(Circulation. 1996;93:153-160.)
© 1996 American Heart Association, Inc.
Articles |
Comparison of Sustained Antithrombotic Effects of Inhibitors of Thrombin and Factor Xa in Experimental Thrombosis
From the Center for Hemostasis, Thrombosis, Atherosclerosis and Inflammation Research (B.J.B., P.W.F., M.L., H.R.B., J.W.t.C.), Academic Medical Center, University of Amsterdam, Netherlands; and Corvas International Inc (G.P.V.), San Diego, Calif.
Correspondence to Bart J. Biemond, MD, Center for Hemostasis, Thrombosis, Atherosclerosis and Inflammation Research, Academic Medical Center, 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 In the pathogenesis of (recurrent) thrombosis, clot-associated thrombin appears to play an important role. Antithrombin III–independent thrombin inhibitors have been shown to neutralize clot-bound thrombin effectively. We compared the sustained antithrombotic effects and the effects on endogenous fibrinolysis of several of these agents with recombinant tick anticoagulant peptide (rTAP), a selective factor Xa inhibitor, and low-molecular-weight heparin (LMWH) in an experimental venous thrombosis model.
Methods and Results Rabbits received either recombinant hirudin (rHir), Hirulog-1, CVS#995 (a novel direct inhibitor of thrombin), rTAP, LMWH, or saline. The effect on thrombus growth was assessed by measuring the accretion of 125I-labeled fibrinogen onto preformed nonradioactive thrombi, and the effect on endogenous fibrinolysis was assessed by measuring the decline in radioactivity of preformed 125I-labeled thrombi in rabbit jugular veins. All direct thrombin inhibitors induced a sustained antithrombotic effect compared with either LMWH and rTAP. In addition, CVS#995 also further decreased thrombus size after stopping its infusion, which was due to a significant enhancement of endogenous fibrinolysis.
Conclusions Direct thrombin inhibition by rHir, Hirulog-1, or CVS#995 induces a sustained antithrombotic effect compared with rTAP and LMWH, which is most likely due to inhibition of clot-bound thrombin. CVS#995 was shown to also enhance the extent of endogenous fibrinolysis to a greater degree compared with rHir and might therefore be an interesting new antithrombotic agent for the treatment of venous and arterial thrombosis.
Key Words: anticoagulants • thrombosis • fibrinolysis
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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Unfractionated heparin is the most commonly used anticoagulant drug in the treatment and prevention of venous and arterial thromboembolic disorders.1 Heparin inhibits thrombin and activated factor X after complex formation with its natural serpin cofactor antithrombin III, which dramatically enhances the affinity of antithrombin III for both enzymes.
Despite the proven clinical efficacy of heparin in the treatment of venous thromboembolism, it has been directly demonstrated by venography that there is a 20% to 30% extension of venous thrombi in patients undergoing treatment with therapeutic doses of unfractionated heparin.2 3 4 5 6 It has been shown that discontinuation of heparin therapy is complicated by recurrent venous thrombosis in approximately 10% of the patients in the following 6 months.4 Furthermore, it has been shown that arterial thrombus formation at sites of vascular damage, such as atherosclerotic lesions in the coronary artery after angioplasty or thrombolysis, is not adequately prevented by the administration of unfractionated heparin.7 Therefore, it has been suggested that clot-bound thrombin, which is not effectively inhibited by the heparin–antithrombin III complex, might be at least partially responsible for continuous fibrin formation and recurrent thrombosis.8
Antithrombin III–independent thrombin inhibitors, such as rHir and its analogues, have been shown to inhibit clot-bound thrombin more effectively than the heparin–antithrombin III complexes by the formation of stable, noncovalent 1:1 stoichiometric complexes.9 Clinical and experimental studies have confirmed that rHir is able to reduce thrombus extension more efficiently than unfractionated heparin in a model of experimental venous thrombosis and showed that it is able to prevent the formation of coronary thrombosis after angioplasty.7 10 11
Recently, several new antithrombotic agents have been developed, such as Hirulog-1 and rTAP,12 13 14 which specifically and independent of plasma cofactors inhibit thrombin and factor Xa, respectively. Hirulog-1 is a synthetic polypeptide consisting of binding regions for the catalytic and exosite binding sites of thrombin.14 Hirulog-1 has been suggested to inhibit clot-bound thrombin more effective than hirudin, presumably due to its reduced molecular size, which facilitates access to this pool of thrombin.15 In clinical and experimental studies, Hirulog-1 has been demonstrated to be a highly effective anticoagulant and antithrombotic agent.16 17 18 19 TAP is a novel serine protease inhibitor originally isolated from the tick Ornithidoros moubata.12 The recombinant form of TAP (rTAP) has been shown to have a strong antithrombotic effect in models of experimental arterial20 21 and venous thrombosis22 mediated though the inhibition of de novo thrombin generation or prothrombinase function. Other than the specific and direct inhibitors of thrombin or factor Xa inhibitors, there has been a great deal of research focused on optimizing the pharmacological profile of standard heparin by fractionating the compound to lower-molecular-weight fragments. These LMWH and synthetic heparinoids have been shown to have a more consistent anticoagulant effect and to induce less bleeding complications than unfractionated heparin in the prevention and treatment of venous thrombosis.23 24 The improved pharmacological profile of the LMWH and heparinoids has been attributed in large part to less protein binding, resulting in a better and more predictable pharmacokinetic profile. However, the mechanism of inhibition remains dependent on antithrombin III. So far, no direct comparison between these new antithrombotic agents has been made in a single experimental model.
In the present study, we compared the antithrombotic effect of LMWH, rHir, Hirulog-1, and rTAP with a newly developed thrombin inhibitor, CVS#995, in a rabbit jugular vein thrombosis model. In vitro, CVS#995 has been demonstrated to inhibit thrombin with a higher affinity and is less sensitive to proteolytic degradation than are similar-sized inhibitors such as Hirulog-1. Because all these new agents have a relatively short half-life in vivo, we chose to investigate the antithrombotic efficacy of each agent during a continuous infusion and after plasma levels have decreased. We focused on the comparison of the sustained antithrombotic capacity of all these new direct thrombin inhibitors with LMWH and rTAP. The effect on thrombus growth was assessed directly after discontinuation of the study medication infusion and 1 or 2 hours afterward. In addition, the effects on endogenous fibrinolysis were compared in the same animal model.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Experimental Preparation
New Zealand White rabbits of approximately 2.5 kg were used for this study. Anesthesia was induced by the administration of 9 mg Ketamin (Aescoket) and 0.5 mL Rompun 2% (Bayer). To maintain adequate anesthesia, intramuscular injections of Ketamin were given repeatedly. The carotid artery and 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 diameter) was introduced for the administration of the study medication and for blood sampling. The jugular veins were cleared over a distance of 2 cm, and all small side branches were ligated. The veins were clamped both proximally and distally.
Thrombus Growth Model
Nonradioactive thrombi were formed in
both jugular veins of the
rabbit by injection of 150 µL homologous rabbit blood aspirated in a
1-mL syringe containing 25 µL human thrombin (Human Thrombin T7009,
Sigma Chemical Co; 150 IU/mL) and 45 µL CaCl2 (0.25
mol/L) into the isolated venous segment. After 30 minutes of aging of
the thrombus, the blood flow was restored by removing the vessel
clamps, and 100 µL 125I-radiolabeled human fibrinogen
(Amersham, approximately 2 µCi) was injected systemically, followed
immediately by the administration of a loading dose and a 2-hour
continuous infusion of the study compound. Blood samples were taken
every hour to calculate the mean plasma radioactivity per milliliter of
blood for each rabbit. One thrombus was removed at the end of the
2-hour infusion (t=120), whereas the contralateral thrombus was removed
1 hour (t=180) or 2 hours (t=240) after discontinuation of the
study
drug administration. Thrombus growth was assessed by measuring the
accretion of 125I-radiolabeled fibrinogen onto the
preformed nonradioactive thrombi. Thrombus growth was measured by
calculating the blood volume accreted onto the clot by comparing
125I-related blood radioactivity with
125I-related thrombus radioactivity. Thrombus growth was
expressed as a percentage of the initial thrombus volume.
Measurement of Endogenous
Fibrinolysis
To measure the extent of endogenous
fibrinolysis, radiolabeled thrombi were formed in the
jugular veins of the rabbit, and the degree of
fibrinolysis was determined by assessing the decline in
initial radioactivity of the preformed thrombus. Therefore, homologous
rabbit blood was mixed with 125I-labeled fibrinogen (final
radioactivity, approximately 10 µCi/mL). An aliquot of 150 µL of
this mixture was then aspirated into a 1-mL syringe containing 25 µL
thrombin (150 IU/mL) and 45 µL CaCl2 (0.25 mol/L) and
quickly injected into the isolated venous segment. After 30 minutes of
aging, the vessel clamps were removed and the infusion of a bolus
injection of study drug was given, followed by a 2-hour continuous
infusion. One thrombus was removed directly after the 2-hour infusion
and the contralateral thrombus was removed 1 hour later. The extent of
endogenous fibrinolysis 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.
Test Compounds
CVS#995 is a synthetic peptide of 19 amino
acids with the
following structure:
(CH3CH2CH2)2-CHCO-D-P-R-
-[COCO]-(G)5-N-G-D-F-(E)2-I-P-E-Y-C-OH
(Mr=2133).25 It contains an activated
carbonyl transition state mimetric (
-keto-amide), which
serves to bridge the catalytic or active site domain with another
sequence that binds to the anion-binding exosite I of
-thrombin. Therefore, CVS#995 is a synthetic
inhibitor of thrombin that couples the stability to
proteolytic degradation by the incorporation of the
-keto-amide functional group with excellent selectivity
resulting from the use of specific binding sequences for the primary
substrate or accessory site on -thrombin. CVS#995 was
synthesized using a combination of solid- and solution-phase
chemistry and was purified by RP-HPLC. The purity of CVS#995 was
determined to be >98% by a combination of analytical criteria that
included RP-HPLC, capillary electrophoresis, FAB mass spectrometry, and
quantitative amino acid analysis. The three-dimensional
crystal structure of CVS#995 bound to human -thrombin confirms
the multisite binding interactions of this inhibitor with
the enzyme, showing the interaction of the catalytic-site
functionality, the tetrahedral transition state mimetic, and the
binding of the carboxyl-terminal sequence to the anion-binding
exosite of thrombin. CVS#995, therefore, is a synthetic thrombin
inhibitor that couples the stability to proteolytic
degradation by the potent transition state intermediate with the
selectivity resulting from the use of accessory binding sites on
thrombin. CVS#995 is a potent inhibitor of amidolytic
substrate hydrolysis by -thrombin
(Ki=2 pmol/L) (G.P. Vlasuk, unpublished
observations). It was shown that the thrombin-induced clotting of
purified fibrinogen could be blocked by CVS#995 in a dose-dependent
fashion. In addition, the ability of CVS#995 to inhibit fluid-phase
and clot-associated thrombin activity was demonstrated in a system
of citrated plasma and thrombin-induced clot formation, as
described previously.9 CVS#995 initially inhibited both
fluid-phase and clot-associated thrombin activity in a
time-dependent fashion, followed by a stable level of inhibition.
Thrombin-induced platelet aggregation could be blocked by
CVS#995 at concentrations of 20 nmol/L or higher. As expected, CVS#995
was absolutely specific for -thrombin with no inhibition of
plasmin, rTPA, or any other serine protease involved in blood
coagulation or fibrinolysis, seen at concentrations
>100 000-fold in excess of that required to fully inhibit
-thrombin.
Because our aim was to study the effect of CVS#995 in a
rabbit model,
we studied the effects of CVS#995 on thrombin-induced formation of
rabbit fibrin and aggregation of rabbit platelets. The results are
provided in Table 1
and indicate that CVS#995 is also
effective in inhibiting thrombin-induced formation of fibrin and
platelet aggregation in rabbits.
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rHir (CGP 39393) with a specific activity of 115 000 ATU/mg was kindly provided by Dr R.B. Wallis, CIBA-GEIGY, Horsham, UK, and obtained from CIBA-GEIGY, Basel, Switzerland.
Hirulog-1 is a synthetic peptide14 that was produced by conventional solid-phase chemistry and characterized as described above for CVS#995.
rTAP was also obtained from Corvas International, San Diego, Calif, and prepared as described previously.26
The LMWH used in this study was Fraxiparin, with a specific activity of 25 000 anti–factor Xa IU/mL, purchased from Sanofi, Paris, France.
All study agents were dissolved in saline.
Treatment Schedules
Seven study groups consisting of eight
rabbits per group were
investigated. The seven study groups were given either (1) CVS#995
administered at a high dose of 1.0 mg/kg bolus injection followed by
the continuous infusion of 5 µg/kg per minute, (2) CVS#995
administered at a median dose of 0.5 mg/kg bolus injection followed by
a continuous infusion of 5 µg/kg per minute, (3) rHir at a dose of
0.5 mg/kg bolus injection followed by a 5 µg/kg per minute continuous
infusion, (4) Hirulog-1 according to the same regimen as rHir, (5) rTAP
administered according to the same regimen as rHir, (6) LMWH at a dose
of 40 anti-factor Xa U/kg bolus followed by a continuous infusion
of 0.33 anti-factor Xa U/kg per minute, or (7) saline control, also
administered as a bolus injection and continuous infusion. The animals
received the loading dose followed by a continuous administration for 2
hours via the carotid artery cannula. The dosages were selected, based
on previous experiments, for their comparable antithrombotic effect
directly at the end of drug infusion. One of the thrombi was removed
and counted directly after the cessation of the continuous infusion,
whereas the contralateral thrombus was taken out 1 hour after the
infusion was stopped. In another seven study groups, treated according
to the same protocol but consisting of six animals per group, one of
the thrombi was again removed immediately after discontinuation of
study medication, but the contralateral thrombi were taken out 2 hours
after cessation of study drug infusion.
The effect on the endogenous fibrinolysis was assessed in an additional experiment consisting of four groups (four rabbits each) treated with either (1) CVS#995 at a high dose of 1.0 mg/kg bolus and a continuous infusion of 5 µg/kg per minute for 2 hours, (2) CVS#995 at a median dose (0.5 mg/kg bolus plus 5 µg/kg per minute continuous infusion), (3) rHir (0.5 mg/kg bolus injection plus 5 µg/kg per minute continuous infusion), or (4) saline. In addition to this, a small number of experiments(n=4) were performed with higher doses of rHir (ie, 1.0 mg/kg bolus injection plus 5 µg/kg per minute continuous infusion or a dose of 5.0 mg/kg bolus injection plus 25 µg/kg per minute continuous infusion). The effect of these agents on endogenous fibrinolysis was assessed by removing one thrombus directly after stopping the infusion and the contralateral thrombus 1 hour later.
Counting of the radioactivity of the thrombi to determine the effect on thrombus growth and fibrinolysis was performed by a second independent investigator.
Blood Sampling and Plasma Preparation
Blood samples were
drawn from the carotid cannula before the
administration of the study medication and 60, 120, and 180 minutes
thereafter. Blood samples (9 vol) were collected in 3.2% citrate (1
vol) for determination of the aPTT, PA activity, and PAI-1 activity.
Platelet-poor plasma was obtained by immediate
centrifugation at 1600g for 20 minutes at
4°C and stored at -70°C until assay.
Pharmacokinetics
To allow us to assess the pharmacokinetics
of CVS#995 and rHir,
an additional six rabbits received an intra-arterial
bolus injection of either CVS#995 or rHir at a dose of 3 mg/kg,
respectively, and a group of six rabbits received a bolus injection of
one of these compounds at a dose of 1 mg/kg. Blood samples (9 vol) to
assess plasma levels of CVS#995 or rHir were collected in 3.2% citrate
(1 vol) that were drawn before and 2, 5, 10, 15, 20, 30, 40, 60, 120,
and 240 minutes afterward. Platelet-poor plasma was obtained by
centrifugation at 1600g for 20 minutes at
4°C. Plasma levels of CVS#995 and rHir were determined by measuring
the inhibition of purified human -thrombin amidolytic activity
in diluted samples of plasma. The plasma levels are expressed in
nmol/L.
Assays
The aPTT was determined by standard methods on a MLA
900C
apparatus using Actin FS as a reagent. PA activity was
measured by amidolytic assay.27 Briefly, 25 µL of plasma
was mixed to a final volume of 250 µL with 0.1 mol/L Tris·HCl, pH
7.5, 0.1% (vol/vol) Tween-80, 0.3 mmol/L S-2251 (Chromogenix), 0.13
mol/L plasminogen (Chromogenix), and 0.12 mg/mL
CNBr-digested fibrinogen fragments (TPA stimulator, Chromogenix).
PA activity was assessed in these samples by spectrophotometric
measurement. PAI-1 activity was measured with an amidolytic method, as
described previously.28 Briefly, plasma 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 (Chromogenix, 0.12 mg/mL)
CNBr fragments of fibrinogen (TPA stimulator, Chromogenix), and 0.1
mmol/L S-2251 (Chromogenix). The PAI-1 activity in the sample was
inversely proportional to the plasmin generated in the incubation
mixture, determined by the conversion of the chromogenic
substrate. Results are expressed in IU, where 1 IU is the amount of
PAI-1 that inhibits 1 IU TPA (first international standard of the World
Health Organization).
To assess the plasma levels CVS#995 and rHir, a
chromogenic
assay was used, measuring the inhibition of purified human
-thrombin in diluted samples of plasma. Briefly, the assay was
conducted by combining in appropriate wells of a Corning microtiter
plate 50 µL of HBSA (10 mmol/L HEPES, pH 7.5, 150 mmol/L sodium
chloride, 0.1% bovine serum albumin), 50 µL of the citrated
plasma sample diluted 1:1000 in HBSA or standard inhibitor
diluted in HBSA (or HBSA alone for Vo measurement), and 50
µL of purified human thrombin (3000 U/mg specific activity; Enzyme
Research Laboratories, Inc) diluted in HBSA. After a 30-minute
incubation at ambient temperature (23°C), 50 µL of the
chromogenic substrate (Pefachrome TPA
[CH3SO2-d-hexahydrotyrosine-glycyl-l-Arg-p-nitroaniline]
reconstituted in deionized water before use; Pentapharm Ltd) was added
to the wells yielding a concentration of 300 µmol/L (5x
Km) and a final total volume of 200 µL. The
initial velocity of chromogenic substrate hydrolysis was
measured by the change in absorbance at 405 nm using a Thermo Max
Kinetic Microplate Reader (Molecular Devices) over a 5-minute period in
which less than 5% of the added substrate was used. The plasma levels
of CVS#995 and rHir were calculated using a standard curve of purified
and quantitated inhibitor made up in control, homologous
citrated plasma covering a broad concentration range followed by
dilution to 1:1000 with HBSA. Under these conditions, the
IC50 for inhibition of purified human -thrombin
(0.25 nmol/L final concentration) by the respective
inhibitor in diluted plasma was not significantly different
from that in HBSA alone. The limit of inhibitor detection
in this assay is 75 nmol/L.
Statistical Analysis
ANOVA (for repeated measures) followed
by a Newman-Keuls test
was applied for statistical analysis. P<.05 was
considered to be statistically significant. All values are 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 the Dutch Law for Animal Experiments.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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The effect of the different antithrombotic agents on thrombus growth is shown in Fig 1
. The administration of CVS#995
resulted in a dose-dependent reduction of thrombus growth compared
with saline control. CVS#995 administered at the high dose (1.0 mg/kg
bolus plus 5 µg/kg per minute continuous infusion) resulted in the
most pronounced antithrombotic effect, resulting in a reduction in
thrombus growth to 25.2±1.8%, whereas CVS#995 administered at the
median dose (0.5 mg/kg bolus plus 5 µg/kg per minute continuous
infusion) reduced thrombus growth to 32.5±1.8% compared with
47.9±2.9% thrombus growth in the saline control animals
(P<.05). The antithrombotic effect of CVS#995 at the median
dose was comparable to the antithrombotic effect of LMWH and rTAP
(thrombus growth, 36.1±3.2% and 35.3±3.0%, respectively;
P=NS). rHir and Hirulog-1 had an equal antithrombotic effect
compared with CVS#995 administered at the median dose (thrombus growth,
29.8±5.6% and 31.0±2.5% versus 32.5±1.8%, respectively;
P=NS) but were slightly more effective in thrombus growth
reduction directly after cessation of their administration than rTAP
and LMWH (P<.05; Fig 1). No difference in bleeding
from
surgical wounds was observed between the various compounds, although
this animal model has not been designed to assess the specific effect
of antithrombotic agents on bleeding.
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-); (2) CVS#995 at a median dose (0.5 mg/kg
plus 5 µg/kg per minute, -
-); (3) rHir (0.5 mg/kg plus 5
µg/kg per minute, -
-); (4) Hirulog-1 (0.5 mg/kg plus 5 µg/kg
per minute, -
-); (5) rTAP (0.5 mg/kg plus 5 µg/kg per minute,
-
-); (6) LMWH (40 anti–factor Xa IU/kg plus 0.33
anti–factor Xa IU/kg per minute, -
-); or (7) saline
(-
-). Thrombus growth is expressed as percentage (mean±SD) of
the initial thrombus volume.
To assess the duration of the antithrombotic effect of these compounds,
the extent of thrombus growth was determined 1 and 2 hours after
termination of the continuous infusion of the test compound (Fig
1
). Thrombus growth in the saline control group significantly
increased over this period, to 65.5±4.3% and 70.1±3.0%,
respectively. The animals treated with LMWH (thrombus growth,
45.7±2.3% and 57.4±3.3%, respectively), rTAP (thrombus growth,
41.6±3.1% and 47.7±2.7%, respectively), rHir (thrombus growth,
35.7±2.7% and 41.6±2.4%, respectively), and Hirulog-1 (thrombus
growth, 37.9±2.1% and 37.2±2.3%, respectively) all demonstrated
an
increased thrombus growth 1 and 2 hours after the end of the infusion,
although to a lesser extent compared with saline (Fig 1). In
accordance
with earlier observations, rHir and Hirulog-1 were shown to have a
significantly higher sustained antithrombotic effect after their
discontinuation than LMWH and rTAP. In contrast, CVS#995 at both doses
not only prevented thrombus growth completely after the end of its
administration but also decreased the amount of labeled fibrinogen
present at the preformed clot significantly. CVS#995 administered
at median dose reduced the amount of accreted fibrinogen from
32.5±1.8% at t=120 to 27.8±2.9% and 27.3±2.3% at
t=180 and
t=240, respectively (P<.05), whereas the higher dose
was shown to have a similar thrombus-reducing effect from
25.2±1.8% at t=120 to 19.3±2.1% and 20.3±1.9% at
t=180 and t=240,
respectively (P<.05). The effect on reducing thrombus size
appeared to level off 1 hour after stopping the administration since
the amount of accreted fibrinogen 2 hours after the end of the infusion
did not significantly differ from the amount measured 1 hour
earlier.
All test compounds induced a very modest and comparable prolongation of
the aPTT (Table 2), but no significant difference in
prolongation between the different antithrombotic agents was
observed.
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Since the administration of CVS#995 not only prevented thrombus growth
completely but also reduced the amount of fibrinogen already accreted
onto the clot, the effect of CVS#995 on the endogenous
fibrinolysis was assessed in an additional study.
CVS#995, administered at the median dose, resulted in an enhanced
endogenous fibrinolysis of 14.4±1.9% at
t=120 and 21.8±2.1% at t=180 compared with
6.9±1.2% and 10.6±1.1%
for rHir and 3.6±1.4% and 5.9±0.8% for saline, respectively
(P<.05; Fig 2). CVS#995, administered at the
higher dose, did not improve the extent of endogenous
fibrinolytic effect any further (thrombolysis,
14.8±2.4% at t=120 and 19.7±2.8% at t=180;
P=NS versus
the median dose; Fig 2), indicating that the
thrombolytic effect of CVS#995 was already maximal at
the median dose. To observe whether the administration of higher doses
of rHir might result in a similar increase in endogenous
fibrinolysis, an additional series of rabbits (n=4)
were studied. However, the administration of rHir at a dose of 1.0
mg/kg bolus plus 5 µg/kg per minute or at a dose of 5.0 mg/kg bolus
plus 25 µg/kg per minute did not induce a further increase in
thrombolysis (thrombolysis at t=120,
6.4% and 7.0%, respectively; thrombolysis at t=180,
9.5% and 10.9%, respectively).
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To assess whether the enhanced extent of endogenous
fibrinolysis could be explained by a systemic
activation of the fibrinolytic system, the plasma levels of PA- and
PAI-1 activity were determined in the animals treated with CVS#995,
rHir, and saline. PA activity levels remained below the detection limit
in all samples (data not shown), and although a statistically
significant difference in PAI-1 activity levels was found between the
animals treated with either CVS#995 or rHir compared with the animals
receiving saline 1 hour after the infusion was started, no difference
in PAI-1 levels was observed between the CVS#995- and rHir-treated
animals during the entire experiment (Table 3).
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To determine whether the difference in sustained antithrombotic effect
between CVS#995 and rHir could be explained by a difference in
elimination, the half-life of both compounds at two different doses
was assessed in an additional study. The results are presented
in Fig 3. No difference in half-life was observed
between the two compounds at a dose of 1 and 3 mg/kg, respectively.
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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Clot-bound thrombin, which has been shown to be inaccessible to the heparin–antithrombin III complex, has been suggested to be partially responsible for recurrent thrombosis after discontinuation of heparin or LMWH treatment and the relative ineffectiveness of heparin in the prevention of rethrombosis after thrombolytic therapy or angioplasty.4 7 8 Experimental and clinical studies with hirudin and hirudin analogues revealed that these antithrombin III–independent direct thrombin inhibitors not only prevented rethrombosis after thrombolytic therapy and angioplasty more effectively than heparin but also had a sustained antithrombotic effect after the end of its administration in a model of experimental venous thrombosis.7 10 11 17 18 19 20 In accordance with these earlier observations, in the present study the antithrombin III–independent direct thrombin inhibitors were shown to have a more intense and sustained antithrombotic effect than LMWH treatment. Recent studies have suggested that Hirulog-1 is able to inhibit clot-bound thrombin more effectively than rHir because of its greater accessibility to this pool of thrombin due to its smaller molecular size15 but also showed that Hirulog-1 is cleared more rapidly from the circulation than rHir because of its higher susceptibility to proteolytic degradation.16 Despite these differences, in the present study both agents had an equal antithrombotic effect during and after their administration. Directly after cessation of its administration, CVS#995, at the median dose, appeared to have a comparable antithrombotic effect to rHir and Hirulog-1 but had a significantly more pronounced and sustained antithrombotic effect after cessation of the infusion. One hour after discontinuation of its administration, CVS#995 not only completely blocked the accretion of radiolabeled fibrinogen but also significantly decreased thrombus size, suggesting a facilitated endogenous fibrinolysis. In contrast, after the discontinuation of either rHir or Hirulog-1, a moderate increase in fibrinogen accretion was demonstrated. Two hours after cessation of the drug administration, no further change in thrombus size was observed in the CVS#995-, rHir–, and Hirulog-1–treated animals. These observed differences between CVS#995 and the other direct thrombin inhibitors are probably due to a combination of greater accessibility to clot-bound thrombin compared with rHir and resistance to proteolytic inactivation compared with Hirulog-1. It should be noted that the pharmacokinetic properties of CVS#995 are similar to those of Hirulog-1 and that the circulating ß-phase elimination half-life of CVS#995 were comparable to the half-life of rHir (G.P. Vlasuk GP, unpublished observations). In accordance, in the present study, no difference in elimination half-life was observed between CVS#995 and rHir. Therefore, the difference in pharmacological effect between CVS#995 and the other agents cannot be explained by an extended half-life of this compound, resulting in a more prolonged maintenance of plasma levels. However, a difference in tissue saturation between the two agents cannot be excluded despite the comparable molecular size of these agents. rTAP, which inhibits factor Xa independent of antithrombin III, was more effective than LMWH in the prevention of thrombus growth after its discontinuation but appeared not to be as effective as CVS#995, rHir, or Hirulog-1 in the dosages studied. Apparently, selective inhibition of factor Xa did not block fibrin deposition as efficiently as inhibition of thrombin. It should be realized, however, that the thrombin-induced clots in this model are relatively thrombin rich and could therefore be more sensitive to thrombin inhibition than inhibition of factor Xa. In addition, careful and extensive comparisons between CVS#995 and other antithrombin agents at a wide range of doses should be made before final conclusions can be reached.
The experiments, which were designed to measure the extent of
endogenous fibrinolysis, suggested that
thrombin inhibition by either CVS#995 or rHir results in a significant
enhancement of the endogenous fibrinolysis
compared with saline. CVS#995 appeared to induce a significantly
stronger enhancement of the extent of endogenous
fibrinolysis than rHir, at least at the doses studied.
These data appear to explain the observed reduction in thrombus size
seen in the fibrin-accretion experiments with CVS#995 compared with
rHir. Experiments with higher doses of rHir revealed that this
treatment did not induce an additional increase in
endogenous thrombolysis. Although these
observations suggest that stable thrombin inhibition enhances the
endogenous fibrinolysis, the mechanism by
which this effect is obtained remains unclear. In vitro experiments
have demonstrated that incubation of endothelial cells
with thrombin induces a significant elevation of PAI-1 antigen levels
in the supernatant and increases the PAI-1 mRNA levels in these
cells.29 30 Therefore, we hypothesized that firm
thrombin
inhibition might stimulate the endogenous
fibrinolysis by lowering PAI-1 secretion of
endothelial cells and possibly by inhibiting the
release of PAI-1 from platelets. However, other than a significant
elevated PAI-1 level in the saline-treated group 1 hour after
starting the infusion, no differences in PAI-1 activity levels were
observed among the different groups in the systemic circulation. The PA
activity level remained below the detection limit in all samples. These
observations, however, do not rule out potential differences in PA and
PAI-1 activity levels at the local level. As alternative explanations
for the difference between CVS#995 and rHir on endogenous
fibrinolysis, it can be hypothesized that CVS#995 may
exert some distinct, yet unidentified, effects on coagulation or
fibrinolytic factors or may evoke a direct effect on
endothelial cells. Another possible mechanism by which
thrombin inhibition could enhance the endogenous clot lysis
might be the inhibition of fibrin cross-linking, which is necessary
for stable clot formation. In vitro studies have demonstrated that
other than the fibrin/fibrin cross-linking, cross-linking of
2-antiplasmin to fibrin monomers is essential for
thrombolysis resistance of fibrin
clots.31 32 These processes are dependent on the
activation of factor XIII to factor XIIIa by
thrombin,33 34 and strong inhibition of thrombin
might
therefore attenuate this process, resulting in clot instability and
enhanced endogenous clot lysis. Another explanation for
enhanced fibrinolysis is the inhibition of thrombus
growth through the stable attenuation of thrombin-mediated fibrin
formation. This inhibition of clot extension might shift the hemostatic
balance toward fibrinolysis, resulting in an overall
reduction in clot size.
In conclusion, the present study in a venous thrombus growth model in rabbits demonstrated that in contrast to rTAP and LMWH, selective thrombin inhibition induces a sustained antithrombotic effect that is most likely explained by neutralization of clot-bound thrombin. Thrombin inhibition by CVS#995 not only was shown to have a strong sustained antithrombotic effect but also appeared to enhance the endogenous fibrinolysis and might therefore be an interesting antithrombotic agent for the treatment of both venous and arterial thrombotic disorders. Although it has been shown that there is good correlation between the results obtained in the rabbit jugular vein thrombosis model and animal models of arterial thrombosis, it should be realized that the results in this study were obtained in a model of venous thrombosis. Therefore, additional comparative studies in different animal models and with different doses of the various compounds need to be performed to further substantiate differences within the growing group of new antithrombotics.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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Dr Büller is the recipient of a fellowship from the Royal Netherlands Academy of Arts and Sciences. We thank Dr Dan Pearson, Michael Weinhouse, and Pureza Vallar at Corvas International Inc for supplying the CVS#995 and Hirulog-1 and Dr Howard Soule for the stimulating discussions. In addition, we would like to thank Huan Tran for his technical expertise.
Received September 12, 1994; revision received July 19, 1995; accepted August 14, 1995.
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References |
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TopAbstractIntroductionMethods Results Discussion References |
|---|
1. Hirsh J. Heparin. N Engl J Med. 1991;324:1565-1574. [Medline] [Order article via Infotrieve]
2. Marder VJ, Soulen RL, Atichartakarn V, Budzynski AZ, Parulekar S, Kim JR, Edward N, Zahari J, Algazy KM. Quantitative venografic assessment of deep venous thrombosis in the evaluation of streptokinase and heparin therapy. J Lab Clin Med. 1977;89:1018-1029. [Medline] [Order article via Infotrieve]
3. Bratt G, Tornebohm E, Gragvist S, Aberg, Lockner D. A comparison between low molecular weight heparin (KABI 2165) and standard heparin in the intravenous treatment of deep venous thrombosis. Thromb Haemost. 1985;54:813-817. [Medline] [Order article via Infotrieve]
4. Brandjes DPM, Heyboer H, Büller HR, de Rijk M, Jagt H, ten Cate JW. Acenocoumarol and heparin compared with acenocoumarol alone in the initial treatment of proximal vein thrombosis. N Engl J Med. 1992;327:1485-1489. [Abstract]
5. Hull RD, Raskob GE, Hirsh J, Jay RM, Leclerc JR, Geerts WH, Rosenbloom D, Sackett DL, Anderson C, Harrison L, Geert M. Continuous intravenous heparin compared with intermittent subcutaneous heparin in the initial treatment of proximal vein thrombosis. N Engl J Med. 1986;315:1109-1114. [Abstract]
6.
Hull RD, Raskob GE, Rosenbloom D, Lemaire J, Pineo GF,
Baylis B, Ginsberg JS, Panju AA, Brill-Edwards P, Brant R.
Optimal therapeutic level of heparin therapy in patients with venous
thrombosis. Arch Intern Med. 1992;152:1589-1595.
7. GISSI-2. A factorial randomized trial of alteplase versus streptokinase and heparin versus no heparin among 12,490 patients with acute myocardial infarction. Lancet. 1990;1:65-71.
8.
Agnelli G, Renga C, Weitz JI, Nenci GG, Hirsh
J. Sustained antithrombotic activity of hirudin after its plasma
clearance: comparison with heparin. Blood. 1992;80:960-965.
9. Weitz JI, Hudoba M, Massel D, Maraganore J, Hirsh J. Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors. J Clin Invest. 1990;86:385-391.
10.
van den Bos AA, Deckers JW, Heyndrickx GR, Laarman GJ,
Suryapranata H, Zijlstra F, Close P, Rijnierse JJMM, Büller HR,
Serruys PW. Safety and efficacy of recombinant hirudin (CGP
39393) versus heparin in patients with stable angina undergoing
coronary angioplasty. Circulation. 1993;88:2058-2066.
11.
Heras MH, Chesebro JH, Penny WJ, Kent RB, Badimon L,
Fuster V. Effects of thrombin inhibition on the development of
acute platelet-thrombus deposition during angioplasty in pigs:
heparin versus recombinant hirudin, a specific thrombin
inhibitor. Circulation. 1989;79:657-665.
12.
Waxman L, Smith DE, Arcuri KE, Vlasuk GP. Tick
anticoagulant peptide (TAP) is a novel inhibitor of blood
coagulation factor Xa. Science. 1990;248:593-596.
13.
Neeper MP, Waxman L, Smith DE, Schulman CA, Sardana M,
Ellis RW, Schaffer LW, Siegl PKS, Vlasuk GP. Characterisation of
recombinant tick anticoagulant peptide: a highly selective
inhibitor of blood coagulation factor Xa.
J Biol Chem. 1990;265:17746-17752.
14. Maraganore JM, Bourdon P, Jablonski J, Ramachandran KL, Fenton JW. Design and characterisation of Hirulogs: a novel class of bivalent peptide inhibitors of thrombin. Biochemistry. 1990;29:7095-7101. [Medline] [Order article via Infotrieve]
15.
Skrzypczak-Jankun E, Carperos VE, Ravichandran KG,
Tulinsky A. Structure of the hirugen and Hirulog-1 complexes of
-thrombin. J Mol Biol. 1991;221:1379-1393. [Medline]
[Order article via Infotrieve]
16. Fox I, Dawson A, Loynds P, Eisner J, Findlen K, Levin E, Hanson D, Mant T, Wagner J, Maraganore J. Anticoagulant activity of Hirulog, a direct thrombin inhibitor, in humans. Thromb Haemost. 1993;69:157-163.[Medline] [Order article via Infotrieve]
17. Cannon CP, Maraganore JM, Loscalzo J, McAlister A, Eddings K, George D, Selwyn AP, Adelman B, Fox I, Braunwald E, Ganz P. Anticoagulant effects of Hirulog, a novel thrombin inhibitor, in patients with coronary artery disease. Am J Cardiol. 1993;71:778-782. [Medline] [Order article via Infotrieve]
18. Yao SK, McNatt J, Anderson HJ, Eidt J, Cui KX, Golino, Glas-Greenwalt P, Maraganore J, Buja LM, Willerson JT. Thrombin inhibition enhances tissue-type plasminogen activator-induced thrombolysis and delays reocclusion. Am J Physiol. 1992;262:374-379.
19. Klement P, Borm A, Hirsh J, Maraganore J, Wilson G, Weitz J. The effect of thrombin inhibitors on tissue plasminogen activator-induced thrombolysis in a rat model. Thromb Haemost. 1992;68:64-68. [Medline] [Order article via Infotrieve]
20.
Schaffer LW, Davidson JT, Vlasuk GP, Siegl PKS.
Antithrombotic efficacy of recombinant tick anticoagulant peptide: a
potent inhibitor of coagulation factor Xa in a primate
model of arterial thrombosis.
Circulation. 1991;84:1741-1748.
21.
Sitko GR, Ramjit DR, Stabilito II, Lehman D, Lynch JJ,
Vlasuk GP. Conjunctive enhancement of enzymatic
thrombolysis and prevention of thrombotic reocclusion
with selective factor Xa inhibitor, tick anticoagulant
peptide. Circulation. 1992;85:805-815.
22. Vlasuk GP, Ramjit D, Fujita T, Dunwiddie CT, Nutt EM, Smith DE, Shebuski RJ. Comparison of the in vivo anticoagulant properties of standard heparin and the highly selective factor Xa inhibitors antistasin and tick anticoagulant peptide (TAP) in a rabbit model of venous thrombosis. Thromb Haemost. 1991;65:257-262. [Medline] [Order article via Infotrieve]
23. Nurmohamed MT, Rosendaal FR, Büller HR, Dekker E, Hommes DW, Vanden Broucke JP, Briët E. Low molecular weight heparin versus standard heparin in general and orthopaedic surgery: a meta-analysis. Lancet. 1992;1:152-156.
24. Prandoni P, Lensing AWA, Büller HR, Carta M, Cogo A, Vigo M, Casara D, Ruol A, ten Cate JW. Comparison of subcutaneous low-molecular weight heparin with intravenous standard heparin in proximal deep-vein thrombosis. Lancet. 1992;1:441-445.
25.
Vlasuk GP, Vallar PL, Weinhouse MI, Bergum PW, Tran HS,
Weitz JI, Tulinsky A, Krishnnan R, Rote WE, Oldeschulte GL, Pearson
DA. A novel inhibitor of thrombin containing
multiple recognition sequences linked by a -keto amide
transition state mimetic. Circulation.
1994;90(suppl I):I-348. Abstract.
26. Laroche Y, Storme V, DeMeutter J, Messens J, Lauwereys M. High-level secretion and very efficint isotropic labeling of tick anticoagulant peptide (TAP) expressed in the methylotrophic yeast, Pischia pastoris. Biotechnology. 1994;12:1119-1124. [Medline] [Order article via Infotrieve]
27. Verheijen JH, Mullaart E, Chang GTG, Kluft C, Wijngaards G. A simple sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurements in plasma. Thromb Haemost. 1982;48:266-269. [Medline] [Order article via Infotrieve]
28. 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]
29. Van Hinsberg VWM, Sprengers ED, Kooistra T. Effect of thrombin on the production of plasminogen activators and PA inhibitor-1 by human foreskin microvascular endothelial cells. Thromb Haemost. 1987;57:148-153. [Medline] [Order article via Infotrieve]
30. Hanss M, Collen D. Secretion of tissue-type plasminogen activator and plasminogen activator inhibitor by cultured human endothelial cells: modulation by thrombin, endotoxin, and histamin. J Lab Clin Med. 1987;109:97-104. [Medline] [Order article via Infotrieve]
31.
Francis CW, Marder VJ. Increased resistance to
plasmic degradation of fibrin with highly cross-linked
-polymer chains formed at high factor XIII
concentrations. Blood. 1988;71:1361-1365.
32.
Van Giezen JJJ, Minkema J, Bouma BN, Jansen
JWCM. Crosslinking of 2-antiplasmin to fibrin is a key factor
in regulation blood clot lysis: species difference. Blood
Coagul Fibrinol. In press.
33. Mosesson MW. Fibrin polymerisation and its regulatory role in haemostasis. J Lab Clin Med. 1990;116:8-17. [Medline] [Order article via Infotrieve]
34.
Sakata Y, Aoki N. Cross-linking of
2-plasmin inhibitor to fibrin by fibrin-stabilizing
factor. J Clin Invest. 1980;65:290-297.
35. Witting WI, Bourdon P, Brezniak DV, Maraganore JM, Fenton JW. Thrombin-specific inhibition by and slow cleavage of Hirulog-1. Biochem J. 1992;283:737-743.
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