(Circulation. 1996;93:1913-1918.)
© 1996 American Heart Association, Inc.
Articles |
Monoclonal Antibody Against Tissue Factor Shortens Tissue Plasminogen Activator Lysis Time and Prevents Reocclusion in a Rabbit Model of Carotid Artery Thrombosis
Presented in part at the 66th Scientific Sessions of the American Heart Association, Atlanta, Ga, November 8-11, 1993, and previously published in abstract form (Circulation. 1993;88[pt 2]:I-615).
From the Department of Internal Medicine, Division of Cardiology, 2nd School of Medicine, University of Naples, Italy, and the Department of Internal Medicine, Division of Cardiology, Yale University, New Haven, Conn (M.D.E., A.B.P.).
Correspondence to Paolo Golino, MD, PhD, Division of Cardiology, 2nd School of Medicine, University of Naples, Via Sergio Pansini 5, 80131 Naples, Italy.
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Abstract |
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Background Tissue factor (TF)–dependent activation of the coagulation is important in the pathophysiology of intravascular thrombus formation. We tested the effects of a monoclonal antibody against TF (AP-1) on lysis time induced by tissue-type plasminogen activator (TPA) and on reocclusion rate in a rabbit model of carotid artery thrombosis.
Methods and Results Intravascular thrombosis was obtained by placing an external constrictor around carotid arteries with endothelial injury. Carotid blood flow velocity was measured continuously with a Doppler flow probe. Thirty minutes after thrombus formation, the rabbits received either AP-1 (0.15 mg/kg IV, n=8) or placebo (n=8). All rabbits also received TPA (80 µg/kg bolus plus 8 µg·kg-1·min-1 infusion for up to 90 minutes or until reperfusion was achieved) and heparin (200 U/kg IV as a bolus). At reperfusion, TPA was discontinued, and the rabbits were followed for an additional 90 minutes. AP-1 shortened lysis time from 44±8 minutes (mean±SEM) in control rabbits to 26±7 minutes in AP-1–treated rabbits (P<.01). Reocclusion occurred in all control rabbits in 10±3 minutes, whereas it occurred in only two of eight AP-1–treated rabbits in 72 and 55 minutes (P<.01). No changes in prothrombin time and ex vivo platelet aggregation in response to various agonists were observed after AP-1 administration, indicating the absence of systemic effects by this antibody.
Conclusions TF exposure and activation of the extrinsic coagulation pathway play an important role in prolonging lysis time and mediating reocclusion after thrombolysis in this model. AP-1, a monoclonal antibody against TF, might be suitable as adjunctive therapy to TPA.
Key Words: thrombolysis • tissue factor • coagulation
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Introduction |
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Coronary thrombolysis is now considered the treatment of choice for selected patients with acute myocardial infarction.1 2 3 In particular, several clinical trials have demonstrated that TPA administered intravenously is a safe and effective agent capable of inducing recanalization of the occluded vessel in most patients.1 4 5 6 7 However, some factors, including the time necessary to achieve effective reperfusion and the occurrence of reocclusion of the infarct-related artery, may significantly blunt the beneficial effects of early thrombolysis. For instance, experimental studies have shown that the extent of myocardial necrosis and thus the impairment of left ventricular function are directly related to the duration of the ischemic period.8 9 10 Furthermore, reocclusion of the infarct-related artery is a relatively frequent phenomenon that occurs after the discontinuation of TPA.1 4 5 6 7 Thus, efforts have been directed toward the identification of adjunctive therapies that may enhance the beneficial effects of thrombolysis.1 3 11 12 13 14 15
TF is a 47-kD membrane-bound glycoprotein essential for activation of the extrinsic coagulation pathway that is constitutively expressed by cells that are not in contact with blood. TF complexes with factors VII and VIIa, permitting enzymatic activation of factors X and IX, the substrates for factor VIIa,16 and ultimately leading to the generation of thrombin.16 Endothelial cells, being in contact with circulating blood, usually do not express significant TF activity. However, TF is found across the arterial wall, its activity increasing from the subendothelium to the adventitia.17 Significant TF activity also has been localized in human atherosclerotic plaques18 and recently in atherectomy specimens obtained from patients with unstable angina.19 In addition, antibodies against TF inhibited in vivo thrombosis.17 Taken together, these data support the hypothesis that TF exposure after arterial damage plays a role in the pathogenesis of acute ischemic coronary syndromes by initiating intravascular thrombus formation.
Thrombogenic factors that primarily determine the occlusion of a coronary artery, including residual stenosis, endothelial damage, and TF exposure, may persist and even become more pronounced during and after thrombolysis, leading to continuous growth of the thrombus and reocclusion of the reperfused artery.20 In particular, generation of thrombin (possibly triggered by TF exposure) seems to have a central role in these phenomena.21 Therefore, the purpose of the present study was to determine the role of TF-mediated activation of the coagulation cascade in prolonging clot lysis time by TPA and affecting reocclusion rate. To achieve these goals, AP-1, a monoclonal antibody against rabbit TF,17 was used in a rabbit model of carotid artery thrombosis.
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Methods |
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Experimental Preparation
This study was performed with a rabbit model of carotid artery thrombosis induced by producing an arterial stenosis superimposed on endothelial damage.22 23 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 that was 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 gentle squeezing of the artery between a pair of rubber-covered forceps. An external plastic constrictor was placed around the damaged site. CBF velocity was measured continuously by a Doppler flow probe positioned proximal to the constrictor. After instrumentation, the rabbits developed cyclic fluctuations of CBF (CFVs) characterized by gradual decreases of flow to almost zero values followed by spontaneous restorations of flow. Previous studies have shown that CFVs are due to recurrent cycles of thrombus formation and subsequent dislodgment.22 23 This pattern of CBF persisted for several minutes until stable occlusion of the vessel (Fig 1
).
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Study Protocol
After a 30-minute period during which CBF was not detectable
because of thrombotic occlusion of the artery, rabbits were assigned to
one of two groups. Group 1 rabbits (n=8), serving as controls, received
a bolus of heparin (200 U/kg) and a bolus of 80 µg/kg human
recombinant TPA (Actilyse, Boehringer Ingelheim) followed by an
infusion of 8
µg·kg-1·min-1
for up to 90 minutes or until reperfusion was achieved. Group 2 rabbits
(n=8) received, immediately before administration of heparin and TPA, a
bolus of AP-1 (0.15 mg/kg IV), a monoclonal antibody against rabbit
TF.17 This dose of AP-1 was chosen because, in a previous
study from our laboratory, it resulted in effective inhibition of
intravascular thrombus formation in about 70% of the
rabbits.17 Effective thrombolysis was
defined as restoration of CBF to 70% or more of the baseline value. At
this time, TPA infusion was discontinued. Reocclusion was defined as
reoccurrence of the failure to detect flow after initial successful
thrombolysis.
The time necessary for adequate thrombolysis was measured. Hemodynamics (arterial blood pressure and heart rate) and CBF velocity were monitored continuously until reocclusion (in which case the observation period was extended for 30 minutes to ensure that thrombus was persistent) or up to 90 minutes if thrombolysis was not achieved.
TF Antibody Preparation
The procedures for AP-1 production, purification,
and characterization were described elsewhere.17 Briefly,
mice were immunized with several injections of purified rabbit brain
TF. After immunization period, rabbits were killed, and cells harvested
from spleen were fused with AG8 myeloma cells. Fused cells were grown
in selective medium to generate hybridoma lines. Culture supernatants
were screened for monoclonal antibodies against rabbit TF by ELISA
assay. The most positive clone selected by secondary ELISA assay was
AP-1. This hybridoma, after further expansion and cloning, was injected
intraperitoneally in mice to obtain large amounts
of TF monoclonal antibody from ascites fluid. The
inhibitory activity of AP-1 on TF-dependent coagulation was
tested in vitro in a two-stage coagulation assay with pure rabbit
TF reconstituted with phospholipids and increasing concentrations of
the antibody. AP-1 inhibited 50% of TF-dependent coagulation at a
concentration of 8 ng/mL.17
Ex Vivo Platelet Aggregation
To determine whether AP-1 affected platelet function
per se, platelet response to various agonists was tested ex vivo
both before and after AP-1 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 turbidometrically on a
Chronolog aggregometer and recorded on a linear recorder. The
aggregometer was calibrated by use of 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 per 1 µL by dilution with PPP as needed.
Aggregation was induced in PRP in response to collagen, ADP, the
thromboxane analogue U46619, and arachidonate
at various concentrations.
Coagulation Studies
Venous blood samples (4.5 mL) for measurements of fibrinogen and
plasminogen were collected in 0.5 mL (0.1 mol/L) sodium
citrate. Samples were placed on ice and centrifuged at
3000g for 10 minutes at 4°C. To inhibit activation of the
fibrinolytic system in vitro, the plasma was then transferred into
polystyrene tubes supplemented with aprotinin at a final concentration
of 200 kallikrein inhibitor units per 1 mL plasma. All
plasma samples were frozen at -20°C until assayed. To determine
the effect of AP-1 administration on PT, blood was collected in sodium
citrate (3.8%) and centrifuged at 2000g for 10
minutes at 4°C to separate the plasma. PT was measured with rabbit
brain thromboplastin on a Cascade TM 480 (Helena Laboratories). All
assays were performed in duplicate. Blood samples for the above
determinations were collected at the following time points: before
thrombus formation (baseline), at the moment of reperfusion, and at the
moment of reocclusion or at 90 minutes of reperfusion.
To determine whether inhibition of the extrinsic coagulation pathway by AP-1 actually results in inhibition of thrombin formation in vivo, plasma FPA levels, which are indexes of thrombin activity, were measured in eight additional rabbits. Carotid artery thrombosis was induced in these rabbits as previously described. Thirty minutes after thrombus formation, the rabbits received heparin and TPA at the same dose described above (n=4) or heparin, TPA, and AP-1 (0.15 mg/kg, n=4). Blood samples were obtained at baseline (ie, before thrombus formation), 30 minutes after thrombus formation, and at the moment of reperfusion. After collection, blood samples were immediately placed on ice and centrifuged at 3000g, and the plasma was stored at -70°C until assayed. FPA levels were measured in triplicate by a radioimmunoassay method with a commercially available kit (Byk-Sangtec) according to the manufacturer's instructions.
Statistical Analysis
All values are expressed as mean±SEM. Lysis times, reocclusion
times, PTs, and fibrinogen and plasminogen levels were
compared between groups by Student's t test for unpaired
observations. One-way and two-way ANOVAs for repeated
measurements were used to compare ex vivo platelet aggregation data
and hemodynamic variables with FPA plasma
concentrations, respectively. When applicable, differences between
groups were tested by 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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Induction of Carotid Thrombosis and Thrombolysis
After arterial injury and placement of the constrictor, CFVs occurred in all rabbits and lasted for several minutes until stable occlusion (Fig 1
). Successful
thrombolysis was achieved in five of eight group 1 rabbits
(control rabbits) and in eight of eight group 2 rabbits (AP-1–treated
rabbits). Rabbits in which reperfusion did not occur after 90 minutes
of TPA infusion were excluded from further statistical
analysis. The time from initiation of TPA to reperfusion was
44±8 minutes in group 1 (range, 34 to 55 minutes) and 26±7 minutes in
group 2 (range, 18 to 37 minutes; P<.01; Fig 2). There was a significant reduction in the total dose
of TPA administered in group 2 compared with control rabbits
(P<.01; Fig 2).
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In all group 1 rabbits that successfully reperfused, CFVs recurred
almost immediately after thrombolysis and persisted for
several minutes until stable reocclusion occurred. Reocclusion time,
defined as the time elapsed between the onset of reperfusion and the
occurrence of a persistent reocclusion, was 10±3 minutes in the five
group 1 rabbits in which carotid arteries underwent reperfusion. In
group 2 rabbits, administration of AP-1 prevented CFVs and reocclusion
in six of eight rabbits. In these six rabbits, a stable and constant
CBF (averaging 93±7% of baseline values) was present at the end
of the experimental period, ie, 90 minutes after TPA discontinuation
(Fig 3). In the other two rabbits, reocclusion occurred
55 and 72 minutes after reperfusion (Fig 3).
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No significant changes were observed in arterial blood pressure and heart rate between the two groups over the course of the experiment (data not shown).
Ex Vivo Platelet Aggregation and Coagulation
Studies
Ex vivo platelet aggregation in response to several agonists
was tested in blood samples obtained from rabbits treated with AP-1
before thrombus formation (baseline) and at the moment of reperfusion.
No differences were observed after AP-1 administration in platelet
aggregation in response to ADP, collagen, arachidonate, or
U46619 (Fig 4). Thus, AP-1 at the doses used in this
study did not affect platelet function.
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To study possible systemic effects of AP-1, which may predispose to an
increased risk of bleeding, PTs were measured in control and
AP-1–treated rabbits from blood samples collected before thrombus
formation (baseline), at the moment of reperfusion, and at the moment
of reocclusion or at 90 minutes of reperfusion. At baseline, PTs
averaged 8.1±0.1 and 8.0±0 seconds in control and AP-1–treated
rabbits, respectively (P=NS). No differences in PTs were
observed between the two groups at reperfusion and at reocclusion or 90
minutes after reperfusion (Fig 5). Thus, the effects of
AP-1 on lysis time and reocclusion rates occurred without adverse
effects on the coagulation system.
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Plasminogen and fibrinogen plasma levels at baseline
averaged 107±5% and 102±6% of normal and 360±20 and 343±23 mg/dL
in control and AP-1–treated rabbits, respectively (P=NS).
As expected, both plasminogen and fibrinogen levels
decreased significantly at the moment of reperfusion, ie, after TPA
administration, and at reocclusion or 90 minutes after reperfusion in
both groups compared with baseline values (P<.01 for both
groups; Fig 5). AP-1–treated rabbits showed a tendency toward a
smaller reduction in plasminogen and fibrinogen levels, but
these differences did not reach statistical significance (Fig 5).
Plasma FPA levels were similar at baseline in the control and
AP-1–treated groups and averaged 6.3±1.6 and 5.3±0.9 ng/mL,
respectively (see the Table). Thirty minutes after
thrombus formation, plasma FPA levels increased markedly in the control
group to 28.9±4.1 ng/mL, whereas in the AP-1–treated rabbits, they
did not change significantly compared with baseline values
(P<.01 versus control rabbits; see the Table). A trend
toward a further increase in FPA levels was observed at the moment of
reperfusion in the control group, but this difference did not reach
statistical significance. In contrast, FPA levels remained stable in
the AP-1–treated group at the moment of reperfusion (Table).
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Discussion |
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The primary findings of this study are that administration of AP-1, a monoclonal antibody against rabbit TF, results in a significant shortening of lysis time by TPA and in a reduction in reocclusion rate after discontinuation of TPA in this rabbit model of carotid artery thrombosis. These data outline the importance of TF exposure, with the consequent activation of the extrinsic coagulation pathway, in causing continuous thrombin formation during thrombolysis and in mediating reocclusion of the vessel after thrombolytic therapy is discontinued.
The extrinsic coagulation pathway is activated when factors VII and VIIa gain access to TF in the vascular subendothelium as a consequence of endothelial damage. Soluble factors VII and VIIa have a low affinity for their substrates, factors X and IX.16 However, when TF binds to factors VII and VIIa, the resulting complex can activate both factors X and IX 1000-fold more efficiently than soluble factors VII and VIIa.16 After formation of the TF–factor VII complex, blood coagulation proceeds through the "common" pathway, ultimately leading to the generation of thrombin.16 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 often is precipitated by disruption of an atherosclerotic plaque.24 Because atherosclerotic plaques are rich in TF-synthesizing cells, like monocytes, foam cells, and mesenchymal-like cells, plaque rupture may result in exposure of significant amounts of TF to circulating blood.18
The importance of TF exposure in triggering intravascular thrombus formation has been suggested in a recent study from our laboratory.17 In an experimental model of recurrent arterial thrombosis similar to that used in the present study, we showed that TF exposure plays an important role in triggering thrombus formation through activation of the extrinsic coagulation pathway17 and that blocking TF activity by AP-1 resulted in complete inhibition of intravascular thrombus formation.17 In addition, Annex et al19 recently showed 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, administration of AP-1 significantly shortened lysis time by TPA compared with control rabbits. Experimental and clinical studies have demonstrated that intravascular thrombi are in a dynamic state for at least several hours after their formation, with new fibrin and platelets being added even during the administration of thrombolytics.25 26 The continuous growth of the thrombus may increase the total mass of the thrombus to be lysed, thus prolonging the time necessary to achieve effective reperfusion of the occluded vessel.20 In addition, reocclusion of the infarct-related artery after successful thrombolytic therapy represents another important factor that may blunt or even negate the benefits of achieving early reperfusion.
The pathophysiological mechanisms responsible for the occurrence of reocclusion of the infarct-related artery continue to be investigated. It has been shown that thrombin plays a central role in mediating both the continuous growth of the thrombus during thrombolytic therapy and the reocclusion of the infarct-related artery.27 In addition to heparin, new direct thrombin inhibitors, including hirudin and Hirulog, have been identified recently.28 These new thrombin inhibitors have the advantage over heparin in that they are independent of antithrombin III, inactivate clot-bound thrombin, and prevent thrombin-induced platelet aggregation.28 Several experimental and clinical studies showed that these agents might be beneficial when administered in conjunction with thrombolytics.29 30 31 32 33 However, a potential limitation of these agents is that new formation of thrombin is not affected.28 This may cause persistent thrombin activity despite the presence of an inhibitor.28 34 These compounds also may carry an increased risk of bleeding when administered in conjunction with thrombolytic therapy, which represents the most troublesome adverse effect.31 32 33
In this regard, a potential advantage of AP-1 is that it inhibits an early step of the extrinsic coagulation pathway, involving binding of factors VII and 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, direct evidence of inhibition of the extrinsic coagulation pathway by AP-1 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 increased markedly in control rabbits during intracarotid thrombus formation and at the moment of reperfusion, indicating the presence of significant thrombin activity despite the administration of heparin. This finding, which also was described in patients with acute myocardial infarction undergoing coronary thrombolysis,27 might reflect the inability of the heparin–antithrombin III complex to inhibit clot-bound thrombin. In contrast, FPA levels in AP-1–treated rabbits did not change significantly compared with baseline values, demonstrating that AP-1, under the experimental conditions of the present study, prevents activation of the extrinsic coagulation pathway, ultimately leading to a reduction in thrombin formation. Inhibition of the extrinsic coagulation pathway by AP-1 also offers an advantage over blocking later steps in the coagulation pathway in that this antibody binds to TF only where arterial damage is present. Consequently, AP-1, at the doses used in this 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.
Study Limitations
A potential limitation should be taken into account in
evaluations of the results of the present study. aPTTs were not
measured in the two groups of rabbits after heparin administration.
Thus, in theory it might be possible that different aPTTs were achieved
in the two groups, which might partially explain the differences
observed in terms of lysis times and reocclusion rates between
the two groups of rabbits. However, we believe that this possibility is
unlikely for the following reasons. First, the randomization schedule
should have minimized differences in terms of response to heparin.
Second, in another study from our laboratory currently in progress, we
have noticed that in all rabbits receiving heparin at the dose of 200
U/kg, aPTTs exceeded 360 seconds (unpublished observations).
Nevertheless, the possibility that aPTTs were different in the two
groups of rabbits after heparin administration cannot be excluded with
certainty and should be considered a potential limitation of the
present study.
Conclusions
In the present study, we have demonstrated that
administration of AP-1, a monoclonal antibody against rabbit TF,
results in a significant shortening of lysis time by TPA in this rabbit
model of carotid artery thrombosis. Furthermore, AP-1 administration
was associated with a significantly lower reocclusion rate when TPA
infusion was discontinued compared with control rabbits. These results
were obtained without affecting platelet function and systemic
coagulation. These data suggest the importance of TF exposure, with the
consequent activation of the extrinsic coagulation pathway, in
mediating continuous thrombin generation that leads to growth of the
thrombus during thrombolysis and causes reocclusion of
the vessel after thrombolytic therapy is discontinued.
Further studies are required to elucidate the potential clinical
applications of AP-1 as adjunctive therapy to
thrombolytic agents.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work 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 NIH grant RO1-HL-39467-01, NIH, Bethesda, Md.
Received September 7, 1995; revision received October 30, 1995; accepted November 15, 1995.
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