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Blood, Vol. 92 No. 6 (September 15), 1998:
pp. 2075-2083
Lysis of Plasma Clots by Urokinase-Soluble Urokinase Receptor
Complexes
By
Abd Al-Roof Higazi,
Khalil Bdeir,
Edna Hiss,
Shira Arad,
Alice Kuo,
Iyad Barghouti, and
Douglas B. Cines
From the Department of Clinical Biochemistry, Hadassah University
Hospital and Hebrew University-Hadassah Medical School, Jerusalem,
Israel; and the Department of Pathology and Laboratory Medicine,
Hospital of the University of Pennsylvania, Philadelphia, PA.
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ABSTRACT |
Single-chain urokinase plasminogen activator (scuPA), the unique
form secreted by cells, expresses little intrinsic plasminogen activator activity. scuPA can be activated by proteolytic cleavage to
form a two-chain enzyme (tcuPA), which is susceptible to inhibition by
plasminogen activator inhibitor type I (PAI-1). scuPA is also activated
when it binds to its cellular receptor (uPAR), in which case the
protein remains as a single chain molecule with less susceptibility to
PAIs. Fibrin clots are invested with PAI-1 derived from plasma and from
activated platelets. Therefore, we compared the fibrinolytic activity
of complexes between scuPA and recombinant soluble uPAR (suPAR) to that
of scuPA, tcuPA, and tcuPA/suPAR complexes. scuPA/suPAR complexes
mediated the lysis of plasma-derived fibrin clots 14-fold more
extensively than did equimolar concentrations of scuPA and threefold
more extensively than did tcuPA or tcuPA/suPAR, respectively. The
enhanced catalytic activity of scuPA/suPAR required that all three
domains of the receptor be present, correlated with its PAI-1
resistance, was not dependent on fibrin alone, and required a plasma
cofactor that was identified as IgG. Human IgG bound specifically to
suPAR and scuPA/suPAR as determined by using affinity chromatography
and immunoprecipitation. Plasma depleted of IgG lost most of its
capacity to promote the fibrinolytic activity of scuPA/suPAR, and the
activity of the complex was restored by adding plasma concentrations of
purified IgG. These studies indicate that scuPA/suPAR can function as a
plasminogen activator in a physiological milieu.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
UROKINASE-TYPE PLASMINOGEN activator
(uPA) has been implicated in several important biological processes
including angiogenesis1-5; wound healing6;
inflammation7; ovulation and placental
development8-10; atherosclerosis, aneurysm, and neointima
formation11-15; and the formation of tumor
metastases.16-19 The results of recent studies also show
that mice with targeted disruption of the uPA gene are fertile, develop
normally, and show little tendency towards spontaneous
thrombosis20,21 but are more prone to form thrombi when
exposed to endotoxin20 or hypoxia22 or when the
uPA gene is disrupted in otherwise healthy tissue-type plasminogen
activator (tPA) / mice.23
Downregulation of uPA expression in wild-type mice contributes to
fibrin deposition in response to endotoxin as well.24 Taken
together, these data suggest an important and unappreciated role for
uPA in fibrinolysis. However, the mechanism by which uPA promotes
fibrinolysis has not been resolved.
uPA is synthesized as a single chain molecule that expresses
little25 or essentially no26 intrinsic
plasminogen activator activity. There is evidence to suggest that the
activity of single-chain uPA (scuPA) may be promoted by fibrin
clots,27,28 but not nearly to the same extent as might be
inferred from the difference in the propensity of transgenic mice
lacking both tPA and uPA to develop thrombosis compared with mice
lacking tPA alone.21,23 scuPA can be activated by
proteolytic cleavage at leu158-ile159
converting it to a two-chain molecule.29 However, two-chain enzyme uPA (tcuPA) is rapidly and essentially irreversibly inactivated by plasminogen activator inhibitor type I (PAI-1), which is present in
plasma in molar excess.30 PAI-1 is present in even higher concentrations in clots as a result of its release locally by activated
platelets31 and vascular cells,32 and its
activity is stabilized by clot-bound vitronectin.33,34
These observations raise a question as to how tcuPA could play such a
seemingly important role in intravascular clot lysis.
One insight into the mechanism by which uPA may contribute to clot
lysis comes from the in vitro observation that scuPA can also be
activated when it binds to its receptor.35,36 Fibrin clots
typically contain neutrophils and macrophages37,38 that express urokinase receptors39 and that accelerate
uPA-mediated clot lysis.37,38,40 In accord with this, lysis
of fibrin clots is impaired when macrophages from patients with
paroxysmal nocturnal hemoglobinemia, which lack uPA receptor (uPAR),
are compared with cells from normal individuals.41 These
studies suggest that the binding of scuPA to uPAR may facilitate clot
lysis at plasma concentrations of the reactants.
scuPA bound to its receptor expresses enzymatic activity comparable
with that of tcuPA although it remains a single chain molecule.35 Further, the enzymatically active complex
between scuPA and uPAR is relatively resistant to PAI-1.42
However, to date, the activation of scuPA by its receptor has been
shown only in the presence of the synthetic plasmin substrate
H-D-norleucyl-hexahydrotyrosyl-lysine-p-nitroanilide D-lactate.36,43 This peptide inhibits plasmin-mediated
conversion of scuPA to tcuPA, but the possibility that it also exerts a
direct effect on the activity of the scuPA/uPAR complex has not been excluded. This leaves open the question as to whether plasma clots contain a physiological activator of the scuPA/uPAR complex and whether
this activator may contribute to the seemingly critical role of uPA in
fibrinolysis.
To address these issues, we asked whether complexes between scuPA and
its receptor are capable of lysing plasma clots in the absence of
potentially activating synthetic peptides. Our data indicate that
plasma clots contain a soluble cofactor that stimulates the
fibrinolytic activity of scuPA/suPAR complexes to levels that exceed
the activity of tcuPA.
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MATERIALS AND METHODS |
Materials.
Recombinant human scuPA, the amino terminal fragment of scuPA (amino
acids 1-143; ATF), and low molecular weight scuPA (amino acids 144 to
411; LMWscuPA) prepared from recombinant human scuPA, recombinant human
soluble uPA receptor (suPAR; amino acids 1-281), and chymotryptic
fragments of suPAR consisting of either domain 1 alone (amino acids
1-87; DI) or domains 2 and 3 (amino acids 88-281; DII-III) were all the
gifts of Drs Jack Henkin and Andrew Mazar (Abbott Laboratories, Abbott
Park, IL) and have been characterized in detail in previous
publications.35,42-45 The plasmin-insensitive scuPA
variant, scuPA-glu158, was kindly provided by Dr Bradford
Schwartz (University of Wisconsin, Madison, WI).46
Lyophilized human fibrinogen essentially free of plasminogen and
thrombin (catalogue # F-4883) and human  -thrombin were obtained from
Sigma (St Louis, MO). Bovine fibrinogen containing trace amounts of
plasminogen was obtained from Calbiochem-Novabiochem (San Diego, CA).
Recombinant active mutant PAI-1 (Catalogue # 1094),
2-antiplasmin, and high molecular weight human tcuPA
(Catalogue # 124) were purchased from American Diagnostica (Greenwich,
CT). Protein G-Agarose was purchased from GIBCO BRL, (Bethesda, MD). Purified human IgG was obtained from Organon Teknika Corp (West Church,
PA). Plasminogen was prepared from normal human plasma as
described.47 To prepare plasma, 450 mL of blood was drawn from healthy volunteers into 63 mL citrate-phosphate-dextrose solution
(CPD; 1.66 g sodium citrate, 61 g dextrose, 206 mg citric acid, and 140 mg monobasic sodium phosphate).
Fibrinolysis assessed by clot size.
In some experiments clots were prepared by adding human -thrombin
(0.2 NIH u/mL final concentration) for 1 hour at 37°C to fibrinogen
containing trace amounts of plasminogen that had been reconstituted to
a concentration of 3 mg/mL in phosphate-buffered saline (PBS), pH 7.4. In other experiments, clots were prepared by adding thrombin (0.4 NIH
U/mL final concentration) to citrated human plasma for 1 hour at
37°C. In a third set of experiments, human or bovine fibrinogen was
resuspended in human serum to a final concentration of 3 mg/mL, and
thrombin (0.4 NIH U/mL) was added as above. In each case, clots were
incubated for 1 hour at room temperature in 24-well tissue culture
plates (Costar, Cambridge, MA). Aliquots of PBS containing scuPA (10 pmol in 10 µL PBS) in the presence or absence of an equimolar
concentration of suPAR was added to the surface of each clot for 2 hours at 37°C by which time digestion was evident. The clots were
then washed several times with PBS, incubated overnight with 0.2% of trypan blue, rinsed four times with PBS, and photographed. The pictures
were scanned by using a Hoefer GS 300 densitometer (Amersham Pharma
Biotech, Piscataway, NJ) and the size of the lytic zones were
calculated by using the NIH Image program. In other experiments, the effect of scuPA, scuPA/suPAR, scuPA/suPAR/ATF, LMWscuPA/suPAR, and
tcuPA on the lysis of plasma clots was compared as described in detail
below.
Fibrinolysis assessed by release of radioactivity.
Purified human fibrinogen was radiolabeled with
125I48 and resuspended to a specific activity
of 30,000 cpm/mL in either PBS containing 3 mg/mL fibrinogen and 0.5 µmol/L Glu-plasminogen or in plasma. Clots were formed from 0.4 mL
soluble fibrinogen or from plasma placed in 16-mm diameter tissue
culture wells (Costar) by adding thrombin (0.2 and 0.4 U/mL final
concentration, respectively). Each plasminogen activator (10 pmol in 10 µL PBS) was then added directly to the center of the formed clot for
2 hours, the clots were washed with PBS, and the radioactivity released
into the lavage solution at specific times was measured. In other
experiments, fibrinolysis was measured as previously
described.49 Briefly, radiolabeled fibrin or plasma clots
were overlaid with serum or PBS containing 25 nmol/L
plasminogen activator (scuPA, scuPA/suPAR complex, or tcuPA) for
varying periods of time at 37°C, and the release of radiolabeled
soluble fibrin degradation products was measured. The percent lysis was
calculated to take into account any theoretical changes in the volume
of distribution of the released cpms; however, no significant
differences were observed when aliquots were taken from different
samples at the various time points or from the same well sequentially.
Effect of PAI-1 on fibrinolysis.
Purified fibrin clots trace labeled with 125I were prepared
as described above. PBS (400 µL) containing 25 nmol/L plasminogen activator (scuPA/suPAR, tcuPA, or tcuPA/suPAR) was added to each clot
in the presence or absence of 50 nmol/L PAI-1 for 200 minutes at
37°C. An aliquot was then removed from the supernatant and counted
for radioactivity.
Preparation of suPAR-Sepharose.
suPAR synthesized in SP2/0 cells was purified as described
previously.42,44,46 suPAR (5 mg/mL in 0.5 mol/L
NaHCO3, pH 8.5) was linked to CNBr-Sepharose (Sigma)
according to the manufacturer's instructions. The resin (1.5 × 50 cm) was washed with 10 column volumes of PBS, 10 column volumes of
PBS containing 1 mol/L KCl, 10 column volumes of PBS, 10 column volumes
of PBS containing 0.2 mol/L glycine, pH 3.0, and finally with
sufficient PBS to re-equilibrate the column to pH 7.4 before use.
The coupling efficiency was greater than 95%, yielding 2.85 mg
suPAR/mL gel.
Binding of plasma proteins to immobilized suPAR.
suPAR-Sepharose (1 mL) was suspended in PBS in the presence or absence
of 100 nmol/L scuPA for 1 hour at 4°C. Ten milliliters of citrated
plasma or PBS was added for 1 hour at 4°C. The beads were
centrifuged, washed four times with PBS and once with PBS/glycine buffer, pH 3.0, and resuspended in PBS containing 4% sodium dodecyl sulfate (SDS). One aliquot of the solubilized proteins was analyzed by
using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described below. From another aliquot, the amino acid
sequences of its constituent proteins were determined (see below). In
another set of experiments, an aliquot of the suspension, before incubation with glycine buffer, was added to a
125I-fibrinogen-derived clot containing 0.5 µmol/L
Glu-plasminogen and the fibrinolytic activity was determined as
described above.
Amino acid sequencing.
Proteins bound to suPAR-Sepharose (50 µL) that had been
preincubated with plasma as described above were analyzed by adding 0.5 mol/L Tris, pH 6.5 containing 10% SDS, 0.1% Coomasie Blue and 5%
 -mercaptoethanol. The samples were boiled for 2 minutes and 25-µL
aliquots were applied to an SDS-12% polyacrylamide gel under reducing
conditions as described.50 Individual bands were then cut
out for sequencing. Sequencing was performed at the Bletterman Macromolecular Research Laboratory of the Hebrew University in the
Interdepartmental Equipment Unit of the Faculty of Medicine using a
Perkin-Elmer Applied Biosystems Division model 429 Precise Microsequencer System (The Perkin-Elmer Corp, Norwalk, CT).
Binding of suPAR and IgG.
Five milliliters of normal human plasma was incubated with 1 mL of
protein G-Agarose for 2 hours at 4°C. The beads were centrifuged at
5,000 rpm for 10 minutes and the plasma was decanted. No residual IgG
was detected in the fall-through fraction on Western blotting. The
capacity of this IgG-depleted plasma and normal plasma to promote clot
lysis by scuPA/suPAR (as described above) and to precipitate suPAR (see
below) were then compared. suPAR was radiolabeled with 125I
(New England Nuclear, Cambridge, MA) as described.51
125I-suPAR (50 nmol/L) was incubated with 0.1 mL normal
citrated plasma (as a source of IgG) or with purified IgG (13 µmol/L)
and 0.5 mL protein G-Agarose for 2 hours at 4°C. The mixture was
centrifuged as above, the precipitate was washed four times with PBS,
and the radioactivity precipitated by protein G-Agarose was measured. Nonspecific binding was determined by using plasma that had been depleted of IgG and by measuring the precipitation of an irrelevant 125I-labeled protein (plasminogen). All experiments were
repeated at least three times.
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RESULTS |
suPAR stimulates the fibrinolytic activity of scuPA on plasma clots.
The purpose of this study was to ask whether complexes formed between
scuPA and its receptor are capable of mediating the lysis of plasma
clots by activating plasminogen. Further, we asked whether
this activity was comparable with that of tcuPA.
To address this question, we began by studying the capacity of scuPA
and scuPA/suPAR to mediate the lysis of fibrin clots formed by adding
thrombin to purified unlabeled bovine
fibrinogen, unlabeled human fibrinogen, or
radiolabeled purified human fibrinogen, each of which contained 0.5 µmol/L human
plasminogen. suPAR inhibited scuPA-mediated plasminogen activator
activity determined by using unlabeled bovine (Fig 1A) and human (not
shown) fibrinogen and 125I-labeled human fibrinogen by
approximately 40% and 50% (Fig 2A), respectively. This inhibition is
similar in extent to that seen previously by using the small
chromogenic peptide S-2251 as the substrate.43
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| Fig 1.
Effect of suPAR on scuPA-mediated clot lysis: zymography.
(A) Lysis of clots prepared from purified bovine fibrinogen. Clots were
prepared by adding thrombin (0.2 NIH U/mL final concentration) to
bovine fibrinogen (3 mg/mL) in PBS for 60 minutes at room temperature.
scuPA (10 pmol in 10 µL PBS or equimolar concentrations of suPAR and
scuPA were added for 2 hours at 37°C and the size of each lytic
area was measured. The lytic areas generated by scuPA were 1.60 and
1.63 cm2, respectively; the corresponding areas for
scuPA/suPAR were 1.19 and 1.05 cm2, respectively. In this
and in each panel below, the experiment shown is representative of
three so performed. (B) Lysis of clots prepared from human plasma.
Clots were prepared by adding thrombin (0.4 NIH U/mL final
concentration) to citrated human plasma for 60 minutes at room
temperature. scuPA, scuPA/suPAR or suPAR (10 pmol in 10 µL) were
added for 2 hours at 37°C. The size of the lytic areas generated by
scuPA/suPAR were 0.94 and 0.90 cm2. Effect of ATF on
suPAR/scuPA-induced lysis of plasma clots. (C) scuPA/suPAR (10 pmol in
10 µL PBS) was added to the plasma clots for 2 hours at 37°C in
the absence or presence of 500 pmol ATF.
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| Fig 2.
Effect of suPAR on scuPA-mediated clot lysis: release of
radioactivity. (A) Lysis of fibrin versus plasma clots. Left side:
clots prepared from purified bovine fibrinogen. scuPA (10 pmol in 10 µL PBS; open bar) or equimolar concentrations of scuPA/suPAR complex
(solid bar) was incubated with clots prepared from
125I-labeled human fibrinogen for 2 hours at 37°C, and
the radioactivity released into the supernatant was measured. The data
are expressed relative to the condition that produced maximal
fibrinolysis. In this and in each panel below, the mean ± standard
error of the mean (SEM) of three experiments is shown. Right side:
plasma clots. Plasma clots, trace labeled with
125I-fibrinogen, were prepared as described in Fig 1B.
scuPA (open bar) or scuPA/suPAR (solid bar) was added for 2 hours at
37°C, and the radioactivity released into the supernatant was
measured. (B) Lysis of plasma clots. Left side: effect of ATF.
125I-labeled plasma clots were incubated with scuPA/suPAR
(10 pmol in 10 µL PBS) alone or in the presence of 500 pmol ATF for 2 hours at 37°C, and the radioactivity released into the supernatant
was measured. Right side: Effect of scuPA-glu158.
scuPA-glu158 (10 pmol in 10 µL PBS; designated *scuPA)
was added to 125I-labeled plasma clots in the absence or
presence of equimolar concentrations of suPAR for 2 hours at 37°C,
and the radioactivity released into the supernatant was measured.
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We next asked whether suPAR has a similar effect on the capacity of
scuPA to mediate the lysis of clots formed by adding thrombin to plasma
(Fig 1B) or to plasma supplemented with trace amount of
125I-human fibrinogen (Fig 2A). The fibrinolytic activity
initiated by scuPA on plasma clots was less than 10% of that seen by
using clots prepared from purified fibrinogen (compare Fig 1A v
1B and Fig 2A, left v right side). However, in contrast to the
modest inhibition seen when purified fibrin was used as a substrate, suPAR caused a 14-fold enhancement of scuPA-mediated fibrinolysis of
plasma clots (Fig 2A). Fibrinolysis was stimulated to the same extent
when plasma clots were incubated overnight with 0.5 nmol/L scuPA/suPAR
compared with scuPA alone (not shown).
The time course of clot lysis by scuPA and scuPA/suPAR was then
examined. Plasma-derived clots were overlaid with serum or with PBS
containing scuPA or scuPA/suPAR. Plasma clots suspended in either
medium were lysed more rapidly by scuPA/suPAR than by scuPA alone
(Fig 3); half-maximal fibrinolysis was
achieved at 54 minutes by scuPA/suPAR compared with 210 minutes
by scuPA. The same result was obtained when clots were overlaid with
plasma (not shown). scuPA/suPAR-mediated fibrinolysis was dose
dependent. Doubling the concentration of the complex decreased the time
needed for half maximal fibrinolysis from 54 to 33 minutes, whereas a 50% decrease in the concentration of the complex prolonged the time to
achieve half maximal fibrinolysis to 102 minutes (not shown).
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| Fig 3.
Time course of lysis of plasma clots by scuPA/suPAR.
125I-labeled clots prepared from plasma were overlaid with
serum and incubated with 25 nmol/L scuPA in the presence (solid
symbols) or absence (open symbols) of equimolar concentrations of suPAR
for 210 minutes at 37°C. The radioactivity released into the
supernatant was measured. The clots were overlaid with serum ( and
 ) or PBS ( and  ), which contained either scuPA alone ( and
) scuPA/suPAR ( and ), suPAR alone ( ) or scuPA/suPAR + 250 nM ATF in PBS (*). The mean ± SEM of three experiments is
shown.
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Four sets of experiments were then performed to ask whether the
stimulatory effect of suPAR on scuPA-mediated lysis of plasma clots was
induced by the cleavage of scuPA to tcuPA by contaminating proteases.
First, the addition of ATF, which inhibits the interaction of scuPA
with suPAR, almost completely blocked the stimulatory effect of suPAR
on scuPA-mediated fibrinolysis (Figs 1C, 2B, and 3). Second, the
plasminogen activator activity of LMWscuPA, which is unable to bind to
suPAR because it lacks the ATF but which is as readily cleaved to a
two-chain enzyme as is full length scuPA, was not affected by the
presence of suPAR (not shown). Third, suPAR enhanced the lysis of
plasma clots mediated by the plasmin-resistant variant,
scuPA-glu158, more than 20-fold (Fig 2B). Fourth, the
plasminogen activator activity of scuPA/suPAR on plasma clots exceeded
that of tcuPA or tcuPA/suPAR (Fig 4).
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| Fig 4.
Comparison of fibrinolysis mediated by tcuPA/suPAR and
scuPA/suPAR. 125I-plasma-derived clots overlaid with serum
were incubated with 25 nmol/L scuPA/suPAR ( ), tcuPA ( ), or
suPAR/tcuPA ( ) at 37°C for the indicated times, and the
radioactivity released into the supernatant was measured. The mean ± SEM of three experiments is shown.
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All three domains of suPAR were required to enhance scuPA-mediated
fibrinolysis. suPAR fragments that contain DI or DII-DIII can bind
scuPA and stimulate its activity in the presence of a small plasmin
substrate.44 However, neither fragment, alone or together,
stimulated scuPA activity in the presence of plasma-derived clot nor
inhibited scuPA-mediated cleavage of the fibrinogen-derived clot (not
shown).
Mechanism of stimulation of suPAR/scuPA by plasma.
These experiments show that suPAR stimulates the enzymatic activity of
scuPA in the presence of clots formed from human plasma, whereas it
modestly inhibits scuPA-mediated lysis of clots formed from purified
fibrinogen. Experiments were then performed to address two nonmutually
exclusive mechanisms to explain these results. First, the possibility
was considered that the activity of scuPA in the absence of suPAR may
depend on its conversion to tcuPA, which is then rapidly inhibited by
PAI-1 in plasma and within the clot. In this case, the enhanced
fibrinolytic activity of scuPA/suPAR on plasma clots may result, in
part, from its relative resistance to PAI-1.42,52
Consistent with this hypothesis, the lysis of 125I-fibrin
clots enriched with PAI-1 (50 nmol/L) by tcuPA and suPAR/tcuPA were
each inhibited approximately 80%, whereas the activity of scuPA/suPAR
was inhibited less than 20% (Fig 5).
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| Fig 5.
Susceptibility of fibrinolysis to PAI-1.
125I-labeled fibrin clots in PBS ( ) or PBS supplemented
with 50 nmol/L PAI-1 (+) were incubated with 25 nmol/L scuPA/suPAR,
tcuPA, or tcuPA/suPAR for 200 minutes at 37°C, and the
radioactivity released into the supernatant was measured. The mean ± SEM of three experiments is shown.
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We then considered a second possibility, ie, that plasma contains one
or more factors that are not present in purified fibrinogen that
promote the activity of the scuPA/suPAR complex directly. To test this
possibility, clots were formed by adding thrombin to purified bovine or
human fibrinogen suspended in human serum. scuPA/suPAR caused the lysis
of this serum-enriched clot to a greater extent than did scuPA alone,
the same pattern seen by using clots prepared directly from plasma (not
shown). Further, lysis of serum-enriched clots scuPA/suPAR was
abolished by ATF. The same results were obtained when scuPA/suPAR or
scuPA were added to fibrin clots covered with serum or plasma compared
with PBS. The stimulatory activity of plasma was not caused by
anticoagulants, because the addition of CPD to clots
formed by fibrinogen did not inhibit fibrinolysis mediated by scuPA nor
did it stimulate fibrinolysis by scuPA/suPAR (not shown).
Isolation of a plasma stimulatory cofactor.
One explanation for the latter set of results is that plasma contains
one or more proteins that bind to suPAR or to scuPA/suPAR and
promote its activity. To identify such a potential cofactor, plasma was
passed over suPAR-Sepharose, or suPAR-Sepharose preincubated with
scuPA, and the tightly adherent proteins (resistant to elution at pH
3.0) were analyzed by SDS-PAGE (Fig 6).
Four distinct bands were identified in the absence (lane 3) or presence
(lane 4) of scuPA. Identical results were obtained when the experiment
was repeated with plasma from four different donors. Bands a, c, and d
were not seen when the incubation was performed in the presence of PBS
(± scuPA) instead of plasma (lanes 2 and 5) indicating the proteins
derived from the added plasma and not from the column itself. None of
these bands were seen when plasma was incubated with a control
Sepharose column (not shown). Only band b was seen when suPAR-Sepharose
was analyzed by SDS-PAGE.
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| Fig 6.
SDS-PAGE analysis of the proteins bound to
suPAR-Sepharose. Lane 1, molecular weight markers; lane 2, suPAR-Sepharose incubated with PBS; lane 3, suPAR-Sepharose incubated
with plasma; lane 4, suPAR-Sepharose incubated with scuPA and plasma;
lane 5, suPAR-Sepharose incubated with scuPA and PBS.
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We then asked whether the ligand(s) that bound to scuPA/suPAR-Sepharose
stimulated the fibrinolytic activity of the complex. To address this
issue, plasma was incubated with scuPA/suPAR-Sepharose and the column
was washed extensively. The washed gel was then added directly to a
fibrinogen-derived clot supplemented with plasminogen. The results
shown in Fig 7 indicate that the
fibrinolytic activity mediated by scuPA/suPAR-Sepharose preincubated
with plasma and then washed was considerably higher than that of
scuPA/suPAR-Sepharose incubated with PBS and washed in the same manner.
suPAR-Sepharose incubated with plasma and washed had no significant
plasminogen activator activity.
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| Fig 7.
Effect of suPAR-plasma suspension on the lysis of fibrin
clots by scuPA/suPAR. An aliquot (10 µL) of the washed
plasma/suPAR-Sepharose suspension (analyzed by SDS-PAGE in Fig 6) was
added to 125I-labeled fibrin clots for 2 hours at 37°C
and the release of radioactivity into the supernatant was measured. The
first lane (solid bar) shows the activity of suPAR/scuPA on the fibrin
clot. The second, middle lane, (open bar) shows the activity of
scuPA/suPAR in the presence of the plasma/suPAR-Sepharose suspension.
The third lane (hatched bar) shows the fibrinolytic activity of the
plasma/suPAR-Sepharose suspension alone. The mean ± SEM of three
experiments is shown.
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We then performed amino-terminal sequence analysis of the two fastest
migrating bands (c and d). The sequences of the proteins were
EVQLVESGGXLVQPGXS and EIVMTQSPXTLS, respectively, where X refers to an
ambiguous signal at that location (Table
1). By using the Wisconsin Package Version 9.0-UNIX, the d sequence was identified as the amino terminus of the variable region of kappa V-III
of human IgG KV3F. The N-terminal amino acid sequence of band c is
identical to that of human IgG heavy chain V-III HV3T (Table 1). Bands
a, b, and c comigrated with bands found on SDS-PAGE analysis of
purified human IgG under reducing conditions (not shown). Band b
migrated at a higher molecular weight than suPAR alone, but identically
with suPAR-Sepharose, and was not found in the eluate from
Sepharose-albumin; therefore, this band appears to represent trace
amounts of the protein-Sepharose complex that dissociated from the
column.
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Table 1.
N-Terminal Amino Acid Sequence of the Proteins Bound to
suPAR-Sepharose and the Isolated Chains of a Human IgG
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Effect of IgG on scuPA/suPAR-mediated fibrinolytic activity.
This unexpected result led us to perform a series of experiments to
determine whether the plasma cofactor activity could indeed be ascribed
to IgG. The first question that was asked was whether IgG bound avidly
to suPAR in a plasma environment. To study this, 125I-suPAR
was incubated with plasma and then with protein G-Agarose. The results
shown in Fig 8A indicate that suPAR was
immunoprecipitated by protein G-Agarose in plasma, whereas under the
same conditions radiolabeled plasminogen was not. When the same
experiment was performed with IgG-depleted plasma, precipitation of
suPAR was reduced by greater than 75% (not shown). Similar results
were obtained when serum or IgG-depleted serum was used instead of plasma or IgG-depleted plasma (not shown). Similar results were also
seen when purified human IgG (13 µmol/L) was substituted for plasma
(Fig 8B). No specific immunoprecipitation of 125I-suPAR was
seen when rabbit serum was used as a source of IgG instead of human
serum (not shown).
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| Fig 8.
(A) Immunoprecipitation of suPAR by plasma IgG.
125I-suPAR or 125I-plasminogen (final
concentration 50 nmol/L) was added to 0.5 mL native plasma [black
bars: (+)] or to IgG-depleted plasma [open bars: ()]. The
mixture was incubated with protein G-Agarose, centrifuged, and washed
repeatedly with PBS, and the precipitated radioactivity was measured.
The mean ± SEM of three experiments is shown. (B) Immunoprecipitation
of suPAR by purified IgG. Protein G-Agarose (0.5 mL) was added to a
mixture containing either 50 nmol/L 125I-suPAR or 50 nmol/L
125I-plasminogen plus 1 mg/mL IgG (black bars) or 1 mg/mL
BSA (open bars), and the precipitated radioactivity was measured as
above. The mean ± SEM of three experiments is shown.
|
|
We then asked whether IgG stimulates the fibrinolytic activity of
scuPA/suPAR, simulating the effect of plasma. We found that IgG indeed
stimulates the fibrinolytic activity of scuPA/suPAR in a dose-dependent
manner (Fig 9), whereas no effect on the
activity of scuPA alone was observed (not shown). Maximal stimulation
was approached at physiological plasma IgG concentrations (Fig 9). The
stimulatory effect of IgG required the presence of scuPA as well as
plasminogen (not shown), excluding either plasminogen activator
activity in the preparation or a direct proteolytic effect on fibrin.
Further, 2PI (1.5 µmol/L) inhibited
scuPA/suPAR-mediated fibrinolysis to the same extent in the presence or
in the absence of IgG (not shown), consistent with fibrinolysis being
mediated through the elaboration of plasmin.
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| Fig 9.
Effect of IgG-addition on lysis of plasma-derived clots.
125I-fibrin clots were incubated with 25 nmol/L scuPA/suPAR
in the presence of the indicated concentrations of purified human IgG
for 2 hours at 37°C, and the radioactivity released into the
supernatant was measured. The mean ± SEM of three experiments is
shown.
|
|
Lastly, we compared the lysis of plasma clots prepared from normal and
IgG-depleted plasma. scuPA/suPAR-mediated fibrinolysis of clots
prepared from IgG-depleted plasma was clearly delayed relative to those
prepared from normal plasma (Fig 10) and
the lytic activity was restored totally when IgG-depleted plasma was supplemented with Sepharose-suPAR-bound IgG or purified IgG (13 µmol/L; not shown).
View larger version (13K):
[in this window]
[in a new window]
| Fig 10.
Effect of IgG-depletion on lysis of plasma-derived
clots. scuPA/suPAR complex (25 nmol/L) was added to
125I-labeled clots formed by the addition of thrombin to
normal plasma or to IgG-depleted plasma (). Fibrinolysis was
determined as described in the legend to Fig 2. The mean ± SEM of
three experiments is shown. The error bars for certain data points were
too small to be appreciated on the graph.
|
|
| |
DISCUSSION |
The results of this study show that scuPA/suPAR mediates the lysis of
plasma clots faster and to a greater extent than does equimolar
concentrations of scuPA or tcuPA alone or tcuPA/suPAR in this
experimental system. The fibrinolytic activity of scuPA/suPAR was not
caused by proteolytic conversion of scuPA to tcuPA for several reasons.
First, ATF, which blocks binding of scuPA to its receptor, totally
inhibited the activity of scuPA/suPAR but did not inhibit the
fibrinolytic activity of tcuPA. Second, suPAR did not stimulate the
fibrinolytic activity of LMWscuPA, which cannot bind to the receptor
but which is readily converted to LMWtcuPA. Third, the activity of
scuPA/suPAR exceeded that of either tcuPA or tcuPA/suPAR. Fourth, suPAR
stimulated the fibrinolytic activity of the plasmin-insensitive scuPA
variant, scuPA-glu158.53
The greater potency of scuPA/suPAR compared with tcuPA to promote the
lysis of plasma clots was explained, in part, by the presence of
fibrinolytic inhibitors in the clot, such as PAI-1, to which the
complex is relatively resistant.42 However, the enhanced
activity is also compatible with the possibility that plasma clots
contain factors that augment the activity of scuPA/suPAR. This latter
possibility is supported by the observation that fibrinolysis mediated
by scuPA/suPAR is actually enhanced when plasma or serum is added to
clots prepared from purified fibrin. This finding also indicates that
the regulator is present in plasma in an active form and is not
activated further by clotting.
The finding that one plasma factor that promotes suPAR/scuPA-mediated
fibrinolysis in vitro is IgG was unexpected. However, this conclusion
was supported by several findings. First, IgG was retained specifically
when plasma was passed over a suPAR-Sepharose column. Second, IgG
specifically immunoprecipitated radiolabeled suPAR but not labeled
plasminogen when studied in parallel. Third, scuPA/suPAR mediated
considerably greater fibrinolytic activity on clots prepared from
normal plasma than from IgG-depleted plasma, whereas no effect of
IgG depletion on the activity of scuPA was observed. Fourth, IgG
stimulated scuPA/suPAR mediated fibrinolysis directly in a
dose-dependent manner but had no effect on the activity of scuPA or
tcuPA. Fifth, IgG restored the fibrinolytic activity of scuPA/suPAR on
clots prepared from IgG-depleted plasma.
There is precedent for finding a link between IgG and the fibrinolytic
system. It has previously been reported that plasminogen binds to
plasmin-cleaved Fab fragments of IgG and IgG-containing immune
complexes and that these fragments provide a template for tPA-mediated
plasminogen activation.54 In our experiments,
125I-plasminogen was not precipitated from plasma by
protein G-Agarose nor did plasminogen copurify with IgG on
suPAR-Sepharose or on scuPA/suPAR-Sepharose, most likely because
sufficient IgG had not undergone proteolytic cleavage or
aggregation54 under these conditions. However, our
findings and those of others54 raise the possibility that
IgG may both promote the adhesion of leukocytes that express both
Fc receptor and uPA receptors to fibrin clots as well as
promoting cell migration by providing a surface on which urokinase and
its substrate can assemble.40,41,55 The finding of a single
amino acid sequence suggests that only a subset of immunoglobulin
molecules associate tightly with suPAR and the mechanism of this
attachment remains unsettled. Thus, it is evident that considerably
more study is required to understand the mechanism by which IgG may
modulate clot lysis and its importance relative to other plasma
proteins that are incorporated into fibrin clots.
Irrespective of the exact mechanism of stimulation and/or the
presence of additional plasma cofactors, our studies show that scuPA/suPAR can mediate the lysis of plasma clots and that the activity
of this complex is regulated by processes that are distinct from those
that control the activity of tcuPA.
| |
FOOTNOTES |
Submitted January 13, 1998;
accepted May 14, 1998.
Supported in part through grants from the National Institutes of Health
(HL40387, HL50970, HL49839), Grant No. 960105000 from the American
Heart Association, and a grant from the Joint Research Fund of the
Hebrew University and Hadassah.
Address reprint requests to Abd Al-Roof Higazi, MD, Department of
Clinical Biochemistry, Hadassah University Hospital, Jerusalem, Israel
91120; e-mail: abd{at}md2.huji.ac.il.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract