(Circulation. 1995;92:1883-1890.)
© 1995 American Heart Association, Inc.
Articles |
Superficial Accumulation of Plasminogen During Plasma Clot Lysis
From Gaubius Laboratory, TNO-PG, Leiden, Netherlands.
Correspondence to Dr D.C. Rijken, Gaubius Laboratory, TNO-PG, PO Box 2215, 2301 CE Leiden, Netherlands. E-mail dc.rijken@pg.tno.nl.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background Binding of plasminogen to partially degraded fibrin is an important step in fibrinolysis, influencing its rate and fibrin specificity. Little is known about the spatial distribution of plasminogen and of plasminogen-binding sites inside thrombi during lysis. In the present study, we investigated this problem, which is important for a better understanding of the local regulation of fibrinolysis and the rate-limiting factors of therapeutic thrombolysis.
Methods and Results An experimental system was used that allowed
continuous visualization and quantification by fluorescence
microscopy of the spatial distribution of
fluorescein-labeled plasminogen inside and
outside model thrombi. Strong superficial accumulation of
plasminogen was observed during lysis of a plasma clot
induced by tissue-type or urokinase-type plasminogen
activators in the surrounding plasma. A distinctly visible
plasminogen-accumulating shell moved continuously with the
reducing surface of the clot. The accumulation decreased in conditions
of exhaustive activation of plasminogen in the outer
plasma. It was found in a purified system that a thin superficial layer
(
50 µm wide) of a plasmin-treated fibrin clot exposes about 2.5
plasminogen-binding sites per fibrin monomer with a
Kd of 2.2 µmol/L. At a
physiological concentration of
plasminogen (1.5 µmol/L) in the outer medium,
plasminogen was concentrated about 10-fold in this layer.
The binding was dose-dependently inhibited by 
-aminocaproic
acid.
Conclusions We conclude that the generation of potent surface-associated plasminogen-binding sites during thrombolysis results in a strikingly high plasminogen concentration at the dynamically changing surface of a lysing clot. The necessity of a continuous plasminogen supply from the plasma supports the use of fibrin-specific and plasminogen-sparing agents for thrombolytic therapy.
Key Words: binding sites • plasminogen • plasminogen activators • fibrinolysis • thrombolysis
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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Dissolution of insoluble fibrin is a key event in thrombolysis. The fibrin matrix of thrombi not only represents the substrate for lysis but also plays an active role because it adsorbs and modulates functional properties of components of the fibrinolytic and antifibrinolytic systems.1 2 3 4 Plasminogen, the precursor of the main fibrinolytic enzyme plasmin, can bind weakly to specific sites exposed on the fibrin network.5 6 This binding contributes to an efficient activation of plasminogen by plasminogen activators.7 8 9 10 11 12 Limited digestion of fibrin by plasmin leads to the generation of new binding sites for plasminogen,13 14 15 16 17 18 19 thereby creating positive feedback for activation of fibrinolysis. Although it is generally believed that this feedback is important for local regulation of fibrinolysis, how this system is organized in a three-dimensional (3D) thrombus has never before been investigated.
Rather contradictory data have been reported regarding quantitative parameters of the binding of plasminogen to partially degraded fibrin. Suenson et al13 reported a Kd value of 10 µmol/L calculated on the basis of the assumption that one binding site is exposed per fibrin monomer; they also reported a Kd of 1.2 µmol/L with 0.48 binding site per fibrin monomer for a gel obtained after thrombin-induced polymerization of fragment X of fibrinogen.14 Tran-Thang et al18 found a Kd of 0.3 µmol/L with 5 binding sites exposed per 100 fibrin molecules. The appearance of low-affinity binding sites was reported with a Kd of 48 µmol/L and 1.1 binding sites per fibrin monomer.17 These data were obtained in different models of 3D fibrin gels. Using an experimental system with fibrin monomer immobilized on a plastic surface, Fleury and Anglés-Cano19 obtained a Kd of 0.66 µmol/L and 2.7 binding sites per fibrin monomer for plasminogen binding to plasmin-treated fibrin. The apparent inconsistency in these data could be, at least partially, a consequence of a nonuniform distribution of plasminogen-binding sites throughout the 3D fibrin clots treated with plasmin from the outside.17 18
In this study, we have investigated the spatial distribution of plasminogen in a plasma clot during its lysis induced by tissue-type and two-chain urokinase-type plasminogen activators (TPA and TCUPA, respectively) in the surrounding plasma and the distribution of plasminogen-binding sites in a plasmin-treated fibrin clot. The results, demonstrating strong and strictly superficial accumulation of plasminogen during clot lysis, have implications in discussions of the use of fibrin-specific versus nonspecific agents for thrombolytic therapy.20 21 22
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Preparations
Glu-plasminogen was a product of Biofine. No contamination by the Lys form of plasminogen or proteins was found in the preparation by SDS-PAGE. To remove possible traces of lysine-like compounds, the plasminogen stock solution was gel-filtered on a Sephadex G-25 column equilibrated with PBS before all experiments. The TPA preparation (Actilyse) was supplied by Boehringer Ingelheim; human urinary TCUPA (Ukidan) was purchased from Serono. Chromogenic substrates, human fibrinogen, and plasmin were products of Kabi. According to manufacturer's specification, specific activity of plasmin was not <15 CU/mg. Fibrinogen was made plasminogen-free by lysine-Sepharose chromatography.23 Fluorescein isothiocyanate (FITC) and porcine carboxypeptidase B were from Sigma Chemical Co. Fluorescein-5-maleimide (FM) was from Pierce. We used also
-aminocaproic acid (EACA, Merck), thrombin (Leo), hirudin
(Pentapharm), and aprotinin (Bayer). Pooled citrated platelet-poor
plasma was used in all experiments involving plasma.
Labeling of Proteins With Fluorescein
Derivatives
Plasminogen (0.5 mL, 40 µmol/L) was gel-filtered
in a centrifuge by use of a microcolumn with Sephadex G-25 (3.5
mL) equilibrated with a buffer containing 30 mmol/L sodium carbonate
and 140 mmol/L NaCl, pH 9.2. FITC (1 mg/mL in dimethyl sulfoxide) was
added to the plasminogen preparation under intensive
stirring to a final concentration of 50 µg/mL. After 1 hour of
incubation in the dark, unreacted FITC was removed by gel filtration on
the same column with Sephadex G-25 reequilibrated with Tris-buffered
saline (TBS) containing 20 mmol/L Tris-HCl and 140 mmol/L NaCl, pH 7.4.
The preparation was stored frozen in small packages that were thawed
only once before each experiment. The
A495/A280 ratio in preparations of
FITC-plasminogen was about 0.7, corresponding to about 2
FITC molecules per molecule of plasminogen. The same
procedure was used for FITC labeling of TCUPA (initial concentration,
250 000 IU/mL) and BSA (3 mg/mL). TPA was labeled with FM, presumably
attached to the sulfhydryl group in position 83,24 as
described elsewhere.25 After labeling, both preparations
of fluorescence-labeled plasminogen
activators retained about 70% of their amidolytic activity
toward specific chromogenic substrates (S-2444 for TCUPA
and S-2288 for TPA) and about 55% of their plasminogen
activating activity, as measured with plasmin-specific substrate
S-2251. The labeled TPA activated plasminogen
approximately 150-fold more efficiently in the presence of a
fibrin-like stimulator.26
Experimental System Design
The experimental system for
visualization of the spatial
distribution of FITC-labeled molecules inside a model thrombus in
conditions of diffusive permeation was described
elsewhere.27 In brief, a plasma or fibrin clot was
situated between two parallel glass slides separated by a 0.2-mm-high
spacer. The geometry of the system allowed us to perfuse the clots with
plasma or buffer in such a way that the perfusing solution flowed
freely around the clot; no hydraulic pressure over the clot that could
force infiltration of the perfusate into the clot was applied.
Non–cross-linked plasma clots were formed by the addition of thrombin
(final concentration, 1.4 NIH U/mL) to plasma. About 1 µL of the
mixture was immediately applied to the chamber and was sucked in
between the two pieces of glass by capillary forces. After 2 minutes, a
clot with an approximate diameter of 2 to 3 mm was formed, attached
firmly to the parallel pieces of glass, and the rest of the chamber
volume (about 25 µL) was filled with plasma containing hirudin (10
ATU/mL). Hirudin was added to prevent clotting of the outer plasma by
the thrombin inside the clot. To obtain cross-linked clots, clotting
was performed in the presence of 30 mmol/L CaCl2,
and the clots were further incubated during 3 hours at 37°C in
recalcified plasma with 300 ATU/mL hirudin. For experiments with
compacted clots, 50 µL of plasma was clotted in a separate tube by
addition of thrombin to a final concentration of 1.4 NIH U/mL; the clot
was compacted by a pipette tip and then placed between two parallel
sheets of glass. After compaction, the clot volume had been reduced
approximately 50- to 100-fold. Fibrin clots were formed from
plasminogen-free fibrinogen (final concentration, 9.2
µmol/L) dissolved in TBS, containing 40 mg/mL BSA (TBS-BSA), by
exactly the same procedure as non–cross-linked plasma clots and were
washed with TBS-BSA.
In experiments with pressure-driven permeation, a modification of the experimental model was used that had a design very similar to that of the system described by Blomback et al.28 Plasma was clotted with 1.4 NIH U/mL thrombin in a chamber with dimensions of 0.2x5x25 mm formed between two parallel sheets of glass and closed on the two long sides by spacers. After clotting, the chamber was situated vertically, and pressure-driven permeation was performed by continuous application of small portions of plasma onto the upper surface of the clot. Thus, the gradient of applied pressure was approximately 1 cm H2O/cm clot (0.75 mm Hg/cm clot). In these conditions, plasma filtered through the clot with an approximate speed of 500 µm/min and dropped down from the lower part of the chamber.
Investigation of the Spatial Distribution of Fibrinolytic
Components During Lysis of a Plasma Clot
To investigate the spatial
distribution of
FITC-plasminogen during lysis, chambers containing plasma
clots were perfused at 37°C with plasma containing TPA or TCUPA and
FITC-plasminogen added as a tracer to a final concentration
of 0.3 µmol/L. When indicated, carboxypeptidase B (final
concentration, 50 µg/mL) or EACA (final concentration, 1 mmol/L) was
added to the perfusing solution. 
2-Plasmin
inhibitor was determined as described by Kluft et
al.29 In experiments with fluorescent derivatives
of plasminogen activators, FITC-TCUPA or FM-TPA
was added to perfusing plasma.
Consecutive images of the spatial distribution of fluorescence during the lysis of plasma clots were obtained by making fluorescence photomicrographs of the same clot in definite time intervals with a fluorescence microscope (Microphot FXA, Nikon) using black-and-white film (400 ASA, 36x24 mm). Perfusions were interrupted for not >3 minutes for photographing.
Accumulation of FITC-Plasminogen in Plasmin-Treated
Fibrin Clot
When indicated, fibrin clots were incubated during 70
minutes at
37°C with plasmin added to the outer medium to a final concentration
of 0.1 CU/mL in TBS-BSA (5 µg/mL). As a result of the treatment
with plasmin, clot diameters reduced by 0.8 to 1.0 mm. Fibrin clots,
either treated with plasmin or not, were washed with TBS-BSA containing
100 KIU/mL aprotinin and then incubated with
FITC-plasminogen in the same buffer at room temperature
during 24 hours, which was sufficiently long to equilibrate the clots
with FITC-plasminogen. Only a negligible portion of
FITC-plasminogen added to the chamber was eventually
accumulated in the clots, so the concentration of
FITC-plasminogen in the outer medium remained practically
constant. For inhibition experiments, various concentrations of
unlabeled plasminogen or EACA were added with
FITC-plasminogen. After a 24-hour equilibration, clots were
photographed as described above.
Quantitative Analysis of FITC-Plasminogen
Binding
After development, the film with negative images of the clots
was scanned along the central long axis of each picture by use of a
scanning densitometer (TLC Scanner CS-910, Shimadzu) with scanning beam
dimensions of 0.5x0.05 mm. To obtain quantitative data about local
concentrations of FITC-plasminogen, a calibration curve was
constructed (not shown), establishing the correlation between the
concentration of FITC-plasminogen in the chamber and the
densitometer signal. In this article, this calibration curve is
reflected by the nonlinear character of the y axis of the
scanning densitometry plots.
The characteristic profile of the spatial distribution of FITC-plasminogen in a lysing clot was a rather sharp peak associated with the clot boundary. The highest point of this peak was taken for determination of the concentration of FITC-plasminogen in the superficial layer.
For construction of binding curves of FITC-plasminogen in the purified system, the local concentration of bound plasminogen was calculated by subtracting the FITC-plasminogen concentration in the outer medium from the local concentration of FITC-plasminogen in the superficial layer. Thus, the outer concentration of the ligand was considered to be equal to the concentration of free ligand inside the clot after equilibration. A GRAPHPAD computer program (ISS) incorporating nonlinear regression analysis was used to calculate quantitative parameters for FITC-plasminogen superficial binding and its inhibition by unlabeled plasminogen and by EACA.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Using an experimental system that allowed us to follow and to quantify the spatial distribution of fluorescence inside and outside a model thrombus,27 we performed two types of experiments: (1) the dynamics of the distribution of fluorescein-labeled plasminogen, TPA, or TCUPA was documented during progressive lysis of a plasma clot surrounded by plasma containing the labeled proteins and (2) after plasmin treatment of a fibrin clot in a buffer system, the lytic process was stopped by addition of aprotinin, and the spatial distribution of plasminogen inside the clot was documented after a 24-hour equilibration of the clot with FITC-labeled plasminogen.
Investigation of the Dynamics of the Distribution of
Fluorescein-Labeled Fibrinolytic Components During Lysis of
the Plasma Clot
Fig 1
(A through C) presents three
consecutive
views of a plasma clot during ongoing lysis induced by TPA added to the
surrounding plasma to a concentration of 1 µg/mL. Tracer
FITC-plasminogen present in the outer plasma was
strongly accumulated in a thin (10 µm wide) superficial layer of
the clot, allowing continuous visualization of the distinct boundary.
Along with progressive lysis, the boundary moved while the radius of
the clot decreased. Under the experimental conditions, no large
plasminogen activation occurred in plasma, as judged by
measurements of 2-plasmin inhibitor
activity, which was never reduced to <65% of its initial level during
the course of the experiment (data not shown).
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Addition of
carboxypeptidase B to the outer plasma during the process
of ongoing lysis almost completely eliminated the bright
plasminogen-containing shell at the surface of the clot
(Fig 1D
). Thus, on the basis of known specificity of the
carboxypeptidase, it is likely that plasminogen-binding
sites are represented by C-terminal lysines of partially
degraded fibrin. Lysis speed decreased drastically after the
carboxypeptidase-induced removal of superficially bound
plasminogen, as seen from comparison of Fig 1C and
1D. The
clot size remained practically unchanged in the presence of the
carboxypeptidase, which was added 72 minutes after the start of lysis.
In a parallel experiment without addition of carboxypeptidase (not
shown), lysis continued with a constantly high speed during at least
120 minutes after the start of lysis. Similarly, addition of EACA in
the millimolar range stopped lysis and simultaneously
destroyed the FITC-plasminogen shell on the surface of a
clot (not shown).
To find out whether the observed superficial
accumulation of
plasminogen also occurs during lysis induced by
non–fibrin-specific plasminogen activators, we
performed similar experiments with TCUPA. Fig 1E and
1F shows images of
a plasma clot after 4 and 30 minutes of lysis induced by 500 IU/mL of
TCUPA in the surrounding plasma. The patterns of spatial distribution
of plasminogen were essentially the same superficial
accumulation as for TPA-induced lysis. However, the accumulation was
significantly lower after 30 minutes of lysis than after 4 minutes, as
manifested by reduced brightness of the clot boundary after 30 minutes
of lysis (Fig 1F). A possible explanation for the observed
reduction of
accumulation is exhaustive activation of plasminogen to
plasmin in the surrounding plasma and consecutive formation of
complexes of plasmin with various inhibitors
(2-plasmin inhibitor,
2-macroglobulin, etc) that may have a reduced ability to
interact with fibrin. Indeed, 2-plasmin
inhibitor was reduced to zero after 30 minutes in the
presence of 500 IU/mL TCUPA in plasma, indicating major exhaustion of
the plasminogen pool. In line with this explanation, the
high accumulation was restored immediately after addition to the same
clot of a new portion of FITC-plasminogen–containing
plasma with freshly added TCUPA (Fig 1G).
According to
our quantitative estimations, the ratio of the local
intensity of fluorescence in the superficial layer to the
fluorescence intensity in the surrounding plasma was about 3 to
4 in the absence of exhaustive activation of plasminogen
(as in Fig 1A through 1C, 1E, and 1G) and decreased to 1.3 to
1.7 when
plasminogen in the outer plasma was depleted (as in Fig 1F).
The technique did not allow us to discriminate between labeled
plasminogen and labeled plasmin-inhibitor
complexes.
As Fig 1H shows, no remarkable accumulation
of
FITC-plasminogen was found in a control experiment
performed in the absence of exogenous plasminogen
activator in the outer plasma.
Fig 2 shows that spatial
distributions of
fluorescent derivatives of the two plasminogen
activators during lysis of a plasma clot were different.
TPA (Fig 2A) exhibited a distribution similar to that of
plasminogen, being dynamically accumulated in a thin
superficial layer. In contrast, TCUPA did not interact with the lysing
clot (Fig 2B). These results indicate that superficial
accumulation of
plasminogen during lysis is not a consequence of a specific
spatial distribution of plasminogen activator
because the superficial accumulation of plasminogen took
place with both activators (Fig 1).
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The following
factors may seriously affect the speed of
thrombolysis in vivo: pressure-driven permeation of solutes
into the
thrombus,30 31 32 33 34
Ca2+-dependent
cross-linking of the fibrin network by factor
XIIIa,35 36
and compaction of a clot as a result of platelet-induced
retraction.37 38 The experiments shown in Fig
3 were performed to find out whether the superficial
accumulation of plasminogen is affected by these
factors.
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In a modification of our experimental system with pressure
applied
across the clot, the lysis was much faster than under conditions of
purely diffusive permeation. The surface of the lysing clot was rather
irregular; more rapid lysis took place in the directions of predominant
flow. These results are in agreement with reports describing channeling
of lysis under conditions of pressure-driven permeation in both in
vitro31 34 and in vivo models.39 As Fig
3A
demonstrates, plasminogen accumulated on this surface in a
way similar to that observed under conditions of purely diffusive
transport. It is noteworthy that the front of the lysis moved
considerably more slowly than the front of the penetration of
FITC-labeled plasminogen, which completely passed through
the part of the clot shown in Fig 3A. In a control experiment
without
the addition of a plasminogen activator, the
original surface of the clot remained unchanged during perfusion and
did not accumulate substantial amounts of FITC-plasminogen
(not shown).
Fig 3B and 3C represents
patterns of
FITC-plasminogen accumulation during lysis of a
cross-linked plasma clot and a compacted plasma clot, respectively.
Both Ca2+-dependent action of factor XIIIa and
compaction of a plasma clot are known to increase resistance to
lysis.35 36 37 38 Indeed, in
both cases we observed a decrease
in lysis speed, which was especially pronounced for compacted clots
(data not presented). Strictly superficial accumulation of
FITC-plasminogen was observed during the lysis of both
cross-linked and compacted plasma clots. Some accumulation of
FITC-plasminogen took place on the surface of a compacted
clot in the absence of added plasminogen
activator (Fig 3D). This accumulation may be attributed to
low-affinity interaction of plasminogen with the intact
fibrin,5 6 which is present in a compacted clot in a
very high concentration (1 mmol/L). However, the patterns of
plasminogen accumulation in both the presence and absence
of TPA were clearly different; in the former case (Fig 3C), the
accumulation was significantly higher and localized to a more narrow
zone on the clot surface.
It is likely that the strictly superficial character of the binding of plasminogen observed in a variety of conditions reflects a similar superficial location of plasminogen-binding sites generated during the lytic process. Because plasma contains a number of components that may affect this binding, we used a purified system for its quantitative characterization.
Distribution of FITC-Plasminogen in the Plasmin-Treated
Fibrin Clot
After plasmin treatment of a fibrin clot in a buffer
system, the
lytic process was stopped by the addition of aprotinin, and the spatial
distribution of plasminogen inside the clot was documented
after a 24-hour equilibration of the clot with FITC-labeled
plasminogen. Fig 4A demonstrates a
strong accumulation of plasminogen in the superficial layer
of the clot. Quantitative representation of these data obtained
by means of scanning densitometry (Fig 4A') indicates
that
FITC-plasminogen was concentrated in this superficial layer
up to about 15 µmol/L, thus providing a 10-fold local accumulation
compared with its concentration in the outer medium, corresponding to
the physiological level of plasminogen
in plasma (1.5 µmol/L). The width of the
plasminogen-accumulating layer was about 50 µm, estimated
as the width of the peak at half-maximal concentration (7.5 µmol/L).
As Fig 4B shows, an intact fibrin clot not treated with plasmin
did not
significantly accumulate FITC-plasminogen on its surface.
In this case, FITC-plasminogen was distributed inside the
clot uniformly, being slightly (1.4-fold) accumulated in the whole
body of the clot compared with its concentration in the outer medium
(Fig 4B'). This finding is consistent with the known low
affinity of Glu-plasminogen for intact
fibrin.5 6 In a similar experiment, FITC-labeled BSA
wasevenly distributed inside and outside the clot,
regardless of whether the latter was pretreated with plasmin (data not
shown).
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The specificity of the binding of FITC-plasminogen in the
superficial layer of a plasmin-treated fibrin clot is demonstrated by
the results in Fig 4C and 4D. The binding was
effectively inhibited by
an excess of unlabeled plasminogen and by EACA.
The quantitative
parameters of the superficial binding of
plasminogen to plasmin-treated fibrin clots were derived
from the results presented in Fig 5. The values
of Kd=2.2 µmol/L and Bmax=23
µmol/L for the concentration of binding sites for
FITC-plasminogen superficial binding were found by
nonlinear regression analysis of the concentration dependence
of FITC-plasminogen binding (Fig 5A). The obtained value
for Bmax corresponds to about 2.5
plasminogen-binding sites per fibrin monomer exposed
locally in the superficial layer. A low-affinity binding was also
detected for which it was possible to determine only the ratio of the
concentration of binding sites to the dissociation constant
Bmax/Kd=0.18. The value
IC50=2.6 µmol/L was obtained for inhibition of
FITC-plasminogen superficial binding by unlabeled
plasminogen (Fig 5B), corresponding to a
Ki=2.3 µmol/L. The Kd
for FITC-plasminogen binding and the
Ki for its inhibition with unlabeled
plasminogen were very similar, implying that the procedure
of labeling with FITC did not noticeably disturb the binding properties
of plasminogen.
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)
and by 
). Nonlinear regression
analysis gave the best fits of the data with sigmoid curves
with IC50=2.6 µmol/L and a Hill coefficient of
-0.91
(r=.98) for unlabeled plasminogen and with
IC50=58 µmol/L and a Hill coefficient of -0.94
(r=.96) for EACA.
Fig 5B also demonstrates the
dose-dependent inhibition of the
superficial binding of FITC-plasminogen by EACA, indicating
that lysine-binding sites of plasminogen were involved in
its superficial accumulation. Nonlinear regression analysis of
these data gave a value of IC50=58 µmol/L. Taking into
account that tranexamic acid, another inhibitor of lysine
binding site-mediated interactions of plasminogen, is
about fivefold more potent than
EACA,5 40 41 our data are
in agreement with the results of Suenson et al,13 who
found that 10 µmol/L tranexamic acid inhibits 50% of
Glu-plasminogen binding to plasmin-treated fibrin.
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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It was first reported by Suenson et al13 and later confirmed and extended by others15 16 17 18 19 that, when fibrin is "nicked" with plasmin, the binding of plasminogen to fibrin increases. Although there is much discrepancy between the data of different research groups regarding quantitative parameters of this binding, it is generally believed that this positive feedback might represent one of the key steps in the local regulation of fibrinolysis. The supposed local character of this mechanism suggests that biochemical approaches dealing with overall binding values without assessment of the distribution of binding sites in a 3D body of a thrombus may not be sufficient for comprehensive understanding of the phenomenon. Clarification of the spatial distribution of plasminogen-binding sites and plasminogen in a thrombus during its lysis is necessary for better understanding of the local regulation of fibrinolysis and its rate-limiting factors.
To investigate this problem, we have used an experimental system incorporating low-magnification fluorescence microscopy for the visualization of the spatial distribution of fluorescent molecules in a model thrombus.27 In contrast to previously described experimental systems in which the binding of labeled plasminogen to model thrombi was investigated after separation of unbound material,13 14 15 16 17 18 19 42 our system allowed us to observe the spatial distribution of plasminogen in the thrombus without disturbing the binding equilibrium. We also were able to follow the dynamic spatial distribution of FITC-plasminogen inside the plasma clot during its lysis.
The main finding of this study is a strong superficial
accumulation of plasminogen accompanying lysis of the clot
surrounded by plasma containing a plasminogen
activator. This accumulation led to formation of a
distinctly visible plasminogen-accumulating shell on
the surface of the plasma clot; the shell moved continuously with the
reducing surface of the clot. According to our estimations, the local
concentration of plasminogen at the surface of the clot was
at least threefold higher than in the surrounding plasma in the absence
of exhaustive activation of plasminogen. In a variety of
experimental conditions explored, lysis was always accompanied by the
superficial accumulation of plasminogen. As shown in
experiments with TPA- and TCUPA-induced lysis (Figs 1 and
2), the
fibrin-binding properties of a plasminogen
activator influenced its own spatial distribution during
lysis of a plasma clot, but the spatial distribution of
plasminogen apparently did not depend on the type of the
plasminogen activator. The superficial
accumulation of plasminogen was observed during lysis of
non–cross-linked, cross-linked, and compacted plasma clots. The
pattern of the superficial accumulation was basically the same under
conditions of diffusive and convective transport of solutes into
clots.
Both carboxypeptidase B and EACA were shown to remove plasminogen from the surface of the lysing clot. Presumably, the carboxypeptidase eliminates plasminogen-binding sites (C-terminal lysines) from the fibrin surface, while EACA occupies lysine-binding sites of plasminogen. Although mechanisms of action of the two agents are different, they cause similar effects manifested by simultaneous elimination of the plasminogen shell on the surface of the lysing clot and cessation of lysis. These findings give rise to a supposition that superficial plasminogen accumulation may be a necessary condition for effective lysis.
The demonstration of the strictly superficial accumulation of
plasminogen gives new insight into the mechanism of
thrombolysis that might be driven primarily by the
activation of the plasminogen accumulated on the surface of
the thrombus during its lysis. This is likely because the
plasminogen concentration on the surface of a clot is
several times higher than in the surrounding plasma and because the
binding of plasminogen to fibrin contributes to efficient
plasminogen
activation.7 8 9 10 11 12
In addition, the
high accumulation of plasminogen is expected to provide a
significant local excess of plasminogen over
2-antiplasmin in the thin superficial layer of the
thrombus, higher than in surrounding plasma, where the molar ratio of
plasminogen to 2-antiplasmin is usually
about 1.5 to 243 and much higher than in a contracted
blood clot.44 These factors can provide conditions for a
local activation of the fibrinolytic system strictly confined in space
by the superficial location of newly generated
plasminogen-binding sites.
The surface of a clot is probably the only place where a really high concentration of the plasminogen-binding sites can be generated. Indeed, Glu-plasminogen at a physiological concentration binds only moderately to a fibrin gel treated throughout with plasmin; binding in the range of 10% to 40% of the plasminogen added inside such a gel was demonstrated by several authors.13 15 16 36 C-terminal lysine residues generated by plasmin cleavage of fibrin chains are probably much more accessible on the surface of a plasmin-treated fibrin gel where molecules are less involved in the maintenance of gel structure and where the lateral packing of fibrin bundles could be disorganized. Plasminogen "bridging" of fibrin fibers45 on the surface of the lysing clot may be an additional mechanism for generation of the plasminogen-rich shell.
Quantitative parameters of the superficial binding of
plasminogen were measured in a purified system. As a result
of a plasmin treatment of a fibrin clot, potent binding sites for
plasminogen were generated only in a thin superficial
layer, not exceeding 50 µm. At a physiological
concentration of plasminogen in the outer medium (1.5
µmol/L), this layer bound about 10-fold more, ie, 15 µmol/L
plasminogen. Analysis of binding isotherms revealed
a Kd of 2.2 µmol/L with about 2.5
plasminogen-binding sites per fibrin monomer exposed
locally in this superficial layer of the plasmin-treated fibrin clot.
The results on the inhibition of this binding by EACA confirmed the
involvement of lysine-binding sites of plasminogen. We
presume that the high plasminogen-binding ability of the
binding sites characterized in a purified system can be modulated by
numerous plasma components, such as carboxypeptidases,
2-plasmin inhibitor, histidine-rich
glycoprotein, and other proteins capable of binding with
either plasminogen or fibrin. This may explain some
quantitative differences in accumulation of
FITC-plasminogen observed in the purified system and in
plasma milieu. Smaller accumulation in the latter case may also be
attributed to the dynamic nature of lysis and to possible differences
in fibrin structure in plasma and fibrin clots.46
The superficial concentration of the fibrinolytic potential demonstrated should be beneficial for lysis of small thrombi with a high surface-to-volume ratio and might be much less effective in the case of massive thrombi. We suppose that this mechanism of lysis can effectively function in vivo in the prevention of thrombosis, creating potent positive feedback for dissolution of small nascent thrombi and early fibrin deposits by use of plasminogen activators that are normally present in plasma in low concentration or locally released from endothelium.47 48
The local accumulation of plasminogen implies the
necessity of the supply of plasminogen from the surrounding
plasma to the surface of the thrombus for its optimal lysis. Our
experiments with lysis induced by a high concentration (500 IU/mL) of a
non–fibrin-specific plasminogen activator
(TCUPA) show that this supply may be restricted in conditions of
systemic activation of plasminogen, leading to a reduction
of accumulation of plasminogen in the superficial layer
(Fig 1E through 1G). This is consistent with findings that
reduced levels of plasminogen in plasma correlate with
decreased
thrombolysis38 49 50 51 52 53
and that
administration of additional plasminogen might be
advantageous for effective lysis when endogenous
plasminogen in plasma is
depleted.45 49 50 51 54 55
The necessity of a continuous
plasminogen supply thus explains why fibrin-specific
plasminogen activators sparing
plasminogen in the circulation are more potent for
thrombolytic therapy than nonspecific
activators.21 22 This phenomenon was explained
previously by Sobel et al,50 who proposed that reduction
of the plasminogen concentration in plasma by nonspecific
plasminogen activators leads to depletion of
plasminogen already present in the clot by a
"plasminogen steal" effect. We believe that both
explanations are relevant and underline that plasminogen in
the circulation should be spared as much as possible during
thrombolytic therapy.
|
Acknowledgments |
|---|
We are grateful to Drs E.J.P. Brommer, F. Haverkate, and J.H. Verheijen for their critical reading of the manuscript.
Received February 19, 1995; revision received April 12, 1995; accepted April 16, 1995.
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References |
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