(Circulation. 1995;92:3032-3040.)
© 1995 American Heart Association, Inc.
Articles |
New Variant of Human Tissue Plasminogen Activator (TPA) With Enhanced Efficacy and Lower Incidence of Bleeding Compared With Recombinant Human TPA
From the Division of Cardiology (C.R.B., R.P.), Department of Internal Medicine, University of Texas Health Science Center, Houston, and the Department of Cardiovascular Research at Genentech, Inc, South San Francisco, Calif.
Correspondence to C.R. Benedict, MD, Department of Internal Medicine, Division of Cardiology, University of Texas Medical School, 6431 Fannin MSB 6.039, Houston, TX 77030.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background The thrombolytic properties of a new variant of tissue plasminogen activator (TPA) (T103N, N117Q, KHRR 296-299 AAAA, or TNK-TPA) with longer plasma half-life, greater fibrin specificity, and increased resistance to inhibition by plasminogen activator inhibitor (PAI-1) were investigated in a rabbit thrombosed carotid artery model.
Methods and Results After 60 minutes of arterial
occlusion, TPA (1.5, 3.0, 6.0, or 9.0 mg/kg as a front-loaded IV
infusion for 90 minutes; n=22) or TNK-TPA (0.38, 0.75, or 1.5 mg/kg as
IV bolus; n=16) was administered. Blood flow through the artery was
monitored for an additional 120 minutes. Bleeding was assessed by
weighing the amount of blood absorbed in a gauze pad placed in a
subcutaneous muscular incision. Recanalization
rates and duration of recanalization were dose
dependent. The doses that produced >80%
recanalization rates with the longest duration of
recanalization were 9.0 mg/kg for TPA and 1.5 mg/kg
for TNK-TPA. At these doses, time to reperfusion (mean±SEM) was
significantly faster (11±2 versus 23±7 minutes) and duration of
recanalization longer (77±9 versus 51±18 minutes)
for TNK-TPA compared with TPA (P<.025). Weights of the
residual thrombi of the TPA group were greater than those of the
TNK-TPA group (P=.004). Concentrations of fibrinogen,
plasminogen, and 
2-antiplasmin at 120
minutes were significantly higher for TNK-TPA–treated animals compared
with TPA-treated animals (P<.001). ANOVA of the blood loss
data determined that there were significant differences between
thrombolytic agents but not between doses. After correction
for saline controls, total blood loss for pooled doses of TPA and
TNK-TPA was 82±6 mg and 40±4 mg, respectively
(P<.01).
Conclusions From these data, we conclude that TNK-TPA, given as a bolus, produces faster and more complete recanalization of occluded arteries in a rabbit experimental model compared with TPA, without increasing systemic plasmin generation or peripheral bleeding. In addition, we observed that TNK-TPA, unlike TPA, did not potentiate collagen-induced aggregation of platelets obtained from human plasma. This lack of effect on platelet aggregation by TNK-TPA potentially could be associated with a decreased risk of reocclusion after successful thrombolysis.
Key Words: thrombolysis • plasminogen activators • occlusion • blood flow
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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For many patients with evolving acute myocardial infarction, thrombolytic therapy is the preferred method of treatment for improving survival and preserving left ventricular function.1 2 3 4 Other animal and patient studies5 6 7 8 9 10 11 12 support the observation that time required for brisk reperfusion of the occluded artery influences the infarct size and preservation of left ventricular function, which may alter survival rates after myocardial infarction. Furthermore, greater preservation of myocardial tissue may also prevent or delay the development of left ventricular dysfunction in these patients in the post–myocardial infarction period. In the GUSTO trial, an aggressive front-loaded protocol for TPA administration was used in an attempt to enhance the rapidity of thrombolysis.4 13 The success of this strategy suggests that variants of TPA with thrombolytic properties that decrease the time required for reperfusion of the occluded artery may offer additional clinical benefits.
Despite proven beneficial effects, thrombolytic therapy is often restricted because of concerns about hemorrhagic complications that can be seen with both TPA and streptokinase.4 The major concern has been the frequency of intracranial bleeding, which has occurred in 1.0% to 1.6% of treated patients.14 15 In the recent GUSTO trial,4 a 0.2% higher hemorrhagic stroke rate was noted in patients receiving TPA relative to those receiving streptokinase.
Recently, a novel variant of human TPA has been described16 that is more potent than native TPA in experimental animal studies and is cleared more slowly from plasma. This variant of TPA may be more amenable for bolus administration, provided that the initial bolus does not produce a "systemic lytic" state with consumption of circulating fibrinogen nor lead to an increase in the incidence of hemorrhagic complications. Using an animal model, we investigated the thrombolytic properties of this new variant of TPA (TNK-TPA) with longer plasma half-life, greater fibrin specificity, and improved resistance to inactivation by PAI-1 compared with native TPA. The data indicate that TNK-TPA given as a single IV bolus is more potent than native TPA in inducing a more rapid and complete thrombolysis of an occluded artery and is associated with less systemic fibrinolysis and lower incidence of bleeding.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Plasminogen Activators
Recombinant TPA (Activase), TNK-TPA, and excipients for TPA or TNK-TPA were provided as frozen, pyrogen-free solutions by Genentech. Aliquots were thawed as needed and any unused portions discarded after 24 hours. Concentrations of the TPA and TNK-TPA stock solutions were 1.0 and 7.7 mg/mL, respectively. Solutions for dosing were made by diluting the stock solutions with excipients such that the volume of solution delivered per kilogram of body weight did not vary with dose. Details of the TNK modification of TPA and the recombinant methods of production are provided elsewhere.16 Briefly, replacement of asparagine in position 117 with glutamine (N117Q) deletes the glycosylation site in kringle 1. The T103N substitution changes threonine in position 103 to asparagine and reintroduces the glycosylation site in kringle 1, but at a different locus. This variation substantially decreases the clearance rate of TNK-TPA from plasma.16 In addition, the amino acids lysine 296, histidine 297, arginine 298, and arginine 299 were each replaced with alanine (K296A, H296A, R298A, R299A) in the protease. This tetra-alanine substitution confers enhanced fibrin specificity and resistance to PAI-1 inhibition.17 The primary structure of TNK-TPA is depicted in Fig 1
. Chinese hamster ovary
cells were stably transfected with plasmid containing the TNK-TPA gene,
amplified in the presence of methotrexate, and grown in serum-free
media for 6 days. The conditioned culture media was concentrated and
diafiltered and lysine affinity chromatography was used
to purify the TNK-TPA.16 Quantitation of TNK-TPA after
purification was done by use of a dual monoclonal assay sensitive to
epitopes in kringle 2 and the protease. For purposes of comparison,
both TPA and TNK-TPA were administered on a weight basis as their
molecular masses were similar (
65 kD).
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Rabbit Model of Carotid Artery Thrombosis
Male New Zealand
White rabbits weighing 3.2 to 3.6 kg were
anesthetized with ketamine (15 mg/kg) and xylazine (15
mg/kg). The right femoral artery was cannulated for recording
of arterial blood pressure by use of a microtransducer
(Electromedics). The right marginal ear vein was cannulated for
administration of fluids and thrombolytic agents. The right
femoral vein was cannulated for drawing blood samples. Then the right
common carotid artery was exposed by a medial longitudinal incision in
the neck and gradual retraction of the facial planes (Fig 2). A
2.5-mm Doppler flow probe was placed on the
carotid artery without constricting the vessel. Proximal to the
Doppler flow probe, a 23-gauge stainless steel needle electrode was
inserted into the carotid artery with minimal trauma. Bleeding was
arrested by use of a piece of gel foam (Upjohn), and the needle was
stabilized by suturing a "surround collar" around the vessel,
which did not narrow the artery. After instrumentation, a 30-minute
control period was allowed. During this time, blood pressure, heart
rate, mean and phasic carotid artery blood flow, and ECG were
continuously monitored. After this period, thrombus formation was
initiated as previously
described.18 19 20 Anodal current
(150 µA) was applied to the needle electrode until a 50% increase in
flow velocity was recorded by the Doppler flow probe. This
corresponds to 50% decrease in cross-sectional area due to
thrombus formation in the lumen.18 19 20
The current was
stopped and the artery was allowed to occlude spontaneously. The
remaining luminal area occluded due to formation of a
fibrin-platelet thrombus at the site of thrombus
initiation.18 19 20 In some animals,
nitroglycerin was given via the carotid artery
(proximal to the site of occlusion) to confirm that the absence of flow
was not due to vasoconstriction of the artery.
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After total occlusion, 1
hour was allowed to elapse for maturation of
the thrombus. Then, rabbits were randomly allocated to one of eight
different groups: (1) excipient (buffer), n=6; (2) TPA 1.5 mg/kg,
n=5;
(3) TPA 3.0 mg/kg, n=5; (4) TPA 6.0 mg/kg, n=6; (5) TPA 9.0
mg/kg, n=6;
(6) TNK-TPA 0.38 mg/kg, n=5; (7) TNK-TPA 0.75 mg/kg, n=5; and
(8)
TNK-TPA 1.5 mg/kg, n=6. Blood samples were drawn at baseline to measure
fibrinogen, plasminogen, 2-antiplasmin,
TPA, and TNK-TPA levels. Template bleeding time and
incisional blood loss from an anterior abdominal surgical
incision (see below) were also assessed. TPA was administered using a
front-loaded protocol similar to that described by Neuhaus et
al,21 ie, the first 15% of the dose was administered over
30 seconds as a loading bolus, the next 35% as an IV infusion over 30
minutes, and the remainder over 60 minutes. TNK-TPA was given as a
single IV bolus over a few seconds. After administration of TPA or
TNK-TPA, the following parameters were monitored: (1) time
to reperfusion of the artery; (2) time to reocclusion; (3) total
duration of carotid artery recanalization; and (4)
template bleeding and blood loss from the abdominal wall incision site.
In addition, sequential blood samples were drawn at 2, 15, 30, 45, 60,
90, and 120 minutes after administration of TPA or TNK-TPA to measure
the fibrinogen, plasminogen, 2-antiplasmin,
TPA, and TNK-TPA antigen levels. At the end of 120 minutes, the
experiment was terminated and the weight of the residual thrombus in
the carotid artery was assessed.
Rabbit Bleeding Assays
We evaluated the incidence of bleeding
by two different methods.
Template bleeding times were measured by use of the Simplate device
(Organon Teknika). Uniform incisions were made on the ventral surface
of the rabbit's ear in such a way as to avoid the superficial veins.
Blood was blotted with filter paper every 30 seconds; care was taken to
avoid the incision. Bleeding time was the interval between the time of
incision until blood did not stain the paper. The incisional bleeding
assay was a modification of previously published
methods.19 20 A 4-cm long, 0.5-cm deep surgical
incision
was made in the anterior abdominal wall, which incised the first layer
of the anterior abdominal wall muscles. A preweighed gauze pad was
placed in the incision for 5 minutes, and the amount of blood absorbed
into the gauze was weighed. Both the bleeding assays were done at
baseline before administration of TPA or TNK-TPA and then 15, 60, and
120 minutes after administration. The increase in blood loss over the
baseline measurement was compared between TPA-treated and
TNK-TPA–treated rabbits.
Platelet Aggregation Studies
The effect of TPA and TNK-TPA on
platelet aggregation was
also examined because previous studies have suggested that platelet
aggregation at the site of thrombolysis contributes to
reocclusion of the vessel.22 23 Platelet aggregation
was done with human platelet–rich plasma prepared from 3.8%
citrated blood
(Na3C6H5O7 · 2H2O,
pH 5.5). Platelet-rich plasma was prepared by centrifuging at
100g for 15 minutes. After the upper two thirds of
platelet-rich plasma was removed, the remainder was
recentrifuged at 600g for 15 minutes to yield
platelet-poor plasma. Platelet count in
platelet-rich plasma was 260 000±25 000/µL. Samples were
kept tightly capped at room temperature until analysis. All
experiments were done within 120 minutes of sample collection. The
effect of varying concentrations of TPA and TNK-TPA on collagen- and
arachidonic acid–sensitized platelet
aggregation response was determined by use of a Biodata PAP-4
platelet aggregometer (BioData Corporation).
Blood Sample Collection
Blood samples were anticoagulated
with K2 EDTA (4.2
mmol/L final concentration).
D-phenylalanyl-L-prolyl-L-arginine
chloromethyl ketone (PPACK, Calbiochem) was added to the samples to a
final concentration of 2 µmol/L to prevent in vitro
plasminogen activation.24 Samples were
centrifuged at 4°C at 1110g for 15 minutes and the
plasma stored at -85°C before analysis.
Assays
Immunoreactive TPA was quantified by use of a dual
monoclonal
ELISA assay. Fibrinogen was measured by use of a semiautomated version
of the Clauss clotting time assay.25
Plasminogen levels were measured by use of a variation of a
method developed by Soria et al.26 After the plasma was
acidified and neutralized to inactivate plasmin
inhibitors, urokinase (Abbott) was used to activate
plasminogen in the presence of the chromogenic
substrate S2251 (Kabi). 2-Antiplasmin activity was
determined by use of the IL TEST chromogenic substrate
assay in an ACL 300+ centrifugal analyzer (Instrumentation
Laboratory). Results for fibrinogen, plasminogen, and
2-antiplasmin are expressed as a percentage of
pretreatment value.
Clearance Calculations
This study was not designed to provide
data for rigorous
pharmacokinetic analysis. However, sufficient blood samples
were drawn to allow estimation of the relative drug exposure of the
different treatment groups. The relation of TPA and TNK-TPA antigen
versus time for TPA and TNK-TPA were used to calculate the AUC from 2
to 120 minutes. Plasma clearance of TPA and TNK-TPA antigen was
calculated by use of the equation: Clearance
(mL · min-1 · kg-1)=Dose
(mg/kg)/AUC
(mg · mL-1 · min-1).
Statistical Analysis
Data are expressed as mean±SEM.
Comparisons between different
doses and groups were conducted by use of ANOVA and Dunnett's test for
multiple comparisons, if indicated.
Animal Welfare Approval
The study protocol was reviewed and
approved by the animal
welfare committee of the University of Texas Health Science
Center.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Thrombolytic Efficacy of TNK-TPA In Vivo
No spontaneous reperfusion was observed in excipient-treated rabbits even after administration of IV nitroglycerin. Recanalization rates, time to reperfusion, and duration of recanalization were dose dependent (Fig 3
). The
doses that produced >80%
recanalization rate with longest duration of
recanalization were 9.0 mg/kg for TPA and 1.5 mg/kg
for TNK-TPA. These were the highest doses used for TPA and TNK-TPA in
the present study. At these doses, time to reperfusion was
significantly faster for TNK-TPA (11±2 minutes) compared with TPA
(23±7 minutes; P<.02). At the next lower dose of TNK-TPA
(0.75 mg/kg), time to reperfusion was significantly faster (13±2
minutes) compared with that observed with the 9.0 mg/kg dose of TPA
(P<.03). Similarly, the duration of
recanalization was significantly greater for
TNK-TPA (77±9 minutes) compared with TPA (51±18 minutes;
P<.025). Maximal carotid artery reperfusion flow rates were
equivalent in animals treated with 9.0 mg/kg of TPA or 1.5 mg/kg of
TNK-TPA (data not shown). Residual thrombus weight in
excipient-treated control animals was 8.6±0.8 mg. Whereas thrombus
weight was lower in animals treated with 6.0 or 9.0 mg/kg of TPA (Fig
4), this difference was not significantly different from
controls. In contrast, with 0.75 or 1.5 mg/kg TNK-TPA, there was a
significant reduction in residual thrombus weight compared with
controls (P<.001).
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Fibrin Selectivity of TPA and TNK-TPA In Vivo
TPA or TNK-TPA
decreased plasma levels of rapidly clottable
fibrinogen, functional plasminogen, and
2-antiplasmin concentrations in a dose- and
time-dependent manner, with 50% or more of the loss occurring in
the first 30 minutes (data not shown). Generally, these plasma proteins
reached their lowest concentrations at the end of the experiment (2
hours after dosing). These data are summarized in Fig 5.
In TPA-treated animals, there was a significant reduction in
fibrinogen, plasminogen, and 2-antiplasmin
levels with increasing doses of TPA. At 9.0 mg/kg of TPA, no
circulating 2-antiplasmin activity was present in
plasma. In contrast, in the TNK-TPA–treated animals, a moderate
decrease in fibrinogen, plasminogen, or
2-antiplasmin levels was observed. At the highest dose
of TNK-TPA (1.5 mg/kg), fibrinogen, plasminogen, or
2-antiplasmin levels were significantly higher compared
with the corresponding values for TPA at either the 6.0 or 9.0 mg/kg
dose (P<.001).
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Clearance of TPA and TNK-TPA
Plasma clearance of TPA or
TNK-TPA administered IV to rabbits was
calculated using the concentration of TPA or TNK-TPA antigen measured
in plasma (Fig 6). We have previously demonstrated a
good correlation between circulating antigen levels and functional
activity for TPA and TNK-TPA.27 After single IV bolus
administration, TNK-TPA clearance approximated a monophasic elimination
profile. As expected from the more rapid clearance of TPA and the
front-loaded infusion used for dosing, plasma disposition of TPA
was more complex. At the two lower doses of TPA (1.5 and 3.0 mg/kg),
plasma concentrations decreased between 0 and 45 minutes, followed by a
period of constant TPA concentrations until the infusion was terminated
at 90 minutes. In contrast, at the two higher doses of TPA (6.0 and 9.0
mg/kg), plasma concentrations remained constant for the entire
90-minute duration of drug infusion. In addition, the rate of decrease
in plasma levels after termination of the infusion at 90 minutes was
reduced compared with that observed for the two lower doses, suggesting
saturation of clearance at the higher doses.
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The AUCs and clearance
values are summarized in the
Table. For TNK-TPA, clearance did not vary with dose. In
contrast, increases in the AUCs were disproportional for increasing TPA
doses. This resulted in decreasing clearance at higher doses. Clearance
of 1.5 mg/kg TNK-TPA (0.6±0.1
mL · min-1 · kg-1) was 14-fold
slower
than clearance of the same dose of TPA (8.6±2.7
mL · min-1 · kg-1). At
approximately
equally effective doses (1.5 mg/kg TNK-TPA and 9.0 mg/kg TPA), the
plasma exposure of the TNK-TPA animals (AUC of 2.9±0.7
mg · mL-1 · min-1) was nearly
50%
that of the TPA-treated group (AUC of 5.1±0.5
mg · mL-1 · min-1).
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Effect of TPA and TNK-TPA on Template Bleeding Times and Incisional
Blood Loss
Control template bleeding time was 2.0±0.2 minutes.
After
administration of aspirin (5 mg/kg IV), template bleeding time
increases to 3.2±0.4 minutes (P<.01) in this model. None
of the doses of TPA or TNK-TPA used in the present study caused a
significant increase in template bleeding time compared with
pretreatment value. The maximum prolongation in template bleeding time
was 2.2±0.3 minutes and 2.1±0.3 minutes for TPA-treated and
TNK-TPA–treated animals, respectively.
The effects of TPA and
TNK-TPA on incisional blood loss are summarized
in Fig 4
. Interestingly, the extent of blood loss of
TPA-treated or
TNK-TPA–treated animals was not dose dependent. Therefore, results for
all doses of each thrombolytic agent were pooled. Mean
total blood loss in the control group was 78±11 mg. With TPA, blood
loss from the incisional site (160±11 mg) was significantly greater
than in controls (P<.001). Administration of TNK-TPA also
increased blood loss (118±9 mg) when compared with the control group
(P<.03). However, blood loss with TNK-TPA was significantly
less than that observed with TPA (P<.01).
Platelet Aggregation in the Presence of TPA and
TNK-TPA
Previous studies have indicated that TPA can potentiate
agonist-stimulated platelet aggregation.22 23 The
effect of different doses of TPA and TNK-TPA on collagen-sensitized
human platelet aggregation is shown in Fig 7. The
platelet aggregation induced by TPA was a dose-dependent
phenomenon. At low (4 and 8 µmol/L) and intermediate (16 µmol/L)
doses of added TPA, collagen-sensitized platelet aggregation
was significantly potentiated. However, at the highest doses of added
TPA, there was minimal platelet aggregation. This is probably due
to the fact that at high doses, TPA generates plasmin, which degrades
fibrinogen, and consequently, stable platelet aggregates do not
form. In contrast, none of the doses of TNK-TPA examined potentiated
the collagen-sensitized platelet aggregation. A similar
difference in the interaction between TPA and TNK-TPA to
arachidonic acid–sensitized platelet
aggregation was also observed (data not shown).
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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The present study indicates that a single bolus IV administration of TNK-TPA will restore blood flow rapidly in a thrombosed carotid artery without increasing systemic plasmin generation relative to a front-loaded dosing regimen of TPA. TNK-TPA was significantly better than TPA with respect to the thrombolysis end points measured, ie, recanalization rates, time to reperfusion, duration of recanalization, and reduction in thrombus mass. It should be noted that these results were achieved at doses of TNK-TPA that were one sixth to one twelfth of the dose used for TPA. Another significant difference between TNK-TPA and TPA in the present study was the amount of blood loss from a deep surgical incision site, which was 35% greater with TPA compared with that observed with TNK-TPA.
The differences in the effect of TPA and TNK-TPA on human platelet aggregation were also noteworthy. Previous studies22 23 have suggested that continued platelet aggregation at the site of thrombolysis contributes to reocclusion of the recanalized vessel. TPA might contribute to this process by facilitating agonist-induced platelet aggregation at the site of ulcerated thrombosed atherosclerotic plaque. Because exposed subendothelial collagen matrix is a major contributor to this process, interaction of platelets with TPA and collagen at the site of thrombosis may paradoxically enhance aggregation and possibly reocclusion. In contrast, TNK-TPA does not enhance collagen- or arachidonic acid–facilitated platelet aggregation and may contribute less to ongoing platelet aggregation at the site of thrombolysis.
Creating variants of TPA for use as a single-bolus thrombolytic agent has been the goal of several mutagenesis efforts.28 29 Slower-clearing variants of TPA would be more convenient to administer and might be associated with enhanced thrombolysis. Because early thrombolysis of the infarct-related artery appears to be associated with increased survival,4 this property may confer additional clinical benefits on these variants. Although many different molecular variants of TPA have been created, most of these also have decreased fibrinolytic activities. The TNK-TPA variant is unique in that potentially beneficial modifications (slower plasma clearance, increased fibrin specificity, and resistance to PAI-1 inhibition) have been engineered into TPA without compromising its intrinsic ability to bind to and lyse fibrin clots in a human plasma milieu.16 For the present study, we chose a rabbit thrombolysis model because in vitro studies have demonstrated that the fibrinolytic activity of TNK-TPA is similar in rabbit and human plasma assays30 and that rabbit and human PAI-1 are 85% homologous and functionally indistinguishable from one another.31
The greater potency observed for TNK-TPA in this study (6- to 12-fold) is in good agreement with results obtained in other animal models of thrombolysis.16 32 33 34 However, the specific properties of TNK-TPA that contribute to this greater potency could vary between models. In ex vivo shunt experiments, exposure to the two thrombolytic agents (ie, AUC) was approximately equal at equipotent doses.16 In the present study, the greatest efficacy of TPA was achieved at doses that resulted in a significant decrease in TPA clearance from circulation. Consequently, at approximately equally effective doses, plasma exposure to TPA was 2- to 4-fold greater than that of TNK-TPA. Therefore, the greater potency (and sustained recanalization) of TNK-TPA in this model may be partially the consequence of properties other than slower plasma clearance. For example, the thrombi generated in this model were a mixture of fibrin, red cells, and platelets. Although not quantitated, the amount of platelet PAI-1 in these thrombi may have contributed to the increased potency of TNK-TPA due to its relative resistance to PAI-1. Interestingly, recent data35 suggest that increased fibrin specificity of TNK-TPA may also play a role in sustaining thrombolysis of platelet-rich clots by avoiding the "plasminogen steal" phenomenon proposed by Sobel et al36 and observed in vivo in a rabbit embolic stroke model.37 Finally, the difference in the effect of TNK-TPA and TPA on collagen-induced platelet aggregation, discussed above, may also have contributed to the sustained recanalization of TNK-TPA in this model.
The causal relation between systemic lytic state and bleeding complication is not well established. Although TPA causes a milder systemic lytic state compared with streptokinase, use of TPA does not always result in decreased incidence of serious bleeding complications.38 39 40 41 In the GUSTO Trial,4 use of TPA was associated with a 0.2% excess incidence of hemorrhagic strokes compared with use of streptokinase. Although TPA is more fibrin specific than streptokinase, the use of TPA at therapeutic doses often results in significant activation of plasminogen and subsequent depletion of fibrinogen. Whereas in some clinical studies a positive correlation between fibrinogen degradation and serious bleeding complication has been noted,42 43 44 other studies have disputed this correlation.45 Based on these discrepancies, it has been suggested that the bleeding associated with thrombolytic therapy may be due to vascular injury and lysis of a preexisting hemostatic plug.46 Until an effective thrombolytic regimen that produces little or no systemic activation of the fibrinolytic system is tested in clinical trials, the relation (if any) between fibrin specificity and bleeding will not be fully evaluated. Thus, it is possible that this new variant of TPA molecule may be associated with lower or higher incidence of bleeding complications compared with TPA. This can be established only by a large and well-controlled clinical trial.
We examined the effect of TPA and TNK-TPA on template bleeding time as
well as on a more rigorous assessment of hemostasis, as indicated by
incisional blood loss.19 20 We have previously shown
that
incisional blood loss correlates with amount of fibrinogen/fibrin that
collects at the site19 and is prolonged by both heparin
and Factor Xa antagonist but not by
aspirin.19 20 Template bleeding time was prolonged by
aspirin but not by TPA or TNK-TPA. This suggests that hemostatic plug
formation, which is probably dependent on normal platelet function,
is not altered significantly by either TPA or TNK-TPA. In contrast,
incisional blood loss was twofold higher with TPA compared with
TNK-TPA. In addition, there was significantly lower consumption of
fibrinogen, plasminogen, and 2-antiplasmin
with TNK-TPA compared with treatment with TPA. However, the lack of
relation between amount of blood lost and extent of
plasminogenemia suggests that blood loss after TPA or
TNK-TPA may not depend exclusively on the degree of systemic lytic
state but also on other less well–characterized vascular
parameters. Irrespective of the underlying mechanism(s)
that may be responsible for the increase in blood loss observed with
these plasminogen activators, bolus
administration of TNK-TPA was always associated with less bleeding when
compared with TPA.
Thus, a combination of increased potency, ability to induce sustained recanalization, and a decreased bleeding tendency makes TNK-TPA a potential candidate for evaluation in human clinical trials. In particular, the possibility of inducing more rapid and complete thrombolysis, combined with ease of administration and lesser tendency to cause bleeding complications, may potentially enlarge the cohort of patients with acute myocardial infarction who may be candidates for thrombolytic therapy.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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The technical assistance of Jerry Todd, Cheryl Pater, and Julie Badillo is gratefully appreciated. We thank Theresa Leftely for typing the manuscript.
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Footnotes |
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Guest Editor for this article was Robert A. O'Rourke, MD, University of Texas Health Science Center, San Antonio.
Received March 22, 1995; revision received June 15, 1995; accepted July 7, 1995.
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References |
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TopAbstractIntroductionMethods Results Discussion References |
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1. Collen D, Topol EJ, Tiefenbrunn AJ, Gold HK, Weisfeldt ML, Sobel BE, Leinbach RC, Brinker JA, Ludbrook PA, Yasuda I, Bulkley BH, Robinson AF, Hutter AM Jr, Bell WR, Spadaro JJ Jr, Khaw BA, Grossbard EB. Coronary thrombolysis with recombinant human tissue-type plasminogen activator: a prospective, randomized, placebo-controlled trial. Circulation. 1984;70:1012-1017.
2.
Chesebro JH, Knatterud G, Roberts R, Borer J, Cohen
LS, Dalen J, Dodge HT, Francis CK, Hillis D, Ludbrook P, Markis JE,
Mueller H, Passamani ER, Powers ER, Rao AK, Robertson T, Ross A, Ryan
TJ, Sobel BE, Willerson J, Williams DO, Zaret BL, Braunwald E.
Thrombolysis in Myocardial Infarction (TIMI) Trial,
phase 1: a comparison between intravenous tissue
plasminogen activator and
intravenous streptokinase.
Circulation. 1987;76:142-154.
3. Anderson JL, Rothbard RL, Hackworthy RA, Sorensen SG, Fitzpatrick PG, Dahl CF, Hagan AD, Browne KF, Symkoviak GP, Menlove RL, Barry WH, Eckerson HW, Marder VJ. Multicenter reperfusion trial of intravenous anisoylated plasminogen streptokinase activator complex (APSAC) in acute myocardial infarction: controlled comparison with intracoronary streptokinase. J Am Coll Cardiol. 1988;2:1153-1163.
4.
The GUSTO Investigators. An international
randomized trial comparing four thrombolytic strategies for
acute myocardial infarction. N Engl J Med. 1993;329:673-682.
5.
Reimer KA, Lowe JE, Rasmussen MM, Jennings RB.
The wave front phenomenon of ischemic cell death, I:
myocardial infarct size vs duration of coronary occlusion in
dogs. Circulation. 1977;56:786-794.
6.
Bush LR, Buja LM, Samowitz Q, Rude RE, Wathen M,
Tilton GD, Willerson JT. Recovery of left
ventricular segmental function after long-term
reperfusion following temporary coronary occlusion in conscious
dogs. Circ Res. 1983;53:248-263.
7. Bergmann SR, Lerch RA, Fox KA, Ludbrook PA, Welch MJ, Ter-Pogossian MM, Sobel BE. Temporal dependence of beneficial effects of coronary thrombolysis characterized by positron tomography. Am J Med. 1982;73:573-581. [Medline] [Order article via Infotrieve]
8. GISSI Study Group. Effectiveness of intravenous thrombolytic treatment in acute myocardial infarction. Lancet. 1986;1:397-401. [Medline] [Order article via Infotrieve]
9. Serruys PW, Simoons ML, Suryapranata H, Vermeer F, Wijns W, van den Brand M, Bar F, Zwaan C, Krauss XH, Remme WJ, Res J, Verheugt FWA, van Domburg R, Lubsen J, Hugenholtz PG. Preservation of global and regional left ventricular function after early thrombolysis in acute myocardial infarction. J Am Coll Cardiol. 1986;7:729-742. [Abstract]
10. Van de Werf F, Arnold AER. Intravenous tissue plasminogen activator and size of infarct, left ventricular function and survival in acute myocardial infarction. Br Med J. 1988;297:1374-1379.
11. The ISAM Study Group. A prospective trial of intravenous streptokinase in acute myocardial infarction (ISAM). N Engl J Med. 1986;314:1465-1471. [Abstract]
12. Ikram S, Lewis S, Bucknall C, Sram I, Thomas N, Vincent R, Chamberlain D. Treatment of acute myocardial infarction with anisoylated plasminogen streptokinase activator complex. Br Med J. 1986;293:786-789.
13.
The GUSTO Angiographic Investigators. The effect
of tissue plasminogen activator, streptokinase,
or both on coronary-artery patency, ventricular
function, and survival after acute myocardial infarction.
N Engl J Med. 1993;329:1615-1622.
14. Smalling RW, Schumacher R, Morris D, Karder K, Fuentes F, Valentine RP, Battey LL Jr, Merhige M, Pitts DE, Liederman HA, Nishikawa A, Adyanthaya A, Hopkins A. Improved infarct-related arterial patency after high dose, weight-adjusted, rapid infusion of tissue-type plasminogen activator in myocardial infarction: results of a multicenter randomized trial of two dosage regimens. J Am Coll Cardiol. 1990;15:915-921. [Abstract]
15. Wu A, van de Werf F, Stassen T, Mattsson C, Pohl G, Collen D. Pharmacokinetics and coronary thrombolytic properties of two human tissue-type plasminogen activator variants lacking the finger-like, growth factor-like, and first kringle domains (amino acids 6-173) in a canine model. J Cardiovasc Pharmacol. 1990;16:197-203. [Medline] [Order article via Infotrieve]
16.
Keyt BA, Paoni NF, Refino CJ, Berleau L, Nguyen H, Chow
A, Lai J, Pena L, Pater C, Ogez J, Etcheverry T, Botstein D, Bennett
WF. A faster-acting and more potent form of tissue
plasminogen activator. Proc Natl
Acad Sci U S A. 1994;91:3670-3674.
17. Paoni NF, Keyt BA, Refino CJ, Chow AM, Nguyen HV, Berleau LT, Badillo J, Pena L, Brady K, Wurm FM, Ogez J, Bennett WF. A slow clearing, fibrin-specific, PAI-1 resistant variant of tPA (T103N, KHRR 296-299 AAAA). Thromb Haemost. 1993;70:307-312. [Medline] [Order article via Infotrieve]
18. Benedict CR, Mathew B, Rex KA, Cartwright J Jr, Sordahl LA. Correlation of plasma serotonin changes with platelet aggregation in an in vivo dog model of spontaneous occlusive coronary thrombus formation. Circ Res. 1986;73:58-67.
19. Benedict CR, Ryan J, Wolitzky B, Gerlach M, Stern D. Active site-blocked factor IXa prevents intravascular thrombus formation in the coronary vasculature without inhibiting extravascular coagulation in a canine thrombosis model. J Clin Invest. 1991;88:1760-1765.
20.
Benedict CR, Ryan J, Todd J, Kuwabara K, Tijburg P,
Cartwright J Jr, Stern D. Active site-blocked factor Xa
prevents intravascular thrombus formation in the coronary
vasculature in parallel with inhibition of extravascular coagulation in
a canine thrombosis model. Blood. 1993;81:2059-2066.
21. Neuhaus K-L, Feuerer WJ, Tebbe S, Niederer W, Vogt A, Tebbe U. Improved thrombolysis with a modified dose regimen of tissue-type plasminogen activator. J Am Coll Cardiol. 1989;14:1566-1569. [Abstract]
22.
Fitzgerald DJ, Wright F, Fitzgerald GA.
Increased thromboxane biosynthesis during
coronary thrombolysis: evidence that platelet
activation and thromboxane A2 modulate the response
to tissue-type plasminogen activation in vivo.
Circ Res. 1989;65:83-94.
23. Eisenberg PR. Pro-coagulant effects of fibrinolytic agents. In: Sobel BE, Collen D, eds. Coronary Thrombolysis in Perspective. New York, NY: Marcel Dekker Inc; 1993:75-99.
24. Mohler MA, Refino CJ, Chen SA, Chen AB, Hotchkiss JA. D-Phe-Pro-Arg-chloromethylketone: its potential use in inhibiting the formation of in vivo artifacts in blood collected during tissue-type plasminogen activator thrombolytic therapy. Thromb Haemost. 1986;56:160-164. [Medline] [Order article via Infotrieve]
25. Clauss A. Gerinnungsphysiologische schnellmethode zur bestimmung des fibrinogens. Acta Haematol. 1957;17:237-240. [Medline] [Order article via Infotrieve]
26. Soria J, Soria C, Samama MM. A plasminogen assay using a chromogenic synthetic substrate: results from clinical work and studies of thrombolysis. In: Davidson JF, Samama MM, Desnoyers PC, eds. Chemical Fibrinolysis and Thrombolysis. New York, NY: Raven Press Publishers; 1978;3:337-346.
27. Berleau L, Nguyen H, Modi N, Bennett W, Keyt BA. Pharmacokinetics and pharmacodynamics of TPA variants in rabbits. Thromb Haemost. 1993;69:1070. Abstract.
28. Higgins D, Bennett W. Tissue plasminogen activator: the biochemistry and pharmacology of variants produced by mutagenesis. Annu Rev Pharmacol Toxicol. 1990;30:91-121. [Medline] [Order article via Infotrieve]
29. Lijnen HR, Collen D. Strategies for the improvement of thrombolytic agents. Thromb Haemost. 1991;66:88-110. [Medline] [Order article via Infotrieve]
30. Refino CJ, Keyt BA, Paoni NF, Badillo JM, Pater CS, Van Peborgh J, Pena L, Berleau LT, Nguyen VH, Bennett WF. A variant of tissue plasminogen activator (T103N, N117Q, KHRR 296-299 AAAA) with a decreased plasma clearance rate is substantially more potent than Activase rt-PA in rabbit thrombolysis model. Thromb Haemost. 1993;69:841. Abstract.
31. Hofmann KJ, Mayer EJ, Schultz LD, Socher SH, Reily CF. Purification and characterization of recombinant rabbit plasminogen activator inhibitor-1 expressed in Saccharomyces cerevisiae. Fibrinolysis. 1992;6:263-272.
32. Refino CJ, Paoni NF, Keyt BA, Pater CS, Badillo JM, Wurm FM, Ogez J, Bennett WF. A variant of t-PA (T103N, KHRR 296-299 AAAA) that, by bolus, has increased potency and decreased systemic activation of plasminogen. Thromb Haemost. 1993;70:313-319. [Medline] [Order article via Infotrieve]
33. Thomas GR, Thibodeaux H, Errett CJ, Badillo JM, Keyt BA, Refino CJ, Zivin JA, Bennett WF. A long–half-life and fibrin-specific form of tissue plasminogen activator in rabbit models of embolic stroke and peripheral bleeding. Stroke. 1994;25:2072-2079. [Abstract]
34. Collen D, Stassen J-M, Yasuda T, Refino CJ, Paoni N, Keyt BA, Roskams T, Guerrero JL, Lijnen HR, Gold HK, Bennett WF. Comparative thrombolytic properties of tissue-type plasminogen activator and of a plasminogen activator inhibitor-1-resistant glycosylation variant, in a combined arterial and venous thrombosis model in the dog. Thromb Haemost. 1994;72:98-104. [Medline] [Order article via Infotrieve]
35. Refino CJ, Badillo JM, Paoni NF, Keyt BA, Nguyen HV, Bennett WF. Increasing the fibrin specificity of t-PA enhances its activity towards platelet rich clots. Fibrinolysis. 1994;8(suppl 1):313.
36. Sobel BE, Nachowiak DA, Fry ETA, Bergman SR, Torr SR. Paradoxical attenuation of fibrinolysis attributable to `plasminogen steal' and its implications for coronary thrombolysis. Coron Artery Dis. 1990;1:111-119.
37.
Thomas GR, Thibodeaux H, Bennett WF, Refino CF,
Badillo JM, Errett CJ, Zivin JA. Optimized
thrombolysis of cerebral clots with tissue-type
plasminogen activator in a rabbit model of
embolic stroke. J Pharmcol Exp Ther. 1993;264:67-73.
38. The TIMI Study Group. The Thrombolysis in Myocardial Infarction (TIMI) trial. N Engl J Med. 1985;312:932-936. [Medline] [Order article via Infotrieve]
39. Verstraete M, Bernard R, Bory M, Brower RW, Collen D, de Bono DP, Erbel R, Huhmann W, Lennane RJ, Lubsen J, Mathey D, Meyer J, Michels HR, Rutsch W, Schartl MM, Schmidt W, Uebis R, von Essen R. Randomized trial of intravenous recombinant tissue-type plasminogen activator versus intravenous streptokinase in acute myocardial infarction. Lancet. 1985;1:842-847. [Medline] [Order article via Infotrieve]
40. White HD, Rivers JT, Maslowski AH, Ormiston JA, Takayama M, Hart HH, Sharpe DN, Whitlock RML, Norris RM. Effect of intravenous streptokinase as compared with that of tissue plasminogen activator on left ventricular function after first myocardial infarction. N Engl J Med. 1989;320:817-821. [Abstract]
41. Magnani B for the PAIMS investigators. Plasminogen Activator Italian Multicenter Study (PAIMS): comparison of intravenous recombinant single-chain human tissue-type plasminogen activator (tPA) with intravenous streptokinase in acute myocardial infarction. J Am Coll Cardiol. 1989;13:19-26. [Abstract]
42. Arnold AER, Brower RW, Collen D, van Es G-A, Lubsen J, Serruys PW, Simoons ML, Verstraete M, for the European Co-operative Study Group for rt-PA. Increased serum levels of fibrinogen degradation products due to treatment with recombinant tissue-type plasminogen activator for acute myocardial infarction are related to bleeding complications, but not to coronary patency. J Am Coll Cardiol. 1989;14:581-588. [Abstract]
43. Rao AK, Pratt C, Berke A, Jaffe A, Ochene I, Schreiber TL, Bell RW, Knatterud G, Robertson TL, Terrin ML, for the TIMI investigators. Thrombolysis in Myocardial Infarction (TIMI) trial—phase I: hemorrhagic manifestations and changes in plasma fibrinogen and the fibrinolytic system in patients treated with recombinant tissue plasminogen activator and streptokinase. J Am Coll Cardiol. 1988;11:1-11. [Abstract]
44.
Fennerty AG, Levine MN, Hirsh J. Hemorrhagic
complications of thrombolytic therapy in the treatment of
myocardial infarction and venous thromboembolism.
Chest. 1989;95:885-895.
45.
Stump DC, Califf RM, Topol EJ, Sigmon K, Thornton D,
Masek R, Anderson L, Collen D, and the TAMI study group.
Pharmacodynamics of thrombolysis with recombinant
tissue-type plasminogen activator:
correlation with characteristics of and clinical outcomes in patients
with acute myocardial infarction.
Circulation. 1989;80:1222-1230.
46. Marder VJ, Sherry S. Thrombolytic therapy: current status. N Engl J Med. 1988;380:1512-1520.
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