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Binding of Fibrin Fragments to One-Chain and Two-Chain Tissue-Type
Plasminogen Activator
By Ahmed A.K. Hasan, Won S. Chang, and Andrei 2.Budzynski
To explore whether fibrin fragments have binding affinity for
the tissue-type plasminogen activator (t-PA) molecule, the
interactions were studied of (DD)E complex and fragments
DD, E,, and E, with one-chain and two-chain t-PA. For this
purpose, a solid-phase binding assay was developed using
microtiter plates with nitrocellulose filters. It was found that
(DD)E complex and fragments DD and E, retained the t-PA
binding function of the parent fibrin molecule, thus demonstrating that t-PA binds to both the D and E domains of fibrin.
Unexpectedly, fragment E, did not bind t-PA. Fibrin fragments had different binding properties for one-chain and
two-chain t-PA. (DD)Ecomplex had the highest and fragment
E, the lowest affinity for one-chain t-PA, both binding curves
being consistent with one class of binding sites. However,
binding of the fragments with two-chain t-PA was distinguished by more than one class of binding sites, with
fragment E, having the highest affinity for this form of the
activator. e-Aminocaproicacid, even at 50 mmol/ L concentration, had only minimal effect on binding of (DD)E complex or
fragment DD to either one-chain or two-chain t-PA. The
potentiatingeffect of fibrin fragments on plasminogenactivation by t-PA was measured by a chromogenic substrate
assay. Fragment DD was the most effective stimulator of
plasminogen activation by t-PA. In conclusion, (DD)E complex and fragment DD retained most of the regulatory
functions of fibrin, which included t-PA binding and t-PAmediated acceleration of plasminogen activation to plasmin.
o 1992by TheAmerican Society of Hematology.
T
pass different domains of the parent molecule,3J1 differences in t-PA binding were expected. On the basis of t-PA
interaction with fibrin,12J3we hypothesized that one-chain
and two-chain t-PA will also interact differently with fibrin
fragments. Having such specific tools as (DD)E complex,
fragments DD, E,, and E,, we addressed the question of
their binding to t-PA and its effect on plasminogen activation. Our previous studies showed that one-chain and
two-chain t-PA have different affinities for the fibrin
To explore which FDPs have affinity for t-PA, we investigated the binding of fibrin fragments with one-chain and
two-chain t-PA by a newly developed solid-phase binding
assay. We studied the potentiating effect of fragments on
plasminogen activation by t-PA using a chromogenic substrate assay. The aim of the work was to correlate the
t-PA-binding parameters of the fragments with the potentiating effect of fibrin fragments on plasminogen activation
and assess the role of fragments in the regulation of
physiologic fibrinolysis.
ISSUE-TYPE plasminogen activator (t-PA) plays an
important role in blood clot dissolution in v i v ~ .t-PA
~,~
converts plasminogen into plasmin, and the rate of plasminogen activation by t-PA is significantly accelerated by
fibrin. Plasmin degrades the fibrin matrix of the clot and
generates different fibrin degradation products (FDPs):
(DD)E complex, and fragments DD, E,, and E,., Fibrin
fragments differ in their fibrin-binding properties and in
their regulatory effects on fibrin polymerization.”6 (DD)E
complex neither binds to fibrin nor inhibits the formation of
a clot; fragment D D binds with high affinity to both
cross-linked and noncross-linked fibrin and inhibits fibrin
monomer polymerization; fragment E, has affinity for
cross-linked fibrin and for fibrin clots both in vitro and in
vivo6s7 but it does not inhibit fibrin polymerization? The
fibrinogen degradation products fragments D, and D, were
reported to potentiate plasminogen activation by t-PA,
whereas fragment E, did not have this effect. Thus, some
fragments can act as effector molecules in the process of
plasmin generation from plasminogen by t-PA, yet the
mechanism of interaction of fragments with t-PA remains
speculative.
Despite experimental attempts, the possible binding sites
for t-PA on the fibrin molecule have not been conclusively
defined.8~~
Studies on the interaction of two-chain t-PA with
different fibrinogen fragments to localize the binding site
for the t-PA molecule concluded that the D domain may
participate in t-PA binding, whereas the E domain would
bind pla~minogen.~
These studies dealt with binding of
two-chain t-PA with different fragments of fibrinogen
rather than of fibrin. The importance of the plasmin
cleavage of the t-PA molecule into two-chain t-PA has not
been addressed. Also, studies in the past on the interaction
of different fibrinogen fragments with t-PA addressed the
process of fibrinogenoly~is~
rather than fibrinolysis, and the
effect of t-PA chain cleavage on modulation of the process
was not explored. A recent report clearly demonstrated that
the t-PA-interacting domain is absent in fibrinogen but
exposed in fibrin.1°
Evidence gathered in the past clearly demonstrated that
plasmic degradation products of fibrin do retain fibrinbinding f ~ n c t i o n .Because
~.~
various fibrin fragments encomBlood, Vol79, No 9 (May 1). 1992: pp 2313-2321
EXPERIMENTAL PROCEDURES
Materials
Human fibrinogen (grade L), plasminogen (20 CUImg), and
plasmin (10 CU/mg) were obtained from Kabi Vitrum (Stockholm,
Sweden). Human a-thrombin, specific activity 3,315 Ulmg, was a
generous gift from Dr John W. Fenton I1 (New York State
Department of Health, Albany). One-chain and two-chain forms of
From the Department of Biochemistry, Temple University School of
Medicine, Philadelphia, PA.
Submitted August 6,1990; accepted December 26, 1991.
Supported by Grant HL 36221 from the National Heart, Lung and
Blood Institute, National Institutes of Health, Bethesda, MD.
Presented in part in abstract form (Circulation 8O:II-642, 1989).
Address reprint requests to Andrei Z. Budzynski, PhD, Department
of Biochemistry, Temple University School of Medicine, 3400 N Broad
St, Philadelphia, PA 191 40.
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.
0 1992 by The American Society of Hematology.
0006-497119217909-0007$3.00/0
2313
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2314
t-PA, purified from a human melanoma cell line, were obtained
from American Diagnostica (New York, NY).The purity of these
preparations had been verified by reduced sodium dodecyl sulfatepolyaclylamide gel electrophoresis (SDS-PAGE) and their specific
activity was 500,000 U/mg.
Trasylol (Aprotinin; Mobay Chemical Corp, New York, NY),
eaminocaproic acid (EACA), and Tween 20 were obtained from
Sigma Chemical Corp (St Louis, MO). Bovine serum albumin
(BSA) (ultrapure grade) was from Boehringer-Mannheim (Indianapolis, IN). Chromogenic substrate S-2251 (D-Val-Leu-Lys-NHpNA) was from Kabi Vitrum. Tissue culture plates with 96 wells
were purchased from Elkay Products, Inc (Shrewsbury, MA).
Sepharose CLdB was obtained from Pharmacia (Piscataway, NJ).
Bio-gel P-6 prepacked, Econo-Pack 10 DG columns were purchased from Bio-Rad (Richmond, CA). The computer program,
Equilibrium Binding Data Analysis (EBDA and Ligand), originally
written by R.J. Munson and D. Rodbard and modified by G.A.
McPherson, was obtained from Elsevier-Biosoft (Cambridge, UK).
Millititer HA plates with 96 wells containing nitrocellulose filters,
HATF type, with a pore size of 0.45 pm were obtained from
Millipore Corp (Bradford, MA). Millititer Vacuum Holder, filtrate
collection tray, and Millititer Filter Punch for solid-phase binding
assay between FDP and t-PA were obtained from the same source.
Methods
Preparation of (DD)E complex. Plasmic degradation product of
cross-linked fibrin, the (DD)E complex, was prepared as described
before., Briefly, 1 g of dry cross-linked fibrin (prepared from Kabi
fibrinogen) and 1 U of plasmin were suspended together as quickly
as possible in 20 mL of prewarmed 0.15 mol/L Tris-HC1 buffer,
containing 5 mmol/L of CaCl, and 0.02% sodium azide, pH 7.4.
The digestion was performed under constant gentle stirring at 37°C
for 21.5 hours for maximum yield of the complex. The reaction was
then stopped by addition of 0.01 mL of Trasylol to a final
concentration of 100 KIU/l U of plasmin. The small amount of
particulate material was removed by centrifugation at 3,OOOg at 4°C
for 15 minutes. Clean supernate was chromatographed on a
Sepharose CL-6B column (2.5 x 190 cm), equilibrated and eluted
with 0.05 mol/L Tris-HCI buffer, containing 0.1 mol/L sodium
chloride, 0.028 mol/L sodium citrate, 25 KIU/mL of Trasylol, and
0.02% sodium azide, pH 7.4. Approximately 250 mg of protein in a
volume of 10 mL was applied in each run with a flow rate of 40
mL/h. Fractions of 8 mL within main peaks were combined and
analyzed on SDS-PAGE and nondissociating PAGE. The main
peak fractions contained pure homogeneous (DD)E complex of
250 Kd.
Preparation of fragment DD. To obtain the maximum yield of
fragment DD; 1g of dry cross-linked fibrin was digested by 10 U of
plasmin for 21.5 hours. The digestion was terminated by 0.1 mL of
Trasylol (100 KIU/l U of plasmin). The centrifuged digest was
chromatographed on a Sepharose CL-6B column (2.5 X 190 cm),
equilibrated, and eluted with 0.05 mol/L Tris-HC1 buffer containing 1 mol/L sodium chloride, 0.028 mol/L sodium citrate, 25
KIU/mL of Trasylol, and 0.02% sodium azide, pH 7.4. The
fractions (8 mL) within the main peak were combined, concentrated, and analyzed. The main peak fractions contained pure
homogeneous fragment DD of 190 Kd.
Preparation offragment of E,. About 50 mg of (DD)E complex
was dissociated with 3 mol/L urea, pH 5.3, at 37°C for 1 hour? The
sample was chromatographed on a Sepharose CL-6B (0.9 X 90 cm)
column, equilibrated and eluted with the same buffer used for the
separation of fragment DD. Fragment DD and E, eluted in two
separate peaks. SDS-PAGE was performed to verify homogeneity
of the preparation. The E, preparation, of 60 Kd was pooled and
concentrated.
HASAN, CHANG, AND BUDZYNSKI
Preparation of fragment E? Fragment E,, of 45 Kd, was obtained from a terminal plasmic digest of cross-linked fibrin by
column gel filtration as described? Briefly, cross-linked fibrin was
digested by 100 U of plasmin/lg fibrin in the absence of Ca2+for 24
hours and gel filtered on the same Sepharose CLdB column used
for separation of fragment DD. Fragment E, was in the second
peak and was separated from fragment DD, present in the first
peak, and (Y polymer remnants, present in the third peak. The
homogeneity of the preparation was tested on SDS-PAGE. The
fractions containing fragment E, were pooled and concentrated.
Preparation of fibrin monomer (FM). FM was prepared as
described b e f ~ r e . ~
Labeling of fibrin fragments. Radioiodination of FDPs, (DD)E
complex, fragments DD, El, and E,, and control protein BSA were
performed by the lodogen method.13 The fragment (0.5 mg) was
incubated with Nal=I for 20 minutes at room temperature. Excess
of radioactive reagent was removed by gel filtration on Econo-Pack
10 DG column equilibrated with 0.01 mol/L sodium phosphate,
0.15 mol/LNaCl, 0.01% Tween 20,25 KIU/mLof Trasylol, pH 7.4.
Labeled fragments were analyzed for homogeneity by SDS-PAGE.
Radiolabeling of t-PA. t-PA was radioiodinated by the iodogen
method and subsequently purified by affinity chromatography on
fibrin-celite column as described by us before.',
Immobilization of &PA. Different amounts of radiolabeled t-PA
(0.1 to 5 pg) in various volumes (20 to 100 p,L) were incubated in
microtiter plate with nitrocellulose filters at 4°C for 24 hours. The
filters were washed once with washing buffer (phosphate-buffered
saline [PBS], 0.1% Tween 20, 25 KIU/mL Trasylol, pH 7.4),
punched out, and counted in a gamma counter. It was determined
that 1 pg of either form of t-PA in 25 p,L of PBS buffer, containing
a 25 KIU/mL of Trasylol, pH 7.4, could be completely immobilized
on a nitrocellulose filter.
Solid-phase binding assay. We developed a solid-phase binding
assay using the principle of membrane binding and filtration of
excess reagents1417to investigate the interaction between FDP and
both forms of t-PA.
The amount of labeled FDP bound to t-PA immobilized on the
nitrocellulose filter was determined by direct measurement of
radioactivity. One-chain or two-chain t-PA (25 p,L, containing 40
p,g/mL in PBS, 25 KIUimL Trasylol, pH 7.4) was absorbed on
nitrocellulose filters in 96-well filtration plates, incubating overnight at 4°C. The next day, the incubation buffer was removed by
vacuum and the bottom of the plate was dried. Any potential
protein binding sites on the filter that remained after immobilization of t-PA were blocked by incubating at room temperature for 1
hour with 0.1% ultrapure BSA in PBS and 25 KIU/mL of Trasylol,
pH 7.4. The solution was removed by vacuum and the filters dried.
The wells were rinsed once with washing buffer (PBS, 0.1% Tween
20, 25 KIU/mL Trasylol, pH 7.4), and once with PBS alone,
vacuum filtered, and dried. Triplicate aliquots (50 pL) of
labeled FDP at 16 different concentrations, ranging from 0.02
pmol/L to 12 pmol/L, in the incubation buffer (PBS, 0.1%
ultrapure BSA, 0.01% Tween 20, 25 KIU/mL Trasylol, pH 7.4)
were incubated in the wells at room temperature for 2 hours. It was
predetermined that equilibrium was achieved under these conditions. Aqueous solution was removed by vacuum, and the wells
were rinsed quickly three times with washing solution and vacuum
dried. Dry filters were punched into plastic tubes using the
Millititer Filter Punch and counted in a gamma scintillation
counter. Simultaneously, and on the same filter plate, 1251-labeled
BSA containing the same amount of protein and radioactivity as
the sample was used in a parallel control experiment to determine
the amount of nonspecifically bound radioactivity. Determination
of nonspecific binding was also performed using lz5I-FDP and
nitrocellulose filters coated with BSA. The results of nonspecific
-
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2315
BINDING OF FIBRIN FRAGMENTS TO t-PA
180
u)
al
c
Fig 1. Binding isotherm of (DD)E complex with
one-chain t-PA. Binding experiments were performed
at a constant amount of one-chain t-PA (14 pmol)
immobilizedon nitrocellulose filter and variable concentrationsof radioiodinated(DD)E complex (0.02 to
12 wmol/L). The fragment bound to t-PA was determined by counting the radioactivity incorporated in
the filter. Inset: Computer ffi Scatchard analysis.
-
0
0
200
FREE, p i c o m o l e s
400
FREE, p i c o m o l e s
binding obtained by the two methods were virtually the same and
showed that nonspecific binding was below 5% of the total input
level of protein. Subtraction of nonspecific binding from total
binding with labeled FDP determined the amount of FDP specifically bound to t-PA. The dissociation constant (kd), overall binding
capacity (Bmax), and the number of classes of binding sites were
calculated from a computer fit to the Scatchard equation. The
method provided optimal (weighted least squares) estimates of the
binding parameters.
Competition of unlabeled FDP with the binding of lZI-FDP to
one-chain or two-chain t-PA was investigated by incubating either
form of immobilized t-PA with a constant amount of radiolabeled
FDP and variable amounts (0.01 to 30 kmol/L) of unlabeled FDP.
Interference of EACA (0.1 to 50 mmol/L) with binding of lZI-FDP
to t-PA was also investigated in a similar manner.
Chromogenic substmate assay. To determine the effect of FDP
on plasminogen activation by t-PA, a chromogenic substrate
(S-2251) assay was performed in a microtiter plate as described
before18J9with some modifications. The assay was performed in a
3oo-pLvol in 0.05 mol/L Tris-HC1,0.15 mol/L NaCI, 0.01% Tween
20, pH 8.0 buffer. The system contained t-PA (5 ng/mL), plasminogen (0.13 pmol/L), S-2251 (0.3 mmol/L), and varying concentrations (0 to 1 kmol/L) of FDP. Each experimental point was
investigated in triplicate. FM, fragment D1,and incubation buffer
were used as controls and appropriate blanks were included. The
0
200
400
reaction mixture was incubated at room temperature (25°C) and
absorbance at 405 nm was measured every 30 minutes using a
multiwell semi-automated plate scanner (Bio-Rad). The change in
absorbance was plotted against time squared and the slope of the
linear part of the curve was taken as a measure of plasminogen
activator activity. Stimulation factors were defined as the ratio of
plasminogen activator activities in the presence of FDP, to the
activity in the absence of FDP.
RESULTS
Analysis of binding data at a constant concentration of
either one-chain or two-chain t-PA and variable concentration of (DD)E complex indicated that at the concentration
range of the fragment used, binding of (DD)E to both
forms of t-PA did not reach saturation (Figs 1 and 2).
Although derivation of Scatchard parameters from binding
data obtained under incomplete ligand saturation has been
questioned by some researchers?O othersz1have shown that
the binding data obtained below saturation levels may
provide meaningful estimates of kd and the nature of the
binding sites. We analyzed the binding data by a computer
fit to the Scatchard equation to quantify the binding of
(DD)E complex to one-chain and two-chain t-PA. Models
Fig 2. Binding isotherm of (DD)E complex with
two-chain t-PA. The procedure for the binding assay
and analysis of the data follows the description given
in the legend for Fig 1. Inset: Computer fit Scatchard
analysis.
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2316
HASAN, CHANG, AND BUDZYNSKI
involving interactions through a single site and multiple
sites were tested. The binding of (DD)E complex to
one-chain t-PA had a complex nature, showing a positive
cooperativity at lower concentration range, and a single
class of binding sites at higher concentration of the ligand
(Fig 1, inset). The best statistical fit was achieved with a
single-site model, characterized by a kd of 8.7 pmol/L (Fig
1, inset and Table 1). The best fit (underlined) has been
selected on the basis of the lowest values of P and mean
square (m.s.) describing the binding of (DD)E complex
with one-chain t-PA, for one-site model (P < g ,m.s. 538),
two-site model (Pm, m.s.m), and three-site model (Pm,
m.s.m). In contrast, the Scatchard plot of (DD)E complex
binding with a two-chain t-PA had a curvilinear form,
implying the presence of more than one class of binding
sites (Fig 2, inset). Ligand analysis derived the best fit with a
two-site model. The best fit (underlined) has been selected
for the binding of (DD)E complexwith two-chain t-PA for a
one-site model (P > .05, m.s. 315), two-site model (P < E,
m.s. E),and three-site model (Pm, m.s.00). Analogous
statistical assessment has been performed for selecting the
best fit to a model for binding of other fibrin fragments with
t-PA. The kd values of the interaction were 0.5 pmol/L and
4.7 pmol/L for the high- and low-affinity site, respectively
(Fig 2, inset and Table 1). All statistical results regarding
the fit of binding data to a one-, two-, or three-site model
are shown in Table 2.
Binding of Fragment DD to t-PA
Data obtained by the solid-phase binding assay at a
constant concentration of t-PA and variable concentrations
of fragment DD showed that binding of fragment DD to
t-PA was dose-dependent and saturable (Figs 3 and 4). The
Scatchard plot of the binding data with one-chain t-PA was
linear and was resolved into a single-site model with a kd of
24 pmol/L (Fig 3, inset and Table 1). On the other hand,
the Scatchard plot of fragment D D binding with two-chain
t-PA was curvilinear, indicating multiple-site interaction.
The best computer fit was achieved with a two-site model
Table 1. Binding Parameters for Fibrin Fragment
interactionWith t-PA
Fragment
Type of t-PA
Model'
(DD)E
One-chain
Two-chain
1 Site
2 Sites
DD
One-chain
Two-chain
1 Site
2 Sites
One-chain
Two-chain
One-chain
Two-chain
1 Site
2 Sites
1 Site
2 Sites
E,
€3
kd (mol/L)
8.7
0.5
4.7
24
0.4
20
>1
>1
x
x 10-6
x 10-6
x
x
x
x 10-2
x 10-2
30 x 10-6
2.2 x 10-9
2.8 x 10-6
Bmax (mol/L)
5.7
2.5
2.0
5.5
8.7
3.2
x
x 10-7
x 10-6
x
x lo-*
x
-
8.3 x 10-7
4.6 x 10-7
2.5 x 10-7
*The information reflects the best fit to a model defined by either 1 or
2 classes of binding sites with different affinities. Best fit for the data
was judged by statistical comparison of fit at P < .05 and lowest mean
square value.
(Fig 4, inset) characterized by a kd of 0.4 pmol/L and 20
pmol/L for the high- and low-affinity sites, respectively (Fig
4,inset and Table 1).
Binding of Fragment E, to t-PA
Binding experiments evaluating the interaction of iodinated fragment E, with one-chain and two-chain t-PA, at
constant concentration of the activator and a variable
concentration of the fragment, showed no measurable
affinity between the components. From the competition
binding assay (data not shown), an approximate kd of
interaction was derived at 50% replacement of the iodinated fragment El by the unlabeled counterpart. The
calculated kd of 10 mmol/L was the same for both forms of
the activator and was regarded rather high and physiologically insignificant.
Binding of Fragment E3 to t-PA
Binding of fragment E3 with one-chain t-PA was dosedependent and saturable. The Scatchard plot was linear
and the kd of the interaction was calculated to be 30
pmol/L (Table 1).In contrast, the interaction between the
fragment and two-chain t-PA was complex and the Scatchard plot had a curvilinear shape (data not shown) which
implied involvement of multiple sites with different affinities. The best fit was obtained with a two-site model. The
high-affinity site had a kd of 2.2 nmol/L and the low-affinity
site had a kd of 2.8 pmol/L (Table 1).
Competition by Unlabeled Fibrin Fragments
Investigation of the effect of unlabeled (DD)E complex
on the interaction of the radioiodinated complex with t-PA
showed that binding of the labeled complex to one-chain
and two-chain t-PA was inhibited in a dose-dependent
manner (Fig 5A). This suggested that the binding of
radioiodinated (DD)E complex to both forms of t-PA was
specific and not caused by nonspecific adsorption or aggregation. In a similar competition assay, unlabeled fragment
DD reversed the binding of the radiolabeled fragment to
one-chain or two-chain t-PA, suggesting that the interaction was specific (Fig 5B). The calculated kds derived from
50% inhibition of binding between fragments, (DD)E or
DD, and t-PA were 10 kmol/L and 30 pmol/L, respectively, and compared well with kd values obtained from the
Scatchard plots (Table 1).
Effect of EACA on Binding Between Fibrin Fragments
and t-PA
To investigate the role of lysine residues in the binding of
(DD)E complex to t-PA and to assess the role of kringle-2
domain in this function, the effect of EACA was studied.
The interaction of (DD)E complex with one-chain and
two-chain t-PA was measured in the presence of up to 50
mmol/L EACA. The results suggest that the EACA was
incapable of displacing (DD)E complex from these plasminogen activators (Fig 6A). As in the case of (DD)E
complex, 50 mmol/L EACA was unable to displace frag-
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2317
BINDING OF FIBRIN FRAGMENTS TO t-PA
Table 2. Statistical Evaluation of Models Assessing the Binding of Fibrin Fragment With t-PA
Model
Fragment
(WE
Type of t-PA
One-chain
Parameter*
One-chain
m
m
m
m
m
-
-
m
m
1,757
13
135
m
D.F.
m.s.
7+ 6< .01
4,097
13
315
Res.
P
10+ 6> .05
6 3 < .05
-
LS
6,846
13
527
3+13-
LS
D.F.
m.s.
Res.
P
DD
Two-chain
PS
D.F.
m.s.
Res.
P
Ei
One-chain
Three-site
m
Res.
P
DD
Two-site
7,240
13
5%
LS
D.F.
M.S.
Two-chain
One-site
> .05
8,247
14
589
10+ 6 > .05
12,52711
1,139
13+ 3-
< .05
3,663
14
262
9+7> .05
m
m
m
m
m
m
m
m
m
m
m
PS
D.F.
No measurable binding
m.s.
Res.
P
E,
Two-chain
IS
D.F.
ms.
Res.
P
E3
One-chain
ZS
D.F.
ms.
Res.
P
E3
Two-chain
LS
D.F.
m.s.
Res.
P
No measurable binding
25,904
11
2.3a
8+ 6> .05
9,251
-
> .05
m
m
m
m
m
m
-
-
m
m
3,622
< .05
-
m
m
m
m
The underlined values of statistical parametersare those which support a specific model for binding.
*The abbreviations are: Ls,weighted sum of squares, evaluates the total squared deviations of the data points from the predicted values on the
curve; D.F., degrees of freedom, the larger the value, the better the fit to the curve; ms., mean square, equals Ls/D.F.; the smaller the value, the
better the fit to the curve; Res., residuals, assess the number of experimental points that occur above (+) the curve or below (-) it. The more equal
distribution of + and - residuals, the better the fit; P, this value determines the level of confidence of fit to the selected model.
mDenotes that the program cannot fit the experimental data to a predicted model making the model impossible to solve. This is possibly due to the
selection of an inappropriate model.
ment DD from one-chain and two-chain t-PA (Fig 6B).
Similar lack of EACA effect on fragment E3was observed
(data not shown).
Effect of FDP on the Activation of Plasminogen by t-PA
Glu-plasminogen activation by t-PA in the presence of
various amounts of FM, (DD)E complex, fragments DD,
D,, and E, was investigated (Fig 7). The activation of
plasminogen by t-PA was stimulated considerably by FDP.
The maximum rate enhancement by FM in this assay was
eightfold. FDPs, fragment DD, and (DD)E complex at 1
p,mol/L accelerated plasminogen activation sevenfold and
sixfold, respectively. The half-maximal acceleration in all
three cases was reached at a concentration of 0.03 pmol/L.
Fragment E, had very little stimulating activity, whereas
fibrinogen fragment D, had almost no effect on plasminogen activation by t-PA. The FDPs fragment DD and (DD)E
complex, in contrast to degradation products derived from
fibrinogen, were stronger stimulators of plasmin generation
from plasminogen by t-PA.
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HASAN, CHANG, AND BUDZYNSKI
90
t
n
-
FREE, picomoles
DISCUSSION
The results demonstrated that (DD)E complex has an
affinity for both one-chain and two-chain t-PA. Analysis of
the binding data of (DD)E complex to one-chain t-PA
showed a complicated interaction and a positive cooperativity. On conversion of one-chain t-PA into its two-chain
counterpart, the binding to (DD)E complex could best be
characterized by a two-site model. The conversion of
one-chain t-PA into two chains did generate a small
number of high-affinity sites. The affinity of t-PA for the
complex (Table 1) was comparable with that of fibrin.12J3
The results suggest that the fibrin-binding sites for t-PA are
retained in the complex. Bosma et aI9 showed that fibrinogen fragment E, did not have any affinity for t-PA. If the E
domain of the (DD)E complex does not participate in this
interaction, then the binding properties of fragment DD
and the (DD)E complex would have been very similar, if
not identical. That has not been found in this work.
Fragment DD bound to both forms of the activator.22The
affinity between the fragment and one-chain t-PA was
lower than that between (DD)E complex and the activator
(Table 1). This fact indicated that the separation of the E
domain from (DD)E complex decreased the affinity of the
remaining fragment D D for one-chain t-PA. Whether this
Fig 4. Binding isotherm of fragment DD with
two-chain t-PA. The procedure for the binding assay
and analysis of the data follows the description given
in the legend for Fig 3. Inset: Computer fit Scatchard
analysis.
.
Fig 3. Binding isotherm of fragment DD with
one-chain t-PA. Bindingexperiments were performed
at a constant amount of one-chain t-PA (14 pmol)
immobilizedon nitrocellulose filter and variable concentrations of radioiodinatedfragment DD (0.02 to 12
Fmol/L). The fragment bound to t-PA was determined by counting the radioactivity incorporated in
the filter. Each point is a mean of three determinations. Inset: Computer fit Scatchard analysis.
was caused by loss of binding sites on the E domain or by
attenuation of the cooperative effect of the E domain on
this interaction remains to be elucidated. Fragment DD
had multiple-site interaction with two-chain t-PA, indicating the presence of more than one class of binding sites.
The high-affinity binding sites for (DD)E complex on
two-chain t-PA were retained in fragment DD (Table 1).
The low-affinity sites for (DD)E complex on two-chain t-PA
became four times weaker for fragment DD.
There was no measurable affinity between fragment E,
and t-PA. The binding between fragment E, and t-PA was
specific with lower affinity for one-chain and higher affinity
for two-chain t-PA. The E domain binding sites seem to
become exposed on degradation of fragment E, by plasmin
to terminal fragment E,. Radioiodinated fragment E, also
failed to bind to sonicated fibrin clot: suggesting an adverse
effect of iodination on fragment E,. This could result from
the blockade of the epitope by the large iodine molecule
attached to tyrosine or histidine residues. Also, the damage
of a crucial histidine residue (p16) cannot be ruled out as
the reason.23
The interaction between fragment E, and one-chain t-PA
was lower than that between (DD)E complex and fragment
DD (Table 1). On conversion of one-chain t-PA into
0
FREE, picomoles
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2319
BINDING OF FIBRIN FRAGMENTS TO t-PA
A
...........
120,
........
8
......I
. . ."."I
U
c
3
0
n
e
c
al
u
L
a2
n
n
Ln
I
N
IOt
-- A
-A
One-chain t-PA
Tvo-chain t-PA
Binding is also mediated through the finger and EGF
domains. It is possible that some specific lysine residues
may be involved that are not sensitive to EACA. In our
experience, 50 mmol/L EACA was also unable to completely replace one-chain and two-chain t-PA from fibrin.13
Absence of any significant effect of EACA on FDP interaction with t-PA can be explained in two ways. (1) Plasmic
degradation of fibrin into FDP exposes new terminal lysine
residues. Binding of FDP with t-PA is mediated through the
newly exposed lysines. EACA did not show any effect on
this interaction because the concentration used was not
high enough to be effective on terminal lysines. (2) Interaction of FDP with t-PA is mediated through specific sites.
Kringle 2 of the t-PA molecule does not seem to play a key
:
0
I
A
0.01
0.1
1
100
10
CONCENTRATION OF (DD)E,pM
120
C
B
a
0
n
r
120
C
80
U
c
L
3
0
al
n
e
W
n
al
n
,4
u
L
P
I
n
-
A One-chain 1-PA
- A Tvo-chain 1-PA
40
Y
al
P
P
__
h
c 80
I
A
ln
cI(
c
40
-- A
-A
I
Ln
0
One-chain t-PA
Tvo-chain t-PA
10
h(
c
0
I
01
0.1
........
I
1
. . . . . . . . . . . ,.,
I
10
CONCENTRATION OF D D , Y H
Fig 5. Reversibility of binding between fibrin fragment and t-PA. A
constant amount of 1251-fragmentwas incubated with one-chain (A)
and two-chain (A)t-PA (14 pmol), immobilizedon nitrocellulosefilter,
in the presence of variable concentrations of unlabeledfragment (0.01
to 30 pmol/L) at room temperature for 1 hour. The amount of
radiolabeled fragment bound to either form of t-PA was determined
by counting the remaining radioactivity on the filter. The amount of
radiolabeled fragment bound by t-PA in the absence of unlabeled
fragment was taken as 100% bound. All values are mean of three
determinations. (A) Effect of unlabeled (DD)E complex on the binding
of labeled (DD)Ewith one-chain (A)and two-chain (A)t-PA. (B) Effect
of unlabeledfragment DD on the binding of labeled fragment DD with
one-chain (A)and two-chain (A)t-PA.
CONCENTRATION OF EACA, pM
B
100
105
lo3
120
'0
C
a
0
n
r
2
80
V
L
__
a2
CT
d
-
n
A One-chain t-PA
- A Tvo-chain t-PA
40
v)
e4
0
10
lo3
5
10
CONCENTRATION OF EACA, pN
two-chain t-PA, the affinity for fragment E, increased many
fold. The binding of fragment E, to two-chain t-PA was the
strongest among all the fragments (Table 1).
EACA, up to 50 mmol/L concentration, inhibited minimally interaction of FDP with t-PA (Fig 6). It has been
postulated that plasmic degradation of fibrin exposes lysine
residues present on kringle 212,24,25
that mediate highaffinity binding. Other investigators concluded that kringle
2 does not mediate any binding between fibrin and t-PA.26,27
Fig 6. Effect of EACA on binding betweenfibrin fragment and t-PA.
A mixture of "1-fragment and EACA (0.1 to 50 mmol/L) was incubated with one-chain (A)and two-chain (A)t-PA (14 pmol), immobilized on nitrocellulose filter. The amount of fragment bound was
determined by counting radioactivity remaining on the filter. The
amount of fragment bound by t-PA in the absence of EACA was taken
as 100% bound. All values are the mean of three determinations. (A)
Effect of EACA on the interactionof (DD)Ecomplex with one-chain (A)
and two-chain (A)t-PA. (B) Effect of EACA on the interaction of
fragment DD with one-chain (A)and two-chain (A)t-PA.
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2320
HASAN, CHANG, AND BUDZYNSKI
1
'
"
'
'
'
'
1
a
7.5
. A FM
5.0
.
-
2.5
oob
0 0
'
I
I
I
0.3
0.6
0.9
O
M
(DD)E
A
El
0 Dl
I
1.2
PROTEIN CONCENTRATION, pM
Fig 7. Potentiating effect of fibrin fragments on plasminogen
activation by t-PA. Various concentrations (0 to 1 pmol/L) of different
fibrin fragments were incubated with a reaction mixture of t-PA (5
ng/mL), plasminogen (0.13 pmol/L), and S-2251 (0.3 mmol/L) in a
multiwell tissue culture plate. FM, fragment D,, and incubation buffer
were used as controls. The reaction mixtureswere incubated at room
temperature (25°C) and absorbance at 405 nm was measured in a
multiwell semi-automated plate scanner at different time intervals.
Each point represents the mean of triplicate determinations. Stimulation factors were determinedand were plotted against the concentration of proteins.
molecule, it was expected that different potentiating effects
on plasminogen activation would be observed.30Fragment
DD and (DD)E complex were strong stimulators of the
plasminogen activation rate and strong ligands for t-PA.
Fragment DD may bind endogenous circulating t-PA and,
having fibrin affinity, deliver it to the clot to increase the
local effective concentration of the activator. Moreover,
fragment DD may inhibit fresh clot formation and platelet
aggregati~n.~,"
Taken together, it seems that fragment DD
is an important component in fibrinolysis and may play a
regulatory role at the site of thrombus formation and
dissolution. On the other hand, the COOH-terminus of the
a-chain and a part of the connector region of the fibrin
molecule do not appear to play any role in the plasminogenactivating process. Fibrin fragments were more potent than
fibrinogen fragments, a fact which suggests that it is the
exposure of binding sites on formation of fibrin from
fibrinogenlothat modulates the process of fibrinolysis.
The significance of different binding properties of FDPs
to the different forms of t-PA may be explained by the
following sequence of events. Both the D and E domains
contain t-PA binding sites for one-chain and two-chain
activators. Lysis of the initial clot is undesirable and the
affinity of t-PA for noncross-linked fibrin clot is low. After
cross-linking, one-chain t-PA binds avidly to fibrin, as does
plasminogen, initiating l y ~ i s . ~ Generation
~,~O
of high molecular weight FDPs, including (DD)E complex, results in
more t-PA binding with positive cooperativity, mobilizing
maximum lytic potential. During fibrinolysis, one-chain
t-PA is converted to its two-chain counterpart, and (DD)E
complex is converted into fragment DD, which has greater
affinity for two-chain than for one-chain t-PA. Further
degradation of fibrin and FDPs results in terminal products, fragments DD and E,. Fragment E, binds strongly
with two-chain t-PA and removes it from the site of lysis.
Fragment DD binds both one-chain and two-chain t-PA.
However, in contrast with fragment E,, fragment DD has an
affinity for the fibrin clot4 and thus may carry t-PA back to
thrombi and enhance fibrinolysis.
role in binding with FDP. That is why the interaction was
not sensitive to EACA and cannot be ascribed only to lysine
residues exposed by plasmin during degradation of fibrin.
Activation rates of plasminogen by t-PA were measured
in the presence of fibrin and FDPs to allow correlation of
rate-enhancing properties and binding. The results of the
stimulation experiments showed that the FDP fragment
DD and (DD)E complex were good rate-enhancers of
t-PA-mediated activation of Glu-plasminogen. It appeared
that fragment E, had a moderate stimulatory capacity,
whereas fibrinogen fragment D, did not have any effect.
The data showed that FDP, like the parent fibrin molecule,
can act as an effector molecule in the process of plasmin
generation from plasminogen by t-PA. Binding of t-PA, and
ACKNOWLEDGMENT
possibly also of plasminogen, and formation of a ternary
complex, may be responsible for the p h e n ~ m e n a . ~As
* , ~ ~ The authors are very thankful to Dr Fredda London for her
critical comments.
these FDPs represent different domains of the fibrin
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From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
1992 79: 2313-2321
Binding of fibrin fragments to one-chain and two-chain tissue-type
plasminogen activator
AA Hasan, WS Chang and AZ Budzynski
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