Donate Help Contact The AHA Sign In Home
American Heart Association
Stroke
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Stroke. 1995;26:1039-1046

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Thomas, G. R.
Right arrow Articles by Bennett, W. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Thomas, G. R.
Right arrow Articles by Bennett, W. F.

(Stroke. 1995;26:1039-1046.)
© 1995 American Heart Association, Inc.

Articles

Intravenous Aspirin Causes a Paradoxical Attenuation of Cerebrovascular Thrombolysis

Presented in part at the annual meeting of the American Society for Pharmacology and Experimental Therapeutics, San Francisco, Calif, July 30-Aug 3, 1993, and published in abstract form in The Pharmacologist. 1993;35:275.

G. Roger Thomas, PhD; Harold Thibodeaux, BS; Carol J. Errett; Martin M. Bednar, MD, PhD; Cordell E. Gross, MD William F. Bennett, PhD

From the Department of Cardiovascular Research, Genentech Inc, South San Francisco, Calif (G.R.T., H.T., C.J.E., W.F.B.), and the Division of Neurosurgery, Vermont Center for Vascular Research, University of Vermont, Burlington (M.M.B., C.E.G.).

Correspondence to G. Roger Thomas, Department of Cardiovascular Research, Genentech Inc, South San Francisco, CA 94080. E mail thomas.roger@gene.com.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Background and Purpose Aspirin treatment is recognized as an advantageous adjunct to thrombolytic agents in myocardial infarct patients. In this study we examined the effects of aspirin on the rate of clot lysis and on the frequency and extent of hemorrhagic transformations in rabbit models of embolic stroke.

Methods Rabbit models of ex vivo platelet aggregation and cutaneous template bleeding times were used to show the anticoagulant effects of aspirin in our experimental paradigm. We monitored tissue-type plasminogen activator (TPA)–induced clot lysis in two rabbit models of embolic stroke by (1) scintigraphically following the dissolution of a 99mTc-tagged clot or (2) using roentgenography to follow the disappearance of an Sn-tagged clot.

Results In animals pretreated (18 hours) with a single administration of aspirin (1, 5, or 20 mg/kg IV) or 1 mg/kg per day for 3 days, the aggregation response of platelets to collagen (3.3 µg/mL) or arachidonic acid (0.5 mmol/L) was attenuated. High-dose aspirin also increased ear template bleeding time from 1.6 to 2.6 minutes. When aspirin (20 mg/kg) was administered 18 hours before embolism and subsequent lysis with TPA (0.3 mg/kg bolus; 3 mg/kg per hour IV), the pretreatment significantly antagonized the rate and extent of TPA-induced clot lysis by up to 70%. This was confirmed in a second embolic stroke model. The suppression of TPA-induced lysis was reversed by administration of the prostacyclin analogue iloprost (10 µg/kg per hour) directly into the cerebral circulation.

Conclusions We conclude that aspirin reduces the effects of TPA in embolic stroke models. This effect may be the result of a loss of endothelial prostacyclin production since the effect is reversed by iloprost.


Key Words: aspirin • prostaglandins • thrombolytic therapy • rabbits


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Aspirin, by virtue of its ability to irreversibly acetylate the cyclooxygenase enzyme,1 is capable of inhibiting both prostacyclin (PGI2) production by endothelial cells2 and thromboxane A2 (TXA2) synthesis by platelets.3 4 At low doses of aspirin an antithrombotic environment can be created by the selective inhibition of TXA2.5 6 Others would argue that both platelet and endothelial cyclooxygenases are equally inhibited, particularly after high doses7 8 9 or single administrations,10 or that any selectivity is a function of the route of administration.11

Although there is still no universal answer to account for the antithrombotic effects of aspirin, it has been used clinically for more than a decade in patients at risk of coronary thrombosis12 and after transient cerebral ischemic attacks to reduce the incidence of further ischemic events.13 14 Recent animal studies have demonstrated that the concomitant administration of aspirin with tissue-type plasminogen activator (TPA) resulted in either no improvement or a worse outcome than with TPA alone. Specifically, a study by Overgaard et al15 concluded that the coadministration of aspirin and TPA in a rat thromboembolic model did not result in a greater reduction in brain infarct size compared with TPA alone. Furthermore, Clark et al16 showed that pretreatment with aspirin, but not heparin, increased the risk of hemorrhagic transformations in a rabbit model of embolic stroke treated with TPA. However, unlike the coronary circulation, no study to date, experimental or clinical, has examined the effect of aspirin on the incidence or rapidity of vascular recanalization in association with thrombolytic therapy in stroke. Thus, a lack of experimental data and the demonstration of regional differences between the cerebral vasculature and systemic vascular beds,17 the anticipated increasing use of thrombolytic therapy for stroke,18 19 20 and the large population base currently using aspirin make it of interest to examine the effects of this cyclooxygenase inhibitor on the effectiveness of TPA for thromboembolic stroke.

Using both gamma scintigraphic (99mTc-tagged) and x-ray (Sn-tagged) imaging of cerebral clots to measure the kinetics of cerebral thrombolysis, we have previously shown that TPA has a narrow dose range for optimum activity on clots of various sizes.21 22 In this study we duplicated the aspirin pretreatment but improved on the TPA dosing regimen of Clark et al16 in an attempt to clarify their findings. Using optimal doses of TPA, we investigated the effects of aspirin on the kinetics of thrombolysis in both small (5 mm) and larger (2.0 cm) clot emboli models. To confirm that therapeutic doses of aspirin were used, initial dose ranging studies were performed in rabbit models of ex vivo platelet aggregation and template bleeding times. Subthreshold, intermediate, and high doses of aspirin were used in our models of TPA-mediated cerebral clot lysis. In this study we examined the effects of a PGI2 analogue on the interaction between TPA and aspirin because one consequence of giving aspirin is the ablation of PGI2 production. We also examined the consequences of aspirin/TPA interactions for cerebral hemorrhage. Preliminary data from this study were presented previously.23


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
All animal studies conformed to the guiding principles of the American Physiology Society and were approved by the Institutional Care and Use Committee of the institution at which the experiments were conducted (Genentech Inc or University of Vermont). A total of 96 male New Zealand White rabbits were used. No animals were lost from the study.

Platelet Aggregation
The dose-dependent effects of aspirin pretreatment on ex vivo platelet aggregation were tested. Animals were pretreated with either sodium carbonate buffer (pH 7.0)7 (1 mL/kg IV; n=6) or aspirin at a dose of 1 (n=6), 5 (n=6), or 20 mg/kg IV (n=6) in a volume of 1 mL/kg. Eighteen hours later, 20 mL of blood was withdrawn from a central ear artery onto 3.15% (wt/vol) sodium citrate (Mallinckrodt Inc), and platelet-rich plasma (PRP) was prepared by centrifuging 1 mL aliquots at 13 000 rpm in an HBI Micro Centrifuge (Accurate Chemical and Scientific Corp) (relative centrifugal field, 13 250g) for 5 seconds. With the use of a dual-channel platelet aggregometer (Chrono-Log Corp), the platelets (300 µL PRP) were challenged with increasing doses of collagen (Chrono-Par 385, Chrono-Log Corp).

Alternatively, in animals in which clot lysis was measured by roentgenography, 0.9 mL whole blood was obtained from the central ear artery and immediately mixed with 0.1 mL sodium citrate (3.8% wt/vol). The citrated whole blood samples (0.5 mL) were diluted with an equal volume of normal (0.9%) saline and allowed to equilibrate to baseline in an impedance aggregometer at 37°C (Chrono-Log Corp). Aggregation was then induced by adding arachidonic acid (Chrono-Par 390, Chrono-Log Corp) at a final concentration of 0.5 mmol/L. Aggregation was observed for 6 minutes and expressed as percent change in impedance.

Template Bleeding Times
Template bleeding times were measured in 12 animals (6 controls, 6 aspirin-treated [20 mg/kg]). Surgicutt blades (International Technidyne Corp) were used to make cuts in the ear 5 mm long and 1 mm deep. The incisions were wicked every 15 seconds with Surgicutt blotting paper (International Technidyne Corp). Only the middle portion of the dorsal surface of the ear was used. To obtain an average value for each animal, six bleeding times per experiment were measured during a 2-hour period.

Measurement of Cerebral Clot Lysis by Gamma Scintigraphy
Whole blood clots comprised 0.5 mL of 99mTc-labeled sulfur colloids (2.5 mCi) (Mallinckrodt Medical Inc), 0.25 mL calcium chloride (1 mol/L) (Sigma Chemical Co), 2 U bovine thrombin (Parke-Davis), and 1 mL whole blood drawn onto sodium citrate (3.8% wt/vol) (Mallinckrodt Inc). The clots, formed in polyethylene (PE 160) tubing (Clay Adams), were then cut into cylinders measuring 5 mm in length and 1.1 mm in diameter, weighed, and washed in PBS containing 1 g/L bovine serum albumin (Sigma Chemical Co). The radioactivity in each clot was measured in a CRC-12 dosimeter (Capintec Inc) and injected into the animal through the internal carotid artery. Clots were not used for embolization if they did not contain 5±1 µCi of radiolabel.

The model of embolic stroke used in these experiments was previously reported in detail.21 Briefly, rabbits were anesthetized with Hypnorm (fluanisone 10 mg/mL and fentanyl 0.2 mg/mL at a dose of 0.7 mL/kg IM) (Janssen Pharmaceuticals), and labeled clots were introduced into the cerebral circulation. External images of the emboli were obtained with the use of an Ohio Nuclear Pho-{gamma} camera (Siemens Medical Services Inc) by means of an energy window that encompassed the emission peaks of 99mTc. Once the placement of the clot in the middle cerebral artery (MCA) was verified, a control 5-minute scintigram was collected and treatment was initiated. Throughout the experiment the data were collected and stored as a series of 5-minute scintigrams. We calculated the kinetics of thrombolysis by analyzing the scintigrams after constructing a region of interest over the embolus. The disappearance of the radiation from the embolic site was normalized for 99mTc decay and calculated as a percentage of the radiation at time zero. Data are presented as percent clot lysis (the inverse of the percent radiation at time zero) ±SEM. Mean arterial pressure (MAP) from the central ear artery was also measured every 5 minutes throughout the experiment with the use of a blood pressure analyzer (Micro Med Inc).

Two series of experiments were conducted. In the first experiment animals were pretreated with either sodium carbonate buffer (pH 7.0)7 (1 mL/kg IV; n=18) or aspirin at a dose of 1 (n=6), 5 (n=6), or 20 mg/kg IV (n=6) in a volume of 1 mL/kg. Eighteen hours later the animals were embolized, and TPA (alteplase [Activase; Genentech Inc]) was administered intravenously as a 0.3-mg/kg bolus followed by a 3.0-mg/kg per hour infusion for 1 hour. In the study that followed, animals were pretreated with either sodium carbonate buffer (pH 7.0)7 (1 mL/kg IV; n=6) or aspirin (20 mg/kg IV; n=12). Again the animals were embolized 18 hours later and given TPA as a 0.3-mg/kg bolus followed by a 3.0-mg/kg per hour infusion for 2 hours. After 1 hour of TPA, animals were randomly assigned to receive either infusions of the PGI2 analogue iloprost (Berlex Bioscience) (10 µg/kg per hour; n=6) or saline (1 mL/kg per hour; n=6). These latter infusions were given directly into the internal carotid artery via the cannula used to introduce the embolus.

Measurement of Cerebral Clot Lysis by Roentgenography
This rabbit model of thromboembolic stroke has been previously described in detail.24 25 26 Briefly, 1 mL of whole blood was withdrawn from the central ear artery and mixed with 50 mg of tin granules (20 µm). The mixture was then incubated overnight at room temperature in polyethylene (PE 90) tubing (Clay Adams) prerinsed with bovine thrombin (Parke-Davis). Immediately before embolization, a 2-cm length was selected.

Fasted New Zealand White rabbits (Charles River, Canada) of either sex, weighing 2.5 to 3.2 kg, were anesthetized with a solution of acepromazine (20 mg, Aveco Co), ketamine (50 mg/kg, Aveco Co), and xylazine (10 mg, Mobay Corp). After anesthetic induction, the femoral artery and vein were cannulated with polyethylene tubing for measurement of MAP and hematocrit, monitoring of arterial blood gases (pH, PO2, and PCO2), drug infusion, and determination of hydrogen washout for the measurement of regional cerebral blood flow (rCBF) by the hydrogen clearance technique. A midline scalp incision was made to expose the calvarium. A craniotomy, performed on the hemisphere to be embolized, was begun immediately posterior to the coronal suture and 10 mm lateral to the midline. Three 30-gauge platinum-iridium electrodes were then placed within the cortex to monitor rCBF in the MCA and posterior cerebral artery distribution. These electrodes were fixed in place with fast-setting epoxy. Flow probes that exhibited a critical reduction in rCBF to less than 15 mL/100 g per minute at the time of the embolization were used for further study. After a midline neck incision was made, the rabbits were tracheostomized and mechanically ventilated (Bird Corp). The right carotid bifurcation was then isolated, the external carotid artery ligated, and the internal carotid artery isolated. The clot embolus was then introduced into the internal carotid artery after an arteriotomy with the use of a 5-mm Beaver blade. The clot was embolized to the anterior circulation of the brain, and a baseline submental-vertex x-ray film was taken to verify intracranial placement of the Sn-tagged clot. Both before clot embolization as well as 1 and 2 hours after clot embolization, rCBF measurements and arterial blood gas and hematocrit levels were obtained. MAP and core temperature were continuously monitored, with MAP held constant between 55 and 60 mm Hg and core temperature held within 1°C of the baseline temperature.

TPA infusion was initiated, and an arteriorrhaphy was then performed with restoration of blood flow through the carotid bifurcation. Sequential x-ray films were taken at 1 and 2 hours after clot embolization. After completion of the TPA infusion, the animal was killed with an overdose of sodium pentobarbital (150 mg/kg; Beuthanasia-D Special, Schering-Plough Animal Health). A calvariectomy was performed, and the brain was harvested and inspected for any residual clot. The whole brain was then x-rayed, and a photograph was taken of the ventral surface of the brain to document the presence of embolus.

In this latter protocol, two series of experiments were also undertaken. In the first experiment, 3 animals were pretreated as previously with aspirin (20 mg/kg IV) 18 hours before instrumentation. In the second experiment, 3 animals were given a low-dose aspirin treatment (1 mg/kg per day IV) for 3 consecutive days. The last dose was given 18 hours before embolization. After embolization all animals received TPA as a 0.3-mg/kg bolus followed by a 3.0-mg/kg per hour infusion for 2 hours.

Intracerebral Hemorrhages
After treatment with either excipient or TPA and 2 hours of gamma scintigraphic imaging, the rabbits were allowed to recover for a period of 24 hours. The animals were then killed with an overdose of sodium pentobarbital (150 mg/kg; Beuthanasia-D Special, Schering-Plough Animal Health) and the brains harvested. After a 2-week fixing period in 10% formalin (Richard-Allan Medical), the brains were observed for gross surface hemorrhages and sectioned into 10x2.5-mm sections. Each section was observed for hemorrhages and scored according to the presence of a hemorrhage on one side or both sides of each section. A hemorrhage apparent on one side was given a score of 1. If the hemorrhage was continuous to the other side of the section, a score of 2 was given. A total score was thus assigned to each brain, and the results were presented as the mean±SEM only of those animals in each group in which hemorrhages occurred. The maximal attainable score was 20.

Statistical Analysis
Statistical analysis of the clot lysis (gamma scintigraphy) and collagen-induced platelet aggregation data was performed with the use of two-way ANOVA for repeated measures to detect significant differences between groups. A two-tailed, unpaired Student's t test was used for the bleeding time data and two-tailed Fisher's exact test for the rate of hemorrhagic transformation. ANOVA and Student's t test were performed on a Macintosh IIci computer (Apple Computer Inc) with the use of STATVIEW SE+ software (Abacus Concepts Inc). Fisher's exact test was calculated as described by Zar.27 In all cases a value of P<.05 was considered significant.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Platelet Aggregation
Aspirin inhibits collagen and arachidonic acid–induced ex vivo platelet aggregation. Inhibition of collagen-induced aggregation was not apparent at 1 mg/kg of aspirin but did occur at doses of 5 mg/kg or greater given 18 hours before blood sampling (P<.001) (Fig 1Down). Higher doses (20 mg/kg) caused greater inhibition of platelet aggregation. Additionally, in animals given 1 mg/kg per day for 3 days, the platelet response to 0.5 mmol/L arachidonic acid was reduced by 65%. These data show that the doses of aspirin used in these studies were pharmacologically active as measured by this ex vivo parameter.



View larger version (12K):
[in this window]
[in a new window]
  		Figure 1. Line graph of platelet-rich plasma taken from animals pretreated with carbonate buffer ({circ}) or 1.0 ({blacktriangleup}), 5.0 ({blacksquare}), or 20 ({bullet}) mg/kg aspirin, showing a dose-dependent inhibition of collagen-induced platelet aggregation. Each point represents the mean±SEM of 6 animals.

Template Bleeding Times
Pretreatment with 20 mg/kg aspirin increased the rabbit ear template bleeding times from a mean value of 1.6±0.1 minutes to 2.6±0.1 minutes (P<.001), showing that in an indirect in vivo assay this high dose of aspirin is pharmacologically active.

Measurement of Cerebral Clot Lysis by Gamma Scintigraphy
When the kinetics of cerebral clot lysis were measured with the use of gamma scintigraphic imaging, aspirin pretreatment (1, 5, or 20 mg/kg) significantly antagonized the thrombolytic activity of a 1-hour TPA regimen on an occlusive embolic clot (P<.001). There was a reduction in both the rate and the extent of clot lysis within the 2-hour experimental observation period (Fig 2Down). In the TPA alone group the time taken for 50% lysis of the clot was 35±5 minutes. In contrast, when TPA was given to animals pretreated with aspirin (20 mg/kg), none achieved 50% lysis within the 2-hour observation period. After 1 hour of TPA treatment, clot lysis in the control group was 71±7%. This lysis proceeded to 93±3% by the end of the experiment. In animals pretreated with 1 mg/kg aspirin, only 24±8% of the clot was lysed at 60 minutes, and at 120 minutes the clot lysis in this group was 39±7%. At the same time points in the group treated with 5 mg/kg aspirin, clot lysis was reduced to 48±10% and 50±12%. Pretreatment with 20 mg/kg aspirin also caused suppression of lysis, resulting in only 23±2% and 27±3% lysis at 60 and 120 minutes, respectively. Thus, 1, 5, and 20 mg/kg aspirin pretreatment suppressed TPA-induced clot lysis by at least 50% in all cases. However, this response was not dose dependent over the range of doses used here.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 2. Line graph shows that cerebral clot lysis induced by tissue-type plasminogen activator (t-PA) (0.3 mg/kg bolus; 3 mg/kg per hour for 1 hour) ({circ}) (n=18) is attenuated in animals pretreated with 1.0 ({triangleup}), 5.0 ({blacksquare}), or 20 ({bullet}) mg/kg aspirin. Each point in the aspirin-treated groups represents the mean±SEM of 6 animals.

To dose iloprost so that a period of TPA treatment alone and a period of cotreatment with TPA and iloprost could be observed, the infusion of TPA had to be extended from 1 hour to 2 hours. As seen in Fig 3Down, in the animals not pretreated with aspirin, the extent of clot lysis is similar irrespective of whether the TPA was administered for 1 or 2 hours (P>.05). In animals given a 2-hour infusion of TPA, the extent of clot lysis was 78±8% at 60 minutes and 93±3% at 120 minutes. In addition, the antagonistic effect of aspirin (20 mg/kg) is unchanged by increasing the TPA infusion to 2 hours, and the clot lysis remains significantly depressed (P<.001). It is also apparent from Fig 3Down that after 1 hour of TPA treatment the animals in the iloprost group were similarly inhibited by having lysed only 18±7% of the clot immediately before being given the PGI2 analogue.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 3. Line graph shows that cerebral clot lysis induced by tissue-type plasminogen activator (TPA; t-PA in the figure) (0.3 mg/kg bolus; 3 mg/kg per hour for 2 hours) ({circ}) is attenuated in animals pretreated with 20 mg/kg aspirin ({bullet}). In animals similarly pretreated with aspirin and treated with TPA, the activity was fully restored during an infusion of iloprost (10 mg/kg per hour) ({triangleup}) directly into the cerebral circulation via the internal carotid artery. Each point represents the mean±SEM of 6 animals.

Coinfusion of TPA and iloprost (10 µg/kg per hour), a dose and route of administration that has no depressor effects in the rabbit, reversed the aspirin-induced antagonism (P<.001), allowing the lysis to occur at its optimal rate, ie, a rate parallel to aspirin-free animals. As seen in Fig 3Up, clot lysis progresses optimally when TPA and iloprost are coadministered (60 to 120 minutes), so that at the end of the experiment (120 minutes) 80±3% of the clot had lysed.

Measurement of Cerebral Clot Lysis by Roentgenography
The observation that aspirin antagonized the clot lysis action of TPA was confirmed in the second model of embolic stroke, which used x-ray imaging of the embolic clot. Throughout these experiments both arterial blood gas (pH range, 7.39 to 7.45; PO2 range, 149 to 171 mm Hg; PCO2 range, 33 to 40 mm Hg) and hematocrit values (29.0% to 34.3%) were maintained within the physiological range. In only one experiment after low-dose aspirin was any clot lysis observed. In that experiment there was a small degree of clot fragmentation. Each of two small clot fragments disappeared over 2 hours, although no lysis of the major clot fragment (>75%) was observed. In all other experiments in animals pretreated with aspirin there was no radiographic evidence of clot lysis (Fig 4ADown). This is in contrast to animals that received TPA alone, in which 16 of 23 animals (70%) demonstrated complete clot lysis within 2 hours (Fig 4BDown). The presence and position of residual clots were verified at the end of each experiment both radiologically and by gross inspection of the base of the brain.



View larger version (138K):
[in this window]
[in a new window]
  		Figure 4. In an animal pretreated with aspirin (1 mg/kg per day for 3 days), failure of tissue-type plasminogen activator (TPA)–induced clot lysis in this experiment is documented in A, demonstrating persistent clot on the submental-vertex x-ray film taken at the conclusion of the experiment. B shows a submental-vertex x-ray film taken after TPA treatment (0.3 mg/kg bolus: 3 mg/kg per hour for 2 hours) documenting complete lysis of the intracranial Sn-tagged clot in a rabbit that did not receive aspirin.

In this study, immediately after clot embolization there was a reduction in rCBF to less than 5 mL/100 g per minute in all animals. In only one animal was there a significant return of cerebral blood flow (CBF) after initiation of TPA therapy. However, this was only observed in the first hour, following which there was further reduction in CBF. This transient return of blood flow in one animal did not correlate with any radiographic evidence for clot lysis.

Intracerebral Hemorrhages
On necropsy of the gamma scintigraphic study animals, it was apparent that aspirin retarded the clot lysis not only during the 2-hour observation period but also during the following 24 hours. As seen in the TableDown, in the two groups that received aspirin (20 mg/kg) and TPA for either 1 or 2 hours, 5 of the 12 animals had visible clots in the MCA 24 hours later. This is in contrast to animals given 1 mg/kg, 5 mg/kg, or no aspirin in which no clot was visible at 24 hours. In the animal groups treated with 20 mg/kg aspirin there was no clear-cut correlation between the MCA clots at 24 hours and the occurrence of cerebral hemorrhage. This suggests that the hemorrhages were not strictly a function of length of ischemia.


View this table:
[in this window]
[in a new window]
  		Table 1. Hemorrhagic Transformation and Residual Clot at 24 Hours

The frequency and the size of hemorrhagic transformations were measured, and the results are shown in the TableUp. Evidence of hemorrhagic transformation was found in approximately one third of the animals given TPA alone either for 1 hour (7/18) or 2 hours (2/6). There was no significant change in the number of hemorrhagic events in any of the treatment groups (P>.05).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The present study examines the effect of aspirin on the kinetics of cerebral clot lysis in two rabbit models of thromboembolic stroke. In these studies aspirin, even at low doses, antagonized TPA-mediated thrombolysis. Reversal of this antagonism was afforded by the coinfusion of the PGI2 analogue iloprost at a dose that did not have gross hemodynamic effects. Although this parameter was not measured, it was unlikely to change the rate of TPA elimination, as previously shown in dogs.28

Our choice of animal model is limited by the species specificity of TPA.29 Given that the response to aspirin is variable between investigators and animal species,7 8 9 30 we confirmed the pharmacological effects of aspirin in our model by demonstrating an inhibition of ex vivo platelet aggregation and an increase in template bleeding time. At the lowest dose of aspirin (1 mg/kg) there was no effect on collagen-induced platelet aggregation, but the antagonism of TPA-induced cerebral clot lysis was pronounced. However, in a second experiment 1 mg/kg of aspirin given on 3 consecutive days did antagonize platelet aggregation induced by arachidonic acid. These apparent inconsistencies possibly reflect the varying susceptibility to aspirin of different agonists of ex vivo platelet aggregation.

Despite species variability, there is general agreement that vasodilator eicosanoids, such as PGI2, contribute to the maintenance of CBF.31 Clinically, aspirin improves or stabilizes declines in cerebral perfusion in patients with multi-infarct dementia,32 possibly due to the elevated levels of TXA2 seen during cerebral ischemia.33 34 On the other hand, aspirin (50 mg/kg) and indomethacin (5 mg/kg) have been shown to decrease brain blood flow by increasing cerebral vascular resistance in the conscious undisturbed rat.35 At these doses of cyclooxygenase inhibitors, it is possible that the production of PGI2, the most abundant prostaglandin in cerebral vessels,36 is reduced. However, it is not clear whether prostanoid control of CBF is wholly dependent on endothelial cyclooxygenase or whether cyclooxygenase located in deeper layers of the cerebral vessels makes a contribution. This becomes an important question because it has been shown that in rabbits given aspirin (10 mg/kg IV), endothelial cells synthesize cyclooxygenase more rapidly than other tissues, including vascular media. At 24 hours after the administration of aspirin, the aortic endothelium had restored prostanoid production to 64% of control, whereas the aortic media had recovered only 33% of its original activity.37 Furthermore, in rabbits given aspirin orally (70 mg/kg), the vasculature took more than 24 hours to recover PGI2 synthetic capacity to control levels.38

Given that aspirin can have significant effects on vascular cyclooxygenase levels that can last for 24 hours or more, a possible explanation for the antagonism of TPA thrombolysis seen in this study may involve a decreased capacity for PGI2 production. Consequently, the cerebral vasculature, when stressed by an embolic stroke, may be unable to efficiently redirect blood flow. Hemodynamic changes can occur either by opening collaterals or by increasing the luminal diameter of the embolized vessel and redirecting blood around the clot. This loss of cerebrovascular regulation would greatly restrict the access of TPA to the embolus, thus reducing the rate at which lysis takes place. In this situation the reversal of the effects of aspirin on clot lysis by iloprost may be explained as a substitution for endogenous PGI2 production. It should also be noted that we have observed a similar aspirin/TPA interaction, although to a much lesser extent, in the rabbit hind limb (data not shown). However, this scenario is not supported by the results of a recent study showing that a dose of 90 mg/kg IV of aspirin given over 20 minutes is required to inhibit cyclooxygenase activity in the cerebral microvessels of normal rabbits.39 It should be noted that one major difference between the two paradigms is that in our study the brain is subjected to a period of cerebral ischemia, a condition known to alter the eicosanoid synthesis profile of brain microvessels toward the lipoxygenase pathway.40 How this affects the response of the vasculature to aspirin is unknown. Nevertheless, and given that there are probably differences in the peak plasma levels of aspirin attained in the two studies (bolus versus infusion), alternative rationales for our observations must be considered.

There is a very narrow range for optimal thrombolytic activity with TPA,21 22 and doses outside this range will result in considerably reduced thrombolysis. Since a shift of TPA activity in either direction can affect the outcome, we investigated the possibility that the presence of aspirin may shift the TPA dose-response curve to the right. These studies demonstrated that aspirin caused greater antagonism of lower doses of TPA, suggesting that "excessive potentiation" of the TPA response did not account for our observations. Conversely, aspirin may reduce either the activity or the plasma concentration of TPA. It has been demonstrated that aspirin can suppress the endogenous production of TPA in normal volunteers after venous occlusion of the forearm.41 However, the increase in factor VIII–related antigen and hemoconcentration induced by venous occlusion were not affected by aspirin.41 Finally, evidence exists in the literature that aspirin has an unexplained, prothrombotic effect in the rabbit that is independent of its inhibition of cyclooxygenase.42 Presently we cannot rule out the possibility that this type of response might play a role in our observations.

The mechanism by which PGI2 or its synthetic analogue, iloprost, might accelerate clot lysis is also open to discussion. Apart from the vasodilator and antiplatelet effects of PGI2 and its metabolite 6-keto-PGE1, they have been shown to stimulate fibrinolysis.43 44 It has been proposed that this may occur through the release of endogenous TPA from the vessel wall by a mechanism akin to that described for human foreskin fibroblasts.45

In the original observation on the effects of aspirin pretreatment on the resultant hemorrhagic transformations, Clark et al16 found that aspirin increased the frequency of cerebral bleeding (hemorrhagic infarctions). In the present study we saw no significant effects of aspirin on the development of hemorrhagic infarctions. Some of the disparities might be explained by the differences in the methodology and the doses of TPA used. Clark et al used 10 mg/kg given over 30 minutes as a 2-mg/kg bolus followed by a 16 mg/kg per hour infusion. In a previous study we have shown that infusions of TPA at this rate for 30 minutes deplete the blood of plasminogen, fibrinogen, and {alpha}2-antiplasmin.22 In the same study we also found that at doses as low as 6.65 mg/kg given over this period there was significant attenuation of TPA activity, presumably due to plasminogen steal.46 Clark et al report that all of the animals showed thrombolysis at 24 hours. However, complete thrombolysis can be achieved in 2 hours when an optimal dosing regimen of TPA is used.21 In the former study the TPA was not administered until 90 minutes after embolization, whereas in the present study that time window was set at 20 minutes. Although we have previously shown that a good correlation between duration of ischemia and hemorrhage only exists when highly fibrin-specific variants of TPA are used,47 it is still possible that differing periods of ischemia in the two studies may in part account for the disparate results on hemorrhagic transformation.

In conclusion, although the precise mechanism by which aspirin antagonizes the action of TPA in the cerebral vasculature is unclear at the present time, the observation is both reproducible and in agreement with previous studies.8 9 Inasmuch as there are differences in the models, the present study and those previously conducted by Clark et al16 and by Overgaard et al15 share the overriding conclusion that aspirin does not enhance the effects of TPA. In these animal models of embolic stroke it may well be detrimental to the eventual outcome. Great caution must be exercised when these results are extrapolated to the human scenario, and at the present time this is probably not possible. New investigations, including a full analysis of the effects of aspirin on the prostaglandin profile in cerebral vasculature under normal and ischemic conditions, are now necessary. These will help to define further the mechanisms responsible for aspirin's antagonism of TPA-mediated thrombolysis of cerebral embolic clots and to fully evaluate the impact of these findings on stroke therapy.


*    Acknowledgments
 
This study was supported in part by the US Public Health Service grants 1-R29-NS-31008-02 and 1-R55-NS-708-01A1. The authors wish to thank Dr Justin A. Zivin and his laboratory at the University of California at San Diego for their assistance and advice during the course of this study. We also wish to thank Jed Ross for assisting with the preparation of the manuscript. The technical advice of Sherry Bullens is also much appreciated. Finally, we wish to thank Dr Joe Hedgpeth of Berlex Bioscience for his generous gift of iloprost.

Received June 8, 1994; revision received December 7, 1994; accepted February 27, 1995.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Roth GJ, Stanford NS, Majerus PW. Acetylation of prostaglandin synthetase by aspirin. Proc Natl Acad Sci U S A. 1975;72:3073-3076. [Abstract/Free Full Text]
  2. Vane JR. Inhibitors of prostaglandin, prostacyclin and thromboxane synthesis. Adv Prostaglandin Thromboxane Res. 1978;4:27-44. [Medline] [Order article via Infotrieve]
  3. Smith JB, Willis AL. Aspirin selectively inhibits prostaglandin production in human platelets. Nature. 1971;231:235-237.
  4. Roth GJ, Majerus PW. The mechanism of the effect of aspirin on human platelets, I: acetylation of a particular fraction protein.J Clin Invest. 1975;56:624-632.
  5. Shaikh BS, Bott SJ, Demers LM. The differential inhibition of prostaglandin synthesis in platelets and vascular tissue in response to aspirin. Prostaglandins Med. 1980;4:439-447. [Medline] [Order article via Infotrieve]
  6. FitzGerald GA, Oats JA, Hawiger J, Maas RL, Roberts LJ, Lawson JA, Brash AR. Endogenous biosynthesis of prostacyclin and thromboxane and platelet function during chronic administration of aspirin in man. J Clin Invest. 1983;71:676-688.
  7. Kelton JG, Hirsh J, Carter CJ, Buchanan MR. Thrombogenic effect of high-dose aspirin in rabbits: relationship to inhibition of vessel wall synthesis of prostaglandin I2-like activity. J Clin Invest. 1978;2:892-895.
  8. Wu KK, Chen Y-C, Fordham E, Ts'ao C-H, Rayudu G, Matayoshi D. Differential effects of two doses of aspirin on platelet-vessel wall interaction in vivo. J Clin Invest. 1981;68:382-387.
  9. Zimmerman R, Thiessen M, Walter E, Morl H. Paradoxical effect of high and low dose aspirin in experimental arterial thrombosis. Artery. 1980;8:422-425. [Medline] [Order article via Infotrieve]
  10. Preston FE, Whipps S, Jackson CA, French AJ, Wyld PJ, Stoddard CJ. Inhibition of prostacyclin and platelet thromboxane A2 after low-dose aspirin. N Engl J Med. 1981;304:76-79. [Abstract]
  11. Cerletti C, Gambino MC, Garattini S, de Gaetano G. Biochemical selectivity of oral versus intravenous aspirin in rats. J Clin Invest. 1986;78:323-326.
  12. ISIS-2 Collaborative Group. Randomised trial of intravenous streptokinase, oral aspirin, both, or neither among 17 187 cases of suspected acute myocardial infarction: ISIS-2. Lancet. 1988;2:349-360. [Medline] [Order article via Infotrieve]
  13. Fields WS, Lemak NA, Frankowski RF, Hardy RJ. Controlled trial of aspirin in cerebral ischemia. Stroke. 1977;8:301-314. [Abstract/Free Full Text]
  14. The Canadian Co-operative Study Group. A randomized trial. N Engl J Med. 1978;229:53-59.
  15. Overgaard K, Sereghy T, Pederson H, Boysen G. Dose response of t-PA and its combination with aspirin in a rat embolic stroke model. Neuroreport. 1992;3:925-928. [Medline] [Order article via Infotrieve]
  16. Clark WM, Madden KP, Lyden PD, Zivin JA. Cerebral hemorrhagic risk of aspirin or heparin therapy with thrombolytic treatment in rabbits. Stroke. 1991;22:872-876. [Abstract/Free Full Text]
  17. Duckworth JW, Wellman GC, Walters CL, Bevan JA. Aminergic histofluorescence and contractile responses to transmural electrical field stimulation and norepinephrine of human middle cerebral arteries obtained promptly after death. Circ Res. 1989;65:316-324. [Abstract/Free Full Text]
  18. Brott T. Thrombolytic therapy for stroke. Cerebrovasc Brain Metab Rev. 1991;3:91-113. [Medline] [Order article via Infotrieve]
  19. Brott TG, Haley EC Jr, Levy DE, Barsan W, Broderick J, Sheppard GL, Spilker J, Kongable GL, Massey S, Reed R, Marler JR. Urgent therapy for stroke, part I: pilot study of tissue plasminogen activator administered within 90 minutes. Stroke. 1992;23:632-640. [Abstract/Free Full Text]
  20. Haley EC Jr, Brott TG, Sheppard GL, Barsan W, Broderick J, Marler JR, Kongable GL, Spilker J, Massey S, Hansen CA, Torner JC, for the t-PA Bridging Study. Pilot randomized trial of tissue plasminogen activator in acute ischemic stroke. Stroke. 1993;24:1000-1004. [Abstract/Free Full Text]
  21. Thomas GR, Thibodeaux H, Bennett WF, Refino CJ, Badillo JM, Errett CJ, Zivin JA. Optimized thrombolysis of cerebral clots with tissue-type plasminogen activator in a rabbit model of embolic stroke. J Pharmacol Exp Ther. 1992;264:67-73. [Abstract/Free Full Text]
  22. Bednar MM, Raymond SJ, Gross CE. Tissue plasminogen activator: comparison of rate and route of administration in a rabbit model of embolic stroke. Neurol Res. 1993;15:405-408. [Medline] [Order article via Infotrieve]
  23. Thibodeaux H, Bennett WF, Errett CE, Thomas GR. Aspirin attenuates tissue-type plasminogen activator (t-PA) lysis of cerebral clots in the rabbit. The Pharmacologist. 1993;35:275. Abstract.
  24. Bednar MM, McAuliffe T, Raymond S, Gross CE. Tissue plasminogen activator reduces brain injury in a rabbit model of thromboembolic stroke. Stroke. 1990;21:1705-1709. [Abstract/Free Full Text]
  25. Gross CE, Bednar MM, Howard DB Sporn MB. Transforming growth factor-ß1 reduces infarct size after experimental cerebral ischemia in a rabbit model. Stroke. 1993;24:558-562. [Abstract/Free Full Text]
  26. Wilson JT, Bednar MM, McAuliffe TL, Raymond S, Gross CE. The effect of the 21-aminosteroid U74006F in a rabbit model of thromboembolic stroke. Neurosurgery. 1992;31:929-934. [Medline] [Order article via Infotrieve]
  27. Zar JH. Biostatistical Analysis. Englewood Cliffs, NJ: Prentice-Hall Inc; 1984:390-395.
  28. Kerins DM, Shuh M, Kunitada S, FitzGerald GA, Fitzgerald DJ. A prostacyclin analog impairs the response to tissue-type plasminogen activator during coronary thrombolysis: evidence for a pharmacokinetic interaction. J Pharmacol Exp Ther. 1991;257:487-492. [Abstract/Free Full Text]
  29. Korninger C, Collen D. Studies on the specific fibrinolytic effect of human extrinsic (tissue-type) plasminogen activator in human blood and in various animal species in vitro. Thromb Haemost. 1981;46:561-565. [Medline] [Order article via Infotrieve]
  30. Housholder GT, Moorrees LL. Fibrinolysis in the rat after short term aspirin therapy. J Oral Surg. 1980;38:412-416. [Medline] [Order article via Infotrieve]
  31. Leffler CW, Armstead WM, Shibata M. Role of eicosanoids in cerebral hemodynamics. In: Phillis JW, ed. The Regulation of Cerebral Blood Flow. Boca Raton, Fla: CRC Press, Publishers; 1993:297-313.
  32. Meyer JS, Rogers RL, McClintic K, Mortel KF, Lotfi J. Randomized clinical trial of daily aspirin therapy in multi-infarct dementia: a pilot study. J Am Geriatr Soc. 1989;37:549-555. [Medline] [Order article via Infotrieve]
  33. Fisher M, Zipser R. Increased excretion of immunoreactive thromboxane B2 in cerebral ischemia. Stroke. 1985;16:10-14. [Abstract/Free Full Text]
  34. Koudstaal PJ, Ciabattoni G, van Gijn J, Nieuwenhuis HK, de Groot PG, Sixma JJ, Patrono C. Increased thromboxane biosynthesis in patients with acute cerebral ischemia. Stroke. 1993;24:219-223. [Abstract/Free Full Text]
  35. Quintana A, Raczaka E, Giralt MT, Quintana MA. Effects of aspirin and indomethacin on cerebral circulation in the conscious rat: evidence for a physiological role of endogenous prostaglandins. Prostaglandins. 1983;25:549-556. [Medline] [Order article via Infotrieve]
  36. Abel-Halim MS, von Holst H, Meyerson B, Sachs C, Änggård E. Prostaglandin profiles in tissue and blood vessels from human brain. J Neurochem. 1980;34:1331-1333. [Medline] [Order article via Infotrieve]
  37. Frazer CE, Ritter JM. Recovery of prostacyclin synthesis by rabbit aortic endothelium and other tissues after inhibition by aspirin. Br J Pharmacol. 1987;91:251-256. [Medline] [Order article via Infotrieve]
  38. Ellis EF, Wright KF, Jones S, Richardson DW, Ellis CK. Effect of oral aspirin dose on platelet aggregation and vascular prostacyclin (PGI2) synthesis in humans and rabbits. J Cardiovasc Pharmacol. 1980;2:387-397.[Medline] [Order article via Infotrieve]
  39. Copeland JR, Willoughby KA, Tynan TM, Moore SF, Ellis EF. Endothelial and non-endothelial cyclooxygenase mediate rabbit pial arteriole dilation by bradykinin. Am J Physiol. 1995;268:H458-H466. [Abstract/Free Full Text]
  40. Asano T, Gotoh O, Koide T, Takakura K. Ischemic brain edema following occlusion of the middle cerebral artery in the rat, II: alteration of the eicosanoid synthesis profile of brain microvessels. Stroke. 1985;16:110-113. [Abstract/Free Full Text]
  41. Levin RI, Harpel PC, Weil D, Chang T-S, Rifkin DB. Aspirin inhibits vascular plasminogen activator activity in vivo. J Clin Invest. 1984;74:571-580.
  42. Buchanan MR, Hirsh J. Effect of aspirin and salicylate on platelet-vessel wall interactions in rabbits. Arteriosclerosis. 1984;4:403-406. [Abstract/Free Full Text]
  43. Korbut R, Bryska-Danek A, Gryglewski RJ. Fibrinolytic activity of 6-keto prostaglandin E1. Thromb Haemost. 1983;50:893. Letter. [Medline] [Order article via Infotrieve]
  44. Moore PK, Hussaini I, Bhardwaj R. Prostanoids stimulate fibrinolysis in the rat. Arch Int Pharmacodyn. 1988;296:155-162.
  45. Crutchley DJ, Conanan LB, Maynard JR. Stimulation of fibrinolytic activity in human skin fibroblasts by prostaglandins E1, E2 and I2.J Pharmacol Exp Ther. 1982;222:544-549. [Abstract/Free Full Text]
  46. Sobel BE, Nachowlak DA, Fry ETA, Bergmann SR, Torr SR. Paradoxical attenuation of fibrinolysis attributable to `plasminogen steal' and its implications for coronary thrombolysis. Coron Artery Dis. 1990;1:111-119.
  47. 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;20:2072-2079.



This article has been cited by other articles:


Home page
 StrokeHome page
C. Cornu, F. Boutitie, L. Candelise, J. P. Boissel, G. A. Donnan, M. Hommel, A. Jaillard, and K. R. Lees
Streptokinase in Acute Ischemic Stroke: An Individual Patient Data Meta-Analysis : The Thrombolysis in Acute Stroke Pooling Project
Stroke, July 1, 2000; 31(7): 1555 - 1560.
[Abstract] [Full Text] [PDF]

Home page
 StrokeHome page
M. M. Bednar and C. E. Gross
Antiplatelet Therapy in Acute Cerebral Ischemia
Stroke, April 1, 1999; 30(4): 887 - 893.
[Abstract] [Full Text] [PDF]

Home page
 StrokeHome page
S. Vanderschueren, I. Van Vlaenderen, and D. Collen
Intravenous Thrombolysis With Recombinant Staphylokinase Versus Tissue-Type Plasminogen Activator in a Rabbit Embolic Stroke Model
Stroke, September 1, 1997; 28(9): 1783 - 1788.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Thomas, G. R.
Right arrow Articles by Bennett, W. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Thomas, G. R.
Right arrow Articles by Bennett, W. F.

Stroke Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 1995 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.