa at various concentrations, 20 pM FXa, 50 µM PCPS, 5 mM CaCl2, 1 µM II(S525C)-fluorescein, in the absence and presence of
2.0 µM AT were monitored until completion at excitation
and emission wavelengths of 490 nm and 538 nm, respectively, with a
515-nm emission filter in place. End point analysis was carried out as
outlined under "Experimental Procedures," and
k2 values were determined. B,
reactions containing various amounts of FVa, 2 nM FXa, 50 µM PCPS, 5 mM CaCl2, and 400 µM S2222 in the absence and presence of 1.0 µM AT were monitored until completion. End point analysis
was carried out as outlined under "Experimental Procedures," and
k2 values were determined.
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|
In the FVa titration in which II(S525C)-fluorescein was the substrate,
a half-maximal effect was obtained at a FVa concentration of ~20
pM. Saturation of the effect was also observed when S2222 was the substrate, but the concentration of FVa at half-maximal effect
was 3.0 nM.
Effects of Heparin on AT Inhibition of Prothrombinase--
Heparin
accelerates inhibition of FXa by AT by several orders of magnitude.
Fig. 8 shows the calculated pseudo first
order rate constants for a variety of heparin concentrations determined by monitoring II(S525C)-fluorescein activation by prothrombinase. The
corresponding k2 value was determined from the
slope of the line to be 1.27 × 105
M
1 s
1. The inset in
Fig. 8 illustrates the pseudo first order rate constants obtained
for analysis of the heparin-dependent AT inhibition of FXa + FVa + PCPS + Ca2+ determined by S2222 hydrolysis. The
corresponding k2 value was determined to be
7.72 × 106 M
1
s
1. The second order rate constant for FXa + Ca2+ + PCPS was also determined in a similar manner and
found to be 1.58 × 107 M
1
s
1. Table III summarizes
the k2 values determined in both the absence and
presence of heparin. Heparin increased the k2
for inhibition of FXa in the presence of Ca2+ and PCPS by
5300-fold. This increase was also observed for FXa + Ca2+ + PCPS + FVa as determined by S2222 hydrolysis. It was impossible to
calculate a -fold difference for prothrombinase inhibition in the
presence of II(S525C)-fluorescein, because prothrombinase appears to be
completely protected at saturating levels of FVa in the absence of
heparin under these circumstances but is inhibited with a second order
rate constant of 1.27 × 105
M
1 s
1 upon heparin addition.
Table III also summarizes the k2 values in the
presence of heparin relative to that for FXa + Ca2+ + PCPS.
The k2 for prothrombinase in the presence of
II(S525C)-fluorescein was found to be 0.8% of that for FXa + Ca2+ + PCPS, implying profound protection of FXa, when it
is incorporated into the prothrombinase complex and in the presence of
prothrombin, even in the presence of heparin. The
k2 for prothrombinase as determined by S2222
hydrolysis, was one-half that obtained with FXa + Ca2+ + PCPS. This value was 60-fold greater than that obtained with prothrombinase plus II(S525C)-fluorescein. The profoundly smaller value
observed when II(S525C)-fluorescein is the substrate again suggests
that the presence of both FVa and prothrombin causes increased
protection of FXa from AT, even in the presence of heparin.

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Fig. 8.
Effects of heparin on the pseudo first order
rate constant. Reactions containing heparin at various
concentrations, 2 µM AT, 60 pM FXa, 50 µM PCPS, 5 nM FVa, 1 µM
II(S525C)-fluorescein, and 5 mM CaCl2 in TBS,
0.01% Tween 80, were monitored until completion at excitation and
emission wavelengths of 490 nm and 538 nm, respectively, with a 515-nm
emission filter in place. Reactions were started by addition of
prothrombinase. End point analysis was carried out as outlined under
"Experimental Procedures" to determine the pseudo first order rate
constants, which were plotted versus [heparin]. The
corresponding slope was used to calculate k2.
Inset, reactions containing heparin at various
concentrations, 2 µM AT, 15 nM FXa, 50 µM PCPS, 50 nM FVa, 400 µM
S2222, and 5 mM CaCl2 in TBS, 0.01% Tween 80, were monitored until completion and treated as above.
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|
Effects of the Concentration of Prothrombin on AT Inhibition
of Prothrombinase--
Reactions containing FXa + FVa + PCPS + Ca2+ and II(S525C)-fluorescein at various concentrations,
in the presence and absence of 2 µM AT and 60 pM heparin, were monitored until completion. End point
analysis was carried out and the k2 values were
determined. Fig. 9 illustrates that
reactions containing II(S525C)-fluorescein at concentrations from 50 to
500 nM exhibited similar k2 values. Results in Table III indicate that the k2 value
in the absence of prothrombin should be 7.72 × 106
M
1 s
1, which is 50-fold higher
than the average k2 value in Fig. 9. Thus,
prothrombin does appear to play a protective role, but, perhaps similar
to FVa, the protective effects of prothrombin saturate at very low
concentrations. Unfortunately, it is impossible to accurately monitor
activation of II(S525C)-fluorescein at concentrations below 50 nM, because the change in fluorescence is too small.

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Fig. 9.
Effects of prothrombin concentration on
k2. Reactions containing 60 pM FXa, 5 nM FVa, 50 µM PCPS, and
II(S525C)-fluorescein at various concentrations, in the presence and
absence of 2 µM AT and 60 nM heparin were
monitored until completion at excitation and emission wavelengths of
490 nm and 538 nm, respectively, with a 515-nm emission filter in
place. End point analysis was carried out as outlined under
"Experimental Procedures," and k2
values were determined.
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|
 |
DISCUSSION |
We have expressed a variant of prothrombin in which the serine of
the thrombin catalytic triad has been replaced with a cysteine that was
subsequently labeled with fluorescein. Characterization of the change
in fluorescence upon cleavage, the rate of activation in the presence
and absence of FVa and the absence of activity upon activation of this
variant showed it to be a good model substrate to investigate its
activation by the enzyme FXa in the presence of the physiological
inhibitor antithrombin. This variant allows easy monitoring of the
conversion of prothrombin to an inactive thrombin and would be a useful
tool for monitoring prothrombin activation in the absence of all
thrombin feedback reactions, such as the activation of FV, and cleavage
of itself.
It has been well established that FXa is less susceptible to inhibition
by antithrombin and the antithrombin-heparin complex when it is
incorporated into the prothrombinase complex (14-20). The extent of
this protection and the mechanism behind it, however, have not been
clearly established. Inhibition of FXa by antithrombin and the
antithrombin-heparin complex under various conditions was
quantitatively determined in this study. In the absence of FVa, the
combination of phospholipid vesicles and prothrombin had no effect on
the second order rate constant of inhibition of FXa + Ca2+
by antithrombin; FVa in the absence of prothrombin showed only a modest
effect, decreasing the rate constant approximately 2-fold. Upon
addition of both FVa and prothrombin, however, the second order rate
constant decreased to an immeasurable level, suggesting that, when both
FVa and prothrombin are present, prothrombinase is extremely protected
from antithrombin, with its half-life increasing from 3 min in the
absence of FVa to greater than 69 min with prothrombin and FVa. Upon
addition of heparin, FVa in the absence of prothrombin was again
observed to decrease the second order rate constant of inhibition by
~50%. In the presence of both FVa and prothrombin, the second order
rate constant decreased to 0.8% of that for FXa + Ca2+ + PCPS, implying profound protection of prothrombinase even in the
presence of heparin. In the presence of heparin at 0.2 unit/ml (40 nM), the half-life increased from 1 s without
prothrombin to 136 s with it.
Prothrombin clearly effects the protection of prothrombinase. Previous
studies have suggested that the thrombin produced at the catalytic
surface consumes antithrombin and therefore lowers its concentration in
the vicinity of the prothrombinase complex (15). In the present study
however, active thrombin was not produced and no change in the
fluorescence of IIa(S525C)-fluorescein was observed upon antithrombin
addition, implying that the variant used in this study does not
associate with and therefore does not consume antithrombin. However,
the remarkable extent to which FXa is protected from inhibition by both
antithrombin and the antithrombin-heparin complex also cannot be
rationalized based on straight forward competition between prothrombin
and antithrombin according to a simple Michaelis-Menten model. Thus,
some other aspect of the conversion of prothrombin to thrombin by
prothrombinase is responsible for the protection. One possibility is
that the interactions of FXa with both prothrombin and antithrombin
require an exosite on FXa distinct from the S1 subsite of the enzyme. That such an exosite exists on FXa is supported by the data of Krishnaswamy and Betz (31). If this rationalizes the protection, the
exosite is not recognized by prothrombin in the absence of FVa, because
prothrombin alone did not protect FXa. Alternatively, the protection is
inherent in the dynamics of the catalytic cycle. Conceivably, for most
of the cycle, FXa is in a conformation or state in which it does not
recognize antithrombin. If this is so, then FXa would be accessible for
only about 1% of the cycle, because the rate constant for inhibition
decreases by a factor of ~100 for inhibition of FXa by the
antithrombin-heparin complex. Regardless of the mechanistic reason for
the protection, the fact that it exists suggests that prothrombin
activation cannot be accounted for by simple substrate competition and
the Michaelis-Menten model.
The serine proteases of other multicomponent enzymatic complexes of the
coagulation cascade (factor VIIa and factor IXa) are also inhibited by
antithrombin. Perhaps they are similarly protected from inhibition in
the presence of the substrate (factor X), and thus the phenomenon
reported here for prothrombinase may apply generally to all of the
reactions of the cascade. If this is the case, the reactions of the
cascade localized at the site of injury would be relatively protected
from antithrombin, whereas enzymes that might escape would be
susceptible to inhibition, thereby both allowing efficient thrombin
formation locally, while preventing or attenuating it systemically.
 |
FOOTNOTES |
*
This work was supported by the Canadian Institute of Health
Research Grant MA-9781 and by National Institutes of Health Grant PHSHL
46703.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.
To whom correspondence should be addressed: Dept. of Biochemistry,
Queen's University, Kingston, Ontario K7L 3N6, Canada. Tel.:
613-533-2957; Fax: 613-533-2987; E-mail:
nesheimm@post.queensu.ca.
Published, JBC Papers in Press, February 27, 2001, DOI 10.1074/jbc.M011586200
 |
ABBREVIATIONS |
The abbreviations used are:
5-IAF, 5-iodoacetamidofluorescein;
bp, base pair(s);
PCR, polymerase chain
reaction;
FVa, Factor Va;
FXa, Factor Xa;
PAGE, polyacrylamide
gel electrophoresis;
AT, antithrombin;
PCPS, unilamellar vesicles of
phosphatidylcholine and phosphatidylserine (75:25);
S2222, N-benzoyl-L-isoleucyl-L-glutamyl-glycyl-L-arginine-p-nitroaniline
hydrochloride;
S2238, H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline
dihydrochloride.
 |
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.