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(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

Claude R. Benedict, MD, DPhil; Canio J. Refino, BS; Bruce A. Keyt, PhD; Rajbabu Pakala, PhD; Nicholas F. Paoni, PhD; G. Rodger Thomas, PhD; William F. Bennett, PhD

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.

	Abstract

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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

	Introduction

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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 studies^{5 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.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
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).

View larger version (44K): [in this window] [in a new window]   Figure 1. Diagram of TNK-TPA structure showing domains, disulfides, and glycosylation sites. Domains or modules of the protein are described as Finger, Growth Factor, Kringle 1, Kringle 2, and Protease. Disulfide linkages are indicated with black bars between cysteines. N-linked glycosylation sites are represented as black Y-shaped structures at Asn 103, Asn 184, and Asn 448. Deletion of the high-mannose carbohydrate at Asn 117 is indicated by hatched Y structure detached from the protein. Tetra-alanine substitutions for K296, H297, R298, and R299 are indicated with thick circles. The active-site serine at position 478 is noted by a star. The arrow indicates the plasmin cleavage site (Arg 275) for conversion of single-chain TPA to two-chain TPA.

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.

View larger version (19K): [in this window] [in a new window]   Figure 2. Representation of the rabbit carotid thrombus formation model. The right common carotid was isolated surgically and a 2.5-mm Doppler flow probe was placed on the artery without constricting the vessel. Proximal to the flow probe, a 23-gauge needle electrode was inserted into the lumen with minimal trauma and stabilized by a collar that was sewn around the vessel without narrowing the artery. This electrode was used to stimulate thrombus formation.

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.

	Results

Top Abstract Introduction Methods Results Discussion References

 
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).

View larger version (31K): [in this window] [in a new window]   Figure 3. Bar graphs show effect of TPA or TNK on incidence of carotid artery recanalization (top), time to reperfusion (middle), and duration of recanalization (bottom). All three parameters were dose dependent for both TPA and TNK-TPA. Numbers above the bars in the top panel represent the ratio of arteries opened to experiments attempted. In the middle panel, nonreperfused arteries are represented by a time of 120 minutes.

View larger version (40K): [in this window] [in a new window]   Figure 4. Bar graphs show effect of TPA or TNK-TPA on residual thrombus mass (top) and blood loss from an abdominal incision site (bottom). There was a greater reduction in residual thrombus mass with TNK-TPA compared with TPA (P<.001), suggesting more complete thrombolysis with TNK-TPA. Incisional blood loss for all the TPA doses was significantly higher compared with that observed with TNK-TPA (P<.02).

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).

View larger version (44K): [in this window] [in a new window]   Figure 5. Bar graphs show maximal decrease in fibrinogen (top), plasminogen (middle), or 2-antiplasmin (bottom) with indicated doses of TPA or TNK-TPA. For comparable thrombolytic doses of TPA or TNK-TPA, there was significantly less fibrinogen (P<.01), plasminogen (P<.02), and 2-antiplasmin (P<.01) in plasma with TPA compared with TNK-TPA.

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.

View larger version (20K): [in this window] [in a new window]   Figure 6. Graphs show changes in plasma levels of TPA or TNK-TPA after IV administration of indicated doses of plasminogen activators. TPA was administered by use of a 90-minute infusion protocol (see "Methods" in text). TNK-TPA was administered as a bolus injection.

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).

View this table: [in this window] [in a new window]   Table 1. AUC and Plasma Clearance of TPA and TNK-TPA in Rabbits

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).

View larger version (23K): [in this window] [in a new window]   Figure 7. Bar graph shows effect of increasing concentrations of TPA and TNK on collagen-induced platelet aggregation. Human platelets in platelet-rich plasma were incubated with indicated concentrations of TPA or TNK-TPA for 1 minute at 37°C, then stimulated with collagen (1 µg/mL final concentration) and the aggregation monitored as an increase in light transmission. Results are mean±SD; n=5.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
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 studies^{22 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.

	Selected Abbreviations and Acronyms

 

AUC = area under the curve GUSTO = Global Utilization of Streptokinase and TPA for Occluded arteries PAI = plasminogen activator inhibitor TNK-TPA = T103N, N117Q, KHRR 296-299 AAAA TPA = tissue plasminogen activator

	Acknowledgments

 
The technical assistance of Jerry Todd, Cheryl Pater, and Julie Badillo is gratefully appreciated. We thank Theresa Leftely for typing the manuscript.

	Footnotes

 
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.

	References

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