From the Department of Clinical Neuroscience (J.M., H.S.M.), King's
College School of Medicine and Dentistry and Institute of Psychiatry;
Department of Cardiovascular Medicine (J.F.M.), University College Medical
School; and Department of Vascular Surgery (P.A.B., S.C.A.F.), King's
College Hospital, London, UK.
Correspondence to Dr Hugh Markus, Department of Neurology, Institute of Psychiatry, De Crespigny Park, London, SE5 8AF, UK. E-mail h.markus{at}iop.bpmf.ac.uk
Methods and Results—Transcranial Doppler
ultrasound recordings from the ipsilateral middle cerebral
artery were made after carotid endarterectomy in 12
control patients and 12 patients receiving intravenous GSNO
from the induction of anesthesia until 2 hours after skin
closure. Recording times were 0.5 to 3.5, 6 to 7, and 24 to 25
hours after skin closure. The Doppler signal was recorded onto
tape, and analysis for ES was performed, with the investigators
blinded to treatment group. All patients received aspirin 300 mg/d
before surgery and 5000 IU of heparin during surgery. The median
(range) number of ES detected during the initial 3-hour postoperative
recording was markedly reduced in the GSNO group compared with
the control group: 7.5 (0 to 61) versus 38.5 (1 to 219)
(P=0.018). This difference persisted until 6 hours after
surgery.
Conclusions—Despite the administration of aspirin and heparin,
frequent embolization occurred and was markedly reduced after the
administration of GSNO. This demonstrates the potential use of
platelet-specific nitric oxide donors in the treatment of
thromboembolic disease. This model of cerebral embolism may allow
determination of the effectiveness of new antiplatelet agents in
humans.
Recently, it was demonstrated that circulating cerebral emboli can be
detected with the use of transcranial Doppler
ultrasonography.5 Emboli appear as high-intensity
transient signals with typical acoustic characteristics. This technique
has been shown to be highly sensitive and specific in validation
studies both in vitro and in animal models.6 7
Embolic signals (ES) have been reported in a wide variety of patient
groups with potential embolic sources such as carotid artery disease,
atrial fibrillation, and cardiac valvular
disease.8 At carotid
endarterectomy, endothelial
denudation takes place, and the outer layers of the
arterial media are exposed, resulting in a potent
thrombogenic surface on which platelet adherence and aggregation
occur. Asymptomatic ES are frequent after carotid
endarterectomy,9 10 and
recent studies demonstrate that a high frequency of ES during the early
postoperative phase correlates with early stroke
risk.11 12 This situation provides a potential
model in which to test the efficacy of new antiplatelet agents. The
frequency of ES in this situation may provide sufficient power to allow
the evaluation of therapies in relatively small numbers of
patients.
In addition to its effects on vascular tone, NO inhibits platelet
aggregation by stimulating soluble guanylate cyclase,
thereby increasing cGMP,13 which leads to reduced
platelet adhesion and aggregation.14 Organic
nitrates, which act through the release of NO, reduce platelet
deposition and thrombus formation after angioplasty in pigs but often
at doses that cause hypotension.15 16 Similarly,
in humans, organic nitrates induce hypotension at doses required for an
antiplatelet effect.17
S-Nitrosoglutathione (GSNO) is a stable
S-nitrosothiol from which NO is released by the action of
enzymes associated with platelet membranes.18
In animals and humans, GSNO has significant antiplatelet action at
doses that cause less hemodynamic effect than
conventional NO donors.19 In a previous study, we
demonstrated that platelet activation occurs after coronary
angioplasty and that this activation can be prevented by the
administration of GSNO.20 GSNO has also been
shown to inhibit platelet activation in the setting of acute
myocardial infarction and unstable angina.21 In
the present study, we examined the hypothesis that GSNO prevents
platelet aggregation and adherence and therefore subsequent
cerebral thromboembolism, as determined with Doppler ultrasound,
immediately after carotid endarterectomy.
GSNO was administered as an intravenous infusion beginning
at the induction of anesthesia at a rate of 2.2 nmol
· kg-1 · min-1
and, if tolerated, increasing to a rate of 4.4 nmol ·
kg-1 · min-1 at 10
minutes and continuing until 2 hours after skin closure. The criterion
for termination of the infusion was a drop in mean arterial
pressure (MAP) of
A commercially available transcranial Doppler machine
(TC 4040; EME/Nicolet Ltd) was used to record from the ipsilateral
middle cerebral artery. A sample volume of 5 mm and a mean
recording depth of 52.77 mm (range, 48 to 56 mm) were
used. Recordings were made to digital audiotape for off-line
analysis. In all patients, a 1-hour recording was made
in the 24 hours preceding surgery. Postoperatively, recordings
were made for 3 hours, beginning 30 minutes from skin closure, and for
1 hour at 6 and 24 hours after skin closure.
Analysis of recordings was performed by an observer
(J.M.) who was blinded to the clinical details, time of
recording, and study group. ES were identified by their
characteristic visual appearance and chirping sound. An intensity
threshold of >7 dB was used because previous studies have shown that
this improves interobserver reproducibility in the detection of
ES.23 24 The intensity of the ES was calculated
by comparison with the built-in intensity scale on the Doppler as
previously described.24 Interobserver
reproducibility of ES detection was determined by 2 observers who
independently analyzed 4 hours of tape recording from 6
patients with symptomatic carotid artery stenosis.
The proportion of specific agreement23 between
observer 1 and observer 2 (H.S.M.) was 0.98 and that between observer 2
and observer 1 was 0.92. For both validation and patient studies, the
timing of all ES was noted, and each signal was saved to the hard drive
for an analysis of intensity.
The number of ES in each group was not normally distributed, and
therefore comparison between the number of ES detected in each group
was performed with the Mann-Whitney U test for
nonparametric data. For comparison of ES intensity, an
unpaired t test was used.
In the control group receiving no GSNO, the median (range) number of ES
detected during the 3-hour postoperative recording period was
38.5 (1 to 219). At 6 hours, the median (range) number of ES per hour
had fallen to 5.5 (0 to 105). By 24 hours, the median (range) number of
ES per hour had fallen to 0 (0 to 30).
On an intention-to-treat analysis, there was a significant
reduction in asymptomatic embolization in the GSNO group
during both the initial 3-hour recording period and hour 6
(Figure 1
Individual numbers of ES in the 2 groups during the first 3 hours are
given in Figure 2
A total of 715 ES were detected in the initial 3-hour postoperative
recording in the control group compared with 197 in the GSNO
group. During this period, ES in the GSNO group were significantly less
intense than those in the control group: the mean (SD) was 12.30 (4.30)
versus 14.27 (4.71) dB (P<0.0001).
In the control group, three patients experienced
perioperative ischemic events. One patient had
a stroke 20 hours after surgery in the ipsilateral internal carotid
artery territory with right facial and arm weakness and dysphasia; this
patient recovered fully over a 3-day period, and a CT brain scan showed
a cortical infarct. Two additional control patients, both of whom had
contralateral carotid occlusion, had strokes in the contralateral
internal carotid artery territory. In 1 patient, aphasia and
hemiparesis were noted on recovery from anesthesia, and he
was left with a residual deficit; a CT scan showed a large area of
infarction in the internal carotid artery watershed areas. The second
patient developed left hemiparesis and coma 3 days after surgery and
died; a brain CT scan showed an intracerebral
hemorrhage. There were no strokes or transient ischemia
attacks in the GSNO group, but 1 patient was noted to have developed
internal carotid artery occlusion on the side of the
endarterectomy on repeat carotid duplex before
discharge.
The patient numbers were too small and the study was not designed to
determine whether there was a significant reduction in clinical events.
However, there were no clinical ischemic events in the GSNO
group. In the control group, there was 1 minor stroke in the
ipsilateral internal carotid territory and 2 strokes in the
contralateral internal carotid artery territory. The latter occurred in
patients with contralateral occlusion; in 1 patient, the pathogenesis
was probably intraoperative hemodynamic
ischemia, whereas in the other, it was hemorrhage,
probably due to a hyperperfusion syndrome. Nevertheless, there
certainly was no increase of events in the GSNO-treated group.
In addition to the lower frequency of ES in the GSNO-treated group, the
mean intensity of the individual ES was lower in the GSNO group.
Theoretically, ES intensity would be expected to increase with
increasing embolic size, and this has been confirmed in experimental
models.6 ES intensity also depends on embolus
composition, with thrombi resulting in more intense ES than
platelet aggregates in experimental models.6
Assuming the embolus compositions were similar in the GSNO-treated and
untreated groups, our results are consistent with emboli in the
GSNO-treated group representing smaller platelet
aggregates. However, there are a number of technical difficulties
associated with the interpretation of ES composition or size on the
basis of intensity alone.6
The present study demonstrates that ultrasonic ES detection allows
the in vivo efficacy of antiplatelet agents to be evaluated in
relatively small numbers of patients. In this study, we tested the
ability of GSNO to reduce embolization from an arterial
luminal surface surgically denuded of endothelium.
However, the same technology may allow the effectiveness of agents to
be tested on embolism resulting from other clinical situations, such as
percutaneous transluminal coronary angioplasty.
The use of this model may allow effective initial evaluation of new
antiplatelet therapies in small studies before their assessment in
large and expensive clinical trials. It should also allow dose-response
studies to be performed before such trials are begun.
Received February 10, 1998;
revision received May 22, 1998;
accepted June 3, 1998.
© 1998 American Heart Association, Inc.
Clinical Investigation and Reports
S-Nitrosoglutathione Reduces the Rate of Embolization in Humans
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—Antiplatelet
agents presently used in the secondary prevention of
cardiovascular disease fail to prevent the
majority of cases of recurrent stroke and systemic embolization. An
evaluation of the efficacy of new agents is hampered by a lack of in
vivo models in humans. Asymptomatic cerebral embolic
signals (ES) may be detected with the use of transcranial
Doppler ultrasonography. These signals are particularly common
after carotid endarterectomy, and this provides a
situation in which new antiplatelet agents can be evaluated. With
this model, we determined the effectiveness of
S-nitrosoglutathione (GSNO), a nitric oxide donor with
relative platelet specificity, in reducing cerebral
embolization.
Key Words: ultrasonics • drugs • platelet aggregation inhibitors • endothelium-derived factors
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Although aspirin is effective in the secondary prevention
of thromboembolic disease,1 many strokes and
systemic embolic events occur despite its use, and there is a need for
more effective antiplatelet agents. Potential agents include
ticlopidine, clopidogrel, the new generation of
glycoprotein IIb/IIIa antagonists, and nitric
oxide (NO) donors. Current methods available for the evaluation of such
agents are not ideal; ex vivo studies such as platelet aggregation
provide an indicator of potential efficacy but may not be completely
representative of biological effectiveness in
vivo.2 Presently available animal
models3 are not always
representative of the situation in humans. Because of
the low incidence of outcome events, large, expensive, multi-center
clinical trials are required in which as many as 19 000 patients may
need to be recruited.4 A reliable model in which
to evaluate the efficacy of new agents in vivo in humans, with small
patient numbers, would be useful in bridging the gap between laboratory
studies and clinical trials.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
We studied 24 patients who were undergoing carotid
endarterectomy for symptomatic internal
carotid artery stenosis of >70% determined angiographically
with the European Carotid Surgery Trial method of
measurement.22 Their demographic characteristics
are summarized in the Table
. The study was approved by the King's
Healthcare Ethical Committee, and informed consent was obtained from
all subjects. All patients were pretreated with aspirin 300 mg/d for
1 week before surgery and were administered 5000 IU of sodium heparin
intravenously during the operation and before carotid
clamping. Patients were allocated either to treatment with GSNO (n=12)
or to no additional treatment (n=12). In view of the potential side
effects of GSNO (eg, hypotension and bleeding), neither the surgeon nor
the anesthetist was blinded to treatment, but all data analysis
was performed with the investigator blinded to the study
group.
View this table:
[in a new window]
Table 1. Patient Characteristics in Control and GSNO-Treated
Groups 10 mm Hg. If MAP returned to the pre-GSNO
level, an additional test infusion was administered and maintained at
the lower rate. If again there was a drop in MAP of 10 mm Hg,
the infusion was stopped permanently.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Details of the patient groups are given in the Table
. All patients
underwent technically successful carotid
endarterectomy. Of the 12 patients allocated to
receive GSNO, 10 tolerated the full dose with no clinically apparent
side effects. In 2 patients, there was a fall in MAP of >10
mm Hg, and the infusion was suspended. The infusion was tolerated at
the half-maximum dose in 1 patient, but in the second patient, even
this infusion rate caused hypotension, and GSNO administration was
stopped. ). The median (range) number of
ES detected in the first 3-hour recording was 7.5 (0 to 61)
(P=0.018 versus controls). During hour 6, the median (range)
number of ES was 0 (0 to 41) (P=0.014 versus controls). By
24 hours, the rate of embolization in both groups was low, and there
was no difference between control and treatment groups: the median
(range) was 0 (0 to 37) (P=0.74 versus controls).
View larger version (17K):
[in a new window]
Figure 1. ES per hour versus time from skin closure
according to treatment group. Solid line indicates median value; boxes,
25th to 75th percentile; and vertical lines, range. . The 2 patients with
frequent ES in the GSNO group were the 2 in whom a full dose of GSNO
could not be given because of hypotension. Exclusion of both patients
who did not tolerate a full dose increased the difference between the 2
groups: at 3 hours in the GSNO group, the median (range) number of ES
was 5.5 (0 to 20) (P=0.005 versus controls); at 6 hours, the
median (range) was 0 (0 to 7) (P=0.003); and at 24 hours,
the median (range) was 0 (0 to 3) (P=0.47).
View larger version (9K):
[in a new window]
Figure 2. Scatter distribution of total ES detected in each
patient during 3-hour postoperative recording according to
treatment group. *Patient in whom infusion was stopped. **Patient
receiving half-maximum dose.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
GSNO resulted in a highly significant reduction in the frequency
of embolization after carotid endarterectomy. This
reduction was maintained at 6 hours after surgery, even though the
infusion was stopped 2 hours after surgery. In the majority of
patients, GSNO was well tolerated, but in 2 individuals, it resulted in
a drop in blood pressure. In 1 patient, it could be continued at half
the maximum dose, but in the other, it had to be stopped. Our data
provide further evidence for the importance of NO in preventing
platelet adhesion and aggregation in vivo and illustrate the
potential use of platelet-specific NO donors that may have
relatively less hypotensive effect, for a given antiplatelet
effect, than conventional NO donors such as
nitroglycerin. GSNO has been administered only as an
intravenous infusion, but future oral analogs, or other
platelet-specific NO donors, may be effective in preventing
thromboembolism. Our results also demonstrate that aspirin alone fails
to prevent many embolic events.
Acknowledgments
This work is supported by a British Heart Foundation project
grant. We thank Dr Dave Madge for providing the GSNO.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
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