(Circulation. 1995;91:1330-1335.)
© 1995 American Heart Association, Inc.
Articles |
Antithrombotic Effects of Thrombolytic Agents in a Platelet-Rich Femoral Vein Thrombosis Model in the Hamster
From the Department of Othopedics and Hand Surgery (J.M.S., A.N.), University Hospital of Umeå, Sweden; Division of Plastic Surgery (A.N.), University of Pittsburgh, Pa; and the Center for Molecular and Vascular Biology (J.M.S., M.H., D.C.), University of Leuven, Belgium.
Correspondence to Désiré Collen, MD, PhD, Center for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg, O & N, Herestraat 49, B-3000 Leuven, Belgium.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background The extent and mechanism of the antithrombotic properties of fibrin-selective and non–fibrin-selective thrombolytic agents have not yet been established.
Methods and Results The antithrombotic, thrombolytic, fibrinogenolytic, and pharmacokinetic properties of the following substances were determined in hamsters in the absence of conjunctive anticoagulant or antiplatelet therapy: recombinant tissue-type plasminogen activator (rTPA), recombinant single-chain urokinase-type plasminogen activator (rscu-PA), two-chain urokinase-type plasminogen activator (UK), with a rTPA deletion mutant lacking amino acids 6 to 173 and a mutation N184E (K2Pt), a rTPA/rscu-PA chimeric plasminogen activator consisting of amino acids 1 to 3 and 87 to 274 of rTPA and amino acids 138 to 411 of rscu-PA (K1K2Pu), streptokinase (SK), and recombinant staphylokinase (STAR). The antithrombotic effect, defined as the intravenous dose required to reduce mural thrombus formation to 50% in a platelet-mediated femoral vein thrombosis model in the hamster, was 6±1, 5±2, 1±0.05, 2.5±0.2, 0.02±0.002, 1±0.09, and 2±0.3 mg/kg (mean±SEM), respectively. The amounts, given as intravenous infusion over 60 minutes that induced 50% clot lysis in a hamster pulmonary embolism model, were 0.18±0.03, 1.1±0.05, 0.9±0.13, 0.34±0.03, 0.04±0.003, 0.05±0.005, and 0.04±0.001 mg/kg, respectively, indicating that for most thrombolytic agents the antithrombotic dose is much higher than their thrombolytic dose. The fibrinogen levels, measured 40 minutes after bolus injection, were reduced to 50% of baseline with 3.1±0.2, 2.5±0.3, 1.2±0.08, 2.0±0.14, 1.7±0.65, 0.54±0.03, and 1.2±0.11 mg/kg, respectively. Mean residence times following intravenous bolus injection were: 18±1, 14±1, 100±10, 80±2, 20±3, and 34±5 minutes for rTPA, rscu-PA, K2Pt, K1K2Pu, SK, and STAR, respectively. Regression analysis revealed a significant correlation of the antithrombotic effect with the fibrinogen breakdown (P=.006) but not with the thrombolytic potency or with the mean residence time.
Conclusions These observations support the hypothesis that thrombolytic therapy with fibrinogen-sparing agents requires the conjunctive use of anticoagulant and/or antiplatelet agents.
Key Words: thrombolysis • pharmacokinetics
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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Thrombolytic therapy has become a standard treatment for patients with acute myocardial infarction.1 To achieve maximal efficacies, combined administration of fibrinolytic and antithrombotic agents is used.2 3 4 5 Although fibrin-specific thrombolytic agents are the most effective in the treatment of myocardial infarction,4 5 incomplete thrombolysis and reocclusion remain significant shortcomings. Platelets have been identified as the predominant cause of delayed reperfusion and subsequent reocclusion.6 7 8 9 10 11 However, the direct effects of available thrombolytic agents on platelet-rich thrombus formation have not been adequately quantified nor their mechanisms elucidated.
The aim of the present study was to quantify and correlate the antithrombotic, thrombolytic, fibrinogenolytic, and pharmacokinetic properties of several plasminogen activators in the same small-animal species.12 13 14 15
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Materials
Thrombolytic Agents
Seven fibrinolytic agents were investigated: rTPA, recombinant tissue-type plasminogen activator (Alteplase, Genentech Inc, courtesy of Dr W.F. Bennett); rscu-PA, recombinant single-chain urokinase-type plasminogen activator (Saruplase, Grünenthal, courtesy of Dr W. Günzler); UK, two-chain urokinase-type plasminogen activator, urokinase, (Serono); K2Pt, an rTPA mutant with deletion of amino acids 6 to 173 and mutation of amino acid N184 to E (purified and characterized as described elsewhere16 ); K1K2Pu, a recombinant TPA/scu-PA chimeric molecule consisting of amino acids 1 to 3 and 87 to 274 of the A-chain of human tissue-type plasminogen activator (TPA) and amino acids 138 to 411 of human single-chain urokinase-type plasminogen activator (scu-PA) (purified and characterized as described elsewhere17 ); SK, streptokinase (Streptase, Behringwerke); and STAR, recombinant staphylokinase (purified and characterized as described elsewhere18 ).
Experimental Animals
Outbred hamsters (245, Pfd, Gold, University of Leuven, Belgium)
with body weights of 80 to 120 g were premedicated with 1.25 mg/kg
atropine IP and anesthetized by an intraperitoneal injection of 50
mg/kg of sodium pentobarbital (Mebumal Vet, ACO Läkemedel). Body
temperatures were maintained at 37°C. Catheters (Portex blue, Hythe)
were inserted in the jugular vein for drug administration and in the
left femoral vein for blood sampling during pharmacokinetic studies.
The experiments were performed according to the guidelines of the
International Society on Thrombosis and
Haemostasis.19
Antithrombotic Effects
The antithrombotic effects of the thrombolytic agents were
studied in a previously described model in the
hamster.12 20 21 Platelet-rich mural
thrombosis in the
right femoral vein was monitored continuously during 40 minutes. rTPA,
rscu-PA, UK, K2Pt, K1K2Pu, SK,
STAR, or saline was randomly assigned and given as a single intravenous
injection via the jugular vein cannula 1 minute before induction of
thrombus formation. At the end of the observation period, a 1-mL blood
sample was drawn from the abdominal aorta into citrate (final
concentration 0.014 mol/L), and the plasma was stored at -20°C until
analyzed. Antigen levels were determined with specific ELISAs for TPA,
u-PA, and STAR as described.17 18 22
Technical Equipment
Platelet-rich mural thrombus
was induced with a modified
atraumatic Acland vascular clamp (ST-RD-S, S&T Micro Lab AG), and the
1-mm femoral veins were mounted on a transilluminator with a 0.5x8-mm
light outlet. An operating microscope (OPMI-I, Zeiss AG) and a video
recording system consisting of a black and white camera (Hamamatsu
CCD/C3007) and a video recorder (Panasonic NVG21 ha) were used for
continuous monitoring of the thrombus formation and degradation. From
the acquired images, platelet accumulation at the site of vessel wall
trauma was quantified with an image processing software program
(TLC-IMAGE, Multihouse TSI with an expanded routine for
this specific application (C.N. Rood) as described
elsewhere.12 20
Thrombolytic
Potency
The dosages that induced 50% clot lysis were determined from
the dose-response curves obtained with UK, K2Pt,
K1K2Pu, SK, and STAR in a hamster pulmonary
embolism model as described elsewhere.22 Briefly, a
50-µL 125I-fibrin–labeled human plasma clot was produced
in vitro and injected into the jugular vein of heparinized hamsters.
Thrombolytic agents or saline were infused intravenously over 60
minutes, and 30 minutes after the end of the infusion the extent of
clot lysis was determined as the difference between the radioactivity
initially incorporated in the clot and the residual radioactivity in
the lungs and the heart at the end of the experiment. At the end of the
infusion and at the end of the experiment, blood samples were collected
as described above.
Fibrinogen Breakdown
The plasma
samples collected at the end of the antithrombotic
experiments (41 minutes after intravenous bolus administration) and at
the end of the clot lysis experiments (30 minutes after the end of the
infusion) were used to determine the residual fibrinogen and
2-antiplasmin levels as described
elsewhere.14
Pharmacokinetics
Hamsters
with a body weight of 100 g received a single IV
injection of 10 µg of either rTPA, rscu-PA, K2Pt,
K1K2Pu, SK, or STAR. Blood samples of 0.2 mL
were collected on 0.014 mol/L sodium citrate (final concentration)
before the injection of the thrombolytic agent and 0.5, 1, 2, 3, 5, 7,
10, 15, 20, 30, 45, 60, and 90 minutes thereafter and centrifuged at
1000g for 10 minutes; the decanted plasma was then stored at
-20°C until analyzed. The plasma levels of rTPA, rscu-PA,
K2Pt, K1K2Pu, and STAR were
measured with ELISAs as described
elsewhere.14 17 18 The
SK concentrations were measured by determining the plasma fibrinolytic
activity with a fibrin plate assay and converted to mass based on a
specific activity of 100 000 U/mg.23
The pharmacological
parameters were calculated as follows. The antigen
levels were plotted on semilogarithmic paper and fitted with a sum of
two exponential terms: C(t) =
Ae-t+Be-ßt
by graphical curve peeling. The drug clearance parameters were
calculated from the coefficients (A and B) and exponents ( and ß)
with standard formulas.24 25 The following clearance
parameters were calculated on the basis of a two-compartment mamillary
model: initial drug concentration in the blood: C0 =
A+B;
volume of the central compartment: Vc = dose/(A+B);
total
volume of distribution: Vd = dose/B; extrapolated area
under the curve: AUC = A/+B/ß; plasma clearance: Clp
= dose/AUC;
initial half-life: t
=
ln2/; final
half-life: tß = ln2/ß.
Additionally, the mean
residence time (MRT), a compartment-independent parameter that
represents the time necessary to clear 62.3% of the injected
substance was derived: MRT =
(VdxAUC)/dose.25
Although this parameter varies with the route of administration, MRT
reflects the average time the drug is detectable in the organism and
thus the fate of the administered substance in the body.
Statistical Analysis
All data are represented as
mean±SEM. The levels of
significance of the observed effects were determined by the Student's
t test for unpaired values. The doses that induced 50%
inhibition of thrombus formation, 50% clot lysis, and 50% fibrinogen
breakdown were calculated by sigmoidal regression analysis
(GRAFIT v 3.0, Erithacus Software Ltd). The obtained values
and MRTs of rTPA, rscu-PA, UK, K2Pt,
K1K2Pu, SK, and STAR were correlated using
ordinary least-squares regression analysis (UNISTAT v
2.0).
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Antithrombotic Effects
Accumulation of a nonocclusive thrombus was observed after vessel wall trauma, reaching a maximal light intensity after 22±4 minutes. The thrombus then gradually disintegrated and embolized followed occasionally (10±4% of the damaged blood vessels) by the formation of a second thrombus within 31±5 minutes. The total AUC within 40 minutes after infliction of the trauma was 9.6±1.1 x 107 A.U. (n=12) in the control group. The appearance of a second thrombus did not result in significant differences in total AUC. Intravenous bolus injection of increasing doses of rTPA, rscu-PA, UK, K2Pt, K1K2Pu, SK, and STAR resulted in a dose-dependent decrease of thrombus formation (Table 1
and the Figure). A 50% reduction (IC50) was
obtained with 6±1, 5±2, 1±0.05, 2.5±0.2,
0.02±0.002, 1±0.09, and
2±0.3 mg/kg rTPA, rscu-PA, UK, K2Pt,
K1K2Pu, SK, and STAR, respectively. UK,
K2Pt, K1K2Pu, SK, and STAR
inhibited thrombus formation at a significantly lower dose
(P<.05) than rTPA and rscu-PA.
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),
rscu-PA (
), urokinase (
), K2Pt (
),
K1K2Pu (
), streptokinase (
), and
staphylokinase (
). A, Dose-dependent effects on thrombus
formation after intravenous bolus injection in hamsters with a
platelet-rich mural femoral vein thrombosis. B, Residual thrombus after
intravenous infusion over 60 minutes in hamsters with a pulmonary
embolism. The results of rTPA and rscu-PA have previously been
reported.
Thrombolytic Potency
A dose-dependent thrombolytic effect was
observed (Table 2 and the Figure
). Fifty-percent
clot lysis
(ED50) was obtained with 0.9±0.13, 0.34±0.03,
0.04±0.003, 0.05±0.005, and 0.04±0.001 mg/kg of UK,
K2Pt, K1K2Pu, SK, and STAR,
respectively. ED50 in the same pulmonary embolism model for
rTPA and rscu-PA, derived from previously published
data,15 was 0.18±0.03 and 1.1±0.05 mg/kg,
respectively.
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Fibrinogen Breakdown
The plasma samples collected at the end
of the antithrombotic and
clot lysis experiments were used to determine the fibrinogenolytic
effect for each thrombolytic agent. In the antithrombotic experiments,
a gradual decrease in fibrinogen and 2-antiplasmin
levels was observed with increasing doses of rTPA, rscu-PA, UK,
K2Pt, K1K2Pu, SK, and STAR,
correlating with the antigen levels obtained in plasma from these
experiments (Table 1 and the Figure). From these
data the dose that
induced 50% fibrinogen breakdown was calculated (Table 3). UK,
SK, and STAR induced fibrinogen breakdown at
significantly lower doses (P<.05) than rTPA and rscu-PA. In
the clot lysis experiments, only UK induced significant fibrinogen
breakdown (P<.01) at doses that induced thrombolysis (Table
2).
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Pharmacokinetics
After intravenous bolus injection of 0.1
mg/kg, rTPA, rscu-PA, SK,
and STAR were cleared with an initial half-life of approximately 2
minutes, while for K2Pt and K1K2Pu
significantly longer values (P<.05) of 4±0.15 and
5±2
minutes were calculated (Table 4). The terminal
half-life for rTPA, rscu-PA, K2Pt,
K1K2Pu, SK, and STAR was 7±0.9, 6±0.7,
53±8,
35±2, 13±2, and 19±0.3 minutes, respectively. MRT, the
time
necessary to clear 62.3% of the injected substance from the
circulation, was 18±1, 14±1, 100±10, 80±2,
20±3, and 34±5 minutes
for rTPA, rscu-PA, K2Pt, K1K2Pu,
SK, and STAR, respectively. MRTs for K2Pt,
K1K2Pu, and STAR were significantly longer
(P<.05) than those of rTPA and rscu-PA.
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Correlation Between Antithrombotic Effect, Thrombolytic Potency,
Fibrinogen Breakdown, and MRT
Least-squares regression analysis of the
results of Table 3
resulted in a significant cross correlation between the antithrombotic
effect, fibrinogen breakdown, and MRT, while no correlation with
thrombolytic potency was observed (Table 5).
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Significant correlations were observed between antithrombotic effect and fibrinogen breakdown (P=.006) and between MRT and fibrinogen breakdown (P=.026). The antithrombotic effect did not correlate with the thrombolytic potency, and its correlation with MRT was not statistically significant (P=.075).
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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The present study was carried out to quantify the antithrombotic effects of several plasminogen activators on platelet-rich thrombus formation in the absence of conjunctive anticoagulant or antiplatelet agents, using a model of platelet-mediated primary thrombus formation in the hamster.20 The antithrombotic effect was then correlated with the thrombolytic potency, the extent of fibrinogen breakdown, and MRTs.
All thrombolytic agents displayed antithrombotic effects. UK,
K1K2Pu, SK, and STAR inhibited 50% thrombus
formation at significantly lower doses than rTPA and rscu-PA. The
thrombolytic potencies of rTPA, rscu-PA, K2Pt, SK, and STAR
were significantly higher (P<.01) than their antithrombotic
effects. Most of the thrombolytic agents inhibited thrombus formation
at doses similar to the systemic fibrinogenolytic effects and exceeded
the thrombolytic potencies (Table 3 and the
Figure).
The antithrombotic effect of the plasminogen activators studied
correlated strongly with systemic fibrinogen breakdown
(P=.006) and weakly with MRT (P=.075) but not
with thrombolytic potency. The only additional significant correlation
was between fibrinogen breakdown and MRT (P=.026) (Table
5).
UK displayed antithrombotic, thrombolytic, and fibrinogenolytic effects
at similar doses, while K1K2Pu exerted an
antithrombotic and thrombolytic effect at a dose significantly lower
(P=.01) than the dose that induced fibrinogen breakdown
(Table 3 and the Figure).
In summary, our data indicate that the antithrombotic effect of most thrombolytic agents parallels their fibrinogenolytic effects and not their thrombolytic potencies. These findings provide indirect support for the conjunctive use of anticoagulants and/or antiplatelet agents during thrombolytic therapy with fibrinogen-sparing agents.
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Acknowledgments |
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This work was supported by a grant from the Belgian government (IUAP 35). The authors express their gratitude to I. Vreys, I. Vanlinthout, A. Bouché, and H. Moreau for their skillful technical assistance. Lars Brännström, Zeiss AB, Stockholm, Sweden, and L.O. Andersson Fine Mechanics, Umeå, Sweden, gave valuable support to the experimental setup.
Received October 31, 1994; revision received January 9, 1995; accepted January 10, 1995.
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