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Stroke. 1997;28:1783-1788

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(Stroke. 1997;28:1783-1788.)
© 1997 American Heart Association, Inc.

Articles

Intravenous Thrombolysis With Recombinant Staphylokinase Versus Tissue-Type Plasminogen Activator in a Rabbit Embolic Stroke Model

S. Vanderschueren, MD, PhD; I. Van Vlaenderen, DVM; D. Collen, MD, PhD

From the Center for Molecular and Vascular Biology, University of Leuven, Belgium.

Correspondence to Désiré Collen, MD, PhD, Center for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg, O & N, Herestraat 49, B-3000 Leuven, Belgium. E-mail Desire.Collen{at}med.kuleuven.ac.be


*    Abstract
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 *Abstract
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 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Background and Purpose Timely intravenous administration of recombinant tissue-type plasminogen activator (alteplase, rTPA) to patients with evolving ischemic stroke improves neurological outcome. The present study was designed to compare the effects of rTPA and recombinant staphylokinase (Sak), a highly fibrin-specific thrombolytic agent, in an experimental model of rabbit embolic stroke.

Methods Groups of 5 to 12 rabbits were given intravenous saline or heparin and aspirin with, in addition, either Sak (1 or 2 mg/kg infused over 30 minutes or 2 mg/kg injected over 1 minute) or rTPA (3 or 6 mg/kg infused over 30 minutes or 6 mg/kg injected over 1 minute). Infusions were started 15 minutes after selective injection of standardized 125I-fibrin labeled rabbit plasma clots into the internal carotid artery.

Results Mean clot lysis over 60 minutes increased from 3.8% after saline to between 27% and 44% after Sak regimens (P=.0001 versus control) and to between 15% and 34% after rTPA regimens (P=.0001). Median volume of the ischemic lesion at 5 hours decreased from 190 mm3 after saline to between 11 and 17 mm3 after Sak (P=.02) and to between 0.5 to 54 mm3 after rTPA (P=.04). Mean neurological impairment at 5 hours (on a scale of 0 to 3) decreased from 2.3 after saline to between 1.3 to 1.6 after Sak (P=.003) and to between 1.1 to 1.9 after rTPA (P=.02). At the highest doses used, fibrinogen depletion was marginal with Sak but total with rTPA. Marked prolongation of ear puncture and cuticle bleeding times was only observed after bolus administration of rTPA.

Conclusions In the present rabbit model of embolic stroke, Sak was significantly more fibrin-specific than rTPA and at least as effective in lysing arterial emboli and limiting ischemia and neurological impairment.


Key Words: thrombolytic therapy • cerebral ischemia • rabbits • animal models


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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Thromboembolic stroke is a leading cause of death and disability, which lacks efficient curative therapy. Recent placebo-controlled trials have shown that timely intravenous administration of thrombolytic agents may alter the natural history of ischemic stroke. Despite an increased incidence of symptomatic intracerebral hemorrhage, the relatively fibrin-specific thrombolytic agent rTPA (alteplase) improved clinical outcome at 3 months if administered within 3 hours of the onset of ischemic stroke1 and before extended infarct signs were detectable on cerebral CT.2 Trials with the non–fibrin-specific agent streptokinase were terminated early because of excess mortality, mainly after hemorrhagic conversion of the cerebral infarct.3 4 5 From these studies it would seem probable that, in addition to time-to-treatment and patient selection (as reflected by the frequency of hemorrhagic transformation in the control group), fibrin selectivity of the agent might affect the likelihood of improved neurological outcome

Thrombolytic therapy for ischemic stroke is based on the premise that timely recanalization of arteries supplying the brain may salvage "the ischemic penumbra,"6 the hypoperfused but potentially viable zone adjacent to the central ischemic area, limit infarct size, and improve functional recovery and survival. This hypothesis is reminiscent of the "open-artery theory" postulated for coronary thrombolysis.7 Yet, current clinical experience suggests that the "time window of opportunity" may be smaller for cerebrovascular than for coronary thrombolysis.

Recombinant Sak is a highly fibrin-selective profibrinolytic agent that has shown promise for coronary and peripheral arterial thrombolysis.8 9 10 11 12 The present study was designed to study the relation between thrombolytic activity, infarct size, and neurological status and, more specifically, to compare the thrombolytic potency and fibrin specificity of Sak and rTPA in a rabbit embolic stroke model.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
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Preparation of the Clots
Radioactive clots were prepared by aspirating, in an 8F catheter (Portex orange, Portex), a mixture of 150 µL pooled rabbit platelet-poor plasma, a trace amount (approximately 1.5 µCi) of 125I-labeled human fibrinogen solution (Amersham), and 35 µL of a mixture of bovine thrombin (15 NIH units/mL) and 0.5 mol/L CaCl2. After incubation at 37°C for 30 minutes, the catheter was flushed with saline and the clot was cut into 3-mm segments. For each experiment, 4 segments were aspirated into a 4F catheter (Portex white) and the radioactivity count of this catheter was determined.

Rabbit Embolic Stroke Model
The animal experiments were conducted according to the guiding principles of the American Physiological Society and the International Committee on Thrombosis and Haemostasis.13

Sixty-two New Zealand White rabbits of either sex, weighing 2.5 to 3.5 kg, were premedicated with an intramuscular injection of 20 mg xylazine (Rompun 2%, Bayer) and 50 mg ketamin (Ketalin, Apharmo). A 0.8-mm cannula (Insyte-W, Becton-Dickinson) was inserted into the right marginal ear vein for maintenance anesthesia with sodium pentobarbital (Nembutal, Sanofi), 5 to 10 mg as needed, and for infusion of study medication. A 1.3-mm cannula (Insyte-W) was placed in the right femoral artery for blood sampling and blood pressure recording. A heating pad was applied to maintain body temperature. The right carotid artery bifurcation was exposed via a midline incision, all minor branches of the right common carotid artery were ligated or cauterized, the right internal carotid artery was identified and its extracranial part exposed, and the main branches of the external carotid artery were ligated. Internal and common carotid arteries were then temporarily clamped with vessel clips. The 4F catheter containing the four 3-mm clots was inserted via the bifurcation of the external carotid artery, with its tip into the ostium of the internal carotid artery. After removal of the vessel clip from the internal carotid artery, the clots were injected selectively and briskly into the internal carotid artery, while the rabbit's reaction was noted. After removal of the 4F catheter, the external carotid artery was closed. Subsequently, the common carotid artery was reopened, thus restoring blood flow toward the internal carotid artery.

The time course of clot lysis was monitored continuously for 75 minutes by external gamma counting, using a 3x0.5-inch sodium iodide/thallium crystal (Bicron), positioned over the rabbit's skull, and connected to a dedicated Canberra-S100 system (Canberra-Packard). Thereafter the operation wounds were closed and the rabbits were allowed to recover from anesthesia.

Study Drugs
Sak (natural variant Sak42D) was produced, purified, and characterized as described elsewhere.14 rTPA (alteplase, Activase) was a kind gift from Genentech, South San Francisco, Calif. Heparin was purchased from Novo Nordisk, and aspirin for intravenous use (Aspegic, lysine acetyl salicylic acid, in vials containing 0.9 g, corresponding to 0.5 g acetyl salicylic acid) was obtained from Synthelabo Benelux.

Fifteen minutes after embolization, infusions were started via the right marginal ear vein. Rabbits were randomly allocated to seven groups: intravenous infusions over 30 minutes starting with a 10% bolus, of saline, Sak (1 mg/kg or 2 mg/kg), or rTPA (3 mg/kg or 6 mg/kg) or bolus infusions over 1 minute of Sak (2 mg/kg) or rTPA (6 mg/kg) (Table 1Down). Sak and rTPA were diluted in physiological saline up to 16.5 mL. The lower dose of rTPA is in the range of the optimal dose (3.3 mg/kg over 30 minutes) derived from previous dose-finding studies in rabbit embolic stroke models.15 The corresponding doses of Sak were determined with the relative molecular weights of rTPA (Mr, 70 000) and Sak (Mr, 15 500) and the equivalence for coronary thrombolysis of 100 mg rTPA versus 30 mg Sak12 in mind.


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  		Table 1. Major End Points

All rabbits allocated to rTPA or Sak were pretreated with 30 mg/kg aspirin, administered after baseline bleeding time determination, and received conjunctive heparin. Heparin was started together with the thrombolytic agent, as a 200 IU/kg bolus, followed by a 100 IU/kg infusion in saline up to 15 mL, for 1 hour.

Outcome Measures
Clot Lysis
Clot lysis was quantitated as the decrease in externally recorded cranial radioactivity count over 60 minutes following study drug initiation and expressed as percent lysis. Additionally, the increase in blood radioactivity was determined on samples taken one minute after embolization and 75 minutes thereafter.

Clinical Outcome
Five hours after embolization the rabbits were scored clinically as: 0, no neurological impairment; 1, mild to moderate neurological impairment; 2, severe neurological impairment (including hemiparesis, persistent sopor, circling behavior); or 3, death within 5 hours of embolization. This semiquantitative classification scheme represents a modification of the score introduced by Bederson et al in a rat middle cerebral artery occlusion model.16 Five hours after the insult, rabbits with no or minimal deficit were well awake and responsive.

Ischemic Lesion
After clinical scoring, the animals were euthanized and the brains were removed. The residual radioactivities of the brain and of the decerebrated skull were measured separately by external gamma counting. Rabbits were only included for further analysis if the former exceeded the latter. This precaution was taken in view of the variability of the rabbit's extracranial circulation17 18 and the ensuing risk of erroneous extracranial deposition of emboli. Consequently, 9 rabbits (1 or 2 out of each of the seven treatment groups) were excluded. The 85% success rate (53 of 62 rabbits) for intracranial embolization equals that of previous reports.17 The brains were then put in a 2% TTC solution (Sigma) at 36°C for 30 minutes. After immersion for 5 minutes in ice water, the brains were cut in coronal slices of approximately 3 mm. After macroscopic examination for hemorrhage, the slices were once again put in 2% TTC at 36°C for 1 hour and thereafter in 10% phosphate-buffered formalin for at least 5 days. The TTC enzymatic tissue staining technique provides a clear macroscopic distinction between infarcted (TTC-negative or unstained) and noninfarcted (TTC-positive or crimson red) areas19 20 (FigureDown). An investigator blinded to treatment assignment and outcome determined the volume of the ischemic lesion. The unstained area of ischemia on each slice was measured planimetrically by means of a computerized image analyzer (Leica), and the volume of ischemic brain, computed from these areas and the thickness of the slices on the basis of a conic section model, was expressed in mm3.



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  		Figure 1. Photograph of the TTC-stained brain of an untreated rabbit (outer view of base). The unstained, pale area represents ischemic damage of the right middle cerebral artery territory.

Hemodynamic and Laboratory Follow-up
Pulse rate and blood pressure were recorded intra-arterially at baseline, at 1 minute after embolization, and at 25 and 55 minutes after study drug initiation. Blood samples were collected in citrated tubes (final concentration: 0.01 mol/L) at baseline and 55 minutes after drug initiation. Laboratory measurements included fibrinogen, {alpha}2-antiplasmin, aPTT, and full blood cell count.

Bleeding Times
Bleeding times were simultaneously determined by puncturing the depilated left ear (EBT) and by transecting the apex of a nail cuticle (CBT), as previously described.21 Bleeding times were recorded at baseline and at 25 minutes after drug initiation. In rabbits receiving bolus infusions, additional bleeding times were determined 5 minutes after the bolus.

Statistical Analysis
Data are expressed as mean±SEM, except for evolving infarct size that is expressed as median and interquartile range. Student's t test was used to compare paired parametric variables. Repeated measures were analyzed using repeated measures of variance and Friedman's nonparametric repeated measures, for parametric and nonparametric variables, respectively. To study correlations, the Spearman rank correlation was used. One-way ANOVA was applied to compare the effects of different agents (saline, Sak, and rTPA) and of different administration schemes (lower-dose continuous infusion, higher-dose continuous infusion, and bolus infusion) on the following dependent variables (assessed posttreatment): percent lysis, clinical score, infarct volume, plasma fibrinogen, plasma {alpha}2-antiplasmin, aPTT, EBT, CBT, pulse, blood pressure, hemoglobin, final white blood cell count, and final platelet count. All tests to determine probability were two-sided; P<.05 was considered to indicate statistical significance.


*    Results
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 *Results
down arrowDiscussion
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Table 1Up shows the major study end points (clot lysis, clinical outcome, and evolving infarct volume) for the seven treatment groups. All thrombolytic regimens produced significant lysis, although mean lysis never exceeded 50%. Relative to saline infusion, and irrespective of the dosing schedule, Sak produced on average 30% and rTPA 24% more lysis (both P<.0001 versus saline). No dose response was apparent for thrombolysis mediated either by Sak or rTPA: doubling of the dose did not increase lytic efficacy. Bolus infusion of 6 mg/kg rTPA over 1 minute was less thrombolytically effective than infusion over 30 minutes. Rabbits given Sak or rTPA had less neurological dysfunction and smaller cerebral infarcts 5 hours after embolization. Compared with saline, Sak reduced mean clinical score by 0.91 (P=.003) and mean volume of ischemic lesion by 530 mm3 (P=.02), and rTPA reduced mean clinical score by 0.65 (P=.02) and mean volume of ischemic lesion by 450 mm3 (P=.04). The differences between the rTPA and Sak groups in clinical score (0.26, P=.3) and evolving infarct size (78 mm3, P=.7) were not significant. Median clinical scores were 1 for groups given 2 mg/kg Sak (either as bolus or infusion) or 3 mg/kg rTPA, and 2 for all other groups.

Percent clot lysis over 1 hour correlated significantly and inversely with evolving infarct size (r=-.58, P=.0001) and with clinical score (r=-.47, P=.0009) at 5 hours. A significant correlation was observed between evolving infarct size and clinical score (r=.67, P<.0001).

Table 2Down shows pre- and posttreatment values of fibrinogen, {alpha}2-antiplasmin, aPTT, and bleeding times. A Sak dose of 1 mg/kg did not alter plasma fibrinogen or {alpha}2-antiplasmin levels, whereas after 2 mg/kg Sak mean plasma fibrinogen dropped to almost 50% of baseline and {alpha}2-antiplasmin levels decreased modestly but significantly. However, hemostatic derangements induced by rTPA were significantly more pronounced: 3 mg/kg rTPA reduced mean circulating levels of fibrinogen and {alpha}2-antiplasmin to 38% of baseline; 6 mg/kg rTPA, while further reducing {alpha}2-antiplasmin, produced virtually complete fibrinogenolysis. Overall, rTPA reduced mean fibrinogen by 76% compared with saline (P=.0001) and by 48% compared with Sak (P=.0001). The effects on these hemostatic markers were comparable, whether the higher doses of Sak or rTPA were given as an infusion or as a bolus. Mean aPTT levels increased more than 2.5-fold in rabbits treated with a thrombolytic agent plus heparin. Compared with saline, mean aPTT was 56 seconds longer after Sak and heparin (P=.03), and 83 seconds longer after rTPA and heparin (P=.001).


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  		Table 2. Coagulation Parameters and Bleeding Times

EBTs or CBTs did not increase significantly after saline or Sak. However, bolus administration of rTPA, in contrast to infusions over 30 minutes, prolonged EBTs and CBTs substantially. EBT was significantly longer after rTPA than after Sak (P=.001), but CBT was not (P=.5).

The evolution of hemoglobin, white blood cell count, and platelets is given in Table 3Down. White blood cell count decreased toward the end of the experiments, in control as well as in actively treated animals, suggesting that leukopenia resulted from the experimental procedure rather than from the infusions. Table 3Down also shows the evolution of pulse rate and blood pressure. Study drugs did not provoke hypotensive reactions.


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  		Table 3. Hematological and Hemodynamic Parameters


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
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Mechanisms of Benefit of Plasminogen Activators in Experimental Stroke
Intravenous infusion of thrombolytic agents 15 minutes after selective injection of homologous plasma clots into the internal carotid artery reduced evolving cerebral infarct size and neurological impairment in a rabbit embolic stroke model. The extent of thrombolysis correlated inversely with size of ischemic lesion and clinical score. Early thrombolysis most likely dissolved the fibrin matrix of obstructive thrombi, restored perfusion, salvaged jeopardized brain tissue, limited cerebral infarct size, and reduced morbidity and mortality. The observed associations, however, do not exclude that thrombolytic agents may also be beneficial in ischemic stroke through other, as-yet-undefined mechanisms. Studies in transgenic mice suggest that the role of endogenous plasminogen activators such as TPA in the brain may be complex and needs further characterization.22

Sak versus rTPA in Rabbit Embolic Stroke
The extent of clot lysis with either rTPA or Sak in the present model was relatively low (<50%) and was not enhanced by doubling the dose. This absence of dose response was not unexpected for rTPA. Indeed, in previous rabbit stroke experiments, an increase in dose of rTPA of more than 3.3 mg/kg15 or 5 mg/kg23 over 30 minutes even resulted in decreased thrombolysis. This effect was explained by the "plasminogen-steal phenomenon"24 : high doses of rTPA convert circulating plasminogen to plasmin, resulting in a decrease of fibrin-associated plasminogen and a reduction of the thrombolytic effect of rTPA. This mechanism, however, cannot fully account for the unexpected lack of dose response when doubling the dose of Sak, because the systemic fibrinolytic effects of even 2 mg/kg Sak were relatively moderate. An alternative explanation of the apparently low thrombolytic plateau involves clot composition and local, rather than systemic, plasmin concentrations. The partial refractoriness to clot lysis may be due to compression of the thrombi during preparation in catheters of decreasing diameter and during injection into the intracranial arteries. Clot compression, as clot retraction, may be associated with extrusion of plasminogen and decreased clot lysability.25 26 During a run-in phase before the present study, the embolization procedure was refined and the size and number of clots were adapted, taking the extensive intracranial collateral circulation of the rabbit into consideration. This procedure yielded reproducible and quantifiable sublethal cerebral infarcts in the majority of control animals.

Bolus administration of thrombolytic agents may improve the ease of administration. In the present model, however, the lytic efficacy of 6 mg/kg rTPA was decreased by 50% when it was infused over 1 minute instead of over 30 minutes, whereas bolus administration of 2 mg/kg Sak did not reduce its lytic potential. In patients with acute myocardial infarction, single boluses of rTPA have also produced lower coronary recanalization rates.27 Initial clinical experience suggests that (double) bolus administration of Sak produces efficient coronary thrombolysis.11 12

The lytic efficacies of infusions over 30 minutes of rTPA of 3 or 6 mg/kg and of Sak doses of 1 or 2 mg/kg were comparable, indicating that in the present model Sak and rTPA are about equipotent on a molar basis. However, the higher doses of rTPA produced profound hypofibrinogenemia, whereas Sak was more fibrin-specific. Intracranial hemorrhages were not observed, possibly due to the intact cerebral vasculature of these healthy rabbits and the short time (15 minutes) between embolization and start of drug infusion. Also, the interval of 5 hours between insult and macroscopic evaluation of the brain tissues may have been too short for the development of gross hemorrhagic conversion. It is increasingly recognized that intracranial hemorrhage complicating coronary thrombolysis in patients frequently originates from vessels with a pathological substrate such as cerebral amyloid vasculopathy28 or changes due to long-standing arterial hypertension.29 The risk of intracranial bleeding after cerebrovascular thrombolysis increases in accordance with the time between therapy and the onset of the ischemic symptoms,30 31 probably as a result of time-dependent loss of vascular integrity. The absence of cerebral bleeding in this model thus affirms that intact vessels protect against bleeding even in case of profound disruption of the circulating hemostatic system.32 33 Whether a systemic plasminolytic state promotes bleeding from damaged vessels is unclear from the present data.

The present study corroborates our previous finding that bolus administration of rTPA at doses that deplete circulating fibrinogen markedly prolongs bleeding times, especially when a short time elapses between bolus infusion and ear puncture or cuticle injury.21 Surprisingly, infusion over 30 minutes of the same dose of rTPA did not prolong bleeding times. The marked bleeding time prolongation by bolus rTPA may be caused by an initial excess of freely circulating rTPA that escapes complexation by plasminogen activator inhibitors. Although this mechanism remains unproven and the clinical significance of bleeding time prolongation and hypofibrinogenemia is still controversial, the present data call into question the efficacy and safety of high-dose intravenous bolus administration of rTPA. In line with this observation, a recent multicenter trial comparing a double bolus of rTPA with accelerated infusion in myocardial infarction patients was terminated early because of inferior benefit-to-risk ratio of double-bolus administration.34

Study Limitations
Although animal models constitute a valid tool to study the mechanism of disease and pharmacological intervention,35 extrapolation to humans should only be made with caution. Rabbits were chosen for practical and theoretical considerations. First, the rabbit embolic stroke model is well established.15 23 36 37 38 39 Second, rabbits are easy to handle and the extracranial carotid circulation can be identified and manipulated without the need for an operation microscope. Third, the fibrin specificity of Sak and rTPA, which is highly species-dependent, is comparable in rabbits and humans.40

Hemostatic markers and bleeding times can at best only be regarded as surrogate markers for thrombolytic bleeding.32 Given the lack of sensitivity of our model for hemorrhagic conversion, the putative safety benefit of improved fibrin specificity could not be validated.

All study animals received aspirin and heparin in addition to either Sak or rTPA. This combined therapy reflects current practice in myocardial infarction management with (relatively) fibrin-specific thrombolytic agents. Conjunctive antithrombotic therapy optimizes coronary thrombolysis with these agents.41 However, the need for conjunctive therapy in cerebrovascular thrombolysis is not established. Recent data in a rabbit embolic stroke model even suggest that aspirin paradoxically attenuates rTPA-mediated lysis.42

In an effort to maximize efficacy, thrombolytic infusions were initiated 15 minutes after embolization of platelet-poor plasma clots into the rabbit's cerebral circulation. Prolonged time-to-treatment38 and increased platelet content of thrombi26 43 are likely to reduce the efficacy of thrombolytic agents.

Because 5 hours may be too early for a definite cerebral infarct to develop, it may be more prudent to consider the infarct zones as ischemic lesions in evolution. Histological assessment was not included in the present study. The 5-hour interval between injury and death was chosen because within this time window the majority of control animals developed severe neurological impairment, which rendered protracted observation undesirable.

Some of the anesthetic drugs used (eg, barbiturates and ketamine) have been claimed to have neuroprotective effects. Yet, they did not prevent control animals from developing significant ischemic lesions. However, a synergistic effect between these anesthetics and thrombolytic agents in reducing ischemia cannot be excluded.44

Conclusions
Very early intravenous administration of rTPA or Sak in rabbits with an experimental embolic stroke reduced infarct size and neurological deficit. In comparison with rTPA, Sak was more fibrinogen-sparing and retained its thrombolytic effects when administered as a bolus, without prolongation of bleeding times. Further preclinical studies with Sak in cerebrovascular thrombolysis appear to be warranted.


*    Selected Abbreviations and Acronyms
 
aPTT = activated partial thromboplastin time
CBT = cuticle bleeding time
EBT = ear bleeding time
rTPA = recombinant tissue-type plasminogen activator
Sak = recombinant staphylokinase
TTC = triphenyltetrazolium chloride solution


*    Acknowledgments
 
S.V. is a research assistant for the National Fund of Scientific Research (NFWO), Belgium. The authors wish to thank Huberte Moreau and Zhang Zhi-yong for their excellent technical assistance and Bart Spiessens for his help with the statistical analyses.

Received December 17, 1996; revision received May 30, 1997; accepted June 3, 1997.


*    References
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 up arrowAbstract
 up arrowIntroduction
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 up arrowResults
 up arrowDiscussion
 *References
 

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