(Hypertension. 1997;29:1314-1321.)
© 1997 American Heart Association, Inc.
Articles |
Cyclosporine Impairs the Ability of Human Platelets to Mediate Vasodilation
From The University of Iowa, Department of Internal Medicine, Iowa City, (H.J.O., T.G.H.) and the University of Nebraska Medical Center, Department of Internal Medicine, Section of Cardiology, Omaha (M.T.O.).
Correspondence to Helgi Óskarsson, MD, Department of Internal Medicine, Division of Cardiovascular Diseases, University of Iowa Hospitals and Clinics, 200 Hawkins Dr, Iowa City, IA 52242. E-mail helgi-oskarsson{at}uiowa.edu
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Abstract |
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Abstract Cyclosporine causes various platelet abnormalities. Whether it affects the ability of platelets to mediate vasodilation is unknown. Platelets were isolated from healthy volunteers and 13 heart transplant patients on cyclosporine. When perfused through preconstricted normal rabbit carotid arteries, activated platelets from transplant patients failed to cause vasorelaxation, whereas normal platelets produced significant vasodilation (-4.0±1.9% versus 30±3% [P<.0001] change in vessel diameter, respectively). When normal platelets were exposed to cyclosporine in vitro, they lost their ability to cause vasodilation in a dose- and time-dependent fashion. However, when activated and perfused through quiescent, N
-nitro-L-arginine–pretreated
arteries, platelets from transplant patients and normal
platelets caused similar degrees of vasoconstriction. The amount of
adenosine triphosphate in the supernatant from
activated cyclosporine-exposed and control
platelets was similar (1.7±0.4 versus 1.5±0.3 µmol/L
[P=NS], respectively). However, concomitant perfusion of
activated platelets from transplant patients impaired
acetylcholine-mediated, endothelium-dependent
vasodilation but perfusion of normal platelets did not. Although
cyclosporine-exposed platelets showed an impaired
ability to produce vasorelaxation, supernatant from the same
platelets caused near normal vasodilation. Human platelets
exposed to cyclosporine have an impaired ability to mediate
vasodilation. This is not due to increased platelet-mediated
vasoconstriction or a decrease in the release of platelet-derived
nucleotides but rather to a short-acting compound released
by cyclosporine-exposed platelets that interferes with
endothelium-dependent vasodilation.
Key Words: cyclosporine • platelets • endothelium • vasoconstriction
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Introduction |
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Cyclosporine has had major impact on the results of organ transplantation. However, its use is associated with significant adverse effects, including cardiovascular complications such as systemic hypertension,1 2 3 renovascular impairment, and thromboembolic events including thrombotic microangiopathy.4 5 The mechanism or mechanisms for these side effects are not clear but are likely to be multifactorial.
Cyclosporine impairs endothelium-dependent vasorelaxation.6 7 8 9 It increases production of thromboxane A210 11 12 and endothelin,13 14 15 which are both potent vasoconstrictors. Cyclosporine causes an increase in sympathetic tone,16 17 and in addition, it increases vascular responses to vasoconstrictors such as phenylephrine, norepinephrine, and angiotensin II.8 18 19 It also causes adverse effects on intravascular hemostatic equilibrium, favoring a prothrombotic state by decreasing the release of prostacyclin8 and nitric oxide (NO)6 7 8 9 from endothelium while increasing thromboxane A2 synthesis and serotonin release by platelets.12 20 21 22 Cyclosporine therapy also increases spontaneous activation of platelets,23 makes platelets hyperaggregable in response to various agonists,12 20 21 22 23 and causes increased expression of fibrinogen receptors on their surface membranes.21 22
However, although cyclosporine has been shown to affect vasomotor responses and platelet function separately, its effect on platelet-mediated vasomotor changes has not been studied. Normally, activated platelets cause vasodilation via secretion of ADP/ATP, which in turn causes endothelium-dependent vasorelaxation,24 25 26 which overpowers the effects of platelet-derived vasoconstrictors such as thromboxane A2 and serotonin. However, it is not known how cyclosporine influences the balance between these vasodilative and vasoconstrictive forces mediated by activated platelets. Thus, the purpose of this study was to elucidate the effect of cyclosporine on platelet-mediated vasomotor tone.
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Methods |
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Heart Transplant Patients and Healthy Subjects
After informed consent was obtained, blood samples were collected from healthy volunteers and 13 patients who had received a heart transplant at least 3 months before blood donation and were currently on cyclosporine as part of their immunosuppressive regimen. All participants were between 18 and 65 years old. No individuals enrolled in the study had total cholesterol greater than 5.7 mmol/L. (We have observed no significant impairment in vasodilation mediated by platelets from healthy volunteers with total cholesterol up to 5.7 mmol/L; data not shown). No participant was a smoker; no one was taking medications known to affect platelet function (except cyclosporine); and no one had creatinine greater than 180 mmol/L (excluding patients with all but mild renal insufficiency, which by itself is not likely to cause significant platelet malfunction) or other disorders known to significantly affect platelet function. The study protocol was reviewed and approved by the Human Investigations Committee of the University of Nebraska Medical Center.
Platelet Isolation
Blood was drawn between 8 and 11 AM. Venous blood
(90 mL) was gently collected from a forearm vein, without venous
stasis, into an acid citrate/dextrose solution (7:1, vol/vol) of the
following composition (mmol/L): sodium citrate 85, citric acid 71, and
dextrose 9.01. The collected blood samples were centrifuged at
100g at room temperature for 15 minutes to prepare
platelet-rich plasma. Platelets were isolated and washed
following a modified method of Mustard27 as previously
described.26 Briefly, platelet-rich plasma as well as
the subsequent resuspended platelet solution was differentially
centrifuged and washed four times in modified Tyrode's buffer
(mmol/L: NaCl 140, NaHCO3 14.3, KCl 3.2,
NaH2PO4 0.5, albumin 0.06, and dextrose
6.6 without Ca2+ and Mg2+). After the final
spin, platelets were resuspended in complete Tyrode's buffer (with
2.6 mmol/L MgCl2 and 4.7 mmol/L
CaCl2), counted by a Coulter counter (Coulter Corp), and
adjusted to the desired final concentration (2x108
platelets/mL).
Platelet Aggregation
Platelet aggregation was determined by the optical method in
a four-channel platelet aggregometer (Bio/Data). Light
transmittance was measured and recorded. Aggregation studies were
performed within 1 hour after preparation in both groups of
participants. Platelets suspended in Tyrode's buffer
(1x108 cells/mL) were activated with thrombin (0.1
U/mL), and aggregation responses were recorded at 4 minutes (at
which time platelets from most platelet donors have reached a
maximal response to thrombin). The results were expressed as the
percent change in light transmittance, with light transmittance of
Tyrode's buffer alone taken as 100% and that of the same Tyrode's
buffer with platelets in suspension taken as 0%.
Vessel Setup
Normal male New Zealand White rabbits (2.5 to 3.0 kg) were
euthanized by an overdose of sodium pentobarbital (50 mg/kg IV).
Heparin (150 U/kg IV) was administered to prevent blood coagulation.
Common carotid arteries were excised and immediately placed in cold
(5°C to 10°C), aerated Krebs' buffer of the following composition
(mmol/L): NaCl 118.3, KCl 4.7, CaCl2 2.5, MgSO4
1.2, KH2PO4 1.2, NaHCO3 25, and
dextrose 11.0. Each artery was cleaned of perivascular tissue, and a 1-
to 1.5-cm-long segment was obtained for use in the study.
Arterial segments were placed in a Plexiglas isolated organ
chamber, cannulated with dual plastic cannulas measuring 1.27 mm
in diameter, and secured with 4-0 silk suture. The carotid
arterial segments were continuously superfused with aerated
Krebs' buffer (95% O2/5% CO2, at pH
approximately 7.4 and 37°C) and perfused intraluminally with aerated
Krebs' buffer at a constant nonpulsating flow of 1.0 mL/min, using a
peristaltic pump (Cole-Parmer).
Dual tubing was joined at a Y-connector downstream from a pump head to accommodate perfusion of chemicals and platelets that could be kept separate until the solutions mixed at the Y-connector. The perfusion time from the Y-connector through a heat exchanger (to maintain temperature at 37°C) to the arterial segment was 90 seconds. Downstream of the vessel, the perfusate flowed through a column of plastic tubing designed to create a continuous intraluminal distending pressure of 60 mm Hg.
Arterial segments were imaged by videomicroscopy. A zoom stereomicroscope (Olympus) connected to a video camera (Panasonic) projected vessel images onto a television monitor and was on-line with an image-acquisition system (Koala Acquisitions Inc). Responses to agonists or activated platelets were observed via the monitor, allowed to reach a stable plateau (3 to 4 minutes), digitally acquired, and archived for subsequent analysis with a quantitative edge detection program (Image, National Institutes of Health). Vascular responses are reported as percent change in vessel diameter.
Materials
Acetylcholine hydrochloride, phenylephrine
hydrochloride, hydrocortisone, 6-mercaptopurine sodium nitroprusside,
and N-nitro-L-arginine were
purchased from Sigma Chemical Co. Cyclosporine was obtained
from Sandoz. Thrombin was obtained from Armour Pharmaceutical Co.
LY-53,857 was purchased from Research Biochemicals International and
SQ-29,548 from BIOMOL Research Laboratories, Inc.
Study Protocol
After cannulation, the vessels were longitudinally stretched to
the approximate in situ length and allowed to equilibrate for 90
minutes before the first intervention. To ensure normal constrictor and
dilator responses, repeated doses of abluminally superfused
phenylephrine (10-6 mol/L) and
intraluminally perfused acetylcholine (10-6
mol/L) were applied until reproducible results were obtained.
Platelets from both healthy volunteers and heart transplant patients were treated the same way. After isolation and preparation into the final buffer, the platelets were kept at room temperature. We have previously found that the ability of the platelets to produce vasorelaxation remains unchanged up to at least 6 hours when kept in the Tyrode's buffer at room temperature. Nevertheless, all studies involving platelet-mediated vasomotor tone were performed within 4 hours of isolation in this protocol.
Platelet-Mediated Vasodilation
For examination of the ability of activated
platelets to mediate vasodilation, arterial segments
were preconstricted via abluminal administration of
phenylephrine (10-5 mol/L) after a
baseline resting diameter was obtained. Once the segments had reached a
stable preconstricted plateau, the intraluminal perfusate was
switched from aerated Krebs' buffer through both perfusion arms to
thrombin (0.2 U/mL) in Krebs' buffer through one perfusion arm and
Tyrode's buffer (with Ca2+ and Mg2+) through
the other perfusion arm, mixing at the Y-connector.
The artery was allowed to equilibrate while being perfused with this
solution (Tyrode's and thrombin solution) for 4 minutes, after which
an image of the vessel was acquired as preconstrictive baseline for
analysis. Subsequently, the arm perfused with Tyrode's buffer
alone was switched to perfusion of platelets (1x108
and/or 2x108 cells/mL) suspended in Tyrode's buffer,
resulting in a one-to-one mix of the thrombin- and
platelet-containing solutions at the Y-connector,
leading to platelet activation and aggregation for 90 seconds
before the thrombin/platelet solution reached the artery. Each dose
of platelets was perfused for 3 to 4 minutes, at which time the
artery had reached a stable plateau in response to the platelets.
An image of the artery was acquired and the diameter compared with the
preconstrictive baseline image. Neither thrombin at the final
concentration (0.1 U/mL), Tyrode's buffer, nor unactivated
platelets alone evoked any noticeable changes in vascular diameter
when perfused through the arterial segment in this
model.
Effect of Cyclosporine, Hydrocortisone, and
6-Mercaptopurine In Vitro on Normal Platelets
For study of the effect of in vitro exposure of these compounds
on the ability of normal platelets to cause vasodilation,
platelets from healthy donors were incubated for 1 to 4 hours in
Tyrode's solution with the desired concentrations of the drugs, while
platelets from the same donors were incubated in Tyrode's buffer
alone. To control for possible intrinsic vasomotor effects of the
drugs, we obtained preconstrictive baseline vessel images after
perfusing the artery for 4 minutes with a mixture of thrombin in
Krebs' and Tyrode's buffers along with the drug being tested each
time, before the corresponding drug/platelet solutions were tested.
In the concentration used, these drugs had no noticeable effects on the
vessel diameter.
Platelet-Mediated Vasoconstriction
For examination of platelet-mediated
vasoconstrictive responses, platelets were perfused
through a quiescent (non-preconstricted) arterial segment.
To eliminate the ability of the vessel to elicit
endothelium-derived NO (EDNO)–dependent vasodilation,
we pretreated the artery with the NO synthase inhibitor
N-nitro-L-arginine (50
µmol/L) both abluminally and intraluminally for 30 minutes before the
platelet experiments. Subsequently, it was maintained in the
abluminal perfusate throughout the platelet perfusion
experiments to ensure continuous inhibitory effect. We have
previously confirmed adequate inhibition of EDNO-dependent vasodilation
with this dose of
N-nitro-L-arginine by showing a
lack of vasorelaxation in response to intraluminally perfused normal
platelets26 as well as acetylcholine
(10-5 mol/L) in a preconstricted artery. Once
the artery had reached a stable quiescent plateau, the intraluminal
perfusate was switched from aerated Krebs' buffer through both
perfusion arms to thrombin (0.2 U/mL) in Krebs' buffer through one
perfusion arm and Tyrode's buffer (with Ca2+ and
Mg2+) through the other. The artery was allowed to
equilibrate with this solution for 4 minutes, after which a baseline
resting image of the vessel was acquired. Subsequently, platelets
suspended in Tyrode's buffer were substituted for the Tyrode's buffer
alone and mixed one-to-one with the thrombin solution at the
Y-connector (final concentrations of
5x107 and 1x108 platelets/mL) and
perfused through the artery. Responses were analyzed as the
percent reduction in vessel diameter compared with resting baseline
diameter. Thrombin at the final concentration (0.1 U/mL), Tyrode's
buffer, or unactivated platelets in Tyrode's buffer alone
evoked no noticeable changes in vessel diameter.
Effects of Thromboxane A2 and
Serotonin Receptor Inhibitors on
Platelet-Mediated Vasomotor Responses
Platelet-mediated vasodilation was studied as
described above but now before and after pretreatment of the artery
with the serotonergic receptor (5-HT2) blocker LY-53,857
(10-5 mol/L) and the thromboxane
A2 receptor antagonist SQ-29,548
(10-5 mol/L). These concentrations of
LY-53,857 and SQ-29,548 completely inhibited the vasoconstriction
caused by intraluminal perfusion of serotonin
(10-5 mol/L) and the thromboxane
A2 analogue U46619 (10-5 mol/L) in
this model (data not shown). LY-53,857 and SQ-29,548 were perfused in
combination intraluminally and superfused abluminally for 40 minutes
before the platelet study. They were subsequently kept in the
superfusate (abluminally) during the platelet perfusion
experiments to ensure continued receptor blockade. Baseline vessel
images were obtained after the artery had been perfused intraluminally
for 4 minutes with the mixture of thrombin in Krebs' and complete
Tyrode's buffer, during continuous abluminal LY-53,857 and/or
SQ-29,548 superfusion, controlling for possible vasomotor effects
elicited by the antagonists themselves in the
superfusate.
Measurement of ATP Released From Activated Platelets
Stirred platelets (1x108 cells/mL) in Tyrode's
buffer (with Ca2+ and Mg2+) were warmed to
37°C in a Chronolog Lumi-aggregometer, with luciferase (2 nmol/L)
added. Subsequently, the platelets were activated with 0.1
U/mL thrombin, and aggregation (light transmittance) and
chemiluminescence (photomultiplier tube) were measured
simultaneously. The results of the chemiluminescence light
signal were compared with an ATP standard curve and converted to
micromoles per liter.
Effects of Intraluminally Perfused Platelets on
Acetylcholine-Mediated Endothelium-Dependent
Vasodilation
Baseline resting and preconstricted vessel diameter were
obtained as described above. Subsequently, during concomitant perfusion
with thrombin in Krebs' and Tyrode's solution (1:1), dose-response
curves to incremental doses (10-8,
10-7, 10-6, and
10-5 mol/L) of abluminally applied
acetylcholine were obtained, with 4 minutes allowed for each dose
response to reach a plateau. Then, after new baseline vessel diameters
had been obtained during perfusion of thrombin-activated
platelets, responses to the same doses of acetylcholine were
studied again while the artery was concomitantly perfused with
thrombin-activated normal or in vivo
cyclosporine-exposed platelets (5x107
platelets/mL).
Vasomotor Responses to the Supernatant From Activated
Platelets
In a preconstricted (phenylephrine,
10-5 mol/L) artery, vasodilative responses to
thrombin-activated (0.1 U/mL) normal and in vitro
cyclosporine-exposed platelets (1x108
platelets/mL) were recorded as described above. Subsequently,
cyclosporine-treated platelets from the same donors
were incubated in Tyrode's buffer at 37°C and activated with
thrombin (0.1 U/mL) for 3 minutes. The platelet aggregates were
centrifuged (150g), and the supernatant was
collected and kept at room temperature for an additional 5 minutes.
Subsequently, vascular responses were recorded during perfusion of
the supernatant through the same preconstricted artery.
Statistical Analysis
All data are presented as mean±SEM. The changes in
vessel diameter are expressed as percent change in diameter, where a
positive number represents vasodilation and a negative number
represents vasoconstriction. The number of experiments (n)
refers to the number of platelet donors. Statistical
analysis was performed with one- or two-way ANOVA, including
repeated measures and pairwise multiple comparisons
(Student-Newman-Keuls and Dunnett's methods) as appropriate. A value
of P<.05 was considered significant.
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Results |
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Effect of In Vivo Exposure of Platelets to Cyclosporine on Their Ability to Cause Vasodilation
Platelets isolated from 13 heart transplant patients on cyclosporine failed to cause vasorelaxation, whereas platelets from 10 healthy volunteers caused significant vasodilation when perfused through a preconstricted normal rabbit carotid artery (Fig 1
). Demographic characteristics of
the two groups are shown in Table 1.
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Effect of In Vitro Exposure of Platelets to
Cyclosporine on Their Ability to Cause
Vasodilation
When platelets from healthy volunteers were incubated in
various concentrations of cyclosporine for 4 hours, they
lost their ability to cause vasorelaxation in a dose-dependent manner
(Fig 2A). Furthermore, when platelets from healthy
donors were incubated for 2 and 4 hours in buffer containing 250 nmol/L
(300 ng/mL) cyclosporine, they lost their ability to dilate
a normal preconstricted artery in a time-dependent fashion (Fig 2B).
All the heart transplant patients who donated platelets were taking
prednisone and azathioprine in addition to cyclosporine.
However, hydrocortisone and 6-mercaptopurine (the active metabolite of
azathioprine) did not impair the ability of normal platelets to
produce vasodilation when tested in vitro (Fig 3).
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Effect of Cyclosporine on Platelet-Mediated
Vasoconstriction
One possible explanation for our observation is that
cyclosporine-treated platelets are unable to cause
normal vasodilation because of overwhelming
vasoconstrictive effects caused by increased release of
platelet-derived vasoconstrictors such as thromboxane
A2 and serotonin.10 11 12 However,
when platelets from heart transplant patients were perfused through
a quiescent (non-preconstricted) artery, in which the NO synthase had
been inhibited, they caused a degree of vasoconstriction similar to
that of platelets from healthy volunteers (Fig 4).
Furthermore, inhibition of thromboxane A2 and
serotonergic (5-HT2) receptors in the artery failed to
restore the ability of cyclosporine-treated
platelets to cause vasodilation in a normal preconstricted artery,
whereas the ability of normal platelets to dilate the artery
remained unchanged (Fig 5).
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Effects of Cyclosporine on ATP Release From
Activated Platelets
Since the effect of cyclosporine cannot be
explained by an overwhelming vasoconstrictive effect,
we proposed that activated cyclosporine-treated
platelets might release less ADP/ATP, which are the main agonists
for platelet-mediated vasorelaxation. However, ATP release from
thrombin-activated normal platelets after 4 hours of
incubation in Tyrode's buffer alone did not differ significantly from
that after incubation in Tyrode's buffer containing 300 ng/mL
cyclosporine (Table 2). The
cyclosporine-treated platelets aggregated more in
response to thrombin activation, consistent with previous
reports.20 21 22 23
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Effects of Cyclosporine-Exposed Platelets on
Acetylcholine-Mediated Vasodilation
In the absence of evidence for augmented
vasoconstrictive forces and results suggesting adequate
release of platelet-derived agonists for
endothelium-dependent vasodilation, we hypothesized
that cyclosporine-treated platelets might release a
compound that interferes with the normally observed EDNO-mediated
vasodilation. Thus, we compared endothelium-dependent
vasodilation caused by incremental doses of abluminally applied
acetylcholine with and without concomitant perfusion of normal and in
vivo cyclosporine-exposed platelets. As shown in Fig 6, cyclosporine-exposed platelets caused
significant impairment in acetylcholine-mediated vasodilation, whereas
normal platelets did not.
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Vasodilator Effects of Platelet Supernatant
In this experiment, we found that whereas perfusion of
activated cyclosporine-exposed platelets caused
mild vasoconstriction, the supernatant from the same platelets
produced normal vasodilation (Fig 7).
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Discussion |
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Cyclosporine has previously been shown to cause platelet abnormalities, including increased platelet aggregability,12 20 21 22 23 spontaneous platelet activation,23 thromboxane A2 and serotonin synthesis,20 21 22 and increased expression of fibrinogen receptors on the platelet surface.21 22 However, this is the first report showing that exposure of human platelets to cyclosporine, both in vivo and in vitro, impairs their ability to cause vasodilation.
Platelets from heart transplant patients, all of whom were on cyclosporine as well as prednisone and azathioprine, were unable to cause vasodilation when activated with thrombin and perfused through normal preconstricted rabbit carotid arteries. Nevertheless, in response to thrombin, the platelets from these patients aggregated to a similar degree as control platelets, confirming an intact activation response to the applied agonist. There was a slight, statistically significant difference in mean age of the heart transplant patients and the control subjects, but this difference is not likely to explain the observed results of the study. Similarly, there was a moderate but not statistically significant difference in total cholesterol levels between the patients and control subjects. Although platelets from patients with hypercholesterolemia have previously been shown to have a suppressed ability to mediate vasodilation,25 the cholesterol level in those patients was significantly higher than in our heart transplant group.
The results from the in vitro experiments substantiate the hypothesis that cyclosporine is responsible for the observed inability of platelets from the heart transplant patients to cause vasodilation. We showed that when platelets from healthy donors were incubated with clinically relevant concentrations of cyclosporine, they lost their ability to relax vessels in a dose- and time-dependent manner, whereas the other two immunosuppressive agents that the patients were on had no adverse effects. In fact, 6-mercaptopurine significantly increased the platelet-mediated vasorelaxation.
To start to address the mechanism for the observed cyclosporine-induced platelet defect, we tested three hypotheses: (1) Vasoconstriction mediated by increased release of vasoconstrictive substances during activation of cyclosporine-exposed platelets masks normal platelet-mediated endothelium-dependent vasodilation; (2) impairment in platelet-mediated vasodilation is due to a decrease in the release of ATP/ADP during activation of cyclosporine-exposed platelets; and (3) activated cyclosporine-exposed platelets release a bioactive substance (or substances) that interferes with the action of and/or decreases the release of EDNO.
Although previous reports have shown increased release of the two main platelet-derived vasoconstrictors, thromboxane A2 and serotonin, during activation of cyclosporine-exposed platelets,20 21 22 we did not find in our model that platelets from patients on cyclosporine produced more vasoconstriction than platelets from healthy volunteers. Furthermore, blocking of serotonergic (5-HT2) and thromboxane A2 receptors in the artery, which should leave the vasodilative forces mostly unopposed, did not improve the ability of cyclosporine-treated platelets to mediate vasodilation. Under the same conditions, normal platelets caused normal vasodilation, and acetylcholine-mediated vasorelaxation was normal. Together, these results allow us to discard the first hypothesis—that augmented vasoconstriction is responsible for the impaired ability of cyclosporine-treated platelets to cause vasodilation.
This study also allowed us to reject the second hypothesis. We tested whether cyclosporine-treated platelets release less ATP during activation than platelets from healthy volunteers. However, although a previous report has described a decrease in ATP/ADP content and release in platelets from patients on cyclosporine,23 we were unable to demonstrate a significant difference between the normal and cyclosporine-treated platelets. In addition, the fact that the supernatant from cyclosporine-treated platelets causes near normal dilation—a response that is inhibited by apyrase, an enzyme that breaks down nucleotides (data not shown)—further supports the hypothesis that sufficient amounts of ADP and ATP are released during activation of cyclosporine-exposed platelets to cause expected vasorelaxation.
In relation to the third hypothesis, concomitant intraluminal perfusion of activated cyclosporine-exposed platelets significantly impaired vasodilator responses to abluminally applied acetylcholine, whereas perfusion of normal platelets did not. These results suggest that a substance (or substances) is released from activated cyclosporine-exposed platelets, but not from normal platelets, that interferes with the vasodilative action of or impairs the release of EDNO. The observation that the supernatant from activated cyclosporine-exposed platelets produced significant vasodilation 5 minutes after platelet activation while the same platelets produced vasoconstriction when activated and perfused through the same vessel suggests that the interfering compound (or compounds) released during activation of the platelets has a short half-life.
Oxygen free radicals are strong candidates for the compound released by activated platelets with a short half-life and ability to interfere with EDNO-dependent vasodilation. Oxygen free radicals are released by activated platelets.28 29 30 31 They may affect platelet activation,30 31 32 and recent data suggest that they play an important role in coronary thrombosis.33 34 Oxygen free radicals also interact with and inactivate NO,35 36 and because of their reactive nature, they generally have a short half-life. The notion that cyclosporine may cause increased release of free radicals by platelets is supported by data showing that cyclosporine-induced impairment of endothelium-dependent vasodilation is mediated by excess free radical production in the vessel wall.9 What appears to be an immediate onset of action of the platelet-derived substance or substances makes it very unlikely that the responsible compound affects expression of the NO synthase gene in the endothelium. Furthermore, we have found that within 1 minute after perfusion of cyclosporine-exposed platelets, the artery responds with normal vasorelaxation to acetylcholine (data not shown), suggesting that the ability of the endothelium to make and release NO is not affected by a brief interaction with activated cyclosporine-exposed platelets. This indicates that the substance (or substances) released by these platelets somehow binds to and inactivates NO after it is released from the endothelium, or alternatively decreases the release of EDNO only for a very short time, in the presence of the platelet-derived substance. In addition, one cannot exclude the possibility that the compound somehow transiently interferes with the action of endothelial agonists such as acetylcholine and platelet-derived ADP/ATP.
Other compounds released from activated platelets, such as eicosanoids and platelet-activating factor, or peptides, such as platelet-derived growth factor, are not likely to mediate such a rapid modulation of EDNO-dependent vasodilation, nor are they known to bind NO.
Significance of Impaired Platelet-Mediated,
Endothelium-Dependent Vasodilation
Activated normal platelets cause vasodilation in large
normal arteries via release of ADP and ATP that stimulate the release
of EDNO.24 25 26 This can be considered a counterregulatory
mechanism, in which the released NO causes vasorelaxation in opposition
to the vasoconstrictive forces elicited by various
vasoconstrictors such as serotonin and
thromboxane A2, also released during
platelet aggregation. Furthermore, NO released into the intimal
space may inhibit smooth muscle cell proliferation37 38
and extracellular matrix synthesis.39 NO is also released
into the vessel lumen, where it inhibits platelet aggregation and
thrombus formation,40 41 42 providing an important negative
feedback on the ongoing platelet activation process.
Thus, the inability of cyclosporine-exposed platelets to mediate this effect creates a condition that favors vasospasm and intravascular thrombosis during platelet activation. This may be of significant clinical consequence and in part explain some of the thromboembolic complications reported with cyclosporine use.4 5 Although it still remains controversial whether cyclosporine therapy may accelerate atherosclerosis, this new finding would also provide one possible mechanism by which cyclosporine might be proatherogenic. The evidence for increased thrombosis within the kidneys of cyclosporine-treated subjects4 also suggest that the observed abnormal platelet-vessel interaction might adversely affect vasomotor control within the renal circulation and therefore indirectly contribute to renovascular hypertension.
Interestingly enough, similar impairment in platelet-mediated vasodilation has previously been reported in platelets from patients with hypercholesterolemia25 and with diabetes mellitus,26 two diseases associated with increased cardiovascular complications. Furthermore, both hypercholesterolemia and diabetes mellitus as well as cyclosporine are associated with endothelial dysfunction,43 44 which can further compromise this important interaction between platelets and endothelium.
Study Limitations
This in vitro model does not contain the multiple components of in
vivo conditions, such as interactions of platelets with leukocytes,
red blood cells, and various components of serum, all of which affect
vessel interactions in a complex manner. Nevertheless, it allows us to
study endothelium-platelet interactions under
controlled and well-defined conditions, providing us with an
opportunity to detect a malfunction in cyclosporine-exposed
platelets that otherwise might have been difficult to appreciate in
a system with multiple variables.
Another criticism of our experiments might be our use of rabbit arteries instead of human vessels in which to test the human platelets. This experimental constraint is the result of the obvious difficulty in obtaining fresh, normal human arteries for experimental use. However, rabbit carotid arteries and human coronary arteries seem to respond similarly to activated human platelets and endothelial agonists in vitro.24 25 26 Furthermore, the rabbit carotid arteries are easy to work with, and they give consistent results even during prolonged experimental protocols.
Conclusion
Human platelets exposed to cyclosporine, either in
vivo or in vitro, have a significantly impaired ability to mediate
vasodilation. This does not appear to be due to increased
platelet-mediated vasoconstriction by
cyclosporine-exposed platelets or a decrease in the
release of platelet-derived nucleotides during
platelet activation but rather to a secondary, as yet unidentified
short-acting compound released by cyclosporine-exposed
platelets that interferes with EDNO-dependent vasodilation.
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Acknowledgments |
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This work was supported in part by a research grant donated by the Emil Petricek Estate (Omaha, Neb); intramural funds from the Department of Internal Medicine, University of Nebraska Medical Center; the Department of Internal Medicine, University of Iowa; and a grant from the Research Foundation in Memory of Helga Jónsdóttir and Sigurlidi Kristjánsson (Reykjavík, Iceland).
Received August 6, 1996; first decision August 30, 1996; accepted November 5, 1996.
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