© 1998 American Heart Association, Inc.
Original Contributions |
Antithrombotic Effects of Recombinant Human, Active Site–Blocked Factor VIIa in a Rabbit Model of Recurrent Arterial Thrombosis
From the Department of Internal Medicine (P.G., M.R., P.C., D.D'A., A.S., A.R., C.B., N.C., F.V., M.C., M.C.), Division of Cardiology, 2nd School of Medicine, University of Naples (Italy), and the Department of Vessel Wall Biology (M.E.), Novo Nordisk A/S, Gentofte, Denmark.
Correspondence to Paolo Golino, MD, PhD, Division of Cardiology, University of Naples "Federico II," via Sergio Pansini 5, 80131 Naples, Italy. E-mail golino{at}ds.cised.unina.it
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Abstract |
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Abstract—The extrinsic coagulation pathway is activated when circulating factor VII (FVII) gains access to tissue factor (TF) exposed as a consequence of vascular injury. Increasing evidence indicates that this TF-dependent activation of the coagulation plays an important role in the pathophysiology of intravascular thrombus formation. In the present study, we tested the effects of recombinant human, active site–blocked activated FVII (FVIIai) in a rabbit model of carotid artery thrombosis. Cyclic flow variations (CFVs), due to recurrent thrombus formation, were obtained in stenotic rabbit carotid arteries with endothelial injury. Carotid blood flow velocity was measured by a Doppler flow probe. After 30 minutes of CFVs, the animals received FVIIai (100 µg · kg-1 · min-1 intracarotid infusion for 10 minutes, n=9). If CFVs were abolished, animals were followed for 30 additional minutes, after which recombinant human activated FVII (FVIIa) was infused into the carotid artery (100 µg · kg-1 · min-1 for 10 minutes) to determine whether FVIIai could be displaced from TF by FVIIa, thus restoring CFVs. To establish the duration of action of FVIIai, an additional group of animals received FVIIai at the same dose as above, and after CFVs were inhibited, they were followed until CFVs were restored or for up to 6 hours. To determine whether CFVs could be restored by epinephrine after their abolition with FVIIai, increasing doses of epinephrine were administered to a third group of 6 animals. FVIIai abolished CFVs in 8 of 9 rabbits (P<.01). This effect was reversible, as FVIIa administration restored CFVs in all animals. Prothrombin times and activated partial thromboplastin times did not change significantly throughout the study. One single 10-minute infusion exerted complete antithrombotic effects for at least 6 hours, despite the fact that at this time point, plasma FVIIai levels were well below threshold concentrations. Epinephrine restored CFVs in 3 of 6 animals in which CFVs were inhibited by FVIIai. FVIIai exerts potent antithrombotic effects in this model; these effects were prolonged even after FVIIai was almost completely cleared from the circulation, probably as a result of the tight binding of FVIIai to TF. Thus, FVIIai might represent an antithrombotic substance of potential interest.
Key Words: thrombosis • tissue factor • factor VII • active site–blocked activated factor VII
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Introduction |
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There is general agreement that intracoronary thrombus formation at sites of complicated atherosclerotic plaques plays an important role in the conversion from chronic to acute coronary artery disease syndromes.1 This hypothesis is also supported by the observation that some chemical mediators released on platelet activation, such as thromboxane A2 and serotonin, are found at increased concentrations in the coronary sinus of patients with unstable angina.2 3 Furthermore, FPA plasma levels, an index of thrombin activity, have been found to be elevated in patients with unstable angina compared with patients with chronic, stable, effort angina.4
Recent evidence indicates that activation of the extrinsic coagulation pathway may play an important role in the pathophysiology of intravascular thrombus formation following arterial injury.5 The extrinsic coagulation pathway is initiated when TF, a 47-kD membrane-bound glycoprotein, is exposed to flowing blood as a consequence of vascular damage.6 TF complexes with FVII and FVIIa, permitting enzymatic activation of factors X and IX, the substrates for factor VIIa, ultimately leading to the generation of thrombin.6 Normally, endothelial cells, being in contact with circulating blood, do not express significant TF activity. However, TF is found across the arterial wall, with its activity increasing from the subendothelium to the adventitia.7 Significant TF activity has also been localized in human atherosclerotic plaques8 and, recently, in atherectomy specimens obtained from patients with unstable angina.9 In addition, we have recently shown that a monoclonal antibody against TF not only inhibits intravascular thrombus formation in a rabbit model of recurrent arterial thrombosis7 but also enhances thrombolysis by t-PA and prevents reocclusion following t-PA discontinuation.10 Taken together, these data support the hypothesis that TF exposure following arterial damage plays a role in the pathogenesis of acute ischemic coronary syndromes by initiating intravascular thrombus formation.
Recently, the availability of recombinant human FVIIa with the active site blocked has permitted further investigation on the role of TF exposure/activation of the extrinsic coagulation cascade in the pathophysiology of intravascular thrombus formation. Recombinant human FVIIai possesses the same affinity for TF as does native FVIIa but, having the active site blocked, is not capable of converting factors IX and X to their activated forms, thus inhibiting the activation of the coagulation cascade at an early step. The purpose of the present study was, therefore, to determine the antithrombotic effects of FVIIai in a rabbit model of carotid artery thrombosis. This model was designed to provide conditions for thrombosis that closely resemble the acute coronary syndromes seen in humans. The results obtained demonstrate that FVIIai exerts potent antithrombotic effects, without incurring potentially harmful systemic effects.
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Materials and Methods |
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Preparation of FVIIa and FVIIai
Recombinant human FVIIa was purified from culture media of a transfected baby hamster kidney cell line as previously described.11 The active site of recombinant FVIIa was blocked by the addition of 2-fold molar excess of FFRcmk. The solution was allowed to incubate for 1 hour at 4°C. Unreacted FFRcmk was separated from FVIIai by Q-Sepharose fast-flow ion-exchange chromatography. FVIIai was eluted with 10 mmol/L CaCl2, and the profile of the eluted peak was narrow and symmetric. The FVIIai solution was adjusted to a protein concentration of
2 mg/mL in 10 mmol/L glycylglycine, 150 mmol/L NaCl,
and 10 mmol/L CaCl2, pH 7.4, sterile-filtered, and
stored at -80°C. The residual FVIIa activity in the FVIIai solution
was <0.1% when measured in an FVIIa-specific amidolytic
assay.12
Experimental Preparation
This study was performed using a rabbit model of recurrent
carotid artery thrombosis as described in detail
elsewhere.13 14 Briefly, New Zealand White rabbits of
either sex were anesthetized with a mixture of ketamine
(35 mg/kg) and xylazine (5 mg/kg) administered intramuscularly.
Anesthesia was maintained during the course of the
experiment by an intravenous infusion of ketamine
sufficient to abolish the corneal reflex. Through a median incision of
the neck, the left or right common carotid artery was exposed and
carefully isolated from the surrounding tissue. Polyethylene catheters
were inserted into a jugular vein and a femoral artery for both drug
administration and blood pressure monitoring. A segment of the exposed
vessel was injured by gently squeezing the artery between a pair of
rubber-covered forceps. An external plastic constrictor was placed
around the damaged site. Carotid blood flow velocity was continuously
measured by a Doppler flow probe positioned proximal to the
constrictor. A small polyethylene catheter (25-gauge outer diameter)
was placed into the carotid artery for the local infusion of drugs.
After instrumentation, the animals developed cyclic fluctuations of
carotid blood flow (CFVs) characterized by gradual decreases of flow to
almost zero values followed by spontaneous or induced restorations of
flow. Previous studies have shown that CFVs are due to recurrent cycles
of thrombus formation and subsequent dislodgment.13 14 15
Study Protocol
The present study comprises three different arms. In the
first arm, the antithrombotic effects of FVIIai, its effects on
systemic blood coagulation parameters (PTs, aPTTs, and FPA
plasma levels), and platelet aggregation were determined. In
addition, in these animals the ability of recombinant human FVIIa to
revert the antithrombotic effects of FVIIai was also tested. The second
arm of the study was included to determine the pharmacokinetics of
FVIIai and to correlate its plasma levels with the antithrombotic
effects. Finally, the third arm of the study was conducted to determine
whether the antithrombotic effects of inhibition of the extrinsic
coagulation pathway can be overridden by stimulating other activating
pathways, namely, epinephrine-induced platelet
activation.
CFV frequency (cycles per hour) and severity (carotid blood flow at its nadir, as a percentage of baseline), heart rate, and arterial blood pressure were continuously measured throughout the experiment. CFVs were monitored for 30 minutes, after which rabbits were assigned to the following groups.
Group 1: Antithrombotic Effects of FVIIai
The experimental protocol followed in this arm of the study is
summarized in Fig 1A
. A first group of 9
animals received an intracarotid infusion of FVIIai at a dose of 100
µg · kg-1 · min-1 for 10
minutes. A pilot study evidenced that an intracarotid infusion of
FVIIai was necessary in order to achieve local concentrations
sufficiently high to displace the tight binding of native FVII/VIIa
from TF. If CFVs were inhibited at the end of the infusion, the animals
were followed for additional 30 minutes to ensure that the
antithrombotic effects were persistent. Thereafter, to show that the
binding of FVIIai to TF was competitive, all animals that showed
inhibition of CFVs received an intracarotid infusion of recombinant
human FVIIa (100 µg · kg-1 ·
min-1 for 10 minutes). If CFVs returned during FVIIa
infusion, they were monitored for an additional 30 minutes. Blood
samples for the measurements of PT and aPTT were obtained at the end of
the 30-minute CFV period and after FVIIai and FVIIa administration. In
addition, blood samples were also obtained before and after FVIIai
administration to measure ex vivo platelet aggregation (see below).
Finally, blood samples for the measurements of FPA plasma levels were
obtained before induction of CFVs (baseline), during CFVs, and after
FVIIai and FVIIa administration.
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Groups 2a and 2b: FVIIai Pharmacokinetic Studies
To determine the pharmacokinetics of FVIIai, as well as the
duration of its antithrombotic effects, additional rabbits were
included in the study. The protocol followed in this arm of the study
is summarized in Fig 1B
. CFVs were initiated as described above, and
they were monitored for 30 minutes. Then, FVIIai was administered as an
intracarotid infusion of 100 µg · kg-1 ·
min-1, as previously described. The infusion was
maintained until CFVs were inhibited (at which time the infusion was
immediately discontinued) or for a maximum of 10 minutes. During FVIIai
infusion, blood samples were obtained every 2 minutes to measure plasma
FVIIai levels. When CFVs were inhibited, these animals were followed
for 3 hours (group 2a, n=8) or for 6 hours (group 2b, n=6). Blood
samples to measure FVIIai plasma levels were obtained every 30 minutes
for the first 3 hours and every hour thereafter.
Group 3: Epinephrine-Induced Restoration of CFVs
To determine whether FVIIai can also protect against
epinephrine-induced restoration of CFVs, 8 additional rabbits
were included in the study. The protocol followed in this arm of the
study is summarized in Fig 1C. CFVs were initiated as described above,
and they were monitored for 30 minutes. Then, FVIIai was administered
as an intracarotid infusion of 100 µg · kg-1
· min-1 for 10 minutes, as previously described. When
CFVs were inhibited, these animals were followed for 30 minutes, after
which an intracarotid infusion of epinephrine was started. This
route of administration was chosen to obtain a high degree of
platelet stimulation in the absence of significant systemic
hemodynamic effects, which, by themselves, may alter
CFVs. Epinephrine was given at an initial dose of 0.01 µg/min
and progressively increased until CFVs were restored or until systemic
effects appeared, ie, when blood pressure started to increase. Once
CFVs were restored, they were observed for 30 minutes.
Ex Vivo Platelet Aggregation
To determine whether FVIIai affected platelet function per
se, platelet aggregation was tested ex vivo both before and after
FVIIai administration. Peripheral venous blood (14 mL) was
collected in a syringe containing 1.5 mL of 3.8% sodium citrate, and
PRP was obtained by centrifugation of blood at
120g for 20 minutes at room temperature. PRP was removed,
and PPP was obtained by further centrifugation at
1000g for 5 minutes. Platelet aggregation was measured
turbidimetrically on a Chronolog aggregometer and recorded on a
linear recorder. The aggregometer was calibrated using PRP and PPP,
and the test was performed on 250 µL PRP in a siliconized cuvette
with continuous stirring. The platelet count in PRP was adjusted to
3x105/µL by dilution with PPP as needed. Aggregation was
induced in PRP in response to various concentrations of ADP and rabbit
thromboplastin.
Coagulation Studies
To determine the effect of FVIIai administration on PT and aPTT,
blood was collected in sodium citrate (3.8%) and centrifuged
at 2000g for 10 minutes at 4°C to separate the plasma. PT
and aPTT were measured in duplicate within 2 hours after blood
collection.
To determine whether inhibition of the extrinsic coagulation pathway by FVIIai actually results in inhibition of thrombin formation in vivo, plasma FPA levels, an index of thrombin activity, were measured by a radioimmunoassay method using a commercially available kit (Byk-Sangtec). Blood samples were collected in prechilled tubes containing an anticoagulant supplied by the manufacturer, immediately placed on ice, and centrifuged at 3000g for 10 minutes at 4°C. The plasma was removed and stored at -70°C until the assay was performed. FPA levels were measured in triplicate according to the manufacturer's instructions.
Measurements of Plasma FVIIai Levels
The concentration of FVIIai in rabbit plasma was determined in
triplicate by an ELISA method using the FVII EIA kit from Dako Corp
according to the manufacturer instructions.
Statistical Analysis
All values are expressed as mean±SEM. The rate of inhibition of
CFVs by FVIIai and the rate of CFV restoration by FVIIa were evaluated
by Fisher's exact test. One-way and two-way ANOVAs with a design for
repeated measurements were used to compare ex vivo platelet
aggregation data, hemodynamic variables, FPA plasma
concentrations, and PTs and aPTTs. When applicable, differences between
groups were tested by a Student's t test for paired or
unpaired samples with Bonferroni's correction. A value of
P<.05 defined significant differences between
populations.
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Results |
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Antithrombotic Effects of FVIIai
After arterial injury and placement of the constrictor, CFVs developed in all group 1 rabbits (n=9). CFV frequency and severity (expressed as carotid blood flow velocity) averaged 17±2 cycles per hour and 13±4% of baseline values, respectively. After intracarotid infusion of FVIIai (100 µg · kg-1 · min-1 for 10 minutes; total dose, 1 mg/kg), CFVs were completely inhibited in 8 of the initial 9 animals (P<.01), as CFV frequency decreased to 0 cycles per hour and carotid blood flow velocity increased to 89±11% of baseline values (Fig 2
). Thirty minutes after CFVs
were inhibited, intracarotid infusion of FVIIa restored CFVs in all
animals (CFV frequency, 19±1 cycles per hour; carotid blood flow
velocity, 5±1% of baseline values; Fig 2), thus indicating that the
binding of FVIIai to TF was reversible, as it could be displaced from
TF by FVIIa. No significant changes in arterial blood
pressure and heart rate were observed throughout the study (Table 1).
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Ex Vivo Platelet Aggregation and Coagulation Studies
Ex vivo platelet aggregation in response to ADP and
thromboplastin was tested in blood samples obtained before (baseline)
and after administration of FVIIai. No significant differences were
observed after FVIIai administration in platelet aggregation in
response to ADP and thromboplastin (Table 2). Thus, FVIIai at the doses used in the
present study did not affect platelet function per se.
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To study possible systemic effects of FVIIai, which may increase the risk of bleeding, PTs and aPTTs were measured in blood samples collected at 30 minutes of CFVs and after FVIIai and FVIIa administration. At the end of the 30-minute CFV period, PTs and aPTTs averaged 8.2±0.6 and 25±3 seconds, respectively. A slight increase in PTs to 10.1±0.6 seconds was observed after FVIIai administration. This increase, however, did not reach statistical significance (P=.09 by ANOVA and Student's t test with Bonferroni's correction). aPTT did not change significantly after FVIIai administration. FVIIa administration resulted in a significant shortening in both PTs and aPTTs with respect only to the values obtained after FVIIai administration.
Plasma FPA levels averaged 6±1 ng/mL before inducing CFVs and
increased significantly to 25±6 ng/mL during CFVs (P<.01
versus baseline, Fig 3), indicating that
induction of recurrent thrombosis was associated with an increase in
thrombin activity. In contrast, a decrease in plasma FPA levels to
values similar to those obtained at baseline was observed when CFVs
were inhibited after FVIIai administration (P=NS versus
baseline, Fig 3). This finding provides direct evidence that thrombin,
generated through activation of the extrinsic coagulation pathway, is
an important mediator of CFVs in this model. After administration of
FVIIa, with CFVs restored, plasma FPA levels increased again to values
similar to those observed during the initial CFVs (Fig 3).
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FVIIai Pharmacokinetic Studies
Plasma FVIIai levels increased progressively during FVIIai
infusion from undetectable levels before starting the infusion to
28.8±3.71 µg/mL at the end of the 10-minute infusion. A slow
decrease in plasma FVIIai concentrations was observed over the 6-hour
observation period. The apparent plasma half-life of FVIIai was 45
minutes (Fig 4). Fig 4 also shows the
correlation between the antithrombotic effects of FVIIai and its plasma
concentrations measured during the experiment. After 8 minutes of
FVIIai infusion, CFVs were inhibited in only 1 of 8 animals in group 2a
and none of group 2b, whereas at the end of the 10-minute infusion,
CFVs were inhibited in a total of 11 of 14 animals (6 of 8 in group 2a,
and 5 of 6 in group 2b). For the 6 rabbits in group 2a in which CFVs
were inhibited, the experiment was terminated after 3 hours from the
end of the infusion. At this time point, CFVs were still inhibited in
all but 1 animal. In this animal, CFVs were spontaneously restored 167
minutes after the end of the infusion. Interestingly, all 5 animals in
group 2b in which FVIIai successfully inhibited CFVs were still
inhibited at 6 hours, despite the fact that at this time point FVIIai
plasma levels were almost undetectable and largely below threshold
concentrations (Fig 4).
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Epinephrine-Induced Restoration of CFVs
Eight animals were included in this arm of the study. Two animals
did not respond to FVIIai administration; therefore, the experiment was
terminated for these animals. Of the remaining 6 animals, 3 animals
experienced restored CFVs after administration of epinephrine
at a mean dose of 0.04±0.01 µg/min (Fig 5). In the other 3 animals,
epinephrine did not restore CFVs, despite the fact that a mean
dose of 0.9±0.1 µg/min was reached (Fig 5). This dose increased the
mean blood pressure by an average of 20% (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The main findings of the present study are that administration of recombinant human FVIIai has potent antithrombotic effects in this rabbit model of recurrent carotid artery thrombosis and that no significant changes in platelet aggregation in response to ADP and thromboplastin or changes in PTs and aPTTs were observed after VIIai administration. Furthermore, an interesting observation of the present study is that the antithrombotic effects of a single 10-minute infusion of FVIIai persisted for at least 6 hours, when the plasma FVIIai levels were largely below threshold concentrations. Taken together, these data outline the importance of TF exposure, with the consequent activation of the extrinsic coagulation pathway, in the pathophysiology of intravascular thrombus formation and indicate that FVIIai might represent a useful antithrombotic intervention.
The extrinsic coagulation pathway is activated when circulating FVII gains access to TF in the vessel wall as a consequence of endothelial damage. Soluble FVIIa has a low affinity for its substrates, factors X and IX.6 However, when TF binds to FVII/VIIa, the resulting complex is capable of activating both factor X and factor IX 1000-fold more efficiently than soluble FVII/VIIa.6 After formation of the complex TF/FVII, blood coagulation proceeds through the "common" pathway, ultimately leading to the generation of thrombin.6 Accumulating evidence indicates that TF-dependent activation of the coagulation cascade is involved in the formation of intravascular thrombi. Coronary thrombosis generally occurs at stenotic sites, and it is often precipitated by disruption of an atherosclerotic plaque.16 Since atherosclerotic plaques are rich in TF-synthesizing cells, like monocytes, foam cells, and mesenchyma-like cells, plaque rupture may result in exposure of significant amounts of TF to circulating blood.8
The importance of TF exposure in triggering intravascular thrombus formation has also been suggested in a previous study from our laboratory.7 In the same experimental model used in the present study, we have shown that TF is normally present across the arterial wall, with its activity increasing from the intima to the adventitia.7 We have also shown that exposure of TF following endothelial injury plays an important role in triggering thrombus formation in this model via activation of the extrinsic coagulation pathway7 and that blocking TF activity by AP-1, a monoclonal antibody against TF, resulted in a complete inhibition of intravascular thrombus formation.7 Furthermore, inhibition of TF-procoagulant activity by AP-1 also resulted in enhancement of the thrombolytic properties of t-PA and in prevention of reocclusion after thrombolysis in an experimental model similar to the one used in the present study.10 In addition, Annex et al9 have recently shown significant TF activity in atherectomy specimens obtained from patients with unstable angina, suggesting that in these patients unstable angina may be precipitated by the activation of the extrinsic coagulation pathway caused by exposure of TF in the subendothelial tissue.
In the present study, recombinant human FVIIai was used as an antithrombotic intervention. The ability of FVIIai to inhibit the procoagulant activity of the complex TF/FVIIa was measured in vitro in a previous study.12 It has been shown that the concentration of FVIIai needed to reduce the FVIIa-dependent factor X activation by 50% (IC50 value) was 0.045±0.012 nmol/L.12 In addition, binding studies carried out with J82 cells, a human bladder carcinoma cell line expressing TF, showed that the IC50 value for FVIIai under these circumstances was 1.1±0.2 nmol/L.12 Thus, the preparation of FVIIai used in the present study showed a potent anti-TF activity in vitro.
The antithrombotic effects of FVIIai were first described by Harker et al.17 These investigators have shown that FVIIai administration prevented thrombus formation in nonhuman primates at sites of carotid endarterectomy. These effects were obtained without significant changes in bleeding time.17 However, in the present study, several original observations can be found that differentiate it from Harker's study. Indeed, the antithrombotic effects of FVIIai observed in the present study were obtained without potentially harmful systemic effects: PTs, aPTTs, and ex vivo platelet aggregation in response to ADP and thromboplastin did not change significantly after FVIIai administration. Of note is the observation that the antithrombotic effects of FVIIai could be promptly reversed by administration of recombinant human FVIIa. This finding indicates that the binding of FVIIai to the exposed vascular TF is reversible and makes this substance particularly attractive for clinical use, as the effects of FVIIai could be rapidly reverted in case of bleeding complications.
Of even more interest is the observation that the antithrombotic
effects of a single 10-minute infusion of FVIIai were prolonged, as
they persisted for at least 6 hours after the infusion was
discontinued. At this time point, plasma FVIIai levels, which averaged
3.1 µg/mL, were largely below threshold concentrations. Indeed, Fig 5
shows that after 8 minutes of infusion, FVIIai plasma levels averaged
23.4 µg/mL. However, at this time point, only 1 animal out of 14
showed inhibition of CFVs. A possible explanation for this phenomenon
may reside in the high affinity of FVII for its cofactor, TF. TF is an
integral membrane single-chain glycoprotein that is
associated with phospholipids. It has an N-terminal extracellular
domain, a membrane-spanning region, and a cytoplasmic region
constituting the carboxyl-terminal end of the protein.18 19
Several studies using a series of site-directed mutants of recombinant
TF, in which alanine residues have replaced selected amino acids in the
TF sequence, have been used to identify a candidate region of TF for
binding FVII.20 21 It has also been shown that a
dansyl-Glu-Gly-Arg chloromethyl ketone–treated FVIIa (another type of
active site–blocked FVIIa, different from the one used in the
present study) possesses a very high affinity for TF
(Ki, 0.7x10-7
mol/L).22 Other experiments, using 125I-labeled
FVIIa, have shown that a 50-fold molar excess of unlabeled FVIIa over
an hour of incubation time was necessary to displace 80% of the
125I-FVIIa previously bound to TF.23 In
addition, compared with native FVIIa, it has been recently shown that
inactivation of the active site of FVIIa with FFRcmk (the same method
used to obtain the FVIIai preparation used in the present study)
resulted in a 5-fold higher affinity for TF and a 2-fold slower
dissociation rate from TF.12 Thus, it can be hypothesized
that FVIIai, once bound to TF, dissociates very slowly from its
cofactor, such that significant antithrombotic effects can be still
achieved even when FVIIai is almost completely cleared from the
circulation. We did not measure FPA plasma levels in group 2b animals
at 6 hours of CFV inhibition. Thus, we cannot directly demonstrate that
the long-lasting antithrombotic effects of FVIIai are indeed due to a
prolonged inhibition of the extrinsic coagulation pathway. However, the
observation recently reported12 that FVIIai has a slower
dissociation rate from its ligand, TF, than does native FVIIa, leading
to a prolonged binding of FVIIai to TF, seems to support our
hypothesis. Nevertheless, lack of FPA measurements in this group of
rabbits represents a possible limitation of the present
study.
An alternative explanation for the prolonged antithrombotic effects of FVIIai may be related to the mechanisms regulating the cell surface TF/FVIIa proteolytic activity. A recent work by Sevinsky et al24 has evidenced that TF procoagulant activity is downregulated in the cells expressing TF by a translocation of the complex TF/FVIIa into noncoated plasmalemma vesicles. Interestingly, this translocation of TF is mediated by cell-associated TFPI, the natural inhibitor of the extrinsic coagulation cascade,25 indicating that formation of the quaternary complex TF/FVIIa/factor Xa/TFPI is necessary for the transport of TF in the cytoplasm.24 Therefore, it can be speculated that the prolonged antithrombotic effects of FVIIai observed in the present study might be explained by an increased translocation of TF from the cell surface to the cytoplasm. However, this possibility seems unlikely, considering the strict requirement for the binding of TFPI to the TF/FVIIa complex. In fact, FVIIai, having the active site blocked, cannot bind TFPI; this should result in inhibition of translocation of the complex to the cytoplasm.
The pathophysiological mechanisms responsible for the formation of intravascular thrombi continue to be investigated. It has been shown that thrombin plays a central role in this phenomenon, as it not only represents the key enzyme of the coagulation cascade but also is a powerful platelet agonist. In addition to heparin, new direct thrombin inhibitors have been recently identified, including hirudin and hirulog.26 These new thrombin inhibitors have the advantage over heparin in that they are antithrombin III independent, inactivate clot-bound thrombin, and prevent thrombin-induced platelet aggregation.26 However, a potential limitation of these agents is that new formation of thrombin is not affected.26 This may cause persistence of thrombin activity despite the presence of an inhibitor.26 27 In addition, these compounds may carry an increased risk of bleeding when administered in conjunction with thrombolytic therapy, which represents the most disturbing adverse effect.27
In this regard, a potential advantage of FVIIai is that it inhibits an early step of the extrinsic coagulation pathway, involving binding of native FVII/VIIa to TF, which ultimately results in inhibition of new thrombin formation, interrupting the positive feedback loop that autoamplifies thrombin generation. In the present study, evidence of inhibition of thrombin formation by FVIIai is provided by the measurements of FPA levels. FPA is cleaved from fibrinogen by the action of thrombin and thus represents an index of thrombin activity. FPA markedly increased in rabbits during the development of CFVs, indicating the presence of high thrombin activity during recurrent thrombus formation in this model. In contrast, FPA levels decreased significantly after FVIIai administration to baseline values, demonstrating that FVIIai, under the experimental conditions of the present study, interrupts the formation of the thrombus and prevents activation of the extrinsic coagulation pathway, ultimately leading to a reduction of thrombin formation. Inhibition of the extrinsic coagulation pathway by FVIIai also offers an advantage over blocking later steps in the coagulation pathway in that this substance binds to TF only where arterial damage is present. As a consequence, FVIIai, at the doses used in the present study, did not exert significant effects on blood coagulation and platelet aggregation. This should ultimately translate into a lower risk of bleeding compared with other antithrombotic interventions.
Conclusions
In the present study, we have demonstrated that administration
of human recombinant FVIIai exerts potent antithrombotic effects in
this rabbit model of recurrent carotid artery thrombosis. Furthermore,
the antithrombotic effects of FVIIai were prolonged and persisted after
the plasma levels of FVIIai decreased almost to baseline values. Thus,
a significant antithrombotic effect can be obtained with FVIIai without
significant changes in coagulation parameters or
platelet function. Further studies are warranted to elucidate the
potential clinical applications of FVIIai as an antithrombotic
agent.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by grant 91.00122.PF41 from the Consiglio Nazionale delle Ricerche (Progetto Finalizzato Prevenzione e Controllo dei Fattori di Malattia), Italy, and by a Research Grant from Novo Nordisk A/S, Gentofte, Denmark.
Received July 16, 1997; accepted October 9, 1997.
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