(Circulation. 1995;91:1175-1181.)
© 1995 American Heart Association, Inc.
Articles |
Thrombolysis and Reocclusion in Experimental Jugular Vein and Coronary Artery Thrombosis
Effects of a Plasminogen Activator Inhibitor Type 1–Neutralizing Monoclonal Antibody
From the Center for Hemostasis, Thrombosis, Atherosclerosis, and Inflammation Research (B.J.B., M.L., J.W.t.C.); the Department of Experimental Cardiology (R.C., M.J.J.); and the Department of Biochemistry (H.P.), Academic Medical Center, University of Amsterdam, the Netherlands.
Correspondence to Bart J. Biemond, MD, Center for Hemostasis, Thrombosis, Atherosclerosis, and Inflammation Research, Academic Medical Center, F4-237, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background Thrombolytic therapy for acute myocardial infarction is often complicated by reocclusion of the initially reperfused artery. Platelets have been shown to play an important role in this process. We determined the contribution of plasminogen activator inhibitor type 1 (PAI-1), stored in the
-granules of
platelets, to thrombolysis resistance and to reocclusion. Methods and Results In a rabbit jugular vein thrombosis model, the effect of a PAI-1–neutralizing monoclonal antibody (CLB-2C8) on thrombolysis and thrombus growth was assessed. The effect on reperfusion, reocclusion, and duration of vessel patency was studied in a canine model of coronary artery thrombosis superimposed on a high-grade stenosis and endothelial damage. In the rabbit jugular vein model, the intravenous administration of 1 mg/kg anti–PAI-1 antibody significantly enhanced the endogenous thrombolysis from 5.5±1.3% in the animals treated with a nonspecific monoclonal antibody (control) to 13.7±2.6% in the animals treated with the anti–PAI-1 antibody. Thrombus growth was reduced significantly, from 41.3±2.6% in the control animals to 22.8±2.8% in the animals treated with the anti–PAI-1 antibody. In combination with a single bolus injection of recombinant tissue-type plasminogen activator (rTPA; 0.25 mg/kg), the anti–PAI-1 antibody reduced thrombus growth significantly, from 21.5±2.7% in the animals treated with rTPA alone to 12.2±2.6% in the animals treated with rTPA and the antibody. No additional effect of the anti–PAI-1 antibody was observed on rTPA-induced thrombolysis. In the canine coronary artery thrombosis model, the administration of a suboptimal dose of rTPA (0.45 mg/kg) induced reperfusion in 7 of the 8 dogs after 19.5±8.2 minutes. Reperfusion was followed by reocclusion in all animals after 3.3±2.6 minutes. Administration of the anti–PAI-1 antibody in combination with rTPA significantly reduced time to reperfusion (8.1±5.2 minutes) and delayed the occurrence of reocclusion to 11.6±12.5 minutes.
Conclusions Administration of the anti–PAI-1 antibody (CLB-2C8) results in increased endogenous thrombolysis and inhibition of thrombus growth in a venous thrombosis model in rabbits and facilitated reperfusion and reduction of reocclusion in a canine model of coronary artery thrombosis.
Key Words: plasminogen activators • occlusions • arteries • thrombosis
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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Thrombolytic treatment significantly reduces mortality in patients with acute myocardial infarction.1 2 The fibrinolytic system is activated by infusion of plasminogen-activating agents such as streptokinase, recombinant tissue-type plasminogen activator (rTPA), or recombinant single-chain urokinase, and this results in rapid clot dissolution.3 However, the success of these thrombolytic strategies has been hampered by thrombotic reocclusion of the initially reperfused vessels in patients with acute myocardial infarction.3 4 5 Mounting evidence indicates that platelets play an important role in both delaying initial reperfusion and mediating subsequent reocclusion under these conditions.6 Experimental studies in a canine model of coronary artery stenosis and thrombosis have demonstrated that reocclusion after thrombolytic therapy is associated with the local aggregation of activated platelets at the site of the stenosis. Inhibition of platelet aggregation by antibodies directed against platelet receptor complex glycoprotein IIb/IIIa largely prevents the occurrence of reocclusion.7 8 Furthermore, platelet aggregation inhibition with aspirin in patients treated with thrombolytic agents reduces the frequency of reocclusions after initially successful reperfusion.9
In a recent study, we identified a platelet component, plasminogen
activator inhibitor 1 (PAI-1), the fast-acting inhibitor of
TPA,10 which is present in large quantities within the
-granules of platelets11 12 and may play a
prominent
role in the process of thrombolysis resistance. PAI-1 belongs to the
serine protease inhibitor ("serpin") family and acts as a
pseudosubstrate for TPA, forming equimolar, inactive PAI-1/TPA
complexes.13 Aggregation of activated platelets,
associated with the release of granular constituents at the site of the
coronary artery stenosis, may cause a high local concentration of
PAI-1, resulting in a thrombus that is more resistant to lysis.
Furthermore, in vitro experiments have revealed that PAI-1 can still
form complexes with TPA when specifically bound to
fibrin.14 Incorporation of a monoclonal antibody, which
blocks the activity of PAI-1, in preformed clots resulted in enhanced
endogenous thrombolysis in vivo.15 Moreover, it
surprisingly reduced thrombus growth irrespective of exogenously
administered TPA.15
The objective of this study was to determine the effect of a systemically administered PAI-1–neutralizing monoclonal antibody (CLB-2C8) on thrombolysis and thrombus extension in the rabbit jugular vein thrombosis model and on vessel patency in an in vivo canine model of coronary thrombosis, reperfusion, and subsequent reocclusion.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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PAI-1 Activity in Plasma and Platelets
Blood was obtained from five healthy volunteers (age, 18 to 32 years), from a mongrel dog, and from three New Zealand White rabbits. For measurement of plasma PAI-1 activity, blood was collected in plastic syringes containing 1 vol EDTA (270 mmol/L), Na2CO3 (1.9 mmol/L), prostaglandin E1 (282 nmol/L, Sigma Chemical Co), and theophylline (30 mmol/L, Sigma). Platelet-poor plasma was obtained by centrifuging the blood samples at 1600g for 30 minutes at 4°C. Plasma samples were stored at -70°C until assayed.
Platelet PAI-1 activity was measured in
blood (9 vol) obtained from
either humans, rabbits, or dogs, drawn in plastic syringes preloaded
with 1 vol 3.6% (wt/vol) trisodium citrate. Platelets were isolated by
gel filtration of platelet-rich plasma, obtained by centrifugation at
180g for 10 minutes at room temperature according to
standard methods.16 The gel-filtered platelets were
incubated with 1 U/mL human thrombin (Roche) for 10 minutes at 37°C
to induce the release of the platelet components. Upon incubation and
subsequent aggregation, the mixture was centrifuged at 4000g
for 30 minutes at 4°C, and the platelet supernatant was isolated and
stored at -70°C until assayed. Previous experiments have shown that
with this method, virtually complete release of PAI-1 present in
the -granules is obtained.15
PAI-1 activity was measured with an amidolytic method as described previously.17 Briefly, plasma and platelet supernatant were incubated with various concentrations of a monoclonal anti–PAI-1 antibody (CLB-2C8) for 1 hour at room temperature. Subsequently, the samples were incubated with a fixed excess of TPA (40 IU/mL) for 10 minutes at room temperature. The residual TPA activity was determined by incubation with 0.13 µmol/L plasminogen, 0.12 mg/mL cyanogen bromide–digested fibrinogen fragments (TPA stimulator), and 0.1 mmol/L S-2251 (all KabiVitrum). The PAI-1 activity in the sample is inversely proportional to the plasmin generated in the incubation mixture, determined by conversion of the chromogenic substrate. Results are expressed in international units (IU), where 1 IU is the amount of PAI-1 that inhibits 1 IU TPA (first international standard of the World Health Organization). CLB-2C8 is a murine monoclonal antibody that has been raised with PAI-1 purified from human HepG2 cells; the corresponding epitope of this antibody has been mapped on PAI-1.18 19 20 CLB-2C8 was selected because of its ability to inhibit the interaction between PAI-1 and the plasminogen activators (TPA and UPA), whereas it does not interfere with fibrin-binding properties of PAI-1.
Rabbit Jugular Vein Thrombosis Model
Experimental
Preparation
New Zealand White rabbits (weight, 
2.5 kg) were
anesthetized
with 9 mg ketamine (Aescoket) and 0.5 mL IM Rompun 2% (Bayer). To
maintain anesthesia, repeated intramuscular injections of ketamine were
given when appropriate. The carotid artery and the jugular veins were
exposed through a medial incision in the neck. The carotid artery was
cleared, and a cannula (baby feeding tube, 1.6 mm OD) was introduced
for administration of the study medication and blood sampling. The
jugular veins were cleared over a distance of 2 cm, and all side
branches were ligated. The veins were clamped both proximally and
distally.
For thrombus growth experiments, thrombi were produced by
injection of
150 µL homologous rabbit blood aspirated in a 1-mL syringe containing
25 µL human thrombin (Human Thrombin T7009, Sigma, 150 IU/mL) and 45
µL CaCl2 (0.25 mol/L) into the isolated venous segment.
This procedure was repeated for the contralateral jugular vein. After
30 minutes of aging of the thrombus, the vessel clamps were removed and
100 µL 125I-labeled human fibrinogen (Amersham, 2
µCi) was injected systemically, followed immediately by intravenous
administration of the study medication. Blood samples were taken every
hour to calculate the mean plasma radioactivity per milliliter of blood
of each rabbit. The thrombi were removed immediately after 60 minutes.
Thrombus growth was determined by measuring the accretion of
125I-labeled fibrinogen onto the preformed nonradioactive
thrombi. Thrombus growth was calculated as the blood volume accreted on
the clot by comparing 125I-related blood radioactivity with
125I-related thrombus radioactivity, and thrombus growth
was expressed as a percentage of the initial thrombus volume.
In the
thrombolysis model, radiolabeled thrombi were produced in the
jugular veins of the rabbit, and the extent of thrombolysis was
determined by measuring the decrease in initial radioactivity of the
preformed thrombi. Homologous rabbit blood was mixed with
125I-labeled fibrinogen (final radioactivity, 25
µCi/mL). An aliquot of 150 µL of this mixture was then aspirated
into a 1-mL syringe containing 25 µL thrombin (150 U/mL) and 45 µL
CaCl2 (0.25 mol/L) and quickly injected into the isolated
venous segment. After 30 minutes of aging, the clamps were removed and
the infusion of study medication was started. Thrombolysis was assessed
by measuring the remaining radioactivity of the thrombus at the end of
the study compared with the initial radioactivity and was expressed as
a percentage of the initial thrombus volume.
Study
Medication
The rabbits were assigned to four groups (four rabbits
each) and
were treated with either (1) rTPA (Actilyse, Boehringer Ingelheim)
administered at a suboptimal bolus dose of 0.25 mg/kg in combination
with CLB-2C8 at a bolus dose of 1 mg/kg; (2) rTPA (0.25 mg/kg) in
combination with a bolus injection of E48 (a murine monoclonal IgG1
antibody raised against malignant human squamous epithelial
cells21 ); (3) a bolus injection of CLB-2C8 (1 mg/kg)
alone; or (4) a bolus injection of E48 (1 mg/kg) alone. The anti–PAI-1
antibody was administered via the carotid cannula immediately before
the bolus injection of either rTPA or saline. The effect on thrombus
growth and thrombolysis was assessed after 60 minutes.
Canine Coronary Artery Thrombosis Model
Experimental
Preparation
Healthy, adult mongrel dogs (weight, 25 to 30 kg) were
used.
Anesthesia was induced with pentobarbital (Nesdonal,
Rhône-Poulenc, 30 mg/kg IV) after premedication with 1 mg/kg
methadon IM (Gist-Brocade) and 0.1 mL/kg Rompun. The dogs were
intubated and artificially ventilated. The right femoral artery and
vein were cannulated for continuous blood pressure monitoring and for
the infusion of study medication, respectively. The chest was opened
via a midsternal thoracotomy, and a pericardial cradle was constructed.
A 2-cm-long segment of the left anterior descending coronary artery
(LAD) was dissected free from the epicardium, and all side branches
were ligated. An ultrasonic flow probe (Transonic) was placed at the
most proximal part of the isolated LAD segment. In addition, the
peripheral part of the segment was constricted to achieve 50% flow
reduction. Then, vessel clamps were placed at the proximal and distal
sides of the prepared LAD segment, which was subsequently traumatized
by external compression with a blunt forceps to disrupt the
endothelium. To induce coronary thrombosis in the isolated segment, a
catheter was inserted into a side branch of the LAD, and a mixture of
400 µL homologous citrated blood, 40 µL 0.25 mol/L
CaCl2, and 60 µL thrombin (Human Thrombin T7009,
Sigma, 1000 U/mL) was injected. After 30 minutes of aging of the
thrombus, the vessel clamps were removed. The occlusion was confirmed
with the flow probe. Procainamide (Astra, 1.0 g) and lidocaine (Astra,
75 mg) were injected as an intravenous bolus injection followed by
continuous intravenous infusion (3
mg · kg-1 · h-1) of lidocaine.
Heparin (Tromboliquine, Organon) was given as a 100-U/kg IV loading
dose, and additional heparin doses (1000 U IV) were given every
hour.
Study Design
The animals were assigned to
four different groups: (1) rTPA
(Actilyse, Boehringer Ingelheim) administered as a bolus injection of
0.45 mg/kg in combination with CLB-2C8 at a bolus dose of 1 mg/kg (8
dogs); (2) rTPA (0.45 mg/kg) in combination with a bolus injection of
saline (8 dogs); (3) no rTPA but a bolus injection of CLB-2C8 (1 mg/kg,
3 dogs); and (4) saline (control, 3 dogs). The study medication was
administered following 10 minutes of stable occlusion after the vessel
clamps were removed. rTPA was administered intravenously by bolus
injection. This bolus injection was repeated every 15 minutes until
reperfusion was achieved or a maximum of four doses were given. The
CLB-2C8 monoclonal antibody was administered as a single intravenous
bolus injection immediately before the infusion of either rTPA or
saline. Blood samples were drawn from the femoral vein catheter before
the surgical procedure; just before the thrombus introduction; and at
30, 60, and 100 minutes thereafter. Coronary blood flow was monitored
continuously at the proximal site of the isolated coronary segment to
document reperfusion and reocclusion. Reperfusion was defined as
reestablishment of coronary flow. Reocclusion was judged to be when the
coronary flow had subsequently decreased to zero. The time that elapsed
from the infusion of study medication until the first recorded
reperfusion was defined as reperfusion time, and the interval between
the first reperfusion and a subsequent reocclusion was defined as
reocclusion time. In addition, the total time of vessel patency
(patency time) and the total number of reocclusions were monitored
during the 70 minutes of observation after the infusion of study
medication.
Statistical Analysis
Statistical analysis of the rabbit
experiments was performed
by ANOVA and a Newman-Keuls test. In the canine experiments, the
Mann-Whitney U test was applied for nonparametric testing. A
value of P<.05 was considered statistically significant.
All values were expressed as mean±SD.
Ethical Considerations
All animal studies were approved by
the Institutional Review
Board for Animal Experiments and were performed according to the
guidelines of the American Physiological Society and Dutch Law for
Animal Experiments.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Inhibition of PAI-1 in Various Species by the Monoclonal Anti–PAI-1 Antibody
We measured the inhibitory effect of increasing concentrations of CLB-2C8 on PAI-1 activity in plasma and platelet releasate from 5 healthy volunteers (age, 18 to 32 years), 3 rabbits, and 1 dog. The results are presented in Fig 1
. PAI-1 activity from
both plasma and platelet releasate was inhibited in a dose-dependent
fashion. Inhibition of PAI-1 activity in plasma and platelet
supernatant by CLB-2C8 appeared to be equally effective, and a plateau
in PAI-1 inhibition was reached at antibody concentrations >50
µg/mL. CLB-2C8 most effectively inhibited human PAI-1; slightly
higher concentrations of CLB-2C8 were needed for inhibition of canine
and rabbit PAI-1.
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) and platelet releasate (
) were
incubated with increasing concentrations of CLB-2C8. PAI-1 activity is
presented as a percentage of the PAI-1 activity in the absence of
CLB-2C8.
Furthermore, we assessed the PAI-1–neutralizing
capacity of a
bolus dose of 1 mg/kg CLB-2C8 in vivo. Blood samples of the dogs were
taken before and after administration of the four different study
medications. The results are presented in Fig 2. The
PAI-1–neutralizing antibody, administered 30 minutes after the
induction of the occluding coronary thrombus, completely abolished
PAI-1 activity in plasma until the end of the experiment. PAI-1
activity remained at a stable level in the control animals during the
entire experiment, whereas the administration of rTPA in combination
with saline induced only a moderate and temporary decrease in PAI-1
activity levels, presumably by the formation of TPA:PAI-1
complexes.
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) (n=8), rTPA in
combination with saline (
)
(n=3), or no rTPA plus no CLB-2C8 (
Thrombolysis and Thrombus Growth of Rabbit Jugular Vein
Thrombosis
We measured the effect of systemic administration of
anti–PAI-1
antibody on thrombolysis and thrombus growth in a rabbit jugular vein
thrombosis model after a period of 60 minutes. The effect of the
antibody on endogenous thrombolysis and thrombus extension and on
thrombolysis and thrombus growth upon administration of rTPA was
determined. Administration of the anti–PAI-1 monoclonal antibody
(CLB-2C8, 1 mg/kg) significantly enhanced endogenous thrombolysis
compared with the E48 antibody control (thrombolysis, 13.7±2.6%
versus 6.1±1.3%; P<.05; Fig 3, top).
However, in the animals receiving thrombolytic therapy consisting of a
bolus dose of 0.25 mg/kg human rTPA, no additional
thrombolysis-enhancing effect of the administered anti–PAI-1 antibody
was observed compared with administration of rTPA in combination with
the E48 antibody (thrombolysis, 26.5±1.8% versus 23.8±1.8%;
P=NS; Fig 3, bottom).
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The effect on
thrombus growth of the anti–PAI-1 antibody was
determined 60 minutes after the bolus injection. Notably, thrombus
extension was strongly reduced in the animals receiving the anti–PAI-1
antibody compared with the E48-treated rabbits (thrombus growth,
22.8±2.8% versus 42.4±3.0%; P<.05; Fig
3
, top). This
finding was observed irrespective of additional thrombolytic therapy.
Thrombus extension upon administration of 0.25 mg/kg rTPA was
significantly reduced by the coinfusion of the anti–PAI-1 antibody
compared with infusion of rTPA in combination with E48 (thrombus
growth, 12.2±2.6% versus 24.0±2.6%; P<.05; Fig
3,
bottom).
Canine Coronary Artery Reperfusion and Reocclusion
Administration of rTPA alone induced coronary reperfusion in 7 of
the 8 animals in this model. Reperfusion was established 19.5±8.2
minutes after administration of rTPA. Administration of the anti–PAI-1
antibody immediately preceding rTPA infusion induced reperfusion in all
animals (8/8) and significantly reduced time to reperfusion compared
with the animals treated with rTPA alone (reperfusion time, 8.1±5.2
versus 19.5±8.2 minutes; P=.018)
(Table). As
in comparable studies,7 8 administration of rTPA
alone
resulted in a pattern of subsequent reperfusions and reocclusions. All
animals with an established reperfusion on administration of rTPA alone
subsequently developed reocclusions after 3.3±2.6 minutes.
Administration of the anti–PAI-1 antibody preceding the rTPA infusion
significantly delayed the onset of reocclusions after the first
reperfusion (reocclusion time, 11.6±12.5 versus 3.3±2.6 minutes;
P=.037). Although administration of the anti–PAI-1
antibody
significantly reduced the number of reocclusions, it did not completely
prevent them. During the 70 minutes of observation, an average of
1.9±1.0 reocclusions of the coronary artery were recorded in the
animals treated with rTPA in combination with CLB-2C8 compared with
5.1±2.3 reocclusions in the animals treated with rTPA alone
(P=.015) (Table). The total duration of periods
with
coronary artery patency during the 70 minutes of observation tended to
be longer in the animals treated with the anti–PAI-1 monoclonal
antibody and rTPA compared with administration of rTPA alone but just
failed to reach statistical significance (total vessel patency,
43.6±21.7 versus 21.7±15.4 minutes, respectively;
P=.055)
(Table).
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None of the animals treated with saline alone (n=3) and none of the animals treated with the anti–PAI-1 antibody alone (n=3) displayed coronary artery reperfusion during the study period.
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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PAI-1, the most important fast-acting inhibitor of the endogenous plasminogen activators, has been suggested to be a potentially important factor in the pathogenesis of coronary artery thrombosis and recurrent myocardial infarction. This was illustrated in recent clinical studies that demonstrated an association between increased plasma levels of PAI-1 and the occurrence of coronary artery thrombosis and recurrent myocardial infarction.22 23 24 25 26 Firm evidence that high PAI-1 levels bear a thrombotic risk has been obtained in transgenic mice, resulting in an increased incidence of venous thrombosis.27 Platelets contain, in their
-granules,
large quantities of PAI-1, which was shown to be immunologically and
probably also functionally identical to plasma/endothelial
PAI-1.28 Activation of platelets induces the release of
these -granules, resulting in a local accumulation of high
concentrations of PAI-1. We recently demonstrated that PAI-1 released
from platelets is preferentially retained within the
thrombus,29 presumably by binding to
fibrin.13 In view of (1) the pivotal role of platelets in
the pathogenesis of reocclusion; (2) the inhibition of local
thrombolysis evoked by the presence of intact platelets and high
concentrations of PAI-1 in
thrombi30 31 32 33 34 35 36 ;
(3) the
observation that reoccluding thrombi are particularly
platelet-rich6 and appeared to be more resistant to
thrombolysis than erythrocyte-rich clots37 ; and (4) the
fact that another anti–PAI-1 antibody, incorporated into preformed
clots, significantly enhanced endogenous thrombolysis and reduced
thrombus growth in the rabbit jugular vein thrombosis
model,15 we hypothesized that PAI-1 might be a platelet
constituent that could play an important mediatory role in
thrombolysis-resistant reocclusions. The present study revealed that systemic administration of a PAI-1–neutralizing monoclonal antibody (CLB-2C8) significantly enhanced endogenous thrombolysis and reduced thrombus growth in the rabbit jugular vein thrombosis model. In this model, no additional thrombolytic effect of administration of the antibody was observed, probably because of the vast excess of administered rTPA compared with the endogenous levels of PAI-1. In a model of canine coronary thrombosis that pathologically resembles the human situation,7 8 administration of CLB-2C8 concurrent with rTPA significantly increased thrombolysis and reduced the number of coronary reocclusions. Time to reperfusion, time to reocclusion, and the number of reocclusions were strongly affected by administration of antibody. Although administration of the anti–PAI-1 antibody completely inhibited PAI-1 activity in vivo, administration of the antibody alone could not induce reperfusion in this model.
Interestingly, others have shown a similar effect on reperfusion and reocclusion of administration of a specific monoclonal antibody against platelet receptor glycoprotein IIb/IIIa in the same experimental animal model.7 8 In that study, the addition of the platelet receptor blocking agent alone was not sufficient to induce spontaneous reperfusion either. The similarity of the effects of both antibodies suggests that enhanced thrombolysis and reduced formation of reocclusion upon thrombolytic therapy can be achieved either by inhibition of platelet aggregation and of the subsequent release of PAI-1 from the platelets (by the anti-platelet glycoprotein IIb/IIIa antibody) or by direct inhibition of PAI-1 activity (by the anti–PAI-1 antibody).
Since administration of TPA alone already induced a significant decrease in the PAI-1 plasma levels, we hypothesize that the profibrinolytic effect of the anti–PAI-1 antibody is more likely to be induced by its local inhibitory effect on PAI-1 present in platelet-rich clots than on circulating PAI-1 in plasma. This is illustrated by the fact that one platelet contains approximately 4000 to 8000 molecules of PAI-1.38 Platelet activation may therefore result in a high local concentration of PAI-1, about 10 000 times higher than the concentration of PAI-1 in plasma. In addition, it has been demonstrated that PAI-1 released upon platelet activation is able to bind fibrin without losing its capacity to neutralize TPA,14 resulting in the formation of lysis-resistant clots. In accordance, in vitro experiments have demonstrated that the lysis resistance of platelet-rich clots depends predominantly on PAI-1 present in these clots.15 31 32 33 34 35 36
Not much is known about possible adverse effects of PAI-1 inhibition, particularly bleeding. Although the two animal models used in this study are not designed to assess effects on bleeding tendency, we did not observe markedly increased bleeding in the animals treated with the anti–PAI-1 antibody. Recently, a homozygous PAI-1 deficiency was described in a young patient with only a relatively mild bleeding tendency.39 Therefore, the inhibition of PAI-1 might be a relatively safe option to prevent coronary reocclusion upon thrombolytic therapy compared with anticoagulant or antiplatelet therapies. However, it should be realized that, in the model used, no antiplatelet therapy and a suboptimal level of anticoagulation were given.40 41 Therefore, it remains to be established whether systemic inhibition of PAI-1 will have an additional therapeutic effect when it is combined with an adequate level of anticoagulation and antiplatelet therapy.
The results of the rabbit experiments showed that systemic administration of anti–PAI-1 was equally effective as the incorporated antibody in the previous study in enhancing the endogenous thrombolysis and reducing thrombus growth.15 Apparently, there is sufficient penetration of the monoclonal antibody into the preformed clot, confirming the relatively dynamic state of thrombi due to continuous endogenous thrombolysis and thrombus growth by platelet accretion and fibrin deposition. In the present study, very high concentrations of the anti–PAI-1 monoclonal antibody were used, which appeared to completely abolish the PAI-1 activity in the circulation. Further studies should focus on the effect of lower concentrations of antibody or the effect of smaller PAI-1–neutralizing Fab fragments of monoclonal antibodies.
In conclusion, this study shows that inhibition of PAI-1 may be a potent adjuvant in the thrombolytic therapy of acute myocardial infarction, although the use of a murine monoclonal antibody in patients bears the risk of immunogenicity, and chimeric anti–PAI-1 antibodies or PAI-1–inhibiting peptides should be developed for eventual clinical applications. Whether administration of specific thrombin inhibitors will have an additional inhibitory effect on the formation of reocclusions remains to be investigated.
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Acknowledgments |
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We thank Charly Belterman for his help with the experiments and Ivo Horn for his help in purifying the monoclonal antibody CLB-2C8.
Received June 20, 1994; revision received September 1, 1994; accepted September 23, 1994.
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References |
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TopAbstractIntroductionMethods Results Discussion References |
|---|
1. Sherry S, Fletcher AP, Alkjaersig N. Developments in fibrinolytic therapy for thromboembolic disease. Ann Intern Med. 1989; 50:560-570.
2. The TIMI Study Group. The Thrombolysis in Myocardial Infarction (TIMI) trial: phase I findings. N Engl J Med. 1985;312:932-936. [Medline] [Order article via Infotrieve]
3. Marder VJ, Sherry S. Thrombolytic therapy: current status. N Engl J Med. 1988;318:1512-1520. [Medline] [Order article via Infotrieve]
4. Chesebro JH, Knatterud G, Roberts R, Borer J, Cohen LS, Dalen J, Dodge HT, Francis CK, Hillis D, Ludbrook P, Markis JE, Mueller H, Passamani ER, Powers ER, Rao AK, Robertson T, Ross A, Ryan TJ, Sobel BE, Willerson J, Williams DO, Zaret BL, Braunwald E. Thrombolysis in Myocardial Infarction (TIMI) trial, phase I: a comparison between intravenous tissue plasminogen activator and intravenous streptokinase. Circulation. 1987;76: 142-154.
5. Topol EJ, Califf RM, George BS, Kereiakes DJ, Abbottsmith CW, Candela RJ, Lee KL, Pitt B, Stack RS, O'Neill WW, and the Thrombolysis and Angioplasty in Myocardial Infarction Study Group. A randomized trial of immediate versus delayed elective angioplasty after intravenous tissue plasminogen activator in acute myocardial infarction. N Engl J Med. 1987;317:581-588. [Abstract]
6. Coller BS. Platelets and thrombolytic therapy. N Engl J Med. 1990;322:33-42. [Medline] [Order article via Infotrieve]
7.
Gold HK, Coller BS, Yasuda T, Saito T, Fallon JT, Guerrero
JL, Leinbach RC, Ziskind AA, Collen D. Rapid and sustained coronary
artery recanalization with combined bolus injection of recombinant
tissue-type plasminogen activator and monoclonal antiplatelet
GPIIb/IIIa antibody in a canine preparation.
Circulation. 1988;77:670-677.
8. Yasuda T, Gold HK, Fallon JT, Leinbach RC, Guerrero JL, Scudder LE, Kanke M, Shealy D, Ross MJ, Collen D, Coller BS. Monoclonal antibody against the platelet glycoprotein (GP) IIb/IIIa receptor prevents coronary artery reocclusion after reperfusion with recombinant tissue-type plasminogen activator in dogs. J Clin Invest. 1988;81:1284-1291.
9. ISIS-2 (Second International Study of Infarct Survival) Collaborative Group. Randomized trial of intravenous streptokinase, oral aspirin, both or neither among 17187 cases of suspected myocardial infarction. Lancet. 1988;2:349-360. [Medline] [Order article via Infotrieve]
10.
Kruithof EKO, Tran-Thang C, Ransijn A, Bachmann F.
Demonstration of a fast-acting inhibitor of plasminogen activators in
human plasma. Blood. 1984;64:907-913.
11. Erickson LA, Ginsberg MH, Loskutoff DJ. Detection and partial characterization of an inhibitor of plasminogen activator in human platelets. J Clin Invest. 1984;74:1465-1472.
12. Sprengers ED, Akkerman JWN, Jansen BG. Blood platelet plasminogen activator inhibitor: two different pools of endothelial cell type plasminogen activator in human blood. Thromb Haemost. 1986;55:325-329. [Medline] [Order article via Infotrieve]
13. Travis J, Salvesen GS. Human plasma proteinase inhibitors. Annu Rev Biochem. 1983;52:655-709. [Medline] [Order article via Infotrieve]
14. Wagner OF, de Vries C, Hohmann C, Veerman H, Pannekoek H. Interaction between plasminogen activator inhibitor type 1 (PAI-1) bound to fibrin and either tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA): binding of t-PA/PAI-1 complexes to fibrin mediated by both the finger and the kringle-2 domain of t-PA. J Clin Invest. 1989;84:647-655.
15. Levi M, Biemond BJ, Van Zonneveld AJ, Ten Cate JW, Pannekoek H. Inhibition of plasminogen activator inhibitor 1 (PAI-1) activity results in promotion of endogenous thrombolysis and inhibition of thrombus extension in models of experimental thrombosis. Circulation. 1992;68:315-320.
16.
Ginsberg MH, Taylor L, Painter RG. The mechanism of
thrombin-induced platelet factor 4 secretion. Blood. 1980;55:661-670.
17. Verheijen JH, Chang GTG, Kluft C. Evidence for the occurrence of a fast-acting inhibitor for tissue-type plasminogen activator in human plasma. Thromb Haemost. 1984;51:392-395. [Medline] [Order article via Infotrieve]
18.
Keijer J, Linders M, van Zonneveld AJ, Ehrlich HJ, de Boer JP,
Pannekoek H. The interaction of plasminogen activator inhibitor 1
(PAI-1) with plasminogen activators (t-PA, u-PA) and fibrin:
localization of interaction sites and physiological relevance.
Blood. 1991;78:1254-1261.
19. Declerck PJ, Collen D. Measurement of plasminogen activator inhibitor 1 (PAI-1) in plasma with various monoclonal antibody-based enzyme-linked immunosorbent assays. Thromb Res. 1990;58(suppl X):3-9.
20. Nielsen LS, Andreasen PA, Grohndal-Hansen J, Huang JY, Kristensen P, Dano K. Monoclonal antibodies to human 54,000 molecular weight plasminogen activator inhibitor from fibrosarcoma cells: inhibitor neutralizing and one-step affinity purification. Thromb Haemost. 1986;55:206-212. [Medline] [Order article via Infotrieve]
21. Quak JJ, Balm AJM, van Dongen GAMS, Brakkee JGP, Scheper RJ, Snow GB, Meijer CJLM. A 22Kd surface antigen detected by monoclonal antibody E48 is exclusively expressed in stratified squamous and transitional epithelia. Am J Pathol. 1990;136: 191-197.
22. Juhan-Vague I, Alessi MC. Plasminogen activator inhibitor I and atherosclerosis. Thromb Haemost. 1993;70:138-143. [Medline] [Order article via Infotrieve]
23. Hamsten A, de Faire U, Walldius G, Dahlen G, Szamosi A, Landou C, Blombäck M, Wiman B. Plasminogen activator inhibitor in plasma: risk factor for recurrent myocardial infarction. Lancet. 1987;2:3-8. [Medline] [Order article via Infotrieve]
24. Hamsten A, Wiman B, de Faire U, Blombäck M. Increased plasma levels of a rapid inhibitor of tissue plasminogen activator in young survivors of myocardial infarction. N Engl J Med. 1985;313:1557-1563. [Abstract]
25. Paramo JA, Colluci M, Collen D, van der Werf F. Plasminogen activator inhibitor in the blood of patients with coronary artery disease. Br Med J. 1985;291:573-574.
26. Prins MH, Hirsh J. A critical review of the relationship between impaired fibrinolysis and myocardial infarction. Am Heart J. 1991;122:545-551. [Medline] [Order article via Infotrieve]
27. Erickson LA, Fici GJ, Lund JE, Boyle TP, Polites HG, Marotti KR. Development of venous occlusions in mice transgenic for the plasminogen activator inhibitor gene. Nature. 1990;346:74-76. [Medline] [Order article via Infotrieve]
28. Erickson LA, Hekman CM, Loskutoff DJ. The primary plasminogen-activator inhibitors in endothelial cells, platelets, serum and plasma are immunologically related. Proc Natl Acad Sci U S A. 1985;82:8-10.
29.
Stringer HAR, van Swieten P, Heynen H, Sixma JJ, Pannekoek H.
Plasminogen activator inhibitor-1 (PAI-1) released from activated
platelets plays a key role in thrombolysis resistance: studies with
thrombi generated in the Chandler loop. Arterioscler Thromb.. 1994;14:1452-1458.
30. de Fouw NJ, de Jong YF, Haverkate F, Bertina RM. The influence of thrombin and platelets on fibrin clot lysis rates in vitro: a study using a clot lysis system consisting of purified human proteins. Fibrinolysis. 1988;2:235-244.
31.
Robbie LA, Booth NA, Croll AM, Bennet B. The roles of
2-antiplasmin and plasminogen activator inhibitor 1
(PAI-1) in the inhibition of clot lysis. Thromb Haemost. 1993;70:301-306. [Medline]
[Order article via Infotrieve]
32. Torr-Brown SR, Sobel BE. Attenuation of thrombolysis by release of plasminogen activator inhibitor type-1 from platelets. Thromb Res. 1993;72:413-421. [Medline] [Order article via Infotrieve]
33. Booth NA, Robbie LA, Croll AM, Bennett B. Lysis of platelet rich thrombi: the role of PAI-1. Ann N Y Acad Sci. 1992;667:70-80. [Medline] [Order article via Infotrieve]
34.
Fay WP, Eitzman DT, Shapiro ADS, Madison EL, Ginsberg D.
Platelets inhibit fibrinolysis in vitro by both plasminogen activator
inhibitor-1-dependent and -independent mechanisms. Blood. 1994;83:351-356.
35.
Braaten JV, Handt S, Jerome WG, Kirkpatrick J, Lewis JC,
Hantgan RR. Regulation of fibrinolysis by platelet-released plasminogen
activator inhibitor 1: light scattering and ultrastructure examination
of lysis of a model platelet-fibrin thrombus. Blood. 1993;81:1290-1299.
36. Potter van Loon BJ, Rijken DC, Brommer EJP, van der Maas APC. The amount of plasminogen, tissue-type plasminogen activator and plasminogen activator inhibitor type 1 in human thrombi and the relation to ex-vivo lysibility. Thromb Haemost. 1992;67:101. [Medline] [Order article via Infotrieve]
37.
Jang IK, Gold HK, Ziskind AA, Fallon JT, Holt RE, Leinbach RC,
May JW, Collen D. Differential sensitivity of erythrocyte-rich and
platelet-rich arterial thrombi to lysis with recombinant tissue-type
plasminogen activator: a possible explanation for resistance to
coronary thrombolysis. Circulation. 1989;79:920-928.
38.
Kruithof EKO, Nicolosa G, Bachmann F. Plasminogen activator
inhibitor 1: development of a radioimmunoassay and observations on its
plasma concentration during venous occlusion and after platelet
aggregation. Blood. 1987;70:1645-1653.
39. Fay WP, Shapiro AD, Shih JL, Schleef RR, Ginsburg D. Complete deficiency of plasminogen activator inhibitor type-I due to a frame-shift mutation. N Engl J Med. 1992;327:1729-1733. [Medline] [Order article via Infotrieve]
40.
Haskel EJ, Adams SP, Feigen LP, Saffitz JE, Gorczynski RJ,
Sobel BE, Abendschein DR. Prevention of reoccluding platelet-rich
thrombi in canine femoral arteries with a novel peptide antagonist of
platelet glycoprotein IIb/IIIa receptors.
Circulation. 1989;80:1775-1782.
41. Prager NA, Torr-Brown SR, Sobel BE, Abendschein DR. Maintenance of patency after thrombolysis in stenotic coronary arteries requires combined inhibition of thrombin and platelets. J Am Coll Cardiol. 1993;22:296-301.[Abstract]
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