From the
Previous studies demonstrated that tissue plasminogen
activator-induced fibrinolysis in vitro is retarded in the
presence of prothrombin (II) activation and that the
anticoagulant-activated protein C appears profibrinolytic by preventing
the formation of thrombin (IIa)-like activity during fibrinolysis. To
disclose the molecular connection between the generation of IIa and the
inhibition of fibrinolysis, a lysis assay that is sensitive to the
antifibrinolytic effect of II activation was developed and was used to
purify a 60-kDa single-chain protein from human plasma. Because the
lysis of a clot, produced from purified components, is retarded when
this protein is present and when II activation occurs in situ,
the protein was named TAFI (thrombin-activatable fibrinolysis
inhibitor). TAFI is cleaved by IIa yielding 35-, 25-, and 14-kDa
products. Amino-terminal sequence analyses identified TAFI as a
precursor of a plasma carboxypeptidase B (CPB). Formation of the 35-kDa
product correlates with both prolongation of lysis time and CPB-like
activity. Prolongation of lysis time saturates at about 125 nM TAFI. Activated TAFI inhibits the activation of Glu-plasminogen
but does not prolong the lysis of clots formed in the presence of
Lys-plasminogen. 2-Guanidinoethylmercaptosuccinic acid, a competitive
inhibitor of CPB, completely inhibits prolongation of lysis by
activated TAFI in a purified system and the prolongation induced by II
activation in barium-adsorbed plasma. This suggests that TAFI accounts
for the antifibrinolytic effect that accompanies prothrombin activation
and that activated protein C appears profibrinolytic by attenuating
TAFI activation.
The activation of II to IIa in vivo is catalyzed by
prothrombinase, a tetrameric complex consisting of the serine protease
factor Xa (FXa),
Activated protein C (APC) is generated by cleavage of the precursor,
protein C, a reaction catalyzed by IIa in complex with thrombomodulin
(10). APC can down-regulate II activation by catalyzing the proteolytic
inactivation of the essential cofactor in prothrombinase,
FVa(11) . Protein S, a cofactor for this reaction, is
responsible for the species specificity of the anticoagulant activity
of APC(12) . APC, however, also appears to up-regulate
fibrinolysis(1, 13, 14, 15, 16) .
We have shown that this effect is not due to a direct profibrinolytic
effect of APC but rather an indirect effect expressed through
inhibition of II activation(1) . This explains why others have
observed that the profibrinolytic effect of APC appears to be
potentiated by protein S in a species-specific manner(17) . Our
data also indicated that II activation, when it occurs during
fibrinolysis and yields either IIa or meizothrombin, is
antifibrinolytic(2) . Those studies also suggested that some
preparations of plasminogen contain a contaminant required to
reproduce, in a system of defined components, the profibrinolytic
effect of APC which is always observed in plasma(1) . Those
studies, however, did not provide a molecular explanation for the
inhibition of fibrinolysis by II activation during fibrinolysis.
This work was undertaken to characterize further the mechanism by
which II activation during fibrinolysis prolongs the lysis time of a
clot. This was accomplished by producing a turbidometric lysis assay
that is both sensitive to a component in plasma which is
thrombin-activated and inhibits fibrinolysis (TAFI) and is buffered
with respect to the inhibitors AT-III and
The cDNA for
TAFI was purified from BAP by differential precipitation with
(NH
We also provide evidence that TAFI is
cleaved by IIa to produce a 35-kDa fragment that correlates with both a
relative increase in lysis time, in a system of defined components, and
CPB activity. Furthermore, we show that the specific carboxypeptidase
inhibitor GEMSA completely inhibits the prolongation of lysis time in a
system of defined components observed in the presence of active porcine
CPB and in a similar system in which TAFI is activated by IIa produced in situ. Since complete inhibition of the prolongation
observed in BAP supplemented with II and PC/PS vesicles in the presence
of FXa occurs with an EC
The inhibition of prolongation of lysis time by GEMSA and the
formation of the 35-kDa fragment of TAFI during fibrinolysis indicate
that carboxypeptidase activity not only prolongs lysis time but also
can be generated in situ. The relatively low abundance of the
35-kDa fragment, in comparison to the fragments migrating between 14
and 21 kDa (Fig. 8), suggests that a steady-state level of
activated TAFI is attained during fibrinolysis. Degradation of
activated TAFI, yielding the small fragments in higher abundance, may
involve any one or a combination of the proteases IIa, plasmin, FXa, or
t-PA which are present in the system. Furthermore, it is unknown if
these or any of the other cleavage products are either partially
processed precursors or degradation products with or without CPB
activity.
The mechanism by which activated TAFI prolongs lysis time
is unclear; however, it is able to modulate t-PA-induced activation of
Glu
Several previous studies indicated that APC influences
fibrinolysis both in vivo and in
vitro(1, 13, 14, 15, 16, 41) .
Furthermore, we have shown previously that II activation during
fibrinolysis in vitro is antifibrinolytic(1) . Since
APC is an anticoagulant and is therefore able to inhibit the activation
of II we concluded that APC appears profibrinolytic by virtue of its
ability to prevent the antifibrinolytic effect generated by II
activation(1) . In addition, the species specificity of the
anticoagulant effect of APC, which is due to the species specificity of
the cofactor protein S, is also exhibited in the profibrinolytic effect
of APC, as shown by Weinstein and Walker(17) . The results of
this study rationalize the apparent antifibrinolytic effect of II
activation by thrombin's ability to activate TAFI, which
subsequently exhibits CPB-like activity and inhibits fibrinolysis.
Thus, the rationale for the profibrinolytic effect of APC lies with its
anticoagulant properties. By inhibiting the activation of II, APC
prevents the subsequent activation of TAFI resulting in a reduced lysis
time when compared with that observed when II activation occurs.
Aliquots from each step of
the purification were dialyzed against HBS. Activity per unit volume of
a sample added to the assay is defined by the expression
activity/µl sample = ((Lt + Xa/Lt - Xa) -
1)/volume sample (µl). A unit of activity is defined as the
activity in 1.0 ml of BAP.
We acknowledge gratefully numerous fruitful
discussions with Dr. Anton Horrevoets.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)a negatively charged
phospholipid surface, calcium ion and the cofactor factor Va
(FVa)(4) . In response to injury, IIa catalyzes the cleavage of
fibrinopeptides A and B from fibrinogen, forming fibrin monomers which
spontaneously polymerize to form the insoluble fibrin network of a
blood clot and prevent the catastrophic loss of blood(5) . The
fibrinolytic cascade works in opposition to the coagulation cascade and
is responsible for the removal of a fibrin clot. Plasmin is the
terminal protease formed by the fibrinolytic cascade and is formed by
the activation of plasminogen by tissue-type plasminogen activator
(t-PA). Plasmin catalyzes the cleavage of fibrin to produce soluble
fibrin degradation products thereby removing a fibrin
clot(6, 7) . Both coagulation and fibrinolysis are in
part regulated by the specific inhibitors antithrombin III (AT-III) and
-antiplasmin, respectively(8, 9) . Both
activation and inhibition of each cascade regulate and maintain a
proper balance between fibrin formation and its dissolution(5) .
-antiplasmin. Using this assay, we purified a 60-kDa
zymogen from plasma which, when activated by IIa, yields an enzyme with
carboxypeptidase B (CPB)-like activity. The generation of this activity
correlates with a prolongation in lysis time and inhibition of
Glu
Pgn activation. Inhibition of IIa generation by APC thus
attenuates activation of TAFI and thereby links the profibrinolytic
effect of APC to its anticoagulant activity.
Materials and Methods
The recombinant
t-PA, Activase, was generously provided by Dr. Gordon Vehar of
Genentech (South San Francisco, CA). The lyophilized powder was
dissolved in water to a final concentration of 1.0 mg/ml and working
stock solutions were prepared as described previously(1) .
Porcine CPB was purchased from Boehringer Mannheim (Laval, PQ) as a
lyophilized powder that was dissolved in water to 5.0 mg/ml and stored
in 20-µl aliquots at -20 °C.
2-Guanidinoethylmercaptosuccinic acid (GEMSA), a specific CPB
inhibitor, was purchased from Calbiochem. Dulbecco's modified
Eagle's medium, nutrient mixture F-12 (1:1) (DMEM/F-12),
Opti-Mem, newborn calf serum, and G418 (Geneticin) were purchased from
Life Technologies Inc., and methotrexate was purchased from David Bull
Laboratories (Vaudreuil, PQ). Phosphatidylcholine/phosphatidylserine
vesicles (PC/PS, 3:1) were prepared according to the method of
Barenholz et al.(18) as described by Bloom et
al.(19) . II and factor X (FX) were purified as described
previously(13) . IIa was prepared from II and isolated by a
modification of the procedure of Lundblad et al.(20) as described previously(21) . FXa was prepared
from FX utilizing the FX activator purified from Russell's viper
venom and isolated as described by Krishnaswamy et
al.(22) . Protein C was purified and activated as described
previously(13) . Factor V (FV) was purified by immunoaffinity
chromatography according to the method of Nesheim et
al.(23) . A concentrate of AT-III was kindly provided by
Alpha Therapeutics (Los Angeles, CA) and was subjected to affinity
chromatography on immobilized heparin as described by Damus and
Rosenberg (8) with the modifications of Nesheim(24) .
Fibrinogen, >95% clottable, was isolated by the procedure of
Straughn and Wagner (25) and subsequently passed over Lys-Sepharose to
remove traces of plasminogen. Glu-plasminogen (GluPgn) was
isolated by Lys-Sepharose chromatography, as described
previously(26) . Glu
Pgn was coupled to CNBr-activated
Sepharose CL-4B (Sigma) by a modification of the procedure described by
Cuatrecasas(27) . Briefly, Sepharose CL-4B (Pharmacia) (5 ml)
was washed at 22 °C with 50 ml of both H
O and 2.0 M Na
CO
under vacuum. The cake of
washed Sepharose CL-4B was then activated with 0.5 g of CNBr (0.5 g of
CNBr dissolved in 0.5 ml of acetonitrile) for 1.0 min at 22 °C and
then washed with 100 ml each of ice-cold 0.1 M
NaHCO
, H
O, and 0.1 M sodium citrate,
pH 6.5, in succession. Five ml of 10 mg/ml Glu
Pgn in 0.1 M sodium citrate, pH 6.5, was then incubated with the activated
resin at 4 °C for 24 h and for a further 24 h after the addition of
1.0 ml of 1.0 M Tris, pH 8.0. This resulted in a 98% coupling
efficiency producing 5.0 ml of a 9.8 mg/ml Pgn-Sepharose resin.
Lys-plasminogen (Lys
Pgn) was prepared as described by Nesheim et al.(26) by incubation of Glu
Pgn (33
µM) with plasmin (0.1 µM) in 6.2 ml of 50
mM Tris, pH 8.0, and 10.0 mM
ACA. The reaction
was stopped after 2.0 h by passing the mixture over
benzamidine-Sepharose, and the flow-through was incubated for 2.0 h at
22 °C with 1.0 µM valyl-phenyl-alanyl-lysyl
chloromethyl ketone (Calbiochem). Lys
Pgn was precipitated with
80% (NH
)
SO
. The pellet was
dissolved in 50% glycerol/H
O and stored at -20
°C. The Lys
Pgn preparation contained no Glu
Pgn as
assessed by acid-urea gel electrophoresis(28) . Plasmin was
prepared by adding 10.0 ml of Lys-Sepharose to 20 ml of 1.0 mg/ml
Glu
Pgn and 50 units/ml human urine urokinase (Calbiochem) in 20
mM Tris-HCl, 150 mM NaCl, pH 8.0 (TBS) and incubating
with gentle agitation at 37 °C for 1.0 h. The resin was then poured
into a column; after washing with 70 ml of TBS, plasmin and residual
plasminogen were eluted with 20 mM
ACA in TBS, collecting
1.0-ml fractions at 22 °C. Protein containing fractions, identified
by absorbance at 280 nm, were pooled and passed over a 2.0-ml p-aminobenzamidine Sepharose 6B (Sigma) column in order to
separate plasminogen from plasmin. The column was washed with TBS,
collecting 1.0-ml fractions at 22 °C until the A
was less than 0.01. Plasmin was then eluted with 20 mM benzamidine in TBS, collecting 1.0-ml fractions.
Protein-containing fractions were identified using the BCA assay
(Pierce Chemical Co.). Appropriate fractions were pooled (8.0 ml), and
plasmin was precipitated by dialysis against 250 ml of ammonium sulfate
at 80% saturation with one change overnight at 4 °C. After
centrifugation (13,000
g, 5 min, 22 °C) of the
retentate, the pellet was dissolved in 50% glycerol/water and stored at
-20 °C until use. From this procedure 400 µl of 17.1
mg/ml plasmin was isolated, amounting to a 34% recovery of 100% active
plasmin as determined by active site titration with p-nitrophenylguanidinobenzoate(29) .
-antiplasmin in the expression vector pPAB (30) was a
kind gift of Sharon Busby of Zymogenetics (Seattle, WA). The expression
vector pNUT and baby hamster kidney cells were provided by Dr. Ross
MacGillivray of University of British Columbia. Baby hamster kidney
cells were cotransfected with pPAB, containing the antiplasmin insert,
and pNUT using Ca
PO
coprecipitation
technique(27, 31) . The DNA (10 µg of each vector)
was precipitated at pH 6.95 and added to the cells and incubated for 4
h. Cells were allowed to recover overnight in DMEM/F-12, supplemented
with 5% newborn calf serum. Positive clones were selected in DMEM/F-12
supplemented with 5% newborn calf serum, 440 µM methotrexate, and 400 µg/ml G418. Positive clones were
screened for antiplasmin production by enzyme-linked immunosorbent
assay using goat polyclonal capture antibody GA2AP-1IG (Affinity
Biologicals, Hamilton, ON) and detected with horseradish
peroxidase-conjugated monoclonal murine antibody (Dr. A. R. Giles,
Queen's University, Kingston, ON). One clone (25 µg of
antiplasmin/ml of medium/24 h from confluent cells on 10-cm
wells) was used for roller bottle production of antiplasmin.
Antiplasmin was routinely isolated from 24-h conditioned medium from
two roller bottles, each containing 150 ml of Opti-Mem supplemented
with 80 µM ZnCl
, 220 µM methotrexate, and 200 µg/ml G418. Conditioned medium was
diluted 1/3 with 20 mM HEPES, pH 7.4 (HB) and passed over a
10-ml Q-Sepharose (Pharmacia) column. The column was washed with 5
column volumes of HB, and antiplasmin was eluted with 0.2 M NaCl in HB. Appropriate fractions were pooled and diluted 1/4 and
passed over a 1.0-ml DEAE fast flow (Pharmacia) column at 22 °C.
The column was washed with 20 ml of HB, and the antiplasmin was eluted
with 0.15 M NaCl in HB. Five mg of antiplasmin is typically
recovered by this procedure, and the antiplasmin inhibits active site
titrated plasmin with a 1:1 stoichiometry.
Lysis Assay and Measurement of the Antifibrinolytic
Effect of Purified TAFI
A concentrated stock solution
(fibrinogen solution) was produced containing 15.4 µM
fibrinogen, 4.2 µM GluPgn, 1.8 µM
-antiplasmin, 52.6 µM PC/PS
vesicles, 44 nM FV, 3.65 µM II, and 5.0
µM AT-III in 20 mM HEPES, 0.15 M NaCl,
pH 7.4 (HBS). The assay was performed by adding 40 µl of the
fibrinogen solution to 160 µl of assay sample or purified TAFI. Two
clots were produced for each sample by pipetting 95 µl of each
assay mixture into wells containing 2.0-µl aliquots of IIa (6.0
nM, final), Ca
(10 mM, final) and
t-PA (441 pM, final) and either buffer or FXa (100
pM, final). Turbidity at 405 nm was then monitored at 2.5-min
intervals in a microtiter plate reader thermostatted at 37 °C.
Lysis time, defined as the time to achieve a 50% reduction of the
maximum turbidity, for each clot was determined from the plot of A
versus time. Relative prolongation
was then defined as the lysis time determined for clots produced in the
presence of FXa relative to the lysis time determined for clots
produced in the absence of FXa.
Purification of TAFI
Two units of fresh
frozen plasma (500 ml), obtained from the Canadian Red Cross (Kingston
General Hospital, ON) anticoagulated with acid citrate dextrose, were
thawed at 37 °C for 0.5 h. The plasma was brought to 80 mM BaCl by dropwise addition of 1.0 M BaCl
with continuous stirring at 4 °C. Barium citrate and adsorbed
vitamin K-dependent proteins were pelleted by centrifugation at 10,000
g for 20 min at 4 °C (these conditions were used
in all subsequent centrifugation steps). An aliquot (10 ml) of the
supernatant was removed and dialyzed against 2.0 liters of HBS with
four changes, for not less than 2.0 h/change, at 4 °C to produce
barium-adsorbed plasma (BAP). To remove remaining Ba
ion, solid (NH
)
SO
was added
directly to the remaining portion of the supernatant to a concentration
of 10% saturation. The mixture was stirred for 0.5 h at 4 °C,
BaSO
was pelleted by centrifugation, and the supernatant
was recovered. The supernatant was brought to 45% saturation
(NH
)
SO
and stirred for 0.5 h at 4
°C. Following precipitation and centrifugation, the pellet was
discarded, and the supernatant was brought to 70%
(NH
)
SO
, stirred for 0.5 h at 4
°C, and centrifuged. The resulting pellet was dissolved in 150 ml
of HB and dialyzed against 4.0 liters of the same buffer at 4 °C
with four changes and minimally 2.0 h between changes. Following
dialysis the retentate was passed over a 450-ml Q-Sepharose column (5.5
19 cm) at 22 °C which had been washed with 0.5 M NaCl, HB, and then equilibrated in HB. The eluate during the load
was collected into a beaker, and the column then was washed with 600 ml
of HB, collecting 10.0-ml fractions. TAFI eluted in a 0-0.2 M NaCl, linear gradient in HB (600 ml/side). The fractions
containing activity were pooled, and the sample was concentrated 5-fold
using an Amicon concentrator with a PY-10 membrane (Amicon, Oakville,
ON). The 15.0-ml concentrate (approximately 100 mg/ml) was then
subjected to gel filtration on three columns linked in tandem (2.5
102 cm) containing AcA-54 equilibrated in HBS. The column was
developed at 4 °C with HBS at a flow rate of 0.42 ml/min,
collecting 4.4 ml/fraction. Fractions containing activity were pooled
and subjected to affinity chromatography at room temperature on a
5.0-ml Pgn-Sepharose CL-4B (9.8 mg/ml resin) column equilibrated in
HBS. After washing with 5 column volumes of HBS and then a similar
volume of HB, TAFI was eluted with 50 mM
ACA, 0.01% Tween
80, HB. Since
ACA is antifibrinolytic, these fractions could not
be assayed. Therefore,
ACA was separated from TAFI by passing the
pooled
ACA eluate over a 1.0-ml DEAE fast flow column equilibrated
in HB and washing with 40 ml of HB. TAFI was then eluted with 0.2 M NaCl, HB. Peak fractions were pooled and stored at 4 °C, and
subsequent experiments were performed within 48 h of isolation.
Amino-terminal Sequence, Amino Acid Composition, and
Extinction Coefficient Determinations
Microamino acid
sequence analyses were performed by the Core Facility for Protein/DNA
Chemistry at Queen's University. Sequences were determined
following electroblotting of bands, resolved by SDS-PAGE, to
polyvinylidene difluoride (Millipore, Bedford, MA)(32) , using
an Applied Biosystems model 475A amino acid analyzer with on-line
phenylthiohydantoin analysis and reverse-phase separation on a C18
column. Quantitative amino acid analysis was performed by HSC
Biotechnology Service Centre (Toronto, ON). To determine the extinction
coefficient of TAFI, duplicate samples of known A, containing norleucine as an internal
standard, were subjected to hydrolysis with 6.0 N HCl followed
by derivatization with phenylisothiocyanate. Separation and
quantitation of the derivatized amino acids were accomplished using the
Picotag processing method and using absorbance at 254 nm, respectively.
The
, using the peptide
molecular weight of 48,442(3) , is 26.4 ± 3.6, and this
value was used to calculate all concentrations for TAFI. (The molar
extinction coefficient is 1.28
10
M
cm
, which is
1.44-fold greater than that calculated from the tyrosine and tryptophan
content(33) .)
Radiolabeling Plasminogen and
TAFI
GluPgn and TAFI were iodinated with
I using IODO-BEADS (Pierce Chemical Co.). IODO-BEADS were
first washed with 1.0 ml of TBS; 500 µl of TBS and 1.0 mCi of
Na
I (ICN, Mississauga, ON) were added and incubated at
room temperature for 5 min. The solution was then removed and added
directly to another tube containing Glu
Pgn (2.0 mg/ml) in 500
µl of TBS, and incubation was carried out for 10 min at room
temperature. The reaction was quenched by the addition of 10 µl of
1.0 M sodium metabisulfite, and unincorporated
I
was removed by gel filtration on a 10.0-ml Sephadex G-25 column
(Pharmacia). The final concentration of
I-plasminogen was
0.89 mg/ml, and the specific radioactivity was 4.5 cpm/ng. Iodination
of TAFI was performed identically except the buffer was HBS and the
final concentration was 0.064 mg/ml with a specific radioactivity of
234 cpm/ng.
Thrombin Cleavage of TAFI
The reaction
was carried out in a 1.5-ml Eppendorf tube containing 100 µl of
TAFI (0.145 mg/ml, 90 µl of 0.153 mg/ml TAFI plus 10.0 µl of
0.064 mg/ml I-TAFI) in HBS. CaCl
(1.0 M) was added to a final concentration of 10 mM. A
10-µl aliquot was removed for the zero time point, and the
remaining 100 µl was brought to 545 nM IIa by the addition
of 9.0 µl of 6.0 µM IIa. The reaction mixture was then
incubated at 37 °C, and at various times a 10.0-µl aliquot was
removed and immediately prepared for SDS-PAGE. Preparation for
electrophoresis included the addition of 10.0 µl of HBS and 5.0
µl of a solution consisting of 25% glycerol, bromphenol blue, 5%
SDS, 75 mM EDTA, pH 7.4, and heating the mixture for 3.0 min
at 90 °C. Approximately 1.5 µg of TAFI and 15,000 cpm were
loaded per well and run under the conditions of Neville (34) on
a 5-15% gradient minigel (Hoefer, model SE250, Technical
Marketing, Ottawa, ON). The gel was stained with Coomassie Blue,
destained, dried in Biowrap (BioDesign Inc., Carmel, NY), and subjected
to autoradiography using Kodak XAR-5 x-ray film overnight at -70
°C. Densitometry was performed using an LKB-2202 densitometer
(Pharmacia).
Activation of TAFI by Thrombin
The effect
of preincubation of TAFI with IIa on lysis time in the presence and
absence of FXa and on the rate of hydrolysis of hippuryl-Arg was
determined. Incubation of 3.14 µM TAFI with 545 nM IIa in 10.0 mM Ca, HBS was carried out
in a final volume of 33 µl for various periods of time (0-2.0
h) at 37 °C. For each incubation time point a clot was produced by
the addition of the fibrinogen solution to wells in which a 1.25-µl
aliquot of the TAFI activation medium had been placed, giving a final
concentration of 40 nM TAFI and 6.8 nM IIa. Included
also in the wells were Ca
(10.0 mM, final)
and t-PA (441 pM, final) plus or minus FXa. Simultaneously, an
aliquot (30 µl) of the TAFI activation medium was removed and added
to 1.0 ml of 0.1 mM hippuryl-Arg in 25 mM Tris, 0.1 M NaCl, pH 7.65, and absorbance at 254 nm was monitored over
time in a Perkin-Elmer Lambda 4b spectrophotometer(35) .
Inhibition of Activated TAFI and Porcine CBB by
GEMSA
Fibrinogen solution diluted 1/5 or a solution of BAP,
diluted 1/3 with HBS plus PC/PS vesicles (10.0 µM, final)
and II (0.73 µM), were supplemented with GEMSA at various
concentrations ranging from zero to 2.0 mM. The correlation
between lysis time obtained in the absence and presence of FXa (100
pM) and the time of incubation of TAFI with IIa was
determined. Lysis times of clots prepared from 95-µl aliquots (see
under ``Lysis Assay'') in the presence and absence of FXa (i.e. plus or minus II activation) were determined for each of
the GEMSA concentrations. These experiments thus constituted an
evaluation of the effect of GEMSA on TAFI-mediated prolongation of
lysis. In a similar series of experiments, fibrinogen solution, diluted
1/5, supplemented with GEMSA at various concentrations, and porcine CPB
(7.4 nM, final), was used in place of TAFI, and lysis times
were determined in the absence of FXa. These experiments were performed
to evaluate the effect of GEMSA on the porcine CPB-mediated
prolongation of lysis.
Activation of TAFI during Fibrinolysis
To
investigate the activation of TAFI in a fibrin clot during
fibrinolysis, two sets of 12 identical clots were formed from
fibrinogen solution containing 110 nMI-TAFI, in
the presence of IIa (6.0 nM, final), Ca
(10.0 mM, final) and t-PA (441 pM, final) in
the wells of a microtiter plate. One set was formed in the presence of
FXa (100 pM) and one set in its absence. At various times a
fibrin clot from each series was solubilized by the addition of 100
µl of 0.2 M acetic acid, thereby quenching all reactions.
Once the clot was solubilized, 55 µl of a solution composed of 5%
SDS, 20% mercaptoethanol, 50 mM EDTA, 20%
glycerol/H
O containing bromphenol blue, and 40 mM Tris-HCl, pH 8.3, was added, and the solution was heated for 5 min
at 90 °C. One clot from each series was allowed to lyse completely,
and the turbidity profile from these clots was used to determine lysis
times. After lysis, this clot was similarly prepared for SDS-PAGE. The
samples were then subjected to SDS-PAGE; the resulting gel was stained,
destained, and dried, and an autoradiogram was produced.
Effect of Lys
To determine the effect of LysPgn on TAFI-induced Prolongation
of Lysis Time
Pgn on
TAFI-induced prolongation of lysis time, two fibrinogen solutions were
produced, each identical, except one contained 0.86 µM
Glu
Pgn and the other 0.90 µM Lys
Pgn. From each
stock solution, six 200-µl samples were produced to which various
concentrations of TAFI were added, such that the final concentrations
ranged from 0 to 31 nM for each group of six. Clots (100
µl) then were formed in the presence and absence of FXa (see under
``Lysis Assay''), and lysis times were determined.
Effect of TAFI on Plasminogen
Activation
Two series of identical clots were formed from
fibrinogen solution containing 140 nM TAFI and 0.47 µMI-plasminogen by adding 95-µl aliquots to
microtiter wells containing either FXa (100 pM, final) or
buffer, and IIa (6.0 nM, final), Ca
(10.0
mM, final) and t-PA (441 pM, final). Each clot was
incubated at 37 °C, and turbidity was monitored to determine lysis
time. At various times between 0 and 200 min the reactions in one clot
from each series (plus and minus FXa) were quenched, and the fibrin was
solubilized by adding 25 µl of 10.0 M urea, 2.0 M acetic acid and heating at 90 °C for 3 min. All samples were
then frozen at -20 °C prior to acid-urea
electrophoresis(28) . Samples were thawed by heating for 3 min
at 90 °C, and a 5.0-µl aliquot was counted in a LKB mini-gamma
counter (Pharmacia) to account for gel loading differences. Twenty
µl from each sample was subjected to acid-urea 7.5% polyacrylamide
gel electrophoresis (28) at 120 V for 1.5 h in a mini-slab gel
electrophoresis apparatus. The gel was stained with Coomassie Blue,
destained, and dried in Biowrap prior to autoradiography. Areas on the
gel corresponding to bands on the autoradiogram were excised, and the
normalized counts were plotted as a function of time. The species that
were excised included Glu
Pgn, Lys
Pgn, and
plasmin-
-antiplasmin complexes.
Assay and Generation of a Standard
Curve
To assess the efficiency of each step in the
purification of TAFI a turbidometric fibrinolysis assay was developed.
The assay utilizes purified components and is based on the prolongation
of lysis which accompanies II activation. Therefore, TAFI activity was
assessed by the prolongation of lysis time in the presence of FXa
relative to the lysis time in the absence of FXa. Neither the
fibrinogen solution nor BAP individually exhibited any relative
prolongation. Prolongation, however, could be reconstituted in clots
formed from BAP supplemented with II and PC/PS vesicles, or from the
fibrinogen solution supplemented with BAP. (Not all isolates of
GluPgn, however, yielded identical lysis times in fibrinogen
solution in the presence and absence of FXa, which suggests that some
plasminogen preparations contain traces of contaminating TAFI, as
inferred previously(1) ). Fig. 1shows the effect of
various volumes (0-25 µl/100-µl clot) of BAP on the lysis
time of clots formed from fibrinogen solution in the presence and
absence of FXa. The differential in lysis times plus and minus FXa
increases linearly over the concentration of BAP studied and provides a
measure of the TAFI concentration.
Figure 1:
Standard curve: effect
of various concentrations of BAP on lysis time of clots produced from
fibrinogen solution in the presence and absence of FXa. Lysis times
were determined for clots (100 µl) that were formed from fibrinogen
solution with various concentrations of BAP (0-25% v/v) in the
presence of IIa, Ca, and t-PA and incubated at 37
°C in the absence (
) or presence (
) of FXa. The
absorbance at 405 nm was monitored at 2.5-min intervals in an
enzyme-linked immunosorbent assay reader, and the time corresponding to
the transition midpoint in the reduction in turbidity is the value
reported as lysis time.
Purification of TAFI
BAP was subjected to
differential ammonium sulfate precipitation, followed by anion exchange
chromatography on Q-Sepharose. TAFI eluted at 50 mM NaCl and
was located in fractions that were typically orange in color. Gel
filtration chromatography was then performed. TAFI eluted on the
trailing edge of the second of two protein peaks. Affinity
chromatography was then performed on Pgn-Sepharose (Fig. 2). The
flow-through contained less than 2.0% of the TAFI activity. TAFI was
eluted as a small trailing protein peak. The eluate was passed over a
1.0-ml DEAE fast flow Sepharose column, and TAFI was eluted with 0.2 M NaCl, 20 mM HEPES, pH 7.4. The protein and TAFI
activity loaded on the column eluted with quantitative recovery in a
single peak of 3.0-ml total volume (Fig. 2, inset). This
procedure produced a 14,300-fold purification with a 12.1% recovery of
TAFI activity (Table) I. The isolated material migrated as a single
band on SDS-PAGE with a mass of 60 kDa (Fig. 3).
Figure 2:
Affinity chromatography of TAFI on
Pgn-Sepharose. The pooled material recovered from gel filtration was
passed over 5 ml of Pgn-Sepharose. The absorbance profile is shown. The
column was washed with HBS (fractions 15-26), then HB (fractions
27-37), and eluted with 50 mM ACA, 0.01% Tween 80
in HB. Fractions 40-60 were pooled and subjected to anion
exchange chromatography on a 1.0-ml DEAE fast flow column at 22 °C,
collecting 1.0-ml fractions. Both the absorbance profile (
) and
the prolongation activity (
) are shown (inset). A peak of
activity, coincident with an absorbance peak, eluted with 0.2 M NaCl in HB.
Figure 3:
Coomassie Blue-stained gel of fractions
from Pgn-Sepharose and DEAE chromatography. Samples were subjected to
SDS-PAGE on a 5-15% gradient gel under nonreducing conditions.
Shown are 20 µg of protein from Pgn-Sepharose load (lane
1); Pgn-Sepharose flow-through (lane 2); and 2.0, 1.5,
and 0.5 µg of protein from the 0.2 M NaCl/HB eluate of
DEAE-Sepharose fractions 41, 42, and 43 (Fig. 2, inset),
respectively (lanes 3, 4, and 5).
Antifibrinolytic Effect of TAFI
Fig. 4shows the lysis time, in both the presence and
absence of FXa, and relative lysis time for each concentration of TAFI.
A dose-dependent increase in lysis time in the absence and presence of
FXa was observed. In the absence of FXa lysis time increased, modestly
and linearly, from 30 to 50 min; however, in the presence of FXa lysis
time increased substantially more, in a saturable manner, from 30 to
130 min. A similar phenomenon was also observed in clots produced from
a fibrinogen solution not containing the inhibitors antiplasmin and
AT-III in the presence of a reduced concentration of t-PA (14.7
pM). The control times without FXa, however, were more
sensitive to the concentration of TAFI. In the absence of the
inhibitors the lysis time with FXa (Lt+FXa) increased, in a
saturable manner, from 55 to 100 min by 125 nM TAFI. The lysis
time in the absence of FXa (Lt-FXa) increased linearly from 55 to
133 min over the concentration range of TAFI. The TAFI-dependent
increase in Lt-FXa in both the presence and absence of the
inhibitors AT-III and antiplasmin indicates that the IIa (6.0
nM), present to initiate clot formation, is capable of
activating TAFI, at least partially. This effect is more pronounced in
the absence of AT-III and antiplasmin than in their presence. The
observation that, in the absence of inhibitors, at high concentrations
of TAFI the Lt-FXa (where the IIa concentration is low, 6.0
nM) exceeds Lt+FXa (where the IIa concentration is high,
maximally 730 nM) suggests that TAFI is both activated and
inactivated by IIa.
Figure 4:
Antifibrinolytic effect of TAFI. Lysis
times were determined for 100-µl clots produced from fibrinogen
solution containing various concentrations of TAFI (0-313
nM), produced in the absence () and presence of FXa
(
), from which relative lysis times were calculated (
).
All clots were formed in the presence of IIa (6.0 nM),
Ca
(10.0 mM), and t-PA (441 pM) and
maintained at 37 °C while absorbance at 405 nm was
monitored.
Thrombin Cleavage of TAFI
A reaction
solution composed of TAFI and I-TAFI in HBS and 10 mM Ca
was incubated at 37 °C with IIa for up to
2.0 h. At various times an aliquot was removed, prepared, and
subsequently subjected to SDS-PAGE followed by autoradiography.
Densitometry of both the Coomassie Blue-stained gel and the
autoradiogram shows that the cleavage patterns of TAFI and
I-TAFI by IIa were identical (data not shown). The
autoradiogram (Fig. 5) shows that TAFI is cleaved to produce
three major species that migrate with a molecular mass of 35, 25, and
14 kDa. Densitometry of the autoradiogram indicated that initially the
35- and 14-kDa bands appear, followed by the 25-kDa band. The intensity
of the 35-kDa band increased to a maximum of 24% of the total
radiolabel by 30 min. This level is maintained until 60 min, after
which it begins to decrease and approaches 14% by 120 min. This is in
contrast to both the 25- and 14-kDa bands, which increase over the
duration of the experiment to 56 and 18%, respectively. The protein
migrating at 60 kDa decreases continuously over the same time period to
13%. The cleavage pattern suggests that the 60-kDa protein is cleaved
to form the 35- and 14-kDa fragments. The 35-kDa fragment can be
proteolyzed further to yield products that migrate at 25 and 14 kDa.
The 60-, 35-, and 25-kDa bands were sequenced, and the first five
residues of each were (FQSGA; 60 kDa), (ASASY; 25 kDa) and (XQAG(A or Y); 35 kDa). The sequences corresponding to the 60-
and 25-kDa proteins were unambiguously determined, whereas the sequence
data of the 35-kDa protein did not permit the identification of the
first amino acid, and there appeared to be two possibilities for the
identity of the fifth amino acid. The sequences unambiguously assigned
correspond to that of plasma CPB(3) . However, our inability to
fit the sequence XQAG(A or Y) to the published sequence of
plasma CPB suggested that the band migrating at 35 kDa is composed of
two unresolved proteins, with respective sequences corresponding to
those of the 60- and 25-kDa proteins. Since serine is not well
quantitated by the sequencing method utilized, the single sequence
obtained may be accounted for by combining the sequences of both the
60- and 25-kDa NH
termini and disregarding the serines. The
cleavage patterns also are consistent with the identification of TAFI
as the plasma CPB precursor reported by Eaton et al. (3).
Figure 5:
Thrombin-catalyzed cleavage of TAFI. The
time course of product formation by IIa-catalyzed cleavage of TAFI was
determined by SDS-PAGE and both Coomassie Blue staining and
autoradiography. I-TAFI was incubated with IIa (545
nM) in the presence of Ca
(10.0
mM). At various times an aliquot was removed, quenched, and
subjected to SDS-PAGE on a 5-15% gradient gel. The gel was
stained and dried and then subjected to autoradiography. The
autoradiogram shows TAFI (lane 1), TAFI with IIa quenched
immediately after the addition of IIa (lane 2), and the
cleavage products after 10, 20, 30, 45, 60, 75, 90, and 120 min of
incubation (lanes 3-10),
respectively.
Activation of TAFI by Thrombin
Activation
of TAFI by IIa was investigated by incubating TAFI with IIa for various
lengths of time and then assaying the activated TAFI for prolongation
activity in the lysis assay and for CPB activity using hippuryl-Arg.
Lysis times are plotted as a function of incubation time of TAFI with
IIa in Fig. 6a. Without incubation of TAFI with IIa,
Lt+FXa was 73 min compared with Lt-FXa of 37 min and
corresponds to a relative prolongation of 1.97. Lt+FXa was
invariant at approximately 70 min for all incubation times less than 45
min. Longer incubation times, however, were accompanied by decreased
values of Lt+FXa. In contrast, Lt-FXa increased to 47 min at
the earliest time point measurable (0.5 min) and attained a value of 70
min by 10 min of incubation, which is equal to the Lt+FXa at that
point. Lt-FXa then paralleled Lt+FXa. These data correspond
to a reduction in the relative lysis time from 2 to 1 by a 10-min
incubation, which is maintained for the remaining periods of
incubation. The relative lysis time, however, does not reflect the
initial activation followed by inactivation observed with the absolute
lysis times. Profiles of both the CPB activity, as reported by rates of
hippuryl-Arg hydrolysis, and Lt-FXa as a function of incubation
time of TAFI with IIa were similar (Fig. 6b), indicating
that prolongation activity correlates with CPB activity. By comparing
both the prolongation activity and the CPB activity with the banding
patterns on gel in Fig. 5, it is apparent that the only band that
has a time course similar to these activities is the band that migrates
at 35 kDa, suggesting that formation of this species correlates with
activation of TAFI.
Figure 6:
Activation of TAFI by thrombin. Activation
of TAFI by IIa was investigated by both prolongation of lysis time (panel a) and cleavage of hippuryl-Arg (panel b).
TAFI (3.14 µM) was incubated with IIa (545 nM) in
the presence of Ca (10.0 mM) at 37 °C
for various periods of time (0-120 min). Shown in panel a are both the absolute lysis times (
,
) and relative
lysis times (squlo]) of clots produced from fibrinogen solution
containing 1.25 µl of the TAFI activation solution, IIa (6.8
nM), and both Ca
(10.0) and t-PA (441
pM). The lysis times were measured in the absence (
) and
presence (
) of FXa (100 pM, final). For each incubation
time with IIa, a third aliquot (30 µl) was removed and added to 1.0
ml of a 1.0 mM hippuryl-Arg solution. Absorbance at 254 nm was
monitored at 37 °C, and rates of hippuryl-Arg hydrolysis were
determined. Shown in panel b are rates of hippuryl-Arg
hydrolysis (
) and lysis times in the absence of FXa (
), each
as a function of incubation time of TAFI with
IIa.
Effect of GEMSA on Prolongation of Lysis Time in
Reconstituted BAP or a System of Purified Components in Which Either
Porcine CPB or TAFI Is Included
GEMSA is a competitive
inhibitor of CPB (3, 36) and therefore was used to
determine whether the prolongation observed by TAFI in a purified
system accounts for a significant proportion of the prolongation of
lysis time observed in clots produced from BAP (diluted with HBS, A = 16) reconstituted with II and PC/PS
vesicles in the presence of FXa. Porcine CPB prolonged the lysis times
of clots produced from the fibrinogen solution without the addition of
FXa. Therefore, prolongation is expressed as the ratio of the lysis
time with porcine CPB to that in its absence. Lysis time in the absence
of porcine CPB was 41 min and increased to 110 min in its presence,
giving a relative prolongation of 2.68. TAFI, in the absence of GEMSA,
produced a 2-fold increase in lysis time from 36 to 72 min when FXa was
included. The Lt+FXa of the clot produced from reconstituted BAP
was 2.17-fold longer than Lt-FXa, 60 min. Shown in Fig. 7is
relative lysis time, normalized to the maximum value, for each set of
conditions. GEMSA completely inhibited the relative prolongation
observed with porcine CPB with an EC
of 0.34
µM. GEMSA also completely inhibited the prolongation of
lysis time induced by FXa, in both the fibrinogen solution with TAFI,
and in reconstituted BAP. The EC
values were 100 and 115
µM, respectively.
Figure 7:
Effect
of GEMSA on prolongation of lysis time. Relative prolongation of lysis
at various concentrations of GEMSA was determined for clots produced
from fibrinogen solution in the presence of 7.4 nM porcine CPB
(), fibrinogen solution in the presence of 40 nM TAFI
± FXa(100 pM) (
), and BAP supplemented with 0.73
µM II, 10.0 µM PC/PS vesicles ± FXa
(100 pM) (
). Relative prolongation was normalized to the
maximum prolongation observed, i.e. that in the absence of
GEMSA, for each set of conditions. Maximum relative prolongations
observed for porcine CPB, fibrinogen solution plus TAFI ± FXa,
and reconstituted BAP ± FXa were 2.68, 2.0, and 2.17,
respectively.
These data indicate that although
activated TAFI and porcine CPB are not equally sensitive to GEMSA,
activated TAFI can be inhibited by a competitive inhibitor of porcine
CPB. These data further support the conclusion that activated TAFI is a
carboxypeptidase. As a result of the near identity of the EC values for complete inhibition of prolongation of lysis by GEMSA,
in both BAP and a system of defined components including TAFI, it is
likely that TAFI accounts for a significant proportion, if not all, of
the prolongation of lysis time observed in clots produced from plasma
in the presence of FXa.
Effect of APC on FXa-dependent Prolongation of Lysis
Time in the Presence and Absence of GEMSA
In a separate
experiment the effect of APC (100 nM) on lysis times of BAP (A = 16) supplemented with 0.73
µM II and 10.0 µM PC/PS vesicles in the
presence and absence of 500 µM GEMSA was determined. Clots
were formed in the presence of IIa (6.0 nM, final) and
Ca
(10.0 mM) plus and minus FXa (100
pM). Lysis time (20 min) was unaffected by either APC or
GEMSA, individually or in combination, in the absence of FXa, and the
2.5-fold prolongation observed in the presence of FXa was not observed
when either APC or GEMSA or both were present. In the presence of FXa,
the reduction in lysis time by APC in the absence of GEMSA was the same
as that observed when GEMSA was included also. Therefore, both APC and
GEMSA can inhibit prolongation of lysis time in reconstituted BAP, and
they both appear to inhibit prolongation through the same mechanism
since the profibrinolytic effect of APC is not observed in the presence
of GEMSA.
Activation of TAFI during
Fibrinolysis
The autoradiograms shown in Fig. 8, a and b, indicate the time courses of
I-TAFI activation during fibrinolysis of clots produced
in the absence and presence of FXa, respectively. The lysis times were
determined to be 60 min in the absence of FXa and 170 min in the
presence of FXa. In the absence of FXa, neither the 35-kDa nor the
25-kDa species is readily apparent until 60 min, whereas in the
presence of FXa the 35-kDa band is apparent by 10 min, and the 25-kDa
band is apparent by approximately 30 min. Since the 35-kDa band
correlates with both prolongation and CPB activity, it is likely that
not only is TAFI activated during fibrinolysis in the presence of FXa,
but also that TAFI accounts for the prolongation observed when II is
activated during fibrinolysis.
Figure 8:
Activation of TAFI in a clot. Two series
of identical clots were produced from fibrinogen solution containing I-TAFI in the presence of IIa, Ca
, and
t-PA, except one series was produced in the absence (panel a)
and one series in the presence (panel b) of FXa. The turbidity
at 405 nm was monitored for each clot at 37 °C. At various times
samples were quenched and subjected to SDS-PAGE on a 5-15%
gradient gel. Shown are the autoradiograms where lanes 1-12 represent the time points 0, 10, 20, 30, 40, 50, 60, 80, 100, 120,
140, and 200 min, respectively. Lysis times, indicated by arrows, were 60 min in the absence of FXa and 170 min in the
presence of FXa.
Effect of Lys
The effect of TAFI on fibrinolysis has thus
far been described using GluPgn on TAFI-induced Prolongation
of Lysis Time
Pgn in the fibrinogen solution.
Glu
Pgn, however, can be cleaved to form Lys
Pgn during
fibrinolysis(37) . Since Glu and Lys
Pgn have different
fibrin cofactor requirements for activation(38) , the effect of
TAFI on fibrinolysis using either Glu or Lys Pgn as the initial source
of plasminogen was investigated. The lysis times for Glu
Pgn in
the absence of FXa averaged 50 min, and there was a TAFI-dependent
increase in lysis time, in the presence of FXa, from 50 to 72 min.
Lysis times for clots produced with Lys
Pgn averaged 47 min in the
absence of FXa and 46 min in the presence of FXa over the range of TAFI
used. Fig. 9shows the relative prolongation observed for each
concentration of TAFI in the presence of Glu or Lys
Pgn. In
contrast to the 1.38-fold prolongation in the presence of Glu
Pgn,
no prolongation was observed in the presence of Lys
Pgn over the
same range of TAFI concentrations. These data indicate that clot lysis
by plasmin generated from Lys
Pgn is unaffected by activated TAFI.
This observation is consistent with the concept that carboxyl-terminal
lysines are less important in the activation of Lys
Pgn than
Glu
Pgn(38) , implies that activated TAFI exerts its effect
prior to the formation of Lys
Pgn or plasmin, and suggests that
activated TAFI inhibits the activation of Glu
Pgn.
Figure 9:
Effect of LysPgn on TAFI-induced
prolongation of lysis time. Relative lysis times were determined for
clots produced from fibrinogen solution containing either Glu
Pgn
(
) or Lys
Pgn (
) in the presence of TAFI and the
presence and absence of FXa.
Activation of
Two series of 12 identical clots
containing I-Glu
Pgn
during Fibrinolysis
I-plasminogen were formed, one in the presence
and one in the absence of FXa. At various times the clots were
solubilized and quenched. One clot from each series was allowed to lyse
completely. Subsequently, each sample was subjected to acid-urea gel
electrophoresis and autoradiography. Fig. 10shows the time
course of Glu
Pgn consumption and concomitant formation of
plasmin-antiplasmin for clots formed in the presence and absence of
FXa, overlaid with their respective turbidometric profiles. The lysis
times were determined to be 44 min in the absence of FXa and 87 min in
the presence of FXa, indicating an approximate 2-fold prolongation. In
both instances the Glu
Pgn consumed equals the sum of the
plasmin-antiplasmin complexes and Lys
Pgn accumulated. Consumption
of Glu
Pgn in the absence and presence of FXa ceased after the
fibrin had been lysed. In the interval from 0 to 20 min, in both
instances, rates of plasminogen activation, as inferred by the slopes
of plasminogen consumption, were approximately equal and relatively
slow. Between 20 and 40 min, however, the rate in the absence of FXa
was approximately 10-fold greater than that in the presence of FXa.
These data show that activated TAFI suppresses fibrinolysis by
attenuating the activation of Glu
Pgn, especially following the
lag phase typically characteristic of plasminogen
activation(39) .
Figure 10:
Activation of I-plasminogen
during fibrinolysis.
I-Plasminogen was used to follow the
activation of plasminogen in clots by autoradiography in the presence
of TAFI (140 nM). One series of clots was formed in the
absence and one in the presence of FXa. At various times clots were
quenched and subjected to acid-urea electrophoresis and
autoradiography. Bands on the gel corresponding to radiolabeled
Glu
Pgn, Lys
Pgn, and plasmin-antiplasmin complexes were
excised, counted, and concentrations of each species were calculated.
Shown is the consumption of Glu
Pgn (
,
), formation
of plasmin-antiplasmin complexes (
,
), and lysis profiles
(- -,--) in the absence (
,
,-
-) and presence (
,
],--) of
FXa.
)SO
, anion exchange chromatography, gel
filtration, and affinity chromatography using Pgn-Sepharose. The
purification scheme resulted in a 12.1% recovery of activity with a
14,300-fold purification. Based on amino-terminal sequence data we have
identified TAFI as a plasma CPB, as described by Eaton et
al.(3) . Utilizing a
of 26.4, a M
of 48,422, and a recovery of
12.1% we calculate that the concentration of TAFI in plasma is
approximately 50 nM.
identical to that of TAFI in a
system of defined components, we conclude that the prolongation
observed in plasma can most likely be attributed to the
carboxypeptidase activity generated by the activation of TAFI by IIa.
Pgn. Since activated TAFI is unable to modulate fibrinolysis
when Glu
Pgn is replaced by Lys
Pgn, it likely does not
effectively inhibit either activation of Lys
Pgn by t-PA or
plasmin activity directly. Furthermore, the substrate specificity of
activated TAFI (i.e. plasma CPB) appears to be limited to
basic amino acids (3, 40). Thus, a reasonable hypothesis is that
activated TAFI modifies the fibrin cofactor by removal of
carboxyl-terminal lysines, thereby inhibiting the activation of
Glu
Pgn. An alternative hypothesis is that the released lysine is
antifibrinolytic. This is unlikely, however, in view of the results
with Lys
Pgn. The lack of an observed prolongation when
Lys
Pgn is the substrate for t-PA is likely a result of its
activation being less dependent on the presence of a cofactor with
carboxyl-terminal lysines(38) . The results with Lys
Pgn
also suggest that the Glu to Lys
Pgn conversion may contribute to
the regulation of fibrinolysis by attenuating the effects of activated
TAFI.
ACA, epsilon-amino
caproic acid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.