(Hypertension. 1995;25:1096-1105.)
© 1995 American Heart Association, Inc.
Articles |
Effect of Circulating Epinephrine on Platelet Function and Hematocrit
From the Department of Internal Medicine, Ullevaal University Hospital, Oslo, Norway (S.E.K.); Division of Hypertension, University of Michigan, Ann Arbor (A.B.W., R.N., A.J.Z., S.J.); and Division of Clinical Pharmacology, Medical University of South Carolina, Charleston (B.E.).
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Abstract We investigated the effect of raising arterial plasma epinephrine within the lower pathophysiological concentration range on various indicators of blood platelet function and hematocrit. Epinephrine was raised over 60 minutes by a stepwise increasing intravenous infusion in 40 healthy men aged 20 to 40 years. Platelet count increased progressively with increasing arterial epinephrine to a maximal change of 69±6 x109/L in EDTA-anticoagulated blood and a maximal change of 42±6 x109/L in acid-citrate-dextrose (ACD)–anticoagulated blood, and the weight of circulating platelets increased by 29% (P<.001). Platelet size increased significantly in EDTA and decreased in ACD, and the difference between EDTA and ACD was significant (P<.0001) for both count and size, suggesting that epinephrine not only recruits platelets into the circulation but also induces some microaggregation in vivo or adhesion ex vivo. Aggregation of platelets in vitro induced by epinephrine decreased (P<.003 for
optical density and
P=.038 for maximal optical density) after epinephrine
infusion compared with saline but did not change when stimulated with
ADP or collagen. These findings suggest a selective downregulation of
the epinephrine-activating mechanisms concomitant with a rise in the
platelet content of epinephrine by 81% (P<.001) and no
change in the platelet sodium-proton membrane exchange. The release of
granular content (ß-thromboglobulin and platelet factor 4) to the
circulation in response to epinephrine was not significant. Thus, under
acute conditions it seems that the platelets may protect themselves
against inappropriate overstimulation by epinephrine. The importance of
platelet epinephrine uptake is still unknown, but sodium-proton
exchange does not seem to be involved in regulating the effects of
circulating epinephrine on platelet function. Epinephrine has a
pronounced effect on raising hematocrit (maximal change of 1.74±0.13
x10-2, P<.0001).
Key Words: catecholamines • hypertension, essential • dopamine • norepinephrine • platelet aggregation • stress • thrombosis
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Introduction |
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Blood platelet dysfunction has received attention as a potential cause of increased cardiovascular morbidity in essential hypertension.1 2 3 4 In a prospective study,5 platelet aggregation characteristics predicted long-term mortality in apparently healthy middle-aged men. Secretion of platelet-derived growth factor may contribute to the smooth muscle hyperplasia of atherosclerotic lesions.6 7 Platelets may accumulate at sites of arterial damage and partial vascular obstruction,8 as evidenced by the platelet thrombi often found on acute lesions of atherosclerotic plaques of the main epicardial arteries in acute myocardial infarction.9 Platelet activation may also play a significant role in patients with persistent unstable angina pectoris.10
Since the first report of raised plasma epinephrine concentrations in essential hypertension,11 the role of epinephrine in the pathogenesis of hypertension has been discussed but still has not been clarified. Hypertensive subjects may respond to environmental stimuli with larger sympathoadrenal responses than normotensive subjects,12 and elevated plasma epinephrine levels in hypertension may be a marker for increased arousal with enhanced neurogenic activity of the type associated with the defense reaction.13 However, even transiently and certainly chronically elevated plasma epinephrine levels deserve consideration as a pathophysiological feature of essential hypertension, because epinephrine has cardiovascular and metabolic (hormonal) effects at concentrations slightly above the normally low resting levels.14 15 16 17 Emotional stress is known to provoke catecholamine release18 19 20 21 and has long been associated with coronary heart disease.22 23
Furthermore, when it was shown three decades ago that epinephrine activates blood platelets in an aggregometer, it was proposed that platelets could be the link between stress and cardiovascular disease.24 25 However, in these studies and a large number of later ones, micromolar concentrations of epinephrine were used in vitro, whereas in vivo only nanomolar concentrations are present. Therefore, it has long been questioned whether epinephrine could activate platelets in vivo. In a recent review, Hjemdahl et al26 concluded that sympathoadrenal activation (eg, mental stress, epinephrine infusion, exercise, and surgical stress) may enhance or reduce platelet aggregability in vitro, whereas in vivo measures of platelet function (platelet count, size distribution) and ex vivo filtragometry more consistently indicate platelet activation.
With this background we felt that the effect of circulating epinephrine on platelet function in humans is still not fully understood. Therefore, in the present study we investigated the effect of intravenous epinephrine infusion and raised arterial plasma epinephrine within the lower pathophysiological concentration range on various indicators of blood platelet function. In addition, we estimated the effect of epinephrine on hematocrit, which also is of increasing interest as a cardiovascular risk factor.27 28 29
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Methods |
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Subjects
Paid volunteers (n=40) were recruited by an advertisement in the local newspaper asking for healthy men 20 to 40 years of age with a body weight less than 95 kg. At the initial screening, medical history, physical examination, electrocardiogram, urinalysis, blood counts, and biochemistries including liver and renal function tests were done to exclude any other concomitant illness, medical treatment, or alcohol or drug abuse. After informed consent was obtained, eligible subjects were given an appointment for the subsequent Clinical Research Center (CRC) study (see below). They were urged not to make any changes in their dietary, smoking, or alcohol habits before the CRC study and were specifically prohibited from taking aspirin or any aspirin-containing over-the-counter medications for at least 10 days before CRC admission (see below). All subjects were white and averaged 32.4±0.9 years of age (range, 20 to 40 years), 81.9±1.3 kg of body weight, and 181.8±1.0 cm of height.
Protocol
The study was approved by the Human Subject Review Committee of
the University of Michigan. Approximately 2 to 4 weeks after the
screening study, subjects were admitted to the CRC. After an overnight
fast and abstinence from alcohol for at least 2 days, subjects were
studied while resting supine in a quiet room where temperature was kept
strictly standardized to 75°F (23.9°C). None of them were regular
smokers; however, six of them reported occasional smoking, and this was
restricted overnight. Only one subject was studied each day, and all
studies were carried out by the same physicians, starting at 8
AM. Under epinephrine-free local anesthesia, 2-inch
polytetrafluoroethylene catheters were initially placed into the left
brachial artery above the elbow (20 gauge) and into an antecubital vein
of the right arm (18 gauge) and kept open with small amounts of 0.9%
NaCl. Thirty minutes after catheter placement and with the subjects
blinded to the order of administration, an intravenous infusion of
0.9% NaCl was given for 60 minutes, immediately followed by an
intravenous infusion of epinephrine for 60 minutes. The rate of 0.9%
NaCl infusion was always equal to the rate of epinephrine infusion with
respect to the amount of fluid given intravenously per minute. The
epinephrine infusion rate was increased stepwise at 10-minute
intervals, starting at a dose of 0.01 µg/kg per minute and reaching
0.06 µg/kg per minute after 50 minutes. This maximal infusion rate
was maintained for 10 minutes and then followed by 20 minutes of 0.9%
NaCl infusion (recovery).
Blood was drawn through the intra-arterial catheter into polypropylene syringes after the first 2 mL was discarded. Blood samples for measurements of platelet count, platelet size, hematocrit, and epinephrine were drawn immediately before and every 10 minutes throughout the epinephrine infusion and additionally 20 minutes before, and except for epinephrine (assay limited to eight samples), also at recovery 20 minutes after the epinephrine infusion period. Blood was drawn immediately before and at the end of the epinephrine infusion for studies of platelet aggregation, plasma concentrations of ß-thromboglobulin (BTG) and platelet factor 4 (PF4), platelet weight per 10 mL blood, platelet contents of catecholamines, and platelet sodium-proton (Na+-H+) exchange.
Analytical Methods
Platelets in plasmas from 2 mL acid-citrate-dextrose
(ACD)–anticoagulated and EDTA-anticoagulated blood samples were
counted and sized with a Coulter model ZM with a 70-µm aperture
tube.30 Platelet-rich plasma was prepared by
centri- fugation at 120g for 10 minutes, separated from
the sedimented red blood cells with a plastic pipette, and transferred
to a capped polystyrene tube. A frequency histogram of platelet size
was displayed on a Channelizer 256 (Coulter Co) previously calibrated
with standard 5.01-µm latex beads (Coulter). Average platelet size
was determined as the peak (mode) of the platelet distribution curve as
determined visually, using a size window of 0 to 28.16 fL over 256
channels (0.11 fL per channel). All determinations of platelet counts
and sizes were done with platelets in isotonic NaCl solution at room
temperature. Both ACD- and EDTA-anticoagulated blood were used because
pilot studies in our laboratory indicated higher estimates of both
platelet count and size in EDTA-compared with ACD-anticoagulated blood.
(Lower count and size in ACD compared with EDTA indicate activated
platelets,31 either lost by adhesion to the glass wall or
clustered in microaggregates larger than 28.16 fL so they could not be
measured by the Coulter system.) Because there is some passive swelling
of the platelets within the first 1 to 2 hours after blood sampling,
all sizing and counting of platelets were performed 2 to 3 hours after
sampling. Hematocrit was determined in these blood samples before
plasmas were spun off by the microhematocrit method using heparinized
capillary tubes (Clay Adams) centrifuged in an MB centrifuge
(International Equipment Co).
Blood (45 mL) for platelet aggregation was mixed with 5 mL of 3.6% ACD solution (20 mmol/L citric acid, 110 mmol/L sodium citrate, 5 mmol/L dextrose) as anticoagulant in polypropylene tubes. The blood was immediately centrifuged at 120g for 15 minutes at room temperature to obtain platelet-rich plasma, which was kept in an atmosphere of 95% O2/5% CO2 to keep plasma pH stable. Platelet concentration was adjusted to 3x108 cells per milliliter with platelet-poor plasma. Changes in optical density were measured with a Chronolog model 540 VS aggregometer and chart recorder as previously reported.32 33 Aliquots of platelet-rich plasma were brought to 37°C and zeroed against autologous platelet-poor plasma solution. The first aliquot of 0.45 mL was mixed with a 0.05-mL volume of 5 mg/mL arachidonic acid to determine whether the subject had taken aspirin in the 10 days before the study (this test was not abnormal in any subject). After the test with arachidonic acid, 0.45-mL aliquots were challenged with a single 0.05-mL volume of epinephrine, ADP, or collagen to obtain final agonist concentrations as shown in the dose-response curves. Incubation was done for a minimum of 3 minutes. For each agonist at each concentration, the data were expressed as the change in optical density or transmittance per minute (slope) and as maximal aggregation (percent) where platelet-poor plasma represents 100% transmittance. Platelet aggregation may have a primary and secondary wave; in the present study, the measurements of slopes were done on the primary wave, and maximal aggregation reflects the intensity of the total wave of aggregation.
Blood for analysis of BTG and PF4 (2.7 mL) was mixed with EDTA and theophylline, supplied with the BTG kit, to which was added 0.1 mg prostaglandin E1 (Sigma Chemical Co). The sample was allowed to cool on melting ice for 15 minutes and was then centrifuged at 4°C for 60 minutes at 2600g. A 0.5-mL midlayer of platelet-poor plasma was collected and stored at -20°C. BTG and PF4 were assayed in the same plasma sample with the use of commercial radioimmunoassay kits (Amersham and Abbott Laboratories, respectively).
Ten milliliters of blood for determination of platelet weight and platelet content of catecholamines and 5 mL for determination of plasma epinephrine were immediately transferred to ice-chilled tubes with EGTA and glutathione as anticoagulant and antioxidant, respectively, and kept on melting ice. Blood was processed and the platelet pellet prepared as previously reported.34 Catecholamines in an extract of the platelet pellet and epinephrine in plasma samples were kept frozen and determined within a few weeks with the radioenzymatic method of Peuler and Johnson.35
Na+-H+ exchange across membranes was measured on platelets from 7 mL ACD-anticoagulated blood as propionic acid–activated volume change by the method described by Grinstein et al36 as previously reported.30 In this technique, platelets are suspended in 140 mmol/L sodium propionate at pH 6.7. Propionic acid penetrates the cell membrane and dissociates within the platelets, causing intracellular acidification. When the intracellular pH falls, the Na+-H+ antiporter is activated, leading to extrusion of intracellular H+ in exchange for Na+ from the outside. Entering Na+ causes osmotic gain of water, resulting in cell swelling. Amiloride blocks Na+-H+ exchange and inhibits that portion of the volume increase that is dependent on the antiporter. Platelet size in sodium propionate with and without amiloride was determined once per minute for 10 minutes with the Coulter model ZM as described above, with platelet concentration standardized to 3x105 platelets per milliliter supporting solution. Solution temperature was maintained at 22° to 23°C. All analyses were completed within 90 minutes of the blood sample being drawn. A peak channel count of 1000 was used to improve the resolution of the platelet distribution mode. Peak platelet volumes in propionate plus amiloride and in propionate alone at 1 and 2 minutes were used to estimate an initial swelling rate over the first 2 minutes as the slope of the regression line for these three time points. This initial rate is in femtoliters per minute and is called the A-1-2 rate (from propionate plus amiloride, minute 1, minute 2). The A-1-2 rate divided by the propionate plus amiloride platelet volume for that subject yields the measure A-1-2 fractional volume change, which gives the rate of volume increase per minute in the first 2 minutes as a fractional increase over baseline volume. The units of this measure are per minute. Amiloride HCl was obtained as a gift from Merck, Sharp & Dohme, and a stock solution (50 mmol/L) was prepared in distilled water by heating and agitation. The final concentration used in the experiments was 100 µmol/L.
Statistics
All data were analyzed using the Michigan interactive data
analysis system (MIDAS) on the Michigan terminal system.
Differences were tested by two-tailed Student's t test for
paired comparison and by repeated-measures ANOVA. Correlation
coefficients (r) were calculated by the least-squares
method. A value of P<.05 was considered to be the limit for
statistical significance; all data are given as mean±SEM.
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Results |
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Arterial plasma epinephrine was raised over 60 minutes by a stepwise increasing intravenous infusion in 40 healthy men aged 20 to 40 years. The initial infusion rate of 0.01 µg/kg per minute lasted for 10 minutes and raised arterial epinephrine concentration from 0.55±0.07 to 1.26±0.12 nmol/L. Further stepwise increments in the epinephrine infusion rate to 0.06 µg/kg per minute produced progressive increases in arterial plasma epinephrine within the lower pathophysiological concentration range to a maximum of 8.40±0.51 nmol/L (Table 1).
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Platelet count did not change in response to intravenous saline but increased progressively with increasing arterial epinephrine concentration to a maximal change of 69±6 x109/L in EDTA-anticoagulated blood (Table 1) and a maximal change of 42±6 x109/L in ACD-anticoagulated blood (Table 2). These dose-related changes in platelet count were observed rather consistently in virtually all subjects and were therefore highly significant (P<.0001). The maximal platelet counts after 60 minutes of epinephrine infusion correlated directly with the arterial epinephrine concentrations (r=.50, P=.001 for EDTA; r=.46, P=.003 for ACD). When the epinephrine infusion was stopped, there was a complete normalization within the 20-minute postinfusion observation period. The increase in platelet count during epinephrine infusion was larger in EDTA- compared with ACD-anticoagulated blood (P=.0938 for change in the period from 40 to 140 minutes and P=.0295 for change in the period from 60 to 120 minutes, but P<.0001 for time points 80 through 120 minutes [paired t test], Fig 1).
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Platelet size increased significantly in EDTA (P<.0001, Table 1) and decreased significantly in ACD (P=.0188, Table 2). The difference in responses to epinephrine infusion between platelet size in EDTA and ACD was highly significant (P<.0001, Fig 2). The magnitude of the responses, however, was rather small, with a maximal change of 0.11±0.03 fL in EDTA and -0.11±0.03 fL in ACD. Saline infusion did not induce any significant change before epinephrine infusion, and in the saline phase after epinephrine, there was a partial recovery toward baseline.
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Aggregation in vitro was performed with platelets adjusted to
3x108 cells per milliliter. Aggregation induced by
epinephrine decreased (P<.003 for optical density and
P<.04 for maximal optical density) after epinephrine
infusion compared with saline infusion (Fig 3) but was
not altered when stimulated in vitro with ADP (Fig 4) or
collagen (Fig 5). Table 3 shows the
differences in in vitro platelet aggregation at each agonist
concentration after intravenous epinephrine infusion compared with
saline infusion.
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The release of granular content (BTG, PF4) to the circulation was not significant for epinephrine infusion compared with saline (Table 4). These results appeared irrespective of some scatter of the data caused by single values in 10 subjects (7 after epinephrine, 3 during saline) above what is considered to be the upper normal limit for plasma BTG of 1.7 mmol/L and which could have been caused by technical artifacts during blood processing. The release of BTG and PF4 was highly significantly intercorrelated (r=.96 for saline and r=.98 for epinephrine, P<.001).
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The weight of circulating platelets (milligrams per 10 mL blood) in EGTA-anticoagulated blood increased by 29% (P<.001, Table 4). The platelet content of epinephrine measured as nanomoles per milligram platelet weight increased by 81% (P<.001, Table 4), whereas the platelet content of norepinephrine or dopamine did not change. The consistency of measurements of platelet catecholamine contents before and after epinephrine infusion was supported by highly significant correlations for norepinephrine (r=.82) and dopamine (r=.80) but also for epinephrine (r=.69, P<.001) despite the increase.
Platelet size was also significantly smaller after epi-nephrine infusion in ACD-anticoagulated blood when the platelets were sized in the Isoton medium30 before Na+-H+ exchange was estimated (preinfusion, 4.22±0.09 fL versus postinfusion, 4.12±0.09 fL, P<.01). Similarly, postinfusion platelets incubated for 10 minutes in 140 mmol/L sodium propionate were smaller in both the presence and absence of amiloride; however, the initial rate of amiloride-sensitive volume increase in response to propionate exposure, an index of Na+-H+ exchange (preinfusion, 0.187±0.008%/min versus postinfusion, 0.190±0.007%/min, P=.56), as well as the net amiloride-sensitive volume increase at the end of 10 minutes, by which time the platelets have achieved a new steady- state size (maximal change preinfusion, 49.9±1.4% versus postinfusion, 50.3±1.6%, P=.68), were not significantly affected by the epinephrine infusion (Fig 6). Values for the preinfusion and postinfusion amiloride-sensitive size changes were highly correlated (r=.75 for slope and r=.76 for maximal size change; P<.001 for both). As we have previously reported,30 there was a correlation between the amiloride-sensitive rate of initial size change and the net amiloride-sensitive size increase at 10 minutes (r=.43, P<.01), and this relationship persisted during the postinfusion period (r=.67, P<.001).
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Changes in amiloride-sensitive platelet volume responses to propionate were not significantly related to in vitro platelet aggregation responses to epinephrine, ADP, or collagen.
Hematocrit did not change in response to intravenous saline but increased progressively with increasing arterial epinephrine concentration slightly above the resting level to a maximal change of 1.74±0.13 x10-2 in EDTA-anticoagulated blood (P<.0001, Table 1) and a maximal change of 1.63±0.14 x10-2 in ACD-anticoagulated blood (P<.0001, Table 2), with no difference between EDTA and ACD. Dose-related changes in hematocrit during the first 30 minutes of epinephrine infusion were observed rather consistently in virtually all subjects and were therefore highly statistically significant at each infused dose (P<.001). However, after 30 to 40 minutes, hematocrit leveled off (Fig 7). When the epinephrine infusion was stopped, there was a rebound effect within the 20-minute postinfusion observation period, with a decrease in hematocrit below the baseline level (change, -0.35±0.11 x10-2 in EDTA and -0.67±0.10 x10-2 in ACD).
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We studied a broad spectrum of platelet function parameters. Together with a low analytical variability for the various tests, a homogenous group of study subjects, and strictly standardized study conditions, these parameters would give a high sensitivity for detecting effects of infused epinephrine on platelet function.
Platelet count increased progressively with increasing arterial epinephrine in both EDTA- and ACD-anticoagulated blood, and the weight of circulating platelets increased by 29%, indicating recruitment of platelets into the circulation. Platelet size increased significantly in EDTA and decreased in ACD. The difference between EDTA and ACD was significant for both platelet count and size, suggesting that epinephrine not only recruits platelets into the circulation but also induces some microaggregation in vivo or adhesion ex vivo. Platelet aggregation in vitro induced by epinephrine decreased after infusion of epinephrine compared with saline but did not change when stimulated with ADP or collagen. These findings suggest a selective downregulation of the epinephrine-activated mechanisms, concomitant with a rise in the platelet content of epinephrine by 81% (P<.001) and no change in the platelet Na+-H+ membrane exchange. The release of granular content (BTG, PF4) to the circulation in response to epinephrine was not significant. Altogether, these measurements and responses, with support in the literature, may give an integrated overview regarding the effects of circulating epinephrine on platelet function. Hematocrit increased significantly at an arterial epinephrine concentration slightly above the resting level to a maximal change of 1.74%.
Since the early studies by Clayton and Cross24 and
O'Brien,25 it has been established that epinephrine (and
norepinephrine) in the presence of fibrinogen and
Ca2+ in vitro induces both primary and secondary
aggregation, potentiates aggregation by unrelated agents, and causes
platelet secretion. These epinephrine-induced platelet responses are
more effectively blocked by yohimbine and rauwolscine than by prazosin,
indicating that epinephrine stimulates human blood platelets by an
2-adrenergic receptor mechanism.37 38 Of
the recently described subtypes of the 2-adrenergic
receptor,39 the human platelet appears to contain only the
2a subtype.40 41 There is no clear evidence
to implicate any other -receptor subtype in platelet responses.
The exact mechanism of 2-receptor–mediated platelet
activation is not known. Inhibition of adenylate cyclase, which is
mediated by a G protein,42 43 is the best studied of the
effects of epinephrine on platelets. High levels of cAMP inhibit
platelet activation via (1) enhancement of calcium sequestration in the
dense tubular system, (2) inhibition of phospholipase C
activation,44 45 and (3) effects distal to changes in
calcium concentration.46 47 Thus, reduction of cAMP levels
by epinephrine could permit platelets to be more easily activated by
other stimuli such as ADP, thrombin, or serotonin. However,
direct reduction of cAMP levels by a nonreceptor agent,
2',5'-dideoxyadenosine, does not result in platelet
aggregation,48 and therefore, while inhibition of
adenylate cyclase is clearly an important modulator of platelet
responses, it is probably not the primary mechanism of
epinephrine-induced platelet activation.49 Furthermore,
although many other platelet activators stimulate phospholipase C, many
data indicate that epinephrine does not directly activate phospholipase
C in human platelets.50 51
Calcium is an important, if not requisite, messenger in platelet activation, and epinephrine-induced stimulation of calcium influx into platelets has been demonstrated by 45Ca2+ tracer methods.52 Surprisingly, increases in cytoplasmic calcium concentrations are not generally observed in responses to epinephrine when fluorescent probes such as quin 2 or fura 2 are used.53 54 55 However, two other calcium probes, chlortetracycline51 and aequorin,56 show increases in calcium concentration on platelet stimulation with epinephrine. These latter probes appear to be reporting a subcompartment of platelet calcium mobilized by epinephrine and possibly important to its effect, but the location of this pool of calcium and its role in platelet activation are not established.
It has also been proposed that 2-adrenergic
receptor–mediated activation of the Na+-H+
antiporter is an important downstream event in secretion and
aggregation.44 57 Amiloride and its analogues, at
concentrations that block Na+-H+ antiporter but
are far above those achieved in vivo, block platelet activation by
epinephrine and ADP.51 Other experimental manipulations
that inhibit Na+-H+ antiporter, such as low
pHo and removal of extracellular sodium, also prevent the
effects of epinephrine.51 Cytoplasmic alkalinization
produced by enhanced Na+-H+ antiporter may
sensitize phospholipase A2. Thus,
2-adrenergic receptors could activate
Na+-H+ antiporter by a direct interaction of
the receptor and the antiporter, by means of a G protein–mediated
mechanism, or as a response to local changes in calcium
concentration.58 However, there has been no direct
demonstration of platelet cytoplasmic alkalinization by epinephrine,
and in the present study, there is no effect of circulating
epinephrine on the Na+-H+ antiporter. Neither
is there any detectable relationship between
Na+-H+ antiporter activity and platelet
aggregation.
Phospholipase A2, which releases arachidonic acid
from platelet lipids to form thromboxane A2
(TXA2), plays an important role in epinephrine-mediated
platelet activation. Inhibitors of cyclooxygenase, which prevent
generation of TXA2 from released arachidonate, block the
secondary wave of epinephrine-induced aggregation. However, these
inhibitors do not prevent the formation of arachidonic acid or
lipoxygenase products that may play a role in the primary wave of
epinephrine-induced aggregation. Stimulation of phospholipase
A2 has generally been considered a distal effect of
receptor activation resulting from cytoplasmic alkalinization or
increased free calcium, but recent evidence suggests that
2-adrenergic receptors may more directly activate
phospholipase A2 via release of G protein
ß
-subunits.59
Although there is a growing understanding of how epinephrine may induce platelet activation in vitro, relatively few studies have addressed the question of whether catecholamines activate human blood platelets in vivo. Levine et al60 found that mental stress associated with public speaking provoked both a rise in plasma catecholamines and platelet release of BTG and PF4. Larsson et al61 reported that both mental stress that increased catecholamine concentrations and epinephrine infusion in a dose of 0.07 µg/kg per minute increased platelet aggregability by 35% as determined by in vivo filtragometry.62 Furthermore, Laustiola et al63 infused epinephrine in doses of 0.1 and 0.2 µg/kg per minute and found a threefold increase in the capacity for TXB2 production by platelets.
On the other hand, Arkel et al64 noted an absent or
decreased slope of the second phase of in vitro aggregation with
epinephrine immediately after the psychological stress of giving an
oral scientific presentation. Similarly, Siess et al65
infused norepinephrine in a dose of 0.07 µg/kg per minute and
reported reduced or unchanged in vitro platelet aggregation. We did not
study washed platelets; possibly, epinephrine preoccupied its own
receptor, and thus the decrease of response to epinephrine may be
caused by the receptor preoccupancy. However,
2-adrenergic receptors exist in states of high and low
agonist affinity.66 Hollister et al67
demonstrated that in response to infusion of either epinephrine or
norepinephrine in a dose of 0.05 µg/kg per minute, platelet
2-adrenergic receptors converted from their
high-affinity to a low-affinity state. Platelet aggregation and
inhibition of adenylate cyclase by epinephrine were also reduced after
infusion.67 According to our present results, such
downregulation after epinephrine infusion may be selective for
epinephrine, as it could not be seen for ADP or collagen. Analogous to
findings in pheochromocytoma,68 it is interesting that ADP
or collagen did not alter platelet aggregation after epinephrine
infusion as an integrated stimulating effect of more than one agonist
might have been expected to do.69
When Lande et al70 infused epinephrine up to 0.04 µg/kg per minute in healthy men, platelet count and size increased, as in the present study, but not plasma concentration of BTG. Epinephrine may expand the platelet pool by recruitment of platelets sequestered in the spleen71 72 ; therefore, recruitment may be a factor in all studies of the relationship between plasma catecholamines and platelet function. However, since larger and metabolically more active platelets73 74 75 should have entered the circulation in all the infusion studies of catecholamines, recruitment probably does not explain the apparently discrepant results reported.
So, how might epinephrine cause platelet activation in healthy humans? Exposure of the surface fibrinogen receptors on platelets is a well-described effect of epinephrine76 77 78 and may explain the changes in aggregation or adhesiveness in vivo in the present and in a previous61 study. Low-grade activation of the cyclooxygenase system59 79 may occur, which could explain the increased capacity for TXB2 production.63 However, it seems that ultimately platelets protect themselves against inappropriate overstimulation by downregulation of receptor-dependent responses and aggregation,64 65 67 and no release occurs as seen presently and previously.70 A protective mechanism seems particularly appropriate from another point of view: it may protect the platelets from indirect stimulation by epinephrine through an effect on lipolysis15 or accelerated blood coagulation80 81 since fatty acids82 and thrombin83 are potent inducers of aggregation in vivo.
BTG is a specific platelet release product, and the plasma
concentration of BTG is regarded as a useful marker of platelet
release.84 85 PF4 is, like BTG, extruded from the platelet
-granules during the release reaction. While the plasma half-life of
BTG is 100 minutes, the half-life of PF4 is only a few minutes, and
measurements of PF4 in the same plasma samples may therefore be
undertaken to separate in vivo from ex vivo platelet
release.85 We found strong correlations between BTG and
PF4 prior to and at the end of epinephrine infusion but no significant
response to epinephrine in the whole group of subjects or when 10
subjects with concomitant very high values of BTG and PF4, indicating
technical artifacts, were excluded. Excess basal activation could
minimize the effects of the epinephrine infusion on BTG and PF4;
however, this was unlikely as baseline concentration of BTG, a protein
with a rather long plasma half-life, was low.85 Baseline
concentrations of arterial epinephrine were also low and comparable to
baseline arterial concentrations in other
studies.17 21 70 86
Although platelets lack enzymes for catecholamine synthesis, the platelet level of catecholamines is much higher than in plasma. The subcellular localization seems to be the serotonin organelles (dense granules).87 Under certain circumstances characterized by elevated plasma norepinephrine levels, such as prolonged sodium depletion86 or pheochromocytoma,34 norepinephrine may be taken up and stored in platelets. Rosen et al88 observed, as we did in the present study, that platelets concentrate epinephrine during intravenous infusion of this catecholamine. However, presently we can only speculate on whether the uptake of epinephrine during infusion is directly involved in concomitant platelet responses.
In the present study, infusion of low-dose epinephrine increased arterial plasma epinephrine to concentrations comparable to those seen during mental stress17 18 19 20 21 or physical activity.89 However, epinephrine normally is released into the systemic circulation through the veins draining the adrenal medulla. Therefore, epinephrine infused intravenously is not equal to adrenal catecholamine release, where a mixture usually of 70% to 85% epinephrine and 15% to 30% norepinephrine is released into the adrenal effluent with accompanying steroid modulation. Besides, infused epinephrine may have indirect effects on platelet function, eg, through lipolysis,15 accelerated blood coagulation,80 81 or increments in plasma norepinephrine.16 Altogether, the in vivo epinephrine effect on platelet function may be extremely complex. In addition, our conclusions apply only to findings under acute conditions and should not be extrapolated to chronic situations, as occur in clinical disorders.
Whole-blood viscosity has been suggested in many reports to be a major
independent risk factor for cardiovascular
disease,27 28 90 91 and it is a correlate of both blood
pressure91 92 and metabolic cardiovascular risk
factors.29 93 Hematocrit is the most important single
determinant of whole-blood viscosity, because viscosity increases
directly with hematocrit throughout its normal range.92
Mental stress can also increase hematocrit,94 95 and in
the present study, hematocrit increased progressively with
increasing arterial plasma epinephrine within the lower
pathophysiological concentration range; thus, sympathetic activity may
also influence this aspect of cardiovascular risk. In fact, in a recent
epidemiological survey in Tecumseh, Mich, high hematocrit was
associated with signs of a hypersympathetic state.93
Sympathetic activation may increase hematocrit through -adrenergic
vasoconstriction and a decrease in plasma volume. Thus,
Cohn96 many years ago demonstrated a prompt decrease in
plasma volume during the intravenous infusion of norepinephrine into
humans.
In conclusion, by evaluating a rather broad spectrum of platelet function parameters, we found that under acute conditions, circulating epinephrine within the lower pathophysiological concentrations recruits platelets into the circulation and induces some microaggregation or adhesion in healthy young men, but there is downregulation of the mechanisms that induce aggregation and significant release does not occur. Thus, it seems that the platelets may protect themselves against inappropriate overstimulation by epinephrine. It can be speculated that the effects of circulating epinephrine on platelet function are related to the rather large uptake of epinephrine into the platelets, whereas platelet N+-H+ exchange does not seem to be involved. Epinephrine has a pronounced effect on raising hematocrit.
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Acknowledgments |
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This study was supported in part by the International Society of Hypertension, Schering-Plough Fellowship Award; the Norwegian Research Council for Science and the Humanities; the National Heart, Lung, and Blood Institute, National Institutes of Health (grant HL-37464); the Clinical Research Center at the University of Michigan (grant 5 M01 RR00042); and the Michigan Diabetes Training and Research Center (grant P 560-DK 20572-12). The authors thank the staff of the Clinical Research Center, the Diabetes Research and Training Center, and the Division of Hypertension of the University of Michigan Hospitals for technical assistance.
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Footnotes |
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Reprint requests to Sverre E. Kjeldsen, MD, PhD, Department of Internal Medicine, Ullevaal Hospital, N-0407 Oslo, Norway. E-mail sverrekj@ulrik.uio.no.
Received September 28, 1994; first decision November 15, 1994; accepted January 18, 1995.
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