From the Departments of Biochemistry and Medicine, Queen's University, Kingston, Ontario, Canada K7L 3N6
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ABSTRACT |
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Thrombin-activable fibrinolysis
inhibitor (TAFI) is a human plasma zymogen similar to pancreatic
pro-carboxypeptidase B. Cleavage of the zymogen by
thrombin/thrombomodulin generates the enzyme, activated TAFI (TAFIa),
which retards fibrin clot lysis in vitro and likely
modulates fibrinolysis in vivo. In the present work we
stably expressed recombinant TAFI in baby hamster kidney cells, purified it to homogeneity from conditioned serum-free medium, and
compared it to plasma TAFI (pTAFI) with respect to glycosylation and
kinetics of activation by thrombin/thrombomodulin. Although rTAFI is
glycosylated somewhat differently than pTAFI, cleavage products with
thrombin/thrombomodulin are indistinguishable, and parameters of
activation kinetics are very similar with kcat = 0.55 s1, Km = 0.54 µM, and Kd = 6.0 nM for
rTAFI and kcat = 0.61 s
1,
Km = 0.55 µM, and
Kd = 6.6 nM for pTAFI. The respective
TAFIa species also were prepared and compared with respect to thermal
stability and enzymatic properties, including inhibition of
fibrinolysis. The half-life of both enzymes at 37 °C is about 10 min, and the decay of enzymatic activity is associated with a quenching
(to ~62% of the initial value at 60 min) of the intrinsic
fluorescence of the enzyme. Stability was highly
temperature-dependent, which, according to transition state
theory, indicates both high enthalpy and entropy changes associated
with inactivation
(
Ho
45 kcal/mol and
So
80 cal/mol/K). Both species of TAFIa are stabilized by the competitive inhibitors 2-guanidinoethylmercaptosuccinic acid and
-aminocaproic acid. rTAFIa and pTAFIa are very similar with respect to kinetics of cleavage of small substrates, susceptibility to inhibitors, and ability to retard both tPA-induced and plasmin-mediated fibrinolysis. These studies provide new insights into the thermal instability of TAFIa, a property which could be a significant regulator
of its activity in vivo; in addition, they show that rTAFI
and rTAFIa are excellent surrogates for the natural plasma-derived species, a necessary prerequisite for future studies of structure and
function by site-specific mutagenesis.
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INTRODUCTION |
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The balance between the activities of the coagulation and fibrinolytic cascades is essential to protect the organism from excessive blood loss upon injury as well as to maintain the fluidity of blood within the vasculature. Imbalances lead to a tendency toward either bleeding or thrombosis, the latter of which is manifested as heart attacks and strokes.
The coagulation and fibrinolytic cascades consist of a series of zymogen to enzyme conversions, terminating in the proteolytic enzymes thrombin and plasmin, which, respectively, catalyze the deposition and removal of fibrin. However, when in a complex with the endothelial cell-surface cofactor thrombomodulin, the specificity of thrombin is changed from fibrinogen to protein C (1), thus changing thrombin to an anticoagulant rather than a procoagulant enzyme.
When formed in the context of a fibrin clot, activated protein C was found to be profibrinolytic as well as anticoagulant (2-5), an observation that results from the ability of activated protein C to prevent the formation of a previously uncharacterized fibrinolysis inhibitor (6). Further investigations in our laboratory led to the isolation of this factor, a 60-kDa glycoprotein present in human plasma at a concentration of approximately 50 nM, which we termed TAFI1 (thrombin-activable fibrinolysis inhibitor (7)). Upon activation of TAFI by cleavage with thrombin, an active enzyme is formed (TAFIa) that possesses carboxypeptidase B-like activity and inhibits fibrinolysis, probably by removal from partially degraded fibrin the C-terminal lysines that contribute to the development of positive feedback in the fibrinolytic cascade (8). Although thrombin itself is a weak activator of TAFI (8), the thrombin-thrombomodulin complex activates TAFI with a 1250-fold higher catalytic efficiency than thrombin alone, suggesting that thrombin/thrombomodulin is the physiological activator of TAFI (8).
We have shown that TAFIa inhibits tPA-mediated fibrinolysis in vitro half-maximally at a concentration of approximately 1 nM (8). This concentration is about 2% the level of the zymogen in plasma, indicating that enough TAFIa could be produced in plasma to have a significant effect on fibrin clot lysis in vivo. In support of this scenario, recent studies utilizing a canine model of thrombolysis indicated that inducible carboxypeptidase activity (probably TAFIa) is increased during thrombosis and thrombolytic therapy (9); in addition, a significant positive correlation is observed between inducible carboxypeptidase activity and the time required for restoration of blood flow (9). These studies strongly imply that regulation of TAFIa activity may play a significant role in modulating hemostasis and thrombolysis in vivo.
The protein that we have termed TAFI has been described previously by
other groups. Hendriks et al. (10) detected an unstable carboxypeptidase B-like activity in human serum; the enzyme
corresponding to this activity was subsequently isolated and named CPU
("unstable" carboxypeptidase (11)). In addition, Eaton et
al. (12) discovered the zymogen as a contaminant in preparations
of 2-antiplasmin and termed it plasma
procarboxypeptidase B (pro-pCPB). These investigators also cloned a
cDNA corresponding to pro-pCPB, the deduced amino acid sequence of
which showed significant homology with pancreatic procarboxypeptidase B
(12). Amino acid sequence analysis of TAFI and fragments derived from
TAFI showed that TAFI and pro-pCPB are the same protein (7).
One of the striking features of the enzyme TAFIa is its inherent instability, a property for which it was given the name carboxypeptidase U (11). The relatively short half-life of the enzyme at body temperature suggests that this inherent instability might be relevant in the down-regulation of the enzyme in vivo. The following studies were undertaken to both investigate the instability of TAFIa and to compare thoroughly the properties of recombinant and plasma-derived TAFI and TAFIa.
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EXPERIMENTAL PROCEDURES |
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Materials--
The synthetic carboxypeptidase substrates
hippuryl-L-arginine (Hip-Arg),
hippuryl-L-lysine (Hip-Lys), and
N-[3-(2-furylacryloyl)]-L-alanyl-L-lysine (FA-Ala-Lys), as well as hippuric acid (HA), -aminocaproic acid (
-ACA), potato carboxypeptidase inhibitor (PCI), and Sepharose CL-4B
were purchased from Sigma. Q-Sepharose Fast Flow anion exchange resin
was from Pharmacia (Upsalla, Sweden). The carboxypeptidase B inhibitor
2-guanidinoethylmercaptosuccinic acid (GEMSA), 1,5-dansyl-Glu-Gly-Arg chloromethyl ketone (dEGRck), and D-Phe-Pro-Arg
chloromethyl ketone (PPAck) were purchased from Calbiochem.
N-Glycosidase F was purchased from Boehringer Mannheim
(Laval, Quebec, Canada). 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). First-strand human liver cDNA preparations were the generous gift of Mona Rahman and Dr. Marlys Koschinsky (Queen's University, Kingston, Ontario). Baby hamster kidney cells and the mammalian expression vector pNUT were provided by
Dr. Ross MacGillivray (University of British Columbia). Newborn calf
serum, Dulbecco's modified Eagle's medium, nutrient mixture F-12
(1:1) (DMEM/F-12), Opti-MEM, UltroSer G,
penicillin/streptomycin/Fungizone mixture (PSF), and reduced
glutathione were obtained from Life Technologies, Inc., and
methotrexate was purchased from David Bull Laboratories (Vaudreuil,
Quebec). For Western blot analysis, a monoclonal antibody (mAB 13)
raised against purified TAFI (13) was employed; a secondary antibody
(goat anti-mouse IgG conjugated to horseradish peroxidase) was obtained
from Sigma. Fibrinogen, Glu-plasminogen, prothrombin, and antithrombin
III were isolated from human plasma, and plasmin and thrombin were
prepared from Glu-plasminogen and prothrombin, respectively, as
described previously (7). TAFI was isolated from human plasma as
described by Bajzar et al. (7), with the exception that the
gel filtration step was omitted. Recombinant human
2-antiplasmin was purified from conditioned medium
harvested from a stably expressing BHK cell line as described
previously (7). Recombinant tPA (Activase) was generously provided by
Dr. Gordon Vehar of Genentech, Inc. (South San Francisco, CA).
Recombinant soluble thrombomodulin (Solulin) was the generous gift of
Dr. John Morser (Berlex Biosciences Inc., Richmond, CA). A
plasminogen-Sepharose column was prepared using purified human
Glu-plasminogen and Sepharose CL-4B according to Bajzar et
al. (7).
Cloning of a TAFI cDNA and Construction of a TAFI Expression
Vector--
Based on the cDNA sequence for plasma
procarboxypeptidase B (pro-pCPB) published by Eaton et al.
(12), three pairs of oligonucleotide primers were designed (see Fig.
1). The sequences of the primers pairs were as follows: primer 1, 5-CTGTTGGGATGAAGCTTTGC-3
and primer
2, 5
-TCGTTGGAAATCTGCTGTTG-3
; primer 3, 5
-CTTGCTGGCAGACGTGGAAG-3
and
primer 4, 5
-GCTGGGAGTATGAATGCATG-3
; primer 5, 5
-GCATACATCAGCATGCATTC-3
and primer 6, 5
-CAATGATTTGGTCTTGCTGG-3
.
By using these primers, three overlapping PCR products were
obtained by PCR amplification using a first-strand human liver cDNA
library (1 µg) as the template. The PCR cycling conditions were as
follows: 5 min at 95 °C followed by 35 cycles of 30 s at
95 °C, 30 s at 52 °C, and 30 s at 75 °C, and a final
15 min at 75 °C. The three primer pairs gave rise to PCR products of
356 base pairs (nucleotides 10-366 of pro-pCPB sequence; Eaton
et al. (12)), 665 base pairs (nucleotides 318-983), and 425 base pairs (nucleotides 953-1378), respectively. The three PCR
products were individually inserted into the EcoRV site of pBluescript SK+ (Stratagene) and analyzed by DNA sequence analysis; the
sequence of the cloned PCR fragments was identical to that reported by
Eaton et al. (12).
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Culture and Transfection of Mammalian Cells-- BHK cells were cultured in 100-mm dishes in DMEM/F-12 containing 5% newborn calf serum in a 37 °C humidified incubator (95% air/5% CO2 atmosphere). Cells were transfected by the method of calcium phosphate co-precipitation (14) using 10 µg of TAFI-pNUT plasmid per 100-mm plate. Six hours following transfection, the cells were washed and fed fresh DMEM/F-12 containing 5% newborn calf serum. The cells were allowed to recover overnight, after which the medium was replaced with DMEM/F-12 containing 5% newborn calf serum and 400 µM methotrexate. After a 2-week selection period, surviving foci were picked and analyzed for expression of TAFI by Western blot analysis.
For Western blot analysis of TAFI expressed from transiently transfected cells, BHK cells were transfected, as described above, with 10 µg/100-mm plate of TAFI-pNUT or revTAFI-pNUT. Following transfection and an overnight recovery period, the medium was replaced with serum-free medium (Opti-MEM; 5 ml/100-mm plate) and culture continued for a further 48 h. At this time, conditioned medium was harvested from the cells and subjected to SDS-PAGE followed by Western blot analysis using a TAFI-specific monoclonal antibody (13).Recombinant Protein Expression--
BHK cell lines stably
expressing TAFI were seeded in triple flasks (500 cm2;
Nunc, Roskilde, Denmark) in DMEM/F-12 containing 1% (v/v) UltroSer G
and 200 µM methotrexate. Once the cells had become
confluent, the medium was changed to Opti-MEM (100 ml/flask) containing
1% (v/v) PSF and 50 µM ZnCl2. Conditioned
medium was harvested at 24-48-h intervals and replaced with fresh
Opti-MEM; the harvested medium was supplemented with Tris, pH 8 (to 5 mM), reduced glutathione (to 0.5 mM), and
dEGRck (to 2 µM) and stored at 20 °C.
Purification of Recombinant TAFI--
Two liters of conditioned
medium was concentrated 10-fold by ultrafiltration using an LP-1 pump
and S1Y30 (30-kDa cutoff) spiral cartridge (Amicon, Oakville, Ontario).
The concentrated medium was then dialyzed against two changes of 4 liters of 20 mM HEPES, pH 7.4 (HB). The retentate was
passed at 22 °C over a 20-ml Q-Sepharose column that had been washed
with HB containing 0.5 M NaCl and then equilibrated with
HB. The column was then washed with 5 column volumes of HB, and rTAFI
was eluted with HB containing 0.2 M NaCl. Fractions
containing activity (assessed by Hip-Arg hydrolysis following treatment
of the fractions with thrombin/thrombomodulin; see below) were pooled
and applied to a 5-ml plasminogen-Sepharose column that had been
equilibrated at 22 °C with HB containing 0.2 M NaCl. The
column was washed with 50 ml of HB containing 0.2 M NaCl
and 25 ml of HB containing 20 mM NaCl and 0.01% (v/v)
Tween 80, and rTAFI was eluted in this buffer containing 100 mM -ACA. Protein containing fractions were then passed
over a 1-ml DEAE-Sepharose Fast Flow column equilibrated with HB at
22 °C. After a wash with 40 ml of HB, rTAFI was eluted with HB
containing 0.15 M NaCl and stored at
70 °C in this
buffer.
Treatment of TAFI with N-Glycosidase F-- Ten micrograms of rTAFI or plasma-derived TAFI (pTAFI) (each in 20 mM HEPES, pH 7.4, 0.15 M NaCl (HBS), 1 mM diisopropyl fluorophosphate) in a volume of 50 µl was denatured by boiling for 5 min in the presence of 1% (w/v) SDS. The reactions were then diluted 10-fold with HBS, and Nonidet P-40 was added to a final concentration of 0.5% (v/v). The reactions were initiated by the addition of 1 unit of N-glycosidase F. Reactions were incubated at 37 °C; aliquots were removed at various times and the reactions stopped by the addition of SDS-PAGE sample buffer (1% (w/v) (final) SDS, 5% (v/v) (final) glycerol, 0.05 mg/ml (final) bromphenol blue) followed by boiling for 5 min. The samples were then subjected to electrophoresis on a 10% polyacrylamide Tris/Tricine gel (15); protein bands were visualized by silver staining (16).
Activation of TAFI and TAFIa Activity Assays--
rTAFI or pTAFI
(1 µM) was incubated for 10 min at 22 °C in the
presence of 5 mM CaCl2, 20 nM
thrombin, and 80 nM thrombomodulin (Solulin) in HBS
containing 0.01% (v/v) Tween 80. Under these conditions, the zymogen
is quantitatively converted to the active enzyme, and the inactivating
cleavage of the active enzyme at Arg-330 is undetectable. Thrombin
activity was then quenched by the addition of the irreversible
chloromethyl ketone inhibitor PPAck (100 nM). Hydrolysis of
the substrates Hip-Arg and Hip-Lys was monitored at 254 nm in a
Perkin-Elmer Lambda 4B spectrophotometer (thermostatted to 22 °C) in
20 mM Tris, pH 7.65, 100 mM NaCl. Extinction
coefficients for the two substrates and the change in extinction
coefficient that occurs upon hydrolysis of the substrates were
determined by titrating Hip-Arg, Hip-Lys, and HA and were as follows:
(Hip-Arg) = 2.149 mM
1 cm
1;
(Hip-Arg
HA) = 0.524 mM
1
cm
1;
(Hip-Lys) = 2.156 mM
1
cm
1;
(Hip-Lys
HA) = 0.517 mM
1 cm
1. Hydrolysis of
FA-Ala-Lys was monitored at 340 nm in a Titertek Twin kinetic plate
reader (22 °C) in HBS containing 0.01% (v/v) Tween 80. Reactions
were performed in a 200-µl volume in Immulon 1 Removawell strips
(Dynatech Laboratories Inc., Chantilly, VA). To determine the change in
absorbance per mol of FA-Ala-Lys that occurs upon hydrolysis of this
substrate, various concentrations of FA-Ala-Lys (in 198 µl) were
placed in successive wells, and measurement of absorbance was
initiated. Two microliters of rTAFIa (60 nM final
concentration) was then added to each well, and the reactions were
allowed to proceed until the substrate was fully consumed. From these
data, the molar change in absorbance upon hydrolysis of FA-Ala-Lys was
determined to be
0.390 mM
1.
Measurement of the Thermal Stability of TAFIa--
TAFIa was
formed from pTAFI and rTAFI by incubation in the presence of
thrombin/thrombomodulin as described above. Following the addition of
PPAck, TAFIa preparations were transferred to wet ice (0 °C) or to
water baths thermostatted at 22, 30, or 37 °C. Timed aliquots were
removed, and the TAFIa activity in them was assessed by measurement of
the rate of FA-Ala-Lys hydrolysis as described above. In some
experiments, saturating concentrations of the competitive inhibitors
-ACA and GEMSA were added to the TAFIa preparations incubated at
37 °C. To assess the stability of the respective zymogens, pTAFI and
rTAFI were incubated at 37 °C for 0, 10, and 60 min, after which
CaCl2, thrombin, and thrombomodulin were added, and the
incubation was continued for a further 10 min at room temperature. At
this time, PPAck was added, and the TAFIa activity was measured as
described above.
In Vitro Fibrinolysis Assays--
To assess the effect of rTAFIa
and pTAFIa on tPA-mediated fibrin clot lysis, 194 µl of a solution
containing fibrinogen (2.9 µM), Glu-plasminogen (0.66 µM), recombinant 2-antiplasmin (0.5 µM), antithrombin III (0.96 µM), and
various concentrations of rTAFIa or pTAFIa (in HBS containing 0.01%
(v/v) Tween 80) was added to microtiter wells containing individual
2-µl aliquots of CaCl2 (10 mM, final),
thrombin (7.7 nM, final), and tPA (442 pM,
final). Lysis of the resultant clots was monitored turbidometrically at
405 nm in a Titertek Twin reader thermostatted at 37 °C as described
previously (7). To assess the effect of rTAFIa and pTAFIa on
plasmin-mediated clot lysis, a solution of 194 µl containing fibrinogen (2.9 µM) and various concentrations of rTAFIa
or pTAFIa was added to microtiter wells containing individual 2-µl
aliquots of CaCl2 (10 mM, final), thrombin (7.7 nM, final), and plasmin (2 nM, final). Lysis of
the resultant clots was monitored turbidometrically as for the
tPA-containing clots.
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RESULTS |
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Expression and Purification of Recombinant TAFI (rTAFI)-- Conditioned serum-free medium (CM) harvested from BHK cells transiently transfected with the TAFI-pNUT expression plasmid was subjected to SDS-PAGE followed by Western blot analysis using a TAFI-specific monoclonal antibody (13) (Fig. 2). Also included in this blot was 10 ng of plasma-derived TAFI (pTAFI) and CM harvested from BHK cells transiently transfected with the pNUT plasmid containing the TAFI cDNA in the reverse orientation (revTAFI-pNUT). An intense immunoreactive band similar in mobility to pTAFI was present in CM harvested from cells transfected with the expression plasmid containing the TAFI cDNA in the forward, but not the reverse, orientation indicating that the former cells were expressing a protein corresponding to rTAFI. Inspection of the blot reveals that rTAFI migrates marginally slower on SDS-PAGE than pTAFI (Fig. 2).
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Comparison of the Biochemical Properties of rTAFI and pTAFI-- Fig. 3 shows the results of SDS-PAGE analysis of purified rTAFI and pTAFI. As predicted by Western blot analysis (Fig. 2), purified rTAFI migrated marginally slower than pTAFI (Fig. 3). However, SDS-PAGE analysis of rTAFI and pTAFI activated by thrombin/thrombomodulin (Fig. 4, upper panel) revealed that the Mr ~35,000 active enzyme (TAFIa) derived by cleavage of the zymogen at Arg-92 (7) and the Mr ~25,000 and Mr ~12,000 fragments derived from cleavage of TAFIa at Arg-330 (7) were of identical size for rTAFI and pTAFI. In addition, the rates of appearance of the Mr ~35,000, Mr ~25,000, and Mr ~12,000 bands (Fig. 4, upper panel) and the rates of appearance of TAFIa activity (as measured by hydrolysis of hippuryl-L-arginine (Hip-Arg)) (Fig. 4, lower panel) were similar for rTAFI and pTAFI.
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Comparison of the Activation of rTAFI and pTAFI by Thrombin/Thrombomodulin-- Although previous studies have shown that TAFI can be activated by thrombin (7), plasmin (12), and trypsin (12), these are all relatively poor activators of the zymogen. Furthermore, at concentrations of these activators required to achieve significant cleavage at Arg-92, TAFIa itself is subsequently cleaved rapidly at Arg-330 to inactivate the enzyme. Recent studies from our laboratory, however, have shown that thrombin complexed with thrombomodulin activates TAFI with a 1250-fold higher catalytic efficiency than thrombin alone without appearing to enhance significantly the rate of the inactivating cleavage (8), suggesting that the physiologic activator is thrombin/thrombomodulin. Therefore, the ability of thrombin/thrombomodulin to activate rTAFI was investigated. The concentrations of substrate (rTAFI or pTAFI) and cofactor (Solulin) were systematically varied, and the initial rates of TAFIa formation were assessed by measurement of N-[3-(2-furylacryloyl)]-L-alanyl-L-lysine (FA-Ala-Lys) hydrolysis. Kinetic data were fit to an equation developed to describe the kinetics of pTAFI activation by thrombin/thrombomodulin (8). Presented in Fig. 6 are the rates of pTAFIa (Fig. 6A) or rTAFIa (Fig. 6B) formation plotted as a function of TAFI concentration. The solid lines in both panels represent the rates calculated from the fit parameters kcat, Km, and Kd. These kinetic constants are presented in Table II and are very similar for rTAFI and pTAFI.
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Comparison of the Enzymatic Properties of rTAFIa and
pTAFIa--
To compare the enzymatic behavior of rTAFIa and pTAFIa,
the ability of the respective enzymes to hydrolyze the synthetic
substrates Hip-Arg (50-600 µM), Hip-Lys (100-1200
µM), and FA-Ala-Lys (0.1-2 mM) was assessed.
Kinetic constants for rTAFIa and pTAFIa for these substrates were
obtained by fitting the rates of substrate hydrolysis to the
Michaelis-Menten equation using nonlinear regression; the results are
presented in Table III. Inhibition
constants (Ki) for three competitive inhibitors of
TAFIa, -aminocaproic acid (
-ACA (19)),
2-guanidinoethylmercaptosuccinic acid (GEMSA (11)), and potato
carboxypeptidase inhibitor (PCI (18)) were also determined (Table III).
The respective inhibitors were titrated (
-ACA, 0.1-5 mM; GEMSA, 2-50 µM; PCI, 2-50
nM) at three different concentrations of substrate (either
Hip-Arg (200, 400, or 600 µM) or FA-Ala-Lys (200, 400, or
600 µM), see Table III), and the data were fit to a
modified form of the Michaelis-Menten equation which describes competitive inhibition. All of the kinetic constants determined were
comparable for rTAFIa and pTAFIa (Table III).
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Comparison of the Stability of rTAFIa and pTAFIa--
Previous
studies have shown that TAFIa activity is not stable (8, 11). Thus, we
measured and compared the stability of rTAFIa and pTAFIa (Fig.
7). The respective zymogens were
activated by thrombin/thrombomodulin; the thrombin was irreversibly
inhibited with PPAck, and the activated enzymes were placed at 0, 22, 30, or 37 °C. Aliquots were removed at various times, and the
FA-Ala-Lys hydrolytic activity in each sample was assessed. Whereas
rTAFIa and pTAFIa are both stable at 0 °C, their activities decay
with similar half-lives of about 120-150, 40-50, and 8-9 min at 22, 30, and 37 °C, respectively (Fig. 7A). The data of Fig.
7A exhibited first-order kinetics of decay. First-order
decay constants were obtained by fitting the data to the equation
[TAFIa] = [TAFIa]0 exp(k·t)
by nonlinear regression. With plasma TAFIa the first-order decay
constants (min
1) at 22, 30, and 37 °C were 0.0040 ± 0.0003, 0.013 ± 0.001, and 0.076 ± 0.004, respectively.
The corresponding values with recombinant TAFIa were 0.0052 ± 0.0003, 0.016 ± 0.001, and 0.088 ± 0.007. The temperature
dependence of the first-order decay constants was interpreted according
to transition state theory whereby the forward rate constant
(kf) for a transition (e.g.
denaturation) from one state to another is given by
kf = (kT/h)exp((
So
Ho
/T)/R)
where k is Boltzmann's constant; T is the
absolute temperature; h is Planck's constant; R
is the molar gas constant, and
So
and
Ho
are the standard entropy
and enthalpy changes for the presumed equilibrium between the initial
state and transition state intermediate (20). The data were fit to the
above equation by nonlinear regression with the values of the rate
constants for decay and temperature as variables and
So
and
Ho
as fit parameters. The
analyses returned values of
So
= 82 ± 19 cal/mol/K,
Ho
= 46 ± 6 kcal/mol for inactivation of plasma TAFIa and
So
= 75 ± 18 cal/mol/K,
Ho
= 43 ± 5 kcal/mol for
recombinant TAFIa. The relatively high positive values for
Ho
imply that inactivation is
not enthalpically favored, possibly because of the need to break
numerous noncovalent bonds. This is offset, however, by a highly
favorable entropy change associated with inactivation. The high
enthalpy change also accounts for the high sensitivity of inactivation
to temperature. According to this interpretation, the process of TAFIa
inactivation involves both the energetically unfavorable disruption of
numerous noncovalent interactions within the protein and the
energetically favorable assumption of a less ordered structure.
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Comparison of Inhibition of Fibrinolysis by rTAFIa and
pTAFIa--
We initially identified TAFI as a factor in human plasma
which, in the context of sustained thrombin generation, was capable of
generating in response to thrombin an activity that retards tPA-mediated fibrin clot lysis in vitro. Thus, the ability
of rTAFIa and pTAFIa to inhibit fibrinolysis was directly compared. Fibrin clots containing plasminogen, tPA, recombinant
2-antiplasmin, antithrombin III, and rTAFIa or pTAFIa at
various concentrations were formed in the wells of microtiter plates,
and lysis of the clots was monitored turbidometrically. A plot of the
time required to achieve 50% clot lysis versus TAFIa
concentration is presented in Fig.
9A. Both rTAFIa and pTAFIa are
capable of retarding clot lysis time up to 2-fold, with both enzymes
achieving their half-maximal effect at a concentration of approximately
1 nM (Fig. 9A).
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DISCUSSION |
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We have expressed a recombinant form of TAFI (thrombin-activable
fibrinolysis inhibitor; also known as CPU (11) and plasma procarboxypeptidase B (12)) in BHK cells. We chose to express rTAFI in
mammalian cells primarily because a number of other proteins involved
in the coagulation and fibrinolytic cascades have been successfully
expressed in BHK cells. These include human prothrombin (22), human
factor VII (23), human factor VIII (24), human antithrombin III (25),
human plasminogen (26), and human 2-antiplasmin (26).
Unlike bacterial expression systems, recombinant proteins expressed in
mammalian cells can be secreted in their native conformation and are
subjected to post-translational modifications such as glycosylation.
In the present study, we found that while the recombinant version of TAFI migrates marginally more slowly on SDS-PAGE than its plasma-derived counterpart, rTAFI is virtually indistinguishable from pTAFI in terms of its ability to be activated by thrombin/thrombomodulin and the stability and enzymatic properties of TAFIa including the ability to inhibit tPA- and plasmin-mediated fibrin clot lysis in vitro.
The mobility difference between rTAFI and pTAFI on SDS-PAGE (Fig. 3) is likely due to differences in the size and/or composition of N-linked glycans. Inspection of the cDNA sequence for plasma procarboxypeptidase B reveals the presence of four potential N-linked glycosylation sites (Asn-22, Asn-51, Asn-63, and Asn-86), all of which are located within the 92-amino acid activation peptide (12). Indeed, there is no apparent difference between the mobility of the Mr ~35,000 TAFIa species derived from rTAFI and pTAFI (Fig. 4). Furthermore, treatment of rTAFI and pTAFI with N-glycosidase F, which specifically removes N-linked glycans, gave rise to terminal products of identical electrophoretic mobility (Fig. 5). Three intermediately glycosylated species are present, indicating that all four potential N-linked sites are utilized in rTAFI and pTAFI.
Differences in the size and/or composition of N-linked glycans are frequently observed in the comparison of a recombinant protein and its naturally occurring counterpart (25, 27-29), which reflect the cell-, tissue-, and species-specificity of glycosylation (30). In some cases such differences have consequences for the functional properties of the recombinant protein. For example, BHK cells secrete a glycoform of ATIII that is not found in plasma and that differs in its affinity for heparin and in its rate of proteinase inhibition, in addition to two other glycoforms that are functionally similar to plasma-derived ATIII (25). However, in other cases differences in glycosylation have no apparent effect on the functional behavior of the recombinant protein. Human factor VIII expressed in BHK cells contains differences in the composition of its N-linked glycans relative to plasma-derived factor VIII (28), yet the two factor VIII preparations are similar with respect to cleavage by thrombin, factor Xa, and activated protein C (24), subunit association and dissociation (24), and pharmacokinetic parameters in baboons (28). Although we did not observe any functional differences between rTAFI and pTAFI attributable to differences in glycosylation, differences in, for example, the pharmacokinetics of rTAFI and pTAFI or in their binding to as yet undescribed substrates cannot be ruled out.
It should be noted that the fully N-deglycosylated TAFI species migrated as doublets, with both members of the doublet in similar proportions in both rTAFI and pTAFI (Fig. 5). These doublets are most likely the result of heterogeneity in usage of O-linked glycosylation sites. It is unlikely that the doublet is due to N-terminal sequence heterogeneity, since TAFI derived from plasma has been shown to possess a unique N-terminal sequence (7, 12).
We found that rTAFI is virtually indistinguishable from pTAFI in terms
of its ability to hydrolyze small peptide substrates and to be
inhibited by the competitive inhibitors -ACA, GEMSA, and PCI (Table
III). These data demonstrate the integrity of the active site of rTAFI
and show that, when produced under the conditions reported in this
study, rTAFI possesses a specific activity similar to that of its
plasma-derived counterpart. Of note is the similarity in the
Ki for PCI, a small (39 amino acid) inhibitor that
has been shown to bind to carboxypeptidase A through regions distinct
from those involved in substrate binding. Together with the similar
kinetic constants obtained for the activation of rTAFI by
thrombin/thrombomodulin (Table II), these data indicate that the
overall structure of TAFI is likely to be faithfully represented by the
recombinant protein.
A striking feature of TAFIa is that its activity is unstable, decaying rapidly at 37 °C both in vitro in the absence of detectable proteolysis and in the serum milieu. In the present study, we found the stability of rTAFIa and pTAFIa to be very similar; although both enzymes were stable at 0 °C, their activities decayed with half-lives of about 10 min at 37 °C, about 40-50 min at 30 °C, and about 120-150 min at 22 °C (Fig. 7). The structural basis for the instability of TAFIa is not clear at present, but a thermodynamic interpretation suggests a highly unfavorable enthalpy change associated with inactivation (which may reflect the requirement to disrupt many noncovalent bonds), offset by a corresponding highly favorable entropy change (which in turn may reflect the adoption of a less ordered structure). Consistent with such changes in structure, we found a marked quenching of the intrinsic fluorescence of TAFIa at 37 °C which correlated temporally with the decay of enzymatic activity (Fig. 8). The quenching of the fluorescence signal is presumably attributable to the exposure to the solvent of residues (largely tryptophans) previously buried in the hydrophobic core of the enzyme. It is noteworthy that this presumptive structural change is not detectable by electrophoresis; the mobility of TAFIa species incubated for 60 min at 37 °C on SDS-PAGE is identical to that of TAFIa species incubated at 0 °C for 60 min (data not shown).
Whether an endogenous inhibitor of TAFIa exists is not known at
present, although data exist that indicate that TAFIa interacts with
2-macroglobulin and pregnancy zone protein (31). The
consequences of these interactions are not known, although binding of
TAFIa to these proteins does not affect its enzymatic activity (31). Because carboxypeptidase U activity (TAFIa) in serum has a half-life similar to that of purified TAFIa shown here (10), the presence of a
fast-acting endogenous inhibitor in vivo is not indicated. Therefore, the intrinsic instability of TAFIa at 37 °C may be physiologically relevant in the down-regulation of TAFIa in
vivo. Furthermore, that the activity of TAFIa is stable at
37 °C in the presence of saturating concentrations of competitive
inhibitors suggests that in the clot milieu, TAFIa activity may be
maintained as long as there is sufficient substrate available.
Although the mechanism by which TAFIa inhibits fibrinolysis has yet to be conclusively determined, it likely involves removal of the C-terminal lysine residues from partially degraded fibrin that are required for maximal stimulation of tPA-mediated plasminogen activation (8). However, additional mechanisms for the antifibrinolytic effect of TAFIa may also be possible, since we have recently observed that TAFIa (albeit at higher concentrations) is capable of attenuating plasmin-mediated fibrinolysis (i.e. when plasminogen activation has been bypassed). Although the mechanism underlying this effect is unclear at present, plasmin itself may be the substrate for TAFIa in this instance (21). Based on the data presented in Fig. 9, which show that rTAFIa is capable of inhibiting both tPA- and plasmin-mediated fibrinolysis in a manner identical to pTAFIa, it is clear that rTAFIa and pTAFIa are comparable in their ability to participate in the reactions involved in the antifibrinolytic effect. These reactions may include hydrolyzing C-terminal basic residues in fibrin and plasmin, as well as inactivation of TAFIa by plasmin cleavage at Arg-330.
In conclusion, these studies show that although plasma and recombinant TAFI exhibit minor differences in glycosylation, recombinant TAFI and TAFIa are excellent surrogates for the natural species and thus their properties can be used to interpret further differences obtained in structure-function studies utilizing site-directed mutagenesis.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Mona Rahman and Dr. Marlys Koschinsky (Dept. of Biochemistry, Queen's University) for the gift of a human liver first-strand cDNA library, Dr. John Morser (Berlex Biosciences, Richmond CA) for the gift of Solulin, and Dr. Gordon Vehar (Genentech, Inc., South San Francisco) for the gift of Activase.
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FOOTNOTES |
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* This work was supported by Heart and Stroke Foundation of Ontario Grant T-2631, National Institutes of Health Grant PO1-HL46703, and Medical Research Council of Canada Grant MT-9781.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 Studentship Award from the Medical Research
Council of Canada.
§ To whom correspondence should be addressed. Tel.: 613-545-2957; Fax: 613-545-2987; E-mail: nesheimm{at}post.queensu.ca.
1
The abbreviations used are: TAFI,
thrombin-activable fibrinolysis inhibitor; rTAFI, recombinant TAFI;
pTAFI, plasma-derived TAFI; TAFIa, activated TAFI; CM, conditioned
medium; Hip-Arg, hippuryl-L-arginine; Hip-Lys,
hippuryl-L-lysine; HA, hippuric acid; FA-Ala-Lys,
N-[3-(2-furylacryloyl)]-L-alanyl-L-lysine;
tPA, (recombinant) tissue-type plasminogen activator; PCI, potato
carboxypeptidase inhibitor; -ACA, epsilon-aminocaproic acid; GEMSA,
2-guanidinoethylmercaptosuccinic acid; PAGE, polyacrylamide gel
electrophoresis; PPAck, D-Phe-Pro-Arg chloromethyl ketone;
dEGRck, 1,5-dansyl-Glu-Gly-Arg chloromethyl ketone; BHK, baby hamster
kidney cells; pro-pCPB, plasma procarboxypeptidase B; Tricine,
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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REFERENCES |
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