805
Platelet-Targeted Fibrinolysis Enhances Clot
Lysis and Inhibits Platelet Aggregation
Christoph Bode, MD; Gabriel Meinhardt; Marschall S. Runge, MD, PhD; Mathias Freitag;
Thomas Nordt, MD; Matthias Arens; John B. Newell, BA;
Wolfgang Kubler, MD; and Edgar Haber, MD
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Background. Although plasminogen activator therapy has been shown to reduce mortality in
patients with severe myocardial infarction, several problems fuel the search for more potent
and specific thrombolytic agents.
Methods and Resuls. To explore the effect of plasminogen activator targeting to platelets, we
covalently linked urokinase that had been modified with N-succinimidyl-3-(2-pyridyldithio)propionate to the Fab' of a monoclonal antibody (7E3) that selectively binds to platelet membrane
glycoprotein (GP) fib/Lla. In an assay measuring (as reflected by plasmin generation) a
plasminogen activator's ability to bind GP fib/lIla immobilized on plastic, urokinase-7E3 Fab'
produced 31-fold more plasmin than did urokinase (p=0.0001). The addition of solubilized GP
IIb/MIIa blocked this enhancement of plasmin generation, indicating that binding was impaired.
Plasmin generation reflecting binding to immobilized intact platelets was 2.4-fold greater for
urokinase-7E3 Fab' than for unconjugated urokinase (p=0.002). In a plasma clot lysis assay,
urokinase-7E3 Fab' was at least 25-fold more potent than either urokinase alone or a mixture of
urokinase and 7E3 (Fab')2 (p<0.009), and potency could be related to platelet concentration in the
clot. Ex vivo, ADP-induced platelet aggregation was inhibited by a urokinase-7E3 IgG conjugate at
a concentration of 8 nM, whereas a mixture of urokinase and 7E3 (Fab')2 in equimolar amounts
required 60 nM and urokinase alone required 1 ,M to achieve the same effect.
Conclusions. Therefore, the targeting of urokinase to the GP lIb/llla platelet receptor both
accelerates clot lysis (when platelets are associated with a fibrin clot) and inhibits platelet
aggregation. (Circulation 1991;84:805-813)
lasminogen activator therapy has been shown
to substantially reduce mortality in patients
with acute myocardial infarction.1-4 However, several problems fuel the search for more
potent and specific thrombolytic agents. Plasminogen
activators appear to be ineffective in approximately
25% of patients,5 and reperfusion of the thrombosed
vessel often does not occur quickly enough to prevent
the formation of a large infarcted area. Furthermore,
P
From Medizinische Klinik III (Kardiologie) der Universitat
Heidelberg (C.B., G.M., M.F., T.N., M.A., W.K.), Heidelberg,
FRG; Cardiology Division, Emory University (M.S.R.), Atlanta,
Ga.; Massachusetts General Hospital (J.B.N., E.H.) and Harvard
Medical School (E.H.), Boston, Mass.; and Bristol-Myers Squibb
Pharmaceutical Research Institute (E.H.), Princeton, N.J.
Supported by a grant from the Deutsche Forschungsgemeinschaft (Bo 726/3-2).
Address for correspondence: Dr. Christoph Bode, Medizinische
Klinik III (Kardiologie), Bergheimerstrasse 58, 6900 Heidelberg,
Germany.
Received September 25, 1990; revision accepted April 2, 1991.
a high rate of early reocclusion6 limits the benefits of
initially successful recanalization.
Arterial thrombi contain a high concentration of
activated platelets, and platelet-rich thrombi have
been shown to be particularly resistant to thrombolysis.7 Platelets also appear to play a key role
when initially reperfused vessels reocclude.8 Recently, a monoclonal antibody (7E3) that blocks the
platelet membrane glycoprotein (GP) lIb/Illa receptor has been developed.9 The combined administration of 7E3 and a thrombolytic agent reduces the rate
of reocclusion1011 and enhances the speed and efficacy of reperfusion12,13 in experimental animals.
Research from our laboratory has shown that
plasminogen activators targeted to the site of a fibrin
deposit by conjugation to an antifibrin antibody have
greatly enhanced potency and specificity.14-16 In the
present study of a urokinase-7E3 Fab' conjugate, we
investigated the effect on thrombolysis and platelet
aggregation of a plasminogen activator targeted to
a platelet antigen.
806
Circulation Vol 84, No 2 August 1991
Methods
Materials
High-molecular-weight two-chain urokinase (Urokinase Medac) was bought from Medac GmbH, Hamburg, FRG. L-Pyroglutamyl-glycyl-L-arginine-p-nitroanilide hydrochloride (S-2444), a chromogenic
substrate for urokinase, was obtained from KabiVitrum, as were H-D-isoleucyl-L-prolyl-L-arginine-pnitroanilide dihydrochloride (S-2288), a chromogenic
substrate for serine proteases, and H-D-valyl-L-leucylL-lysine-p-nitroanilide dihydrochloride (S-2251), a
chromogenic substrate for plasmin. Affinity-purified
polyclonal rabbit anti-human albumin IgG was purchased from Boehringer Mannheim, FRG. N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP) was purchased from Pierce Chemical, Rockford, Ill., and '251labeled human fibrinogen was purchased from
Amersham Buchler GmbH, Braunschweig, FRG.
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Fresh-frozen plasma and platelet-rich plasma were
purchased from the University of Heidelberg blood
bank or the German Red Cross, Baden-Baden, FRG.
Plasminogen was purified from fresh-frozen plasma on
lysine-Sepharose according to established procedures,17 and purity was assessed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and by chromogenic substrate assay (S-2251).
Bio-Beads SM-2 were obtained from Bio-Rad. p-Aminobenzamidine was coupled to CH-Sepharose 4B as
described by Holmberg et al.18 Monoclonal antibody
7E3 (Fab')2 was a gift from Centocor, Malvern, Penn.
The fibrin-specific monoclonal antibody (59D8) has
been described.19 All other chemicals came from Sigma
Chemical Co., St. Louis.
Punification of GP IIb/IIIa
GP 1Ib/IJIa was purified from platelet-rich plasma as
described.20 Before using GP Ilb/lIla in the assay
procedures, we quantitatively purified it of Triton
X-100 by gel filtration on a Bio-Beads SM-2 column
(25 xl cm).21 As an alternate, GP lIb/llla was purified
in small amounts on a lysine-Sepharose 4B column to
which the peptide Gly-Arg-Gly-Asp-Ser-Pro-(Cys) had
been coupled with maleimidobenzoylsuccinimide ester
(MBS) in a modification of the method described by
Pytela et a122 or on a lysine-Sepharose column to which
7E3 Fab' had been coupled with SPDP. Purity of the
resulting protein preparation was assessed by SDSPAGE under reducing and nonreducing conditions.
The protein was stored at -20°C in tris-(hydroxylmethyl)-aminomethane buffered saline (TBS)-CaCl
(0.15 M NaCl, 20 mM Tris, 1 mM CaCl, pH 7.4).
Protein Concentrations
Protein concentrations were determined by either the method of Lowry et a123 or optical density
at 280 nm.
Electrophoresis
SDS-PAGE was performed according to the
method of Laemmli,24 and proteins were visualized
with Coomassie brilliant blue G-250 according to
Neuhoff et a125 or by silver staining.26
Preparation of 7E3 Fab'
Antibody 7E3 has been described.9 7E3 Fab' was
prepared by reducing 7E3 (Fab')2 (10 mg, 2 mg/ml
0.1 M sodium phosphate, pH 6.8) at room temperature for 18 hours in 1 mM 2-mercaptoethylamine, 1
mM EDTA, and 10 mM sodium arsenite, followed by
the addition of solid Ellman's reagent to a concentration of 5 mM. After 30 minutes at room temperature, excess reagent was removed by gel filtration on
a Sephadex G-25 column (30x2 cm) equilibrated
with 0.1 M sodium phosphate (pH 6.8). In this
protected form, 7E3 Fab' has remained structurally
and functionally intact at 4°C under sterile conditions
for more than 3 months. The thiol form of 7E3 Fab'
was regenerated by treatment with 10 mM 2-mercaptoethylamine for 30 minutes at room temperature,
followed by gel filtration.
Punification of Urokinase
Urokinase was separated from human serum albumin (added as a stabilizer) by affinity chromatography on a cyanogen bromide-activated Sepharose 4B
column to which polyclonal rabbit anti-human albumin antibody had been coupled. The urokinasealbumin mixture was repeatedly chromatographed
until the urokinase in the fall-through fraction was
electrophoretically devoid of albumin. The column
could be regenerated by elution of albumin with 0.2
M glycine (pH 2.75). As an alternate, functionally
intact urokinase was selected by affinity chromatography on benzamidine-Sepharose 4B.18
Preparation of Urokinase-7E3 Fab' Conjugate
Urokinase (4 ml) in NaPi buffer (3.0 mg/ml) in 0.14
M NaCl, 3.7 mM sodium phosphate, and 1 mM KCl
(pH 7.4) was mixed with SPDP (20 mM in absolute
ethanol, fivefold molar excess added dropwise to the
stirred solution), and the reaction was allowed to
proceed for 30 minutes at room temperature. The
reaction mixture was then dialyzed overnight against
several changes of 0.1 M sodium phosphate and 0.1 M
NaCl (pH 7.4). Analysis for 2-pyridyldisulfide content27 typically showed 0.5-2.0 residues per urokinase
molecule. The substitution level was kept intentionally
low to minimize loss of urokinase activity and avoid
the formation of higher-molecular-weight aggregates.
The thiol form of 7E3 Fab' (5.0 ml at 1.0 mg/ml in
sodium phosphate, pH 6.8) was mixed with SPDPmodified urokinase and allowed to react for 20 hours
at room temperature. The reaction was halted by the
addition of a 100-fold molar excess (compared with
protein concentrations) of iodoacetamide in 0.1 M
sodium phosphate (pH 8.0).
Urokinase-7E3 IgG conjugate and urokinase59D8 IgG conjugate were synthesized as described.28
Bode et al Platelet-Targeted Plasminogen Activator
Punification of Urokinase-7E3 Fab' Conjugate
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The desired conjugate was purified from the reaction mixture in two sequential affinity chromatography steps. First, selection for plasminogen activator
binding to benzamidine-Sepharose retained conjugated and unconjugated plasminogen activator but
not uncoupled 7E3 Fab' (which was also recovered).
The eluate (elution buffer of 0.1 M sodium acetate
and 0.4 M sodium chloride, pH 4.0) was neutralized
with 3 M Tris-HCl (pH 8.0) and passed through a
column of Sepharose conjugated to goat anti-mouse
Fab antibody. This column retained only the desired
urokinase-7E3 Fab' conjugate, which was eluted
with 0.2 M glycine (pH 2.8), neutralized with 3 M
Tris-HCl (pH 8.0), and dialyzed against PBSA (0.01
M sodium phosphate, 0.14 M sodium chloride, 0.01%
Tween 80, and 0.01% sodium azide). Conjugate
could be stored in this buffer under sterile conditions
at 4°C for at least 4 weeks.
Urokinase-7E3 IgG conjugate was purified according to this procedure, and urokinase-59D8 IgG
conjugate was purified as described.28
Quantitation of Urokinase Activity
Urokinase activity was determined as described.29
To obtain a comparison of the enzymatic properties
of urokinase and its conjugate, not only with regard
to cleavage of a chromogenic substrate but also with
regard to plasminogen activation, enzyme activity was
monitored with the plasmin-sensitive chromogenic
substrate S-2251. Cell culture plates (96-well, Linbro) were coated with TBS-CaCl containing 5%
(wt/vol) bovine serum albumin (BSA) to inhibit nonspecific absorption. The plates were then extensively
rinsed with water and dried. Plasminogen activator
solution (50 gl) containing various amounts of enzyme was added to the wells. Then, a substrate
solution consisting of (1:3 vol/vol) 4 mM S-2251 in
H20 and plasminogen (0.75 CU/ml) in S-2251 assay
buffer (50 mM Tris and 110 mM sodium chloride)
was mixed, and 100 gl was added to each well. The
plasmin-dependent conversion of substrate S-2251
was read at 405 nm in a conventional enzyme-linked
immunosorbent assay reader (Titertek Multiscan
Plus, Flow, Lugano, Switzerland). Medac Urokinase
was used as standard.
Plasminogen Activation as a Function of Binding to
Whole GP IIb/IIIa Complex
Microtiter plates (96-well, U-shaped, Beckton Dickinson) were coated overnight at 4°C with 100 ,l GP
IIb/lIIa solution (50 gg/ml). The plates were extensively rinsed with water, and 200 ,1 TBS-CaCl (pH 7.4)
containing 1% BSA was added to block nonspecific
binding. After 6 hours at room temperature, the plates
were rinsed again. TBS-CaCl (100 ,ul) containing different amounts of urokinase or urokinase-7E3 Fab'
conjugate was then added to each well, and the plates
were allowed to incubate for 16 hours at 4°C. The
plates were rinsed again, and 100 ,ul of S-2251 substrate
807
solution (described above) was added to each well. The
increase in absorbance at 405 nm was measured after
90 and 120 minutes. Inhibition assays were performed
with the following modification. Before urokinase or
urokinase-7E3 Fab' conjugate was added to the wells,
100 ,ul of one of three TBS-CaCl buffer solutions (pH
7.4) was placed in each well: buffer alone, buffer
containing GP lIb/llla at 0.6 mg/ml, or buffer containing BSA at 0.6 mg/ml.
Plasminogen Activation as a Function of Binding to
Whole Platelets
Platelet concentrates were stored at -80°C. After
thawing, they were centrifuged at 1,800g for 15
minutes at 4°C. The pellet, containing the platelets,
was washed three times in TBS-EDTA (0.15 M NaCl,
20 mM Tris, and 1 mM EDTA, pH 7.4). Platelets in
the final pellet were resuspended in TBS-CaCl to a
concentration of approximately 100,000 platelets/,al.
Platelet suspension (100 pl) was added to each well
of a 96-well microtiter plate, and the plates were
centrifuged at 2,000g for 10 minutes.30 After the
plates had been washed twice with water, 200 pAl
TBS-CaCl containing 5% BSA (wt/vol) was added to
each well. After 6 hours at room temperature, the
plates were washed again with water. At this point,
the coated plates could be stored for as long as 1
week at 4°C. Then, plasminogen activator in TBSCaCl containing 2.5% BSA was added to each well
and incubated for 3 hours at room temperature or
overnight at 4°C. After extensive rinsing with water,
S-2251 substrate solution was added, and the increase in absorbance at 405 nm was measured after
90 and 120 minutes.
Clot Lysis Assay in Human Plasma
The method of Lijnen et a131 was used with some
modification. Five units of fresh-frozen plasma obtained from the local blood bank were pooled, aliquoted, and refrozen. Immediately before each experiment, the activities of urokinase and urokinase-7E3
conjugate were calibrated as described above. The
plasminogen activation potential of the urokinase-7E3
conjugate was determined in the absence of the antibody target molecule and related to that of a standard
concentration of urokinase. Appropriate dilutions were
made so that the plasminogen activation potential was
identical for each sample. The dilutions of the urokinase-7E3 conjugate were tested again in triplicate
against the dilutions of urokinase (in triplicate). Only
when all dilutions showed equal plasminogen activation
potential was the assay performed.
Platelet concentrates were washed as described
above at room temperature. The final pellet was
resuspended in the same volume of plasma. As an
alternate, the number of platelets was determined,
and a desired platelet concentration was achieved
by dilution with plasma. Platelet-rich clots were
made by adding to the platelet suspension in the
following sequence: '251-labeled human fibrinogen
(500,000 cpm/ml of suspension), 0.5 M CaCl (final
808
Circulation Vol 84, No 2 August 1991
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concentration in plasma, 0.05 M), and thrombin (8
National Institutes of Health [NIH] units/ml of
plasma). Immediately after the addition of thrombin, the solution was drawn into Silastic tubing
(inner diameter, 4 mm) and allowed to clot for 30
minutes at 37°C. The tubing was then cut into
2.5-cm sections, yielding clots of approximately 0.2
ml. The clots were removed from the tubing, and
each was placed into a plastic vial and washed with
3 ml of 0.15 M NaCI. The clots were subsequently
counted and suspended in 2 ml of fresh-frozen
plasma (of the same pool from which they had been
made; clots made of plasma containing 14 x 106
platelets were incubated in platelet-poor plasma).
In some experiments, we obtained platelet-free
plasma by centrifuging fresh-frozen plasma at
20,000 rpm for 20 minutes before adding it to the
trace-labeled clots. Figure 7 summarizes the results
of these experiments. Experiments were started by
the addition of plasminogen activators (or TBS as
control) in 200 g1 of TBS. At various intervals,
aliquots of fresh-frozen plasma were removed from
each tube for determination of radioactivity. Lysis
was determined from the calculated total amount of
radioactivity released from the clot and radioactivity initially incorporated into the clot.
Platelet Aggregation
Platelet aggregation was measured in an aggregometer (Bio/Data PAP-4). Aggregation was stimulated
with ADP (final concentration, 2 ,uM). Fresh platelets
were collected on citrate from young healthy adults by
venipuncture. Platelet-rich plasma was obtained by
centrifugation at 900 rpm in plastic tubes at room
temperature for 15 minutes in a laboratory centrifuge
(Sorvall RT 6000). The effects on platelet aggregation
of urokinase, 7E3 (Fab')2, a mixture of urokinase and
7E3 (Fabl)2, and urokinase-7E3 Fab' conjugate were
measured after a 5-minute preincubation at 37°C of the
substance to be tested with platelet-rich plasma. To
quantify the inhibitory effect of the different substances
on platelet aggregation, we investigated the minimal
concentration of a substance capable of preventing final
transmittance of more than 10%. All assays were
performed within 60-180 minutes of platelet collection.
Results
Characterization of Urokinase-7E3 Fab' Conjugate
The nature of the disulfide-linked urokinase-7E3
Fab' conjugate is defined in part by its purification.
Because of the two-step affinity purification procedure, the conjugate must have both the ability to bind
to benzamidine (i.e., have serine protease activity)
and the ability to bind to a goat anti-mouse Fab
antibody (i.e., contain a Fab portion). When the
conjugate is electrophoresed under reducing conditions, disulfide bonds are reduced, resulting in the
visualization of the individual components: heavyand light-chain urokinase at 34 and 21 kDa, respectively, and heavy- and light-chain Fab' at 28 and 25
2.5
2.0
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0.5
2.5
Plasminogen Activator (units)
10
FIGURE 1. Plot of plasminogen activation as a function of
binding to immobilized target molecule glycoprotein IIb/IIIa.
Plasmin-dependent cleavage of chromogenic substrate S-2251
was recorded as the rate of change in absorbance at 405 nm
after 90 minutes (o, urokinase; 0, urokinase-7E3 Fab'
conjugate) and 120 minutes (o, urokinase; *, urokinase-7E3
Fab' conjugate). Each point represents mean of three independent experiments. Error bars, SD.
kDa, respectively (not shown). Molecules of an apparent molecular weight of approximately 100 kDa
were visualized under nonreducing conditions, indicating that predominantly a 1:1 complex of Fab' and
urokinase had formed. The yield after double-affinity
purification varied between 3% and 5%, calculated
on the basis of antibody-coupled urokinase activity.
Plasminogen Activation in the Absence of Target
Molecule GP IIb/IIIa
The plasminogen activator activity of urokinase
was compared with that of the urokinase-7E3 Fab'
conjugate in the absence of the antibody target
molecule GP JIb/lIla by measuring plasmin generation in a plasminogen solution. Plasmin-dependent
cleavage of chromogenic substrate S-2251 was recorded as the rate of change in absorbance at 405 nm
for the concentrations of activators actually used in
the tests. The enzymatic activities of urokinase and
urokinase-7E3 Fab' conjugate were comparable at
all concentrations tested and at all time points after
an appropriate calibration procedure.
Plasminogen Activation as a Function of Binding to
GP IIb/IIIa Complex
Urokinase and urokinase-7E3 Fab' conjugate were
compared in the presence of GP IIb/Illa as follows.
After the plasminogen activator activity of each species had first been assessed in the absence of GP
JIb/IIIa, as described under "Methods," samples with
identical enzymatic activity were then assayed with the
target molecule GP IIb/Illa. Figure 1 shows that after
90 minutes, considerably more plasmin had been
generated by the urokinase-7E3 Fab' conjugate than
by the parent molecule urokinase in the presence of
GP lIb/Illa. Compared with urokinase at 90 and 120
Bode et al Platelet-Targeted Plasminogen Activator
809
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0
0.5
10
2
0.1
50
Plasminogen Activator (units)
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FIGURE 2. Plot of plasminogen activation as a function of
binding to immobilized target molecule glycoprotein (GP) Ilb!
IIIa in the presence and absence of solubilized GP Ilb/IIIa.
Plasmin generation was determined at 90 minutes (o, urokinase;
0, urokinase-7E3 Fab' conjugate; *, urokinase-7E3 Fab'
conjugate plus 0.6 mg/ml GP IIb/IIIa). Each point represents
mean of three independent experiments. Error bars, SD.
minutes, urokinase-7E3 Fab' conjugate had generated 35-fold (p=0.0015) and 31-fold (p=0.0001)
more plasmin, respectively (Figure 1). The probable
reason for this difference is the binding of the conjugate to the target molecule through the 7E3 antibody
combining site, whereas urokinase, which is incapable
of specific binding, is washed out.
To determine whether the enhanced efficacy of
urokinase-7E3 Fab' conjugate was caused by antigen
binding, we added free GP IIb/lIla to the assay
solution. Figure 2 shows that after the addition of
free GP JIb/llla, the conjugate is equipotent to
urokinase alone. BSA alone had no effect (data not
shown).
2.0
In
0
aw
10
FIGURE 4. Plot ofplasminogen activation by urokinase and
urokinase-7E3 Fab' conjugate as a function of binding to
whole, immobilized platelets. Plasmin generation was determined at 210 minutes (o, urokinase; 0, urokinase-7E3 Fab'
conjugate). Each point represents mean of two independent
experiments. Error bars, SD.
As a further validation of specificity, a conjugate
of urokinase and intact antifibrin monoclonal antibody 59D8 was synthesized and compared with a
urokinase-7E3 IgG conjugate (both antibodies are
of the same isotype and were tested at equipotency
in the S-2251 assay) in an assay measuring plasminogen activation in the presence of GP JIb/llla.
Figure 3 shows that urokinase-59D8 is equipotent
to urokinase, whereas urokinase-7E3 is more potent. This indicates that simple linkage of urokinase
to an antibody of the IgG1 isotype does not enhance
plasminogen activation in the presence of GP IIb/
Illa. Targeting by the fibrin-specific site in 59D8
would not be expected in this assay in the absence
of fibrin.
Plasminogen Activation as a Function of Binding to
Intact Platelets
In this assay, we investigated plasminogen activation in the presence of intact platelets adhered to the
bottom of a 96-well microtiter plate. As shown in
Figure 4, urokinase-7E3 Fab' was 2.4-fold more
potent than urokinase (p=0.002). Enhancement of
activity was less than that observed with purified GP
JIb/lIla, probably because of a lower density of
epitopes for the antibody.
2.5
0
0.5
2.5
Plasminogen Activator (units)
1.5
1.0
0
c:
.5
16
31
62
125
250
Plasminogen Activator (units)
FIGURE 3. Plot of comparison of urokinase-7E3 IgG conjugate with a conjugate of urokinase and the fibrin-specific
monoclonal antibody 59D8; the antibodies are of the same
isotype. Plasmin generation was determined at 90 minutes (o,
control conjugate; 0, urokinase-7E3 IgG conjugate; urokinase). Each point represents mean of six independent experiments. Error bars, SD.
u,
Thrombolysis of Plasma Clots by Urokinase-7E3
Fab' Conjugate
Figure 5 compares the activity of urokinase-7E3
Fab' with that of urokinase in an in vitro human
plasma clot lysis assay. Because interassay variability
is quite large with this assay, intra-assay comparisons
were made whenever possible. At the concentrations
tested, urokinase showed very little activity against
these platelet-rich clots (approximately 14.4x 106
platelets/mm3), whereas the conjugate was 970-fold
as active (p=0.03). An equimolar mixture of uroki-
Circulation Vol 84, No 2 August 1991
810
30
100
80
20
en
c)
._o
030
0,O-
60
40
10
20
0
0
10
31
20
50
100
Plasminogen Activator (units/mi)
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FIGURE 5. Plot of lysis ofplatelet-rich plasma clots containing approximately 14.4x106 platelets/mm3. Lysis at 4 hours
(a) and 18 hours (a) is shown for urokinase-7E3 Fab'
conjugate. Lysis at 18 hours is shown for an equimolar mixture
of urokinase and 7E3 (Fab')2 (A), urokinase (o), and saline
control (.). Each point represents mean of three independent
experiments. Error bars, SD.
nase and 7E3 (Fab')2 was not significantly different
from urokinase in lytic activity (p=0.19). The relatively small difference between lysis results at 4 hours
and those at 18 hours was regularly observed and may
be explained by assuming that the most resistant
parts of the thrombus remain intact for the longest
time. Also, activator inactivation and/or plasmin activation may play a part in generating this effect.
To quantify the effect of platelet concentration on
plasma clot lysis, we clotted fresh-frozen plasma that
contained 2.0 +0.8 x 103 (platelet free), 19.7 +1.2 x 103
(platelet poor), or 14.4+0.68x106 platelets/mm3. For
assay, the clots were suspended in platelet-poor
fresh-frozen plasma containing 19.3 ± 1.7 x103 platelets/mm3. Figure 6 (top panel) shows that urokinase7E3 Fab' was twice as potent as urokinase in lysing
clots made of platelet-poor plasma (p=0.0001) and
much more potent than urokinase in lysing clots
made of plasma containing the highest platelet concentration (Figure 6, bottom panel). Urokinase-7E3
and urokinase were equipotent in the lysis of clots
made of virtually platelet-free plasma. The concentration of platelets in the clot did not affect the
degree of lysis by urokinase at the lowest and intermediate concentrations, whereas at the highest concentration lysis by urokinase could not be effectively
measured. Thus, it appears that platelet concentration is a major determinant of the efficacy of the
fibrinolytic activity of urokinase-7E3.
Figure 7 shows that the enhancement of fibrinolytic activity by urokinase-7E3 Fab' relative to urokinase was independent of platelet age. Plasma clots
were made from fresh blood (drawn immediately
before the experiment) and stored platelet-rich
plasma (from outdated platelet-rich plasma packs)
containing 283 + 41.6x 103 and 307 9.1 x 103 platelets/mm3, respectively, and they were incubated in
50 62.5 75
100 125
Plasminogen Activator (units/mi)
250
15 -
.)
10 I
-JO
5
0
-
25
25
50
50
75
100
125
75
100
125
Plasminogen Activator (units/mi)
FIGURE 6. Plots of lysis of plasma clots containing various
concentrations ofplatelets in the presence of urokinase-7E3
Fab' conjugate. Top panel: Lysis at 4 hours of clotted plasma
that contained 2.0±0.8 x103 platelets/mm3 (o, urokinase; A,
urokinase-7E3 Fab' conjugate) and 19. 7+1.2x 103 platelets/
mm3 (, urokinase; 0, urokinase-7E3 Fab' conjugate). Bottom panel: Lysis at 8 hours of clotted plasma that contained
14.4+0.68x 106 platelets/mm3 (o, urokinase; 0, urokinase7E3 Fab' conjugate). Each point represents mean of three
independent experiments. Error bars, SD.
platelet-free plasma. There was no significant difference between fresh and stored platelets with regard
to fibrinolysis by urokinase-7E3 Fab' or urokinase.
Platelet Aggregation
The inhibitory effects on platelet aggregation of
urokinase, 7E3 (Fab')2, an equimolar mixture of urokinase and 7E3 (Fab')2, and a urokinase-7E3 IgG conjugate were tested ex vivo. Urokinase-7E3 IgG conjugate completely inhibited platelet aggregation at a
concentration of 8 nM, whereas a mixture containing
an equimolar amount of 7E3 (Fab')2 and urokinase
required a concentration of 60 nM to achieve the same
effect. 7E3 (Fab')2 alone also required 60 nM to inhibit
aggregation. Urokinase was least effective, requiring 1
,M to achieve inhibition (Figure 8).
Discussion
After purification involving two sequential and
different affinity chromatography steps, urokinase-
Bode et al Platelet-Targeted Plasminogen Activator
to the concentration of platelets in the clot. Clots
greatly enriched in platelets could not be lysed at the
concentrations of urokinase tested, whereas lysis was
readily apparent at equivalent concentrations of the
urokinase-7E3 Fab' conjugate. Thus, compared with
urokinase, the relative thrombolytic potency of the
urokinase-7E3 Fab' conjugate increased with the
number of platelets in the clot. However, our results
confirm findings that platelet-rich clots are particularly resistant to thrombolysis,7 as can be deduced
from the reduced absolute rates of thrombolysis
achieved by both urokinase and urokinase-7E3 Fab'
conjugate. Platelet aggregation was more effectively
inhibited by the conjugate than by urokinase, 7E3
(Fab')2, or an equimolar mixture of urokinase and
7E3 (Fab')2.
It is likely that antibody targeting to the GP
lIb/llla receptor accounts for the increased fibrinolytic potency observed for urokinase-7E3 Fab'. As
would be expected in an antigen-antibody interaction, soluble GP JIb/llla competitively inhibited the
increase in plasminogen activation observed in the
presence of immobilized receptor. Enhanced plasminogen activation could only be demonstrated with
a conjugate containing the GP lIb/IIIa-specific antibody 7E3; it was not observed with a conjugate
containing an antibody of different specificity (antifibrin antibody 59D8), albeit of the same isotype. In
addition, the urokinase-7E3 Fab' conjugate had no
advantage over urokinase with regard to lysis of
plasma clots made from platelet-free-plasma.
It is of interest that the epitope to which the
antibody binds is stable to storage; no difference was
observed in the lysis of clots containing fresh or old
platelets. This observation suggests that if the prin-
80
60
C,nU)
40
20
0
16
62
31
125
Plasminogen Activator (units/mi)
FIGURE 7. Plot of lysis after 4 hours of clots containing stored
(, urokinase; *, urokinase-7E3 Fab' conjugate) and fresh (o,
urokinase; A, urokinase-7E3 Fab' conjugate) platelets. Each
point represents mean of three independent experiments. Error
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
bars, SD.
7E3 Fab' conjugate was shown to have a molecular
size of approximately 100 kDa and an approximately
1:1 molar ratio of urokinase and 7E3 Fab'. Compared with uncoupled urokinase in a quantitative in
vitro assay measuring (as reflected by plasmin generation) a plasminogen activator's ability to bind to GP
Ilb/llla or whole platelets immobilized on plastic,
urokinase-7E3 Fab' conjugate generated more plasmin in the presence of immobilized GP 1Ib/IIIa or
whole platelets than did urokinase. Urokinase-7E3
Fab' conjugate proved to be more potent than urokinase or an equimolar mixture of urokinase and 7E3
(Fab')2 in the lysis of platelet-rich plasma clots in
human plasma, and the rate of lysis could be related
(nM)
(nM) B
A
811
60
0
30
0
250
15
0)
mi
100
C
0
-
D
As'-
c
-_
0
30
(C
W)
20) 50
15
0)11
100
1 min
.
L
J
FIGURE 8. Plots of inhibition of platelet aggregation induced by ADP (at 2 pM) in platelet-rich plasma obtained in one
representative experiment (offive with platelets obtained from different donors) at different concentrations of urokinase (A), 7E3
(Fab')2 (B), a mixture of urokinase and 7E3 (Fab')2 (C), and urokinase-7E3 IgG conjugate (D).
812
Circulation Vol 84, No 2 August 1991
ciple of platelet-targeted fibrinolysis were applied in
vivo, old and new thrombi would be equally susceptible to lysis. The striking effect of the conjugate in
preventing platelet aggregation does not appear to be
the result of blockade of the GP Ilb/lIla receptor
because the conjugate is far more potent than 7E3
alone. The high concentration of urokinase at the
receptor site may activate plasmin locally, resulting in
the lysis of fibrinogen bound to the receptor or even
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the receptor itself.
Plasminogen activator-antifibrin antibody conjugates or recombinant proteins have been shown to be
more effective than the parent plasminogen activators in the lysis of in vitro clots and in vivo venous
thrombi.1516 Platelet-directed plasminogen activators
such as the one we described not only bind to a
different target in the thrombus but also address two
significant residual problems in thrombolytic therapy:
the platelet-rich arterial thrombus that is resistant to
lysis7,8 and early platelet-mediated reocclusion. Figure 6 (bottom panel), showing lysis by the antibody
conjugate of a clot containing a high concentration of
platelets under conditions in which urokinase was
completely ineffective, supports the potential therapeutic role of a platelet-targeted agent. Further
studies will show whether the promising in vitro
properties of this molecule will result in superior
thrombolysis in vivo.
8.
9.
10.
11.
12.
13.
Acknowledgments
14.
We thank Kwan Y. Hui, PhD, for synthesizing the
peptide Gly-Arg-Gly-Asp-Ser-Pro-(Cys); Harvey Berger, MD, for helpful discussions about the manuscript;
and Ms. Simone Bauer for expert technical assistance.
15.
16.
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KEY WORDS * urokinase * plasminogen activator * fibrinolysis
* thrombolysis * platelets
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Platelet-targeted fibrinolysis enhances clot lysis and inhibits platelet aggregation.
C Bode, G Meinhardt, M S Runge, M Freitag, T Nordt, M Arens, J B Newell, W Kübler and E
Haber
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Circulation. 1991;84:805-813
doi: 10.1161/01.CIR.84.2.805
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