(Circulation. 1995;91:1336-1340.)
© 1995 American Heart Association, Inc.
Articles |
Intravenous Cocaine Induces Platelet Activation in the Conscious Dog
From the Charles A. Dana Research Institute and the Harvard-Thorndike Laboratory of the Department of Medicine, Beth Israel Hospital and Harvard Medical School, Boston, Mass (A.D.K., R.P.S., J.A.W.); the New England Regional Primate Research Center, Southborough, Mass (R.P.S.); and the Hematology Division, The Toronto Hospital, Toronto, Ontario (E.L.Y.).
Correspondence to J. Anthony Ware, MD, Cardiovascular Division, Beth Israel Hospital, 330 Brookline Ave, Boston, MA 02215.
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Abstract |
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Background Cocaine consumption has been associated with thrombosis of coronary and peripheral arteries. Since cocaine has been found to induce platelet activation in vitro, we sought to establish whether cocaine induced platelet activation in vivo.
Methods and Results Chronically instrumented, conscious dogs were infused with cocaine (1 mg/kg), norepinephrine (0.2 to 0.4 mg/kg), or saline intravenously over 1 minute. Activated canine platelets were identified in whole blood collected from an indwelling aortic catheter by flow cytometric detection of the binding of a monoclonal antibody directed against the activation-dependent antigen P-selectin. Infusion of cocaine resulted in an elevation of mean arterial pressure (91±3 to 128±7 mm Hg [P<.001]) and heart rate (87±9 to 125±11 beats per minute [P<.01]). A similar change (P=NS) in mean arterial pressure followed norepinephrine infusion (100±5 to 137±13 mm Hg [P<.04]), whereas saline infusion had no effect. Cocaine resulted in a substantial but delayed increase in platelet P-selectin expression (14±7% [P<.08], 31±12% [P<.04], and 55±22% [P<.04] at 17, 22, and 27 minutes after drug infusion, respectively). The magnitude of this increase was similar to that found in blood treated ex vivo with the agonists ADP or PAF (23±7% and 53±15%, respectively). No significant increase in P-selectin expression was detected in the blood of animals that received norepinephrine or saline. Serum cocaine concentrations were highest immediately after infusion (538±55 ng/mL at 2 minutes) but declined rapidly (185±22 and 110±25 ng/mL at 17 and 32 minutes after infusion); in contrast, the increase in benzoylecgonine concentrations was delayed (from <25 ng/mL in all but one animal [34 ng/mL] at 2 minutes to 46±4 and 71±11 ng/mL at 17 and 32 minutes, respectively, after infusion).
Conclusions Intravenous cocaine induces activation of individual circulating platelets; this effect is not reproduced by infusion of norepinephrine at doses sufficient to exert similar hemodynamic effects. The delay in detection of activated platelets after treatment with cocaine may result from the adhesion and subsequent detachment of activated platelets; alternatively, cocaine metabolites, rather than the drug itself, may induce platelet activation.
Key Words: cocaine • norepinephrine • proteins • cells
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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Many myocardial infarctions that follow cocaine ingestion and in which no significant fixed coronary artery stenosis has been found have been postulated to result from coronary vasospasm, adrenergic increases in myocardial oxygen demand, and/or transient thrombosis.1 2 Other myocardial infarctions occur in the presence of significant atherosclerotic narrowing and/or endothelial dysfunction, which appear to be promoted by cocaine use and are often complicated by thrombus formation.2 An attractive hypothesis to explain this association of cocaine with thrombosis of coronary as well as peripheral3 4 5 6 arteries is that cocaine either directly or indirectly induces the activation of platelets. The identification of coronary thrombi composed primarily of platelets in patients after cocaine use7 lends further support to this notion.
Cocaine has been shown to reduce the threshold of rabbit8 and human9 platelets to aggregation by conventional agonists, as assessed by turbidometric methods. Addition of cocaine to whole blood from some normal donors induces the expression of the platelet alpha granule protein P-selectin and association of fibrinogen with the platelet surface, both of which denote platelet activation, and enhances the ability of subthreshold concentrations of the physiological agonists ADP and epinephrine to induce these responses.10 Although these observations strongly suggest that cocaine induces platelet activation in vitro, whether such an effect can be observed in vivo is not known. Therefore, the purpose of this study was to determine whether cocaine induced in vivo activation of platelets. To test this hypothesis, we administered cocaine intravenously to conscious (nonanesthetized), chronically instrumented dogs and measured the expression of P-selectin11 in whole blood arterial samples.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Surgical Preparation and Instrumentation
The dogs used in this study were maintained in accordance with the Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources and the Harvard Medical School Standing Committee on Animal Care. The techniques for surgical instrumentation of the dogs have been described previously.12 13 Briefly, 2 or more weeks before the infusion of cocaine, mongrel dogs of either sex (weight, 21 to 34 kg) were sedated with xyalazine (10 mg/kg) and anesthetized with halothane (1 vol%). With a sterile technique, a Tygon catheter was implanted in the descending aorta and externalized through a left thoracotomy, which was then closed. Aortic blood pressures were obtained from the chronically implanted catheter and measured with strain-gauge manometers (Statham Instruments) calibrated with a mercury manometer. Heart rate was determined by a cardiotachometer triggered from the aortic pressure pulse. Hemodynamic measurements and blood samples were obtained while the unrestrained dogs were lying quietly. All experiments were conducted while the dogs were healthy and at least 2 weeks after recovery from surgery to preclude an effect of surgery on platelet physiology.
In the experimental protocol, dogs received either intravenous cocaine (1 mg/kg) (n=7) or an equivalent volume of saline (n=3) over 1 minute via a patent limb vein catheter. Hemodynamic recordings were made continuously, with analyses performed at 0.5, 1, 2, 5, 10, 15, 20, 25, and 30 minutes. Systemic blood pressure data were recorded on a multichannel tape recorder (Honeywell) and a direct-writing oscillograph (Gould-Brush). Heart rate was determined by a cardiotachometer triggered from the aortic pressure pulse and recorded continuously. Blood samples, processed as described below, were obtained at baseline and 2, 7, 12, 17, 22, and 27 minutes after completion of the cocaine infusion. In those dogs treated with cocaine, samples for plasma cocaine concentration analysis were obtained at 2, 17 (n=7), and 32 (n=3) minutes after the drug infusion. The aortic catheter was flushed with nonheparinized saline between sample acquisitions.
Cocaine is known to increase blood pressure, a primary determinant of shear stress, in this conscious canine model. Additionally, cocaine has been shown to have a sympathomimetic effect both via the central nervous system and as a result of the peripheral release of both norepinephrine and epinephrine.14 15 Since both sympathetic agents16 and shear stress17 18 are known stimulators of platelet activation in vitro, we sought to control for these effects of cocaine. In three additional control animals, we infused norepinephrine, 0.2 to 0.4 µg/kg over 1 minute, which we had previously found to have a pressor effect comparable to that of cocaine.12 Hemodynamic analysis and blood sampling were performed in a manner similar to that in the cocaine- or saline-treated dogs.
Blood Sampling and Treatment
Blood was sampled from the
aortic catheter without aspiration.
The initial 3 mL of sample was discarded, and the remainder was
anticoagulated with 3.8% trisodium citrate (Sigma Chemical Co) (1:9
dilution). Samples were fixed immediately with a 1:1 dilution of 1.5%
paraformaldehyde in phosphate buffer at 4°C10 and
processed after 2 hours of fixation. To provide a positive control,
duplicate baseline samples from some animals were treated with ADP 10
µmol/L (Sigma) and platelet activating factor (PAF) 8 µmol/L
(Biomol Research Labs) without stirring and incubated for 2 minutes at
room temperature before fixation.
Flow Cytometric Evaluation of P-Selectin Expression
The
murine monoclonal antibody KC4.1, which binds to canine
P-selectin,19 was conjugated to biotin by standard
methods. Streptavidin conjugated to fluorescein isothiocyanate (FITC)
then determined KC4.1-biotin binding to platelet P-selectin. To confirm
that antibody binding was specific to platelet activation and did not
reflect an artifact of sample processing or microaggregate formation,
the binding of isotype-matched control antibodies as well as
streptavidin-FITC was determined.
Platelet P-selectin expression was detected by a technique similar to that described previously.10 After cell fixation with paraformaldehyde, which was used to prevent time-dependent P-selectin expression,20 5-µL aliquots of whole blood were diluted in 50-µL aliquots of PBS. Preliminary experiments demonstrated consistent antibody binding after a 2-hour fixation period. Samples were then incubated with a saturating concentration of KC4.1-biotin for 20 minutes at room temperature, followed by centrifugation at 800g for 1 minute and washing with 50 µL of phosphate buffer. After treatment with streptavidin-FITC and 20 minutes of incubation, the samples were diluted with phosphate buffer to a final volume of 600 µL and examined immediately by flow cytometry.
After that procedure, samples were analyzed in a FacStar Plus flow cytometer (Becton Dickinson). FITC was excited with a 5-W argon laser at 200-mW power with a wavelength of 488 nm and detected with a 530±11-nm bandpass filter. Platelets were distinguished from other blood cells on the basis of their forward and right-angle light scatter,19 and an electronic "gate" was set around these particles. The number of particles in this gate was then compared with that in an erythrocyte gate to establish a platelet count relative to the erythrocyte count so that the total number of circulating platelets could be estimated. KC4.1 binding was then determined by analyzing 5000 platelets per sample at a rate of <1000 cells per second. Light scatter and fluorescence data were obtained with gain settings in a logarithmic mode, and data were analyzed on a Hewlett-Packard Consort 30 software package. Fluorescence was expressed as mean log fluorescence per cell, which was converted to a linear scale and calibrated with standard fluorescent beads (Flow Cytometry Standards Corp).21
Serum Cocaine and Catecholamine Analysis
Blood samples for
serum concentrations of cocaine and its
primary metabolites, benzoylecgonine and ecgonine methyl ester, were
measured in serum treated immediately with sodium fluoride and
potassium oxalate, effective inhibitors of the
pseudocholinesterases,22 which rapidly metabolize cocaine
in blood. These concentrations were determined by gas
chromatography/mass spectrometry.23 Plasma norepinephrine
and epinephrine levels were determined at baseline, 5 minutes (peak
effect), and 30 minutes after cocaine administration in three animals
by the radioenzymatic assay of Peuler and Johnson24 so
that changes in plasma catecholamines that followed administration of
cocaine in this model could be determined.
Statistical Analysis
All data are reported as the
mean±SEM. Statistical significance
was assessed by Student's paired t test and
repeated-measures ANOVA.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Infusion of cocaine into the dogs resulted in an immediate increase in blood pressure and heart rate (Fig 1
). The
mean arterial pressure increased significantly, from 91±3 mm Hg at
baseline to a peak of 128±7 mm Hg (P<.001) at 1 minute
after cocaine infusion, and gradually returned to 102±3 mm Hg by 30
minutes. Likewise, the increase in heart rate observed after
intravenous cocaine occurred rapidly, with an increase from the
baseline value of 87±9 beats per minute noted at 2.5 minutes after the
infusion (125±11 beats per minute [P<.01]),
returning to
near baseline values (94±10 beats per minute) by 30 minutes after the
infusion. In contrast, the control group of animals treated with equal
volumes of intravenous saline demonstrated no real change in mean
arterial pressure (97±3 mm Hg) or heart rate (100±2 beats per
minute) at any of the monitored time points after the control infusion.
However, infusion of 0.2 to 0.4 µg/kg norepinephrine resulted in
blood pressure changes similar (P=NS) to those observed
after intravenous treatment with cocaine, inducing an immediate
increase in the mean arterial pressure from 100±5 mm Hg at baseline
to 137±13 mm Hg at 30 seconds (P<.04), followed by a
gradual decline toward baseline.
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P<.04, *P<.005. Each point
represents the mean±SEM for three to seven separate
determinations. Bottom, Heart rate (beats per minute) at baseline and
after infusion of cocaine (1 mg/kg), norepinephrine (0.2 to 0.4
µg/kg), or saline. *P<.01. Each point represents
the mean±SEM for three to seven separate determinations.Changes in plasma catecholamine levels after cocaine administration accompanied the observed hemodynamic changes. Plasma norepinephrine increased from a baseline of 213±64 to 474±79 pg/mL and decreased to 289±85 pg/mL at 30 minutes. Plasma epinephrine levels demonstrated a similar change: baseline, 191±89 pg/mL, rising to 463±73 pg/mL at 5 minutes after infusion and falling to 236±67 pg/mL at 30 minutes. These levels are similar to those that our laboratory has noted previously.13
The infusion of cocaine resulted in a significant but delayed
increase in platelet P-selectin expression. Binding of the monoclonal
antibody KC4.1 increased gradually, with a 14±7% increase
(P<.08) at 17 minutes, a 31±12% increase
(P<.04) at 22 minutes, and a 55±22% increase
(P<.04) 27 minutes after infusion (Fig 2).
One animal showed no significant increase in platelet P-selectin
expression after cocaine. This increase in P-selectin expression was
not associated with a significant change in platelet count (platelets
were 4.8% of particles before infusion and 3.3% of particles after
infusion [P=NS]). In contrast, infusion of normal
saline
or norepinephrine did not induce significant changes in KC4.1 binding.
Additionally, P-selectin expression was not increased in one dog
infused with epinephrine (0.2
µg · kg-1 · min-1), which,
like
norepinephrine, induced hemodynamic effects similar to those of cocaine
(data not shown). The isotype-specific antibody and streptavidin-FITC
control failed to bind to platelets, indicating that our findings are
not the result of microaggregate formation or nonspecific binding of
antibodies or fluorophores. For comparison, in vitro incubation of
preinfusion canine blood with the standard platelet agonists ADP (10
µmol/L) and PAF (8 µmol/L) resulted in a 23±7%
(P<.03) and 53±15% (P<.01) increase in KC4.1
binding, respectively. Thus, the infusion of cocaine was associated
with a sustained significant expression of P-selectin on circulating
blood platelets; the extent of P-selectin expression was similar to
that observed when canine platelets were exposed to physiological
agonists in vitro.
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In preliminary studies to investigate the mechanisms for the delay in P-selectin expression, the time course of changes in serum concentrations of cocaine and its primary metabolites, benzoylecgonine and ecgonine methyl ester, were measured. Serum cocaine levels were highest immediately after receipt of cocaine (mean concentration of 538±55 ng/mL at 2 minutes [n=7]) but steadily declined after the infusion (185±22 ng/mL at 17 minutes [n=7] and 110±25 ng/mL at 32 minutes [n=3]). In contrast, the rise in serum benzoylecgonine concentration was delayed; the concentration of this metabolite was below the limits of detection of the assay (<25 ng/mL) at 2 minutes in all (n=7) but one animal (34 ng/mL) but increased with time (46±4 ng/mL at 17 minutes [n=7] and 71±11 ng/mL at 32 minutes [n=3]). The serum concentration of ecgonine methyl ester was below the limits of detection (10 ng/mL) in two animals, with a mean of 15±1 ng/mL (n=5) at 2 minutes after infusion, increasing to 20±3 ng/mL at 17 minutes (n=7). Thus, the times of peak levels of the cocaine metabolites coincided with those of platelet activation.
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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The results of this study indicate that intravenous cocaine induces significant expression of P-selectin on the surface of circulating platelets in dogs. This conclusion is strengthened by the fact that no anesthetics were present at the time of blood sampling in these chronically instrumented animals. The expression of P-selectin on the platelet surface results from activation-induced alpha granule secretion, a process that reflects coalescence of the alpha granule with the internal canicular system, which becomes externalized and thus becomes a marker for platelet activation; P-selectin also mediates interaction between platelets and neutrophils.25 Both activation of platelets and formation of platelet-neutrophil conjugates can promote development of thrombi.26
The mechanism by which cocaine causes platelet activation is not yet clear. In this study, we considered whether catecholamines such as norepinephrine might activate platelets directly or by induction of hemodynamic stress. To test these possibilities, we studied the effect of an intravenous infusion of norepinephrine in a dose that previously had been shown to mimic the pressor effects of the cocaine infusion used in these experiments.12 Infusion of norepinephrine resulted in a peak blood pressure that was higher, although of shorter duration, and a less pronounced increase in heart rate than did infusion of cocaine. Despite the similar peak pressor effects of cocaine and norepinephrine, platelet P-selectin expression was noted only after cocaine infusion. While a single bolus of norepinephrine is unlikely to mimic accurately the complex hemodynamic and sympathomimetic effects of cocaine in vivo, our findings suggest that neither the release of norepinephrine nor the resultant hemodynamic effects are sufficient to explain the ability of cocaine to activate platelets, and an additional, possibly direct, effect of the drug on platelets is likely. It is possible that the catecholamine and hemodynamic changes associated with cocaine administration "prime" platelets to facilitate a direct effect of cocaine; alternatively, cocaine may serve to reduce the threshold for shear stress and catecholamines to induce platelet activation. In fact, we10 and others9 have shown that cocaine can promote the agonist effect of epinephrine and other agonists on blood platelets in vitro, and thus both a direct and an enhancing effect may contribute to platelet activation in vivo.
In the present study, cocaine-induced platelet P-selectin
expression was detected only after a significant time delay (
17
minutes after the drug infusion). The reason for this delay is not
known. Although P-selectin expression is a late platelet-activation
event when compared with conformational changes in glycoprotein
IIb/IIIA or fibrinogen binding, in vitro studies demonstrated
P-selectin expression to be maximal 5 minutes after exposure to
cocaine.10 It is possible that the drug activated
platelets immediately and that such activation led to their initial
adhesion to leukocytes and/or endothelium with later detachment and
entry into the systemic circulation. Alternatively, platelet activation
might be induced by metabolites of cocaine rather than the primary form
of the drug. In our experiments, serum cocaine levels fell rapidly
after infusion of the drug, and serum catecholamine levels were near
baseline at the time that P-selectin expression was first detected;
however, the hydrolytic metabolite benzoylecgonine, which was not
detectable in the first minutes after the infusion, continued to rise
thereafter. Thus, it is possible that benzoylecgonine or ecgonine
methyl ester, the pseudocholinesterase-derived metabolite of cocaine,
or their derivatives induce platelet activation, rather than the
primary drug. This hypothesis is supported by our previous in vitro
findings that the response of platelets to cocaine was notable in
plasma or washed preparations only in the presence of millimolar
concentrations of the drug, whereas P-selectin expression by platelets
in whole blood was detected with 100-fold lower concentrations of the
drug10 ; since erythrocytes contain
pseudocholinesterases,27 they may participate in the
poorly understood enzymatic mechanism by which benzoylecgonine is
formed.28 Thus, future investigations of the effects of
cocaine metabolites on platelets in both in vitro and in vivo models
are warranted.
There are several limitations to our study. The peak serum cocaine levels achieved in these experiments resulted from the maximum dose that we could use without inducing agitation in our animals and are similar to those noted in experimental human subjects receiving similar intravenous doses of the drug, although the kinetics of metabolism differ.29 Furthermore, only the lower range of serum cocaine concentrations obtained from a series of patients after cocaine-related death (100 ng/mL to 24 µg/mL) overlapped the concentration achieved in our model.30 No thrombotic or other complications were noted in our dogs; it is possible that higher or repeated doses of cocaine may induce macroscopic thrombi. Furthermore, caution is warranted in extrapolating the findings of this study to the problem of cocaine-associated thrombosis in patients. Because of significant species variation in platelet physiology, the effect of cocaine on human platelets in vivo may differ significantly. Also, this model addresses acute cocaine intoxication and may prove relevant to the effects seen in first-time or intermittent cocaine users. Whether similar effects on platelet function result from chronic cocaine ingestion is unknown and provides an interesting topic for future investigations.
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Acknowledgments |
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This work was supported by US Public Health Service grants HL-38820 and DA-06306 to Dr Ware and HL-33107 to Dr Stephen Vatner. Dr Ware is the recipient of a Research Career Development Award in Thrombosis (HL-02271). Dr Shannon is the recipient of a Clinician Scientist Award from the American Heart Association. Dr Kugelmass is the recipient of an Individual National Research Service Award (HL-08816). We wish to thank Drs Richard Kuntz and Karen Keaney for their statistical assistance, as well as Claudia Cabral, MS, for assistance with flow cytometry, which was performed at the Beth Israel Hospital Flow Cytometry facility. We also wish to thank Dr Vatner for his support and Thomas Manders for his technical assistance. Serum cocaine, ecgonine methyl ester, and benzoylecgonine concentrations were performed at the Center for Human Toxicology, University of Utah, under contract of the National Institute for Drug Abuse.
Received October 6, 1994; revision received December 29, 1994; accepted January 9, 1995.
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U. Wilbert-Lampen, C. Seliger, T. Zilker, and R. M. Arendt Cocaine Increases the Endothelial Release of Immunoreactive Endothelin and Its Concentrations in Human Plasma and Urine : Reversal by Coincubation With {sigma}-Receptor Antagonists Circulation, August 4, 1998; 98(5): 385 - 390. [Abstract] [Full Text] [PDF]
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J. E. Hollander, D. E. Brooks, and S. M. Valentine Assessment of Cocaine Use in Patients With Chest Pain Syndromes Arch Intern Med, January 12, 1998; 158(1): 62 - 66. [Abstract] [Full Text] [PDF]
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R. P. Shannon, P. Lozano, Q. Cai, W. T. Manders, and Y.-t. Shen Mechanism of the Systemic, Left Ventricular, and Coronary Vascular Tolerance to a Binge of Cocaine in Conscious Dogs Circulation, August 1, 1996; 94(3): 534 - 541. [Abstract] [Full Text]
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