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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 2019-2025
Binding of Tissue-Plasminogen Activator to Fibrin: Effect of
Ultrasound
By
Farhan Siddiqi,
Tatjana M. Odrljin,
Philip J. Fay,
Christopher Cox, and
Charles W. Francis
From the Vascular Medicine Unit, Department of Medicine and
Biostatistics Department, University of Rochester School of Medicine & Dentistry, Rochester, NY.
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ABSTRACT |
Ultrasound reversibly alters the structure of polymerized fibrin, an
effect that could influence tissue-plasminogen activator (t-PA)
binding. We have, therefore, characterized the effects of ultrasound on
binding of t-PA to fibrin using a novel system in which radiolabeled,
active-site blocked, single chain tissue-plasminogen activator flowed
through a fibrin gel at constant rate, and specific binding was
determined by monitoring incorporation of radiolabel. Results using
polymerized fibrin were compared with those using a surface of fibrin
immobilized on Sepharose beads in a similar system. Interaction of t-PA
with surface-immobilized fibrin involved two classes of binding sites
(Kd = 31 nmol/L and 244 nmol/L) and a maximum binding ratio of 3.8 mol t-PA/mol fibrin. Ultrasound increased Kd for the high affinity site
to 46 nmol/L (P < .0001), but it had no significant effects
on the Kd 244 nmol/L site nor on Bmax. Tissue-plasminogen
activator binding to noncrosslinked fibrin involved two sites with Kds
of 267 nmol/L and 952 nmol/L, while a single Kd 405 nmol/L site was
identified for crosslinked fibrin. Ultrasound had no significant effect
on the binding affinity for noncrosslinked fibrin, but Bmax
was increased in the presence of ultrasound, from 31 µmol/L to 43 µmol/L (P < .0001). Ultrasound decreased the Kd for
crosslinked fibrin to 343 nmol/L (P = .026) and also
increased Bmax from 22 µmol/L to 25 µmol/L
(P = .015). Ultrasound also affected the kinetics of t-PA
binding to fibrin, significantly accelerating the rate of dissociation
by 77% ± 5% for noncrosslinked fibrin and by 69% ± 3% for
crosslinked fibrin (P < .001 for each). These results
indicate that ultrasound exposure accelerates t-PA binding, alters
binding affinity, and increases maximum binding to polymerized fibrin,
effects that may result from ultrasound-induced changes in fibrin
structure.
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INTRODUCTION |
FIBRINOLYSIS IS CAREFULLY regulated by
efficiently generating the serine protease plasmin at sites of fibrin
formation, and limiting its systemic activation. Several mechanisms
contribute to the localized activation of fibrinolysis including the
specific binding of reactants to fibrin, including t-PA, the enzyme
which converts plasminogen to plasmin.1-6 In addition to
binding t-PA, fibrin also facilitates the t-PA-mediated conversion of
plasminogen to plasmin.1,7,8 Specificity for binding to
fibrin rather than to its circulating precursor, fibrinogen, depends on
unique structural features that arise from the conversion of fibrinogen to fibrin. The structure of fibrin may, however, also limit binding because it is polymeric, composed of two-stranded protofibrils aggregated laterally to form larger fibers varying in
width.9,10 Previous studies examining binding of t-PA to
fibrin have used several approaches to overcome the problem of binding
to such a polymeric solid. These include binding of t-PA to fibrin
immobilized on a plastic surface,5,6 and also mixing t-PA
and fibrinogen before clotting.1-3 In another approach,
access of t-PA to binding sites on polymerized fibrin was facilitated
by sonicating the fibrin to form a suspension.4 The latter
most closely approximates important pathologic conditions in which t-PA
is administered in pharmacologic doses into the blood outside of a clot
as treatment for obstructive thrombosis. The particles in the sonicate
were, however, still large and could restrict access of enzyme,
possibly explaining the failure to achieve saturation binding in that
study.4
Fibrinolysis is accelerated in the presence of high frequency, low
intensity ultrasound (US) both in vitro11-17 and in
vivo.17-21 The enhanced fibrinolysis results from
accelerated enzymatic degradation rather than from mechanical
disruption of fibrin. The effects are non-thermal and may be mediated
in part by increasing transport of reactants into the fibrin matrix.
Thus, US increases clot uptake of t-PA in the absence of
flow,22 and it also increases pressure-mediated flow
through fibrin.23 The latter finding suggests that US
alters fibrin structure, which is the primary determinant of flow
resistance, and scanning electron microscopy has documented reversible
alteration in fiber density and diameter caused by US.24
Because changes in fibrin structure could affect t-PA binding, we have
investigated the effect of US on interactions of t-PA with fibrin,
using a permeation system to maximize access of t-PA to binding sites
with the gel matrix. Since permeation plays an important role in
transport of fibrinolytic reactants into thrombi in vivo, this system
approximates t-PA delivery during pharmacologic administration. Binding
of t-PA to unpolymerized fibrin used a similar system with fibrin
immobilized on Sepharose beads. Using this approach, we have
characterized the binding of t-PA to non-crosslinked fibrin,
crosslinked fibrin and non-polymerized fibrin, and examined the effects
of US on binding parameters.
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MATERIALS AND METHODS |
Tissue plasminogen activator.
Recombinant t-PA (Activase; Genentech, South San Francisco, CA) was
inactivated by incubation with a five-fold molar excess of
D-Phe-Pro-Arg-chloromethylkeytone (PPACK) (Bachem, Torrence, CA), and
excess PPACK was removed by gel filtration chromatography on Sephadex
G-10 (Bio-Rad Laboratories, Hercules, CA). The inactivated t-PA showed
no enzymatic activity when incubated with the chromogenic substrate
H-D-Ile-L-Pro-L-Arg-P-nitroanalid (S-2288) (Kabi Vitrum, Stockholm,
Sweden). Radio-iodination of inactivated t-PA was performed using the
iodogen method,25 and unbound 125I was removed
by Sephadex G-10 chromatography. The radiolabeled, inactivated t-PA was
affinity purified using a column of fibrin celite as described
previously4 and modified with one additional step of
washing the fibrin-bound celite column with 0.5 mol/L arginine
hydrochloride before t-PA application to remove unbound proteins. The
fibrin-binding form of inactive t-PA that eluted with 0.5 mol/L
arginine hydrochloride comprised approximately 90% of the total
inactivated, labeled t-PA, and this was used for binding experiments.
The t-PA had a specific activity of 4.1 × 107 cpm/mg, and
its mobility on sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) was identical to active, unlabeled t-PA.
Preparation of polymerized fibrin.
Fibrin gels were prepared in segments of plastic transfer pipettes with
a 4 mm internal diameter (Samco, Marsh Biomedical Products, Rochester,
NY) using a modification of previously described methods.26,27 To increase adhesion of fibrin to the
plastic, a 10 mm segment of the tube was scratched symmetrically using a 20 gauge needle and then lined with a thin layer of fibrin prepared by addition of human thrombin (Cal-Biochem, LaJolla, CA) at a concentration of 1 U/mL in 0.5 mL fibrinogen (Kabi Vitrum) at a
concentration of 6 mg/mL in 0.018 mol/L sodium citrate, 0.132 mol/L
sodium chloride, pH 7.4. Following clotting, the fibrin was expelled
from the tube under high pressure leaving a thin coating of fibrin over
the scratched segment.
Non-crosslinked fibrin clots for perfusion were formed within the 10 mm
scratched and fibrin-lined segment. Fibrinogen (6.0 mg/mL) in 0.018 sodium citrate, pH 7.4 containing 0.057 mol/L sodium chloride and 100 kallikrein inhibitory U/mL aprotinin (Trasylol® FBA Pharmaceuticals,
Westhaven, CT) was clotted by addition of human thrombin to a
concentration of 0.5 U/mL, and 0.1 mL of the mixture was rapidly added
to the scratched segment and incubated for one hour at 37°C before
perfusion. Crosslinked fibrin clots were prepared in the same way with
the addition of 20 mmol/L calcium chloride and 5 U/mL of factor XIII
(Behring, Marburg, Germany). The polypeptide chain composition of
non-crosslinked and crosslinked fibrin was characterized by SDS-PAGE,
which confirmed cleavage of fibrinopeptides and demonstrated the
absence of  or  chain crosslinking in non-crosslinked fibrin and
extensive crosslinking for crosslinked fibrin.
Surface-immobilized fibrin preparation.
Fibrin immobilized on Sepharose beads was also used for perfusion
experiments. Purified IgG of monoclonal antibody J88B, reactive with an
epitope within the sequence arg63-met78 of the chain of human
fibrinogen28 was kindly provided by Dr P.J.
Simpson-Haidaris, Rochester, NY and immobilized by incubating 1 mL of
hydrated, activated CNBr Sepharose (Pharmacia Biotech, Piscataway, NJ)
with 1 mL of IgG in 0.2 mol/L sodium bicarbonate buffer, pH 8.3 and gently mixing for 18 hours at 4°C. Residual unbound sites were blocked by incubation in 1 mol/L glycine, and the suspension was then
washed twice with 50 mL each of 0.2 mol/L sodium bicarbonate buffer pH
8.3 containing 1 mol/L sodium chloride and then with 50 mL of 0.1 mol/L
sodium acetate buffer pH 4.0 containing 0.5 mol/L sodium chloride. The
suspension was then packed in a column and equilibrated with 0.05 mol/L
tris buffer, pH 7.4 containing 0.13 mol/L sodium chloride and 1%
bovine serum albumin. A 1 mL volume of fibrinogen (20 mg/mL) was
perfused through the column, and it was then washed with 4 mL of the
equilibrating buffer to remove any unbound fibrinogen. The eluate was
collected and the protein concentration determined, indicating that 2.5 mg of fibrinogen was bound per 1 mL of beads. To convert fibrinogen to
fibrin, 0.24 mL of suspension was incubated with 1 mL of 0.5 U/mL of
human thrombin.
Binding measurements.
Radiolabeled, active-site blocked t-PA at selected concentrations from
1.8 to 1,000 µg/mL in 0.05 mol/L tris buffer, pH 7.4 containing 0.13 mol/L sodium chloride and 1% bovine serum albumin was perfused through
fibrin gels or fibrin-bound Sepharose at a constant rate of 15 µL/min
using a pump, and aliquots of 50 µL or 100 µL were collected to
calculate binding. For each perfusion, equilibrium binding was
considered to occur when there was no additional binding of t-PA to the
fibrin column, and each column was used to derive a single equilibrium
binding point (Fig 1). Perfusions at each
t-PA concentration were done in triplicate, and nonspecific binding was
determined using the same system with a 1,000-fold molar excess of
unlabeled t-PA. Linear regression using Scatchard analysis was
performed using the Ligand program.29 Non-linear regression
analysis was also used, fitting data to an equation of the form B = B1F/K1F + B2F/K2 + F,
where B the bound concentration and F free.
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| Fig 1.
Binding of t-PA to non-crosslinked fibrin in a single
perfusion. Radiolabeled, active-site blocked t-PA was perfused at a constant rate of 15 µL/min through a column of fibrin. The amount of
bound t-PA was calculated by measuring radioactivity in aliquots of
effluent from the column. Equilibrium was considered to occur when
there was no additional binding of t-PA to the fibrin column as
indicated by the flat portion of the curve. A single point representing
mean equilibrium binding taken from that part of the curve was used for
binding isotherms (Fig 3) and Scatchard analysis (Fig 4).
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This provided a good estimate of Bmax from B1 + B2. It also determined Kd when a single binding site was
identified and the higher affinity site if two binding sites were
found. It was not accurate in calculating the Kd of the lower affinity
site when two were identified because the relative affinities were not
sufficiently different for resolution, and results are presented using
both types of analysis. Rates of dissociation were determined for each perfusion by fitting the dissociation of radiolabeled t-PA after addition of a four-fold excess of unlabeled t-PA to f(x) = ae bx using b as the apparent rate constant.
Ultrasound exposure.
All perfusions were performed in temperature-controlled water at 25°C
in a tank fitted with a piezoelectric transducer as described.11 The US transducer had an area of 4 cm2 and operated at a frequency of 1 MHz. The US field at
the site of exposure was adjusted to 2 W/cm2 for each
experiment, and US was generated in cycles of 5 ms on and 5 ms off. For
experiments with US exposure, a 3 cm rubber block was placed behind the
perfusion apparatus as an acoustic absorber to minimize standing waves
and a US blocking material was placed after the rubber to block
insonification of control samples.
Statistics.
Equilibrium binding data in the presence and absence of US was
compared. Linearlized data of bound versus bound/free from the
Scatchard analysis was analyzed for each site by linear regression to
derive the best fit line. The resulting lines for binding in the
presence and absence of US were compared using analysis of covariance
to determine if the slopes were significantly different. This provided
a statistical comparison of Kd values for each site. Each regression
analysis included an examination of residuals as a check on the
required assumptions of normally distributed errors with constant
variance. The significance of differences in Bmax in the
presence and absence of US was determined from the nonlinear regression
by comparing the estimated values using an approximate t test.
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RESULTS |
Perfusion of t-PA through fibrin led to a progressive increase in
binding until a stable level was achieved and addition of a fourfold
molar excess of unlabeled t-PA then resulted in displacement of bound,
radiolabeled t-PA after a lag period representing time for perfusion
through the fibrin gel as shown in Fig 2using non-crosslinked fibrin. The overall pattern was similar when the
experiment was conducted in the presence of 1 MHz US at 2 W/cm2, but there were differences in both the amount and
rate of binding. Binding increased more rapidly in the presence of US,
and equilibrium was achieved faster. For example, in the experiment
shown in Fig 2 total bound t-PA was approximately 4.5 x
109 mol in the presence of US, but only 3.0 × 109 mol in its absence. Dissociation of bound t-PA after
addition of excess unlabeled ligand was also more rapid as indicated by the slope of the dissociation curve, and the baseline level was reached
sooner in the presence of US despite an initial higher level of
equilibrium binding. The level of baseline binding observed in the
presence of excess unlabeled t-PA was the same with or without US.
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| Fig 2.
Binding of t-PA to non-crosslinked fibrin during
perfusion. Radiolabeled, active-site blocked t-PA was perfused at a
constant rate of 15 µL/min through a column of non-crosslinked
fibrin. Aliquots of 100 µL were collected to calculate binding. At
the arrow, a 1,000-fold molar excess of unlabeled t-PA was added to the
perfusate. Results are shown for binding without ultrasound (dotted
line) and in the presence of 1 MHz ultrasound at 2 W/cm2
and 50% duty cycle.
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These data were analyzed to compare rates of association and
dissociation of t-PA and fibrin in the presence and absence of US. A
total of 33 surface-immobilized fibrin, 32 non-crosslinked fibrin, and
28 crosslinked fibrin perfusion experiments with US and an equal number
of perfusions without US were compared over a range of t-PA
concentrations from 1.8 µg/mL to 1,000 µg/mL. These were used for
quantitative analysis of both equilibrium binding and dissociation
rates. The dissociation rate constant was calculated using time as a
variable; however, the rates of association and dissociation are
dependent on the flow rate in the system. The association of t-PA with
fibrin was, therefore, limited in all experiments by the flow rates.
Therefore, the rate of binding and dissociation was dependent on the
flow rate and does not represent the true rate constant. Since flow was
constant in all experiments, however, these apparent rate constants can be used for comparison of relative rates with and without US. Each
dissociation was fit to a single exponential decay model of f(x) = aebx with b as the apparent rate constant. For all
perfusions, the apparent rate constants without US were normalized to
100%, and the change in the rate of US-dependent dissociation was then
determined. Surface-immobilized fibrin showed no significant change in
binding kinetics in the presence of ultrasound. However, for both
non-crosslinked and crosslinked polymerized fibrin US exposure resulted
in significant increases in the rates of dissociation of 77% ± 5%
and 69% ± 3%, respectively (P < .001). The increases in
dissociation rates with US were independent of t-PA concentration over
the range tested.
Equilibrium binding constants were also determined from analysis of the
perfusion experiments, considering that the mean of the measured points
on the flat portion of the binding curve represented equilibrium
binding at a single concentration of t-PA (Fig 1). Experiments over a
range of t-PA concentrations from 1.8 µg/mL to 1,000 µg/mL were
then used to determine binding isotherms (Fig 3). For fibrin immobilized on Sepharose
beads, saturation was achieved at approximately 1 µmol/L t-PA with
little difference in binding in the presence or absence of US (Fig 3A).
Compared with fibrin monomer, saturation occurred at higher t-PA
concentrations for both non-crosslinked fibrin (Fig 3B) and crosslinked
fibrin (Fig 3C), and there was an increase in maximum binding for both in the presence of US. Binding of t-PA was modeled quantitatively using
Scatchard analysis and nonlinear regression, comparing binding in the
presence and absence of US. Binding to fibrin monomer (Fig 4A) was described best with a two-site
model with Kds of 31 nmol/L and 244 nmol/L (Table
1). Ultrasound had a small effect on the binding of t-PA to fibrin monomer, increasing Kd from 31 nmol/L to 46 nmol/L for the high affinity site (P < .0001), which was also reflected in the change of slope in the reciprocal binding plot
(Fig 4A), but US had no effect on the Kd 244 nmol/L site. Bmax was 67 µmol/L in the absence and 69 µmol/L in the
presence of US (P = .09). Similar results were obtained when
the data was analyzed using nonlinear regression with a high affinity
Kd 31 nmol/L site identified, Bmax of 67 µmol/L, and a
maximum molar binding ratio of 3.8 ± .03 mol t-PA/mol fibrin.
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| Fig 3.
Fibrin binding isotherms in the presence and absence of
ultrasound. Equilibrium binding to fibrin of t-PA at a single
concentration was determined from individual perfusion experiments as
in Fig 1. One perfusion experiment generated a single point for binding in either the absence ( ,  ,  ; -----) or presence ( ,  ,
 ; _____) of ultrasound. (A) Surface-immobilized
fibrin. (B) Non-crosslinked fibrin. (C) Crosslinked fibrin.
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US had greater effects on t-PA binding to polymerized fibrin. Scatchard
analysis of binding to noncrosslinked fibrin (Fig 4B) identified two
sites with Kds of 267 nmol/L and 952 nmol/L. Using nonlinear
regression, the Kd of the higher affinity site was similar, 239 nmol/L.
Exposure to US caused no significant changes in affinity as reflected
by the apparently parallel lines in the Scatchard plot (Fig 4B); and
analysis of covariance confirmed the visual impression that there was
no statistically significant difference in the slopes of lines
characterizing the two binding sites in the presence and absence of US.
There was, however, a significant change in maximum binding with US as
reflected by both an increase in x-intercept for the lines describing
both binding sites and by the apparent increase in concentration of bound t-PA at saturation in the binding isotherms (Fig 3B). With US
exposure Bmax increased from 31 µmol/L to 43 µmol/L
(P < .0001) corresponding to a 36% increase in the molar
binding ratio from 1.77 ± .04 to 2.42 ± .06. t-PA bound to a single
Kd 405 nmol/L site on crosslinked fibrin, as determined by Scatchard
analysis or 350 nmol/L by nonlinear regression. There was no
significant difference in affinity in the presence and absence of US as
indicated by parallel Scatchard plots (Fig 4C) and by analysis of
covariance. There was, however, a significant increase in maximum
binding evident from binding isotherms (Fig 3C) and an increase in
Bmax from 22 µmol/L to 25 µmol/L (P = .015)
estimated from nonlinear regression and increase in maximum molar
binding ratio from 1.27 ± .06 to 1.44 ± .02 mol t-PA/mol fibrin.
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DISCUSSION |
The insoluble, polymeric structure of fibrin complicates
characterization of ligand binding because it may limit access to potential binding sites. Therefore, we chose to examine t-PA binding to
fibrin using a constant flow permeation system that results in
saturation (Figs 1 and 2) and also prevents depletion of free ligand. A
further advantage is that it more closely approximates the interaction
of t-PA with fibrin during pharmacologic administration when a large
performed intravascular thrombus is exposed to circulating t-PA in the
blood. Binding of t-PA to fibrin is important in regulating the rate of
fibrinolysis, increasing the catalytic efficiency kcat/Km, for conversion of plasminogen to
plasmin several hundred-fold in the presence of fibrin as compared with
fibrinogen.1,8
Our findings indicate that binding of t-PA to surface-immobilized
fibrin is best described by two binding sites with Kds of 31 nmol/L and
244 nmol/L. The monoclonal antibody used to immobilize fibrinogen
reacts with a site not known to be involved in t-PA binding, and
treatment with thrombin most likely resulted in conversion of
fibrinogen to surface-immobilized fibrin monomer, but the absence of
polymerization was not confirmed. Binding to polymerized noncrosslinked fibrin also involved two sites, but of lower affinity (267 nmol/L and
952 nmol/L), while a single, Kd 405 nmol/L site was identified for
crosslinked fibrin. These results are in general agreement with
previous studies, but there are important differences that may be
related to the experimental approach. Higgins and Vehar2 clotted a solution of fibrinogen and t-PA and identified a single Kd
380 nmol/L binding site with greater affinity for single chain than for
two chain t-PA. Such a system eliminates the problem of transport of
soluble ligand into an insoluble fibrin matrix, although the
equilibrium may be affected by the concurrent processes of
polymerization and t-PA binding. Different results were obtained by
Larsen et al3 who used a similar method but varied the
concentration of fibrin instead of t-PA and observed a single Kd of
approximately 15 µmol/L site. Hussain et al4
characterized the binding of t-PA to preformed polymerized fibrin, the
same problem approached in this report. Recognizing the transport
problem, they mixed t-PA with a finely sonicated suspension of fibrin.
Similar to our findings, they found two distinct sites for
non-crosslinked fibrin with Kds of 320 nmol/L and 1,500 nmol/L and a
single, 580 nmol/L site for crosslinked fibrin. They concluded that
crosslinking prevented binding to a lower affinity binding site only
available on noncrosslinked fibrin, but a particular problem was the
inability to achieve saturation of binding, and this limited the
analysis. Both Fleury et al5 and Grailhe et al6
examined t-PA binding to a nonpolymerized monolayer on plastic and
identified a single high affinity site with Kds of 3 nmol/L and 1 nmol/L, respectively.
Previous reports have identified binding sites for t-PA on fibrin and
localizing them in the regions of 148-16030-32 and
311-379.33,34 Since fibrin is dimeric, a total of four potential binding sites per molecule are, therefore, available. This is
consistent with our identification of two distinct sites and a maximum
molar binding ratio of 3.8 for fibrin monomer. Polymerized fibrin
presents a more complex substrate than fibrin monomer for binding.
Following thrombin cleavage of four fibrinopeptides from fibrinogen,
the resulting monomers aggregate in a half-staggered overlap pattern to
form two-stranded protofibrils which then aggregate laterally to form a
branching network of fibers each composed of 14 to 22 protofibrils.9,10 Consequently, all potential binding sites
on fibrin are not equally available, and t-PA may have greater
accessibility to binding sites exposed on the surface of fibers than to
similar sites within the interior. As with fibrin monomer, two sites of
differing affinities were identified for non-crosslinked fibrin,
consistent with binding to the sites on and chains. The lower
affinities, however, suggest that polymerization may alter the
conformation of binding sites, lowering affinity or reducing the
accessibility of t-PA. Our results with crosslinked fibrin are
consistent with those of Hussain et al4 who found a single
binding site suggesting that structural changes accompanying crosslinking eliminate one binding site.
Because of the effect of US on fibrin structure24 and on
fibrinolysis,11-17 we sought to determine whether t-PA
binding to fibrin was altered by US. It was found that US had little
effect on binding to surface-immobilized fibrin with no change in
Bmax, a small increase in Kd from 31 nmol/L to 46 nmol/L,
and no effect on the kinetics of binding. US also had little effect on
binding affinity for non-crosslinked fibrin and only a small effect on crosslinked fibrin affinity. The reason for the small but significant changes in Kd are unclear but could reflect modest changes in conformation of polymerization sites induced by US. Effects were more
apparent, however, on Bmax and also on the apparent rate constant for t-PA binding, which increased by 78% and 69% for non-crosslinked and crosslinked fibrin, respectively. These data are
consistent with an effect of US on increasing accessibility of t-PA to
binding to sites unavailable in polymerized fibrin that are fully
accessible on fibrin monomer.
The overall rate of fibrinolysis is determined by several factors
including fibrin structure, plasminogen, and t-PA concentration and the
presence of enzyme inhibitors. The transport of t-PA into the clot and
binding to fibrin is an important determinant of the rate as indicated
by both experimental results35-40 and mathematical modeling.41,42 Diamond and Anand42 have
presented a model of fibrinolysis using multicomponent
convection-diffusion equations. The model predicts that transport of
reactants into the fibrin matrix is a rate-limiting step in
fibrinolysis. The model also analyzes the effects of steric hindrance
of t-PA binding to fibrin within fibers as compared with that on the
surface of fibers. Increasing binding of t-PA by improved penetration
into fibrin fibers accelerates fibrinolysis up to four-fold in the
model. Therefore, both the increased permeation through fibrin in the presence of US, as shown previously23 and the increased
binding of t-PA to fibrin reported herein may contribute to the
acceleration of fibrinolysis with US.
Other data also indicates that US causes a reversible alteration in
fibrin structure. The flow of fluid through fibrin gels under constant
pressure increases reversibly with US exposure.23 Because
flow at constant pressure is a function of resistance, which is
determined by fiber structure,43 this finding indicates that US causes a reversible alteration in fibrin structure. This was
confirmed by electron microscopic observations demonstrating that
noncrosslinked fibrin that is fixed during US exposure shows an
increased fiber density and a decrease in mean fiber
diameter.24 This effect is reversible, with return of fiber
structure to baseline when the acoustic field is removed. Fully
crosslinked fibrin showed less change in fiber density and size,
consistent with the smaller effect on Bmax demonstrated.
Therefore, experiments with flow resistance, electron microscopic
observations, and t-PA binding data are all consistent with a
reversible alteration in the polymerization state of fibrin fibers
caused by US. Together, these findings support the novel concept that
US can exert bioeffects through a nonthermal mechanism by altering the
structure of a biopolymer and influencing its interaction with
physiologically important ligands.
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FOOTNOTES |
Submitted June 9, 1997;
accepted November 3, 1997.
Supported in part by Grants No. HL-30616 and HL-50497 from the National
Heart, Lung and Blood Institute, National Institutes of Health,
Bethesda, MD.
Address reprint requests to Charles W. Francis, MD, Vascular Medicine
Unit, PO Box 610, 601 Elmwood Ave, Rochester, NY 14642.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Carol Weed for her help in the preparation of the
manuscript.
| |
REFERENCES |
1.
Rijken DC,
Hoylaerts M,
Collen D:
Fibrinolytic properties of one-chain and two-chain human extrinsic (tissue-type) plasminogen activator.
J Biol Chem
257:2920,
1982[Free Full Text]
2.
Higgins DL,
Vehar GA:
Interaction of one-chain and two-chain tissue plasminogen activator with intact and plasmin-degraded fibrin.
Biochemistry
26:7786,
1987[Medline]
[Order article via Infotrieve]
3.
Larsen GR,
Henson K,
Blue Y:
Variants of human tissue-type plasminogen activator.
J Biol Chem
263:1023,
1988[Abstract/Free Full Text]
4.
Husain SS,
Hasan AAK,
Budzynski AZ:
Differences between binding of one-chain and two-chain tissue plasminogen activators to non-cross-linked and cross-linked fibrin clots.
Blood
74:999,
1989[Abstract/Free Full Text]
5.
Fleury V,
Loyau S,
Lijnen R,
Nieuwenhuizen W,
Angles-Cano E:
Molecular assembly of plasminogen and tissue-type plasminogen activator on an evolving fibrin surface.
Eur J Biochem
216:549,
1993[Medline]
[Order article via Infotrieve]
6.
Grailhe P,
Nieuwenhuizen W,
Angles-Cano E:
Study of tissue-type plasminogen activator binding sites on fibrin using distinct fragments of fibrinogen.
Eur J Biochem
219:61,
1994
7.
Camiolo SM,
Thorsen S,
Astrup T:
Fibrinogenolysis and fibrinolysis with tissue plasminogen activator, urokinase, streptokinase-activated human globulin and plasmin.
Proc Soc Exp Biol Med
38:277,
1971
8.
Hoylaerts M,
Rijken DC,
Lijnen HR,
Collen D:
Kinetics of the activation of plasminogen by human tissue plasminogen activator. Role of fibrin.
J Biol Chem
257:2912,
1982[Abstract/Free Full Text]
9.
Hantgan R,
McDonagh J,
Hermann J:
Fibrin assembly.
Ann NY Acad Sci
408:344,
1983[Medline]
[Order article via Infotrieve]
10.
Weisel JW:
Fibrin assembly. Lateral aggregation and the role of the two pairs of fibrinopeptides.
Biophys J
50:1078,
1986
11.
Francis CW,
Onundarson PT,
Carstensen EL,
Blinc A,
Meltzer RS:
Enhancement of fibrinolysis in vitro by ultrasound.
J Clin Invest
90:2063,
1992
12.
Blinc A,
Francis CW,
Trudnowski JL,
Carstensen EL:
Characterization of ultrasound-potentiated fibrinolysis in vitro.
Blood
81:2636,
1993[Abstract/Free Full Text]
13.
Luo H,
Steffen W,
Cercek B,
Arunasalam S,
Maurer G,
Siegel RJ:
Enhancement of thrombolysis by external ultrasound.
Am Heart J
125:1564,
1993[Medline]
[Order article via Infotrieve]
14.
Olsson SB,
Johansson B,
Nilsson A-M,
Olsson Ch,
Rouer A:
Enhancement of thrombolysis by ultrasound.
Ultrasound Med Biol
20:375,
1994[Medline]
[Order article via Infotrieve]
15.
Harpaz D,
Chen X,
Francis CW,
Marder VJ,
Meltzer RS:
Ultrasound enhancement of thrombolysis and reperfusion in vitro.
JACC
21:1507,
1993 [Abstract]
16.
Tachibana K:
Enhancement of fibrinolysis with ultrasound energy.
J Vasc Intervent Radiol
3:299,
1992[Medline]
[Order article via Infotrieve]
17.
Lauer CG,
Burge R,
Tang DB,
Bass BG,
Gomez ER,
Alving BM:
Effect of ultrasound on tissue-type plasminogen activator-induced thrombolysis.
Circulation
86:1257,
1992[Abstract/Free Full Text]
18.
Kudo S:
Thrombolysis with ultrasound effect.
Tokyo Jikeikai Med J
104:1005,
1989
19.
Hamano K:
Thrombolysis enhanced by transcutaneous ultrasonic irradiation.
Jikeikai Med J
106:533,
1991
20.
Kornowski R,
Meltzer RS,
Chernine A,
Vered Z,
Battler A:
Does external ultrasound accelerate thrombolysis? Results from a rabbit model.
Circulation
89:339,
1994[Abstract/Free Full Text]
21.
Kashyap A,
Blinc A,
Marder VJ,
Penney DP,
Francis CW:
Acceleration of fibrinolysis by ultrasound in a rabbit ear model of small vessel injury.
Thromb Res
76:475,
1994[Medline]
[Order article via Infotrieve]
22.
Francis CW,
Blinc A,
Lee S,
Cox C:
Ultrasound accelerates transport of tissue plasminogen activator into clots.
Ultrasound Med Biol
21:419,
1995[Medline]
[Order article via Infotrieve]
23.
Siddiqi F,
Blinc A,
Braaten J,
Francis CW:
Ultrasound increases flow through fibrin gels.
Thromb Haemost
73:495,
1995[Medline]
[Order article via Infotrieve]
24. Braaten JV, Siddiqi F, Francis CW: Ultrasound reversibly
disaggregates large fibers in fibrin gels. Thromb Haemost (in press)
25.
Fraker PJ,
Speck JR Jr:
Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril.
Biochem Biophys Res Commun
80:849,
1978[Medline]
[Order article via Infotrieve]
26.
Carr ME Jr,
Shen LL,
Hermans J:
Mass-length ratio of fibrin fibers from gel permeation and light scattering.
Biopolymers
16:1,
1977[Medline]
[Order article via Infotrieve]
27.
Blombäck B,
Okada M:
On pores in fibrin gels.
Thromb Res
16:141,
1982
28.
Odrljin TM,
Rybarcyzk BJ,
Francis CW,
Hamaguchi M,
Lawrence SO,
Simpson-Haidaris PJ:
Calcium modulates plasmin cleavage of the fibrinogen D fragment chain N-terminus: Mapping of monoclonal antibody J88B to a plasmin sensitive domain of the chain.
Biochim Biophys Acta
1298:69,
1996[Medline]
[Order article via Infotrieve]
29.
Munson PJ,
Rodbard D:
Ligand: A versatile computerized approach for characterization of ligand-binding systems.
Analyt Biochem
107:220,
1980
30.
Voskuilen M,
Vermond A,
Veeneman GH,
van Boom JH,
Klasen EA,
Zegers ND,
Nieuwenhuizen W:
Fibrinogen lysine residue A157 plays a crucial role in the fibrin-induced acceleration of plasminogen activation, catalyzed by tissue-type plasminogen activator.
J Biol Chem
262:5944,
1987[Abstract/Free Full Text]
31.
Nieuwenhuizen W,
Voskuilen M,
Vermond A,
Hoegee-de Nobel B,
Traas DW:
The influence of fibrin(ogen) fragments on the kinetic parameters of the tissue-type plasminogen-activator-mediated activation of different forms of plasminogen.
Eur J Biochem
174:163,
1988[Medline]
[Order article via Infotrieve]
32.
Schielen WJ,
Voskuilen M,
Adams PJ,
Tesser GI,
Nieuwenhuizen W:
The sequence -(312-324) is a fibrin-specific epitope.
Blood Coag Fibrinolysis
1:521,
1991
33.
Schielen WJG,
Adams HPHM,
Van Leuven K,
Voskuilen M:
Structural requirements of fibrinogen A alpha-(148-160) for the enhancement of the rate of plasminogen activation by t-PA.
Blood
77:2169,
1991[Abstract/Free Full Text]
34.
Yonekawa O,
Voskuilen M,
Nieuwenhuizen W:
Localization in the fibringen -chain of a new site that is involved in the acceleration of the tissue-type plasminogen activator-catalyzed activation of plasminogen.
Biochem J
283:187,
1992
35.
Kandarpa K,
Drinker PA,
Singer SJ,
Caramore D:
Forceful pulsatile local infusion of enzyme accelerates thrombolysis: In vivo evaluation of a new delivery system.
Radiology
168:739,
1988[Abstract/Free Full Text]
36.
Prewitt RM,
Gu S,
Greenberg D,
Chan SM,
Schick U,
LaPoiknte H,
Ducas J:
Effects of flow on recombinant tissue plasminogen activator-induced pulmonary thrombolysis.
J Appl Physiol
71:1441,
1991[Abstract/Free Full Text]
37.
Bookstein JJ,
Saldinger E:
Accelerated thrombolysis. In vitro evaluation of agents and methods of administration.
Invest Radiol
20:731,
1985[Medline]
[Order article via Infotrieve]
38.
Blinc A,
Planinsic G,
Keber D,
Jarh O,
Lahajnar G,
Zidansek A,
Demsar F:
Dependence of blood clot lysis on the mode of transport of urokinase into the clot a magnetic resonance imaging study in vitro.
Thromob Haemost
65:549,
1991[Medline]
[Order article via Infotrieve]
39.
Blinc A,
Keber D,
Kahajnar G,
Stegnar M,
Zidansek A,
Demsar F:
Lysing patterns of retracted blood clots with diffusion or bulk flow transport of plasma with urokinase into clotsa magnetic resonance imaging study in vitro.
Thromb Haemost
68:667,
1992[Medline]
[Order article via Infotrieve]
40.
Wu J-H,
Siddiqui K,
Diamond SL:
Transport phenomena and clot dissolving therapy: An experimental investigation of diffusion-controlled and permeation-enhanced fibrinolysis.
Thromb Haemost
72:105,
1994[Medline]
[Order article via Infotrieve]
41.
Zidansek A,
Blinc A:
The influence of transport parameters and enzyme kinetics of the fibrinolytic system on thrombolysis: Mathematical modelling of two idealised cases.
Thromb Haemost
65:553,
1991[Medline]
[Order article via Infotrieve]
42.
Diamond SL,
Anand S:
Inner clot diffusion and permeation during fibrinolysis.
Biophys J
65:2622,
1993[Abstract/Free Full Text]
43.
Blömback B,
Okada M:
Fibrin gel structure and clotting time.
Thromb Res
25:51,
1982[Medline]
[Order article via Infotrieve]
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Abstract
Introduction
Abstract
, 
,