Donate Help Contact The AHA Sign In Home
American Heart Association
Hypertension
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Hypertension. 1995;25:1096-1105

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

(Hypertension. 1995;25:1096-1105.)
© 1995 American Heart Association, Inc.

Articles

Effect of Circulating Epinephrine on Platelet Function and Hematocrit

Sverre E. Kjeldsen; Alan B. Weder; Brent Egan; Richard Neubig; Andrew J. Zweifler; Stevo Julius

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.).


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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 {Delta}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


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
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).


View this table:
[in this window]
[in a new window]
  		Table 1. Platelet Size, Platelet Count, and Hematocrit in EDTA-Anticoagulated Blood and Plasma Epinephrine During Infusion With Saline and Epinephrine

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).


View this table:
[in this window]
[in a new window]
  		Table 2. Platelet Size, Platelet Count, and Hematocrit in Acid-Citrate-Dextrose–Anticoagulated Blood During Infusion With Saline and Epinephrine



View larger version (33K):
[in this window]
[in a new window]
  		Figure 1. Line graph shows that the increase ({Delta}) in platelet count during epinephrine infusion was significant (P<.0001) compared with baseline for both EDTA- and acid-citrate-dextrose (ACD)–anticoagulated blood and larger in EDTA- compared with ACD-anticoagulated blood (P=.0938 for change in 40 to 140 minutes and P=.0295 for change in 60 to 120 minutes, but P<.0001 for time points 80 through 120 minutes [paired t test]).

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.



View larger version (30K):
[in this window]
[in a new window]
  		Figure 2. Line graph shows that the increase in platelet size in EDTA-anticoagulated blood (P<.0001) and the decrease in platelet size in acid-citrate-dextrose (ACD)–anticoagulated blood (P<.02) during epinephrine infusion were significant compared with baseline and that the difference in response to epinephrine infusion between platelet size in EDTA and ACD was significant (P<.0001).

Aggregation in vitro was performed with platelets adjusted to 3x108 cells per milliliter. Aggregation induced by epinephrine decreased (P<.003 for {Delta}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.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 3. Line graph shows that aggregation induced by epinephrine decreased after epinephrine infusion compared with saline infusion for both {Delta}optical density and for maximal optical density (n=40).



View larger version (16K):
[in this window]
[in a new window]
  		Figure 4. Line graph shows that aggregation induced by ADP did not change after epinephrine infusion compared with saline infusion (n=40).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 5. Line graph shows that aggregation induced by collagen did not change after epinephrine infusion compared with saline infusion (n=40).


View this table:
[in this window]
[in a new window]
  		Table 3. Differences in In Vitro Platelet Aggregation at Each Agonist Concentration After Intravenous Epinephrine Infusion Compared With Intravenous Saline Infusion

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).


View this table:
[in this window]
[in a new window]
  		Table 4. Plasma Concentrations of Platelet Release Factors, Platelet Weight, and Platelet Content of Catecholamines After Intravenous Infusion of Saline and Epinephrine

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).



View larger version (56K):
[in this window]
[in a new window]
  		Figure 6. Bar graph shows that the amiloride-sensitive platelet volume increase in response to propionate exposure, an index of sodium-proton exchange (bottom), and line graph shows that the actual platelet volume increase at the end of 10 minutes, by which time the platelets have achieved a new steady-state size (top), were not significantly affected by epinephrine infusion.

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).



View larger version (32K):
[in this window]
[in a new window]
  		Figure 7. Line graph shows that hematocrit increased progressively with increasing arterial epinephrine concentration slightly above the resting level with no difference between EDTA- and acid-citrate-dextrose (ACD)–anticoagulated blood.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
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 {alpha}2-adrenergic receptor mechanism.37 38 Of the recently described subtypes of the {alpha}2-adrenergic receptor,39 the human platelet appears to contain only the {alpha}2a subtype.40 41 There is no clear evidence to implicate any other {alpha}-receptor subtype in platelet responses.

The exact mechanism of {alpha}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 {alpha}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, {alpha}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 {alpha}2-adrenergic receptors may more directly activate phospholipase A2 via release of G protein ß{gamma}-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, {alpha}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 {alpha}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 {alpha}-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 {alpha}-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.


*    Acknowledgments
 
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.


*    Footnotes
 
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.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Hamet P, Tremblay J, Sugimoto H. Platelets in hypertension and peripheral vascular disease. In: Longenecker GL, ed. The Platelets. Physiology and Pharmacology. New York, NY: Academic Press Inc; 1985:367-381.
  2. De Clerck F. Blood platelets in human essential hypertension. Agents Actions. 1986;18:563-580. [Medline] [Order article via Infotrieve]
  3. Nyrop M, Zweifler AJ. Platelet aggregation in hypertension and the effects of antihypertensive treatment. J Hypertens. 1988;6:263-269. [Medline] [Order article via Infotrieve]
  4. Kjeldsen SE, Neubig RR, Weder AB, Zweifler AJ. The hypertension-coronary heart disease dilemma: the catecholamine-blood platelet connection. J Hypertens. 1989;7:851-860. [Medline] [Order article via Infotrieve]
  5. Thaulow E, Erikssen J, Sandvik L, Stormorken H, Cohn PF. Blood platelet count and function are related to total and cardiovascular death in apparently healthy men. Circulation. 1991;84:613-617. [Abstract/Free Full Text]
  6. Weksler BB, Nachman RL. Platelets and atherosclerosis. Am J Med. 1981;71:331-333. [Medline] [Order article via Infotrieve]
  7. Ross R. The pathogenesis of atherosclerosis: an update. N Engl J Med. 1986;314:488-500. [Medline] [Order article via Infotrieve]
  8. Turitto VT. Blood viscosity, mass transport, and thrombogenesis. Prog Hemost Thromb. 1982;6:139-177. [Medline] [Order article via Infotrieve]
  9. Stehbens WE. Relationship of coronary-artery thrombosis to myocardial infarction. Lancet. 1985;2:639-642. [Medline] [Order article via Infotrieve]
  10. Hamm CW, Lorenz RL, Bleifeld W, Kupper W, Wober W, Weber PC. Biochemical evidence of platelet activation in patients with persistent unstable angina. J Am Coll Cardiol. 1987;10:998-1004. [Abstract]
  11. Franco-Morselli R, Elghozi JL, Joly E, DiGiuilio S, Meyer P. Increased plasma adrenaline concentrations in benign essential hypertension. Br Med J. 1977;2:1251-1254.
  12. Eliasson K, Hjemdahl P, Kahan T. Circulatory and sympathoadrenal responses to stress in borderline and established hypertension. J Hypertens. 1983;1:131-139. [Medline] [Order article via Infotrieve]
  13. Hjemdahl P. Plasma catecholamines as markers for sympathoadrenal activity in human primary hypertension. Pharmacol Toxicol. 1988;63(suppl 1):27-31.
  14. Clutter WE, Bier DM, Shah SD, Cryer PE. Epinephrine plasma metabolic clearance rates and physiologic thresholds for metabolic and hemodynamic action in man. J Clin Invest. 1980;60:94-101.
  15. Staten MA, Matthews DE, Cryer PE, Bier DM. Physiological increments in epinephrine stimulate metabolic rate in humans. Am J Physiol. 1987;253:E322-E330. [Abstract/Free Full Text]
  16. Kjeldsen SE, Petrin J, Weder AB, Julius S. Contrasting effects of epinephrine on forearm hemodynamics and arterial plasma norepinephrine. Am J Hypertens. 1993;6:369-375. [Medline] [Order article via Infotrieve]
  17. Rostrup M, Westheim A, Kjeldsen SE, Eide I. Cardiovascular reactivity, coronary risk factors, and sympathetic activity in young men. Hypertension. 1993;22:891-899. [Abstract/Free Full Text]
  18. Dimsdale J, Moss J. Plasma catecholamines in stress and exercise. JAMA. 1980;243:340-342. [Abstract]
  19. Hjemdahl P, Freyschuss U, Juhlin-Dannfelt A, Linde B. Differentiated sympathetic activation during mental stress evoked by the Stroop test. Acta Physiol Scand Suppl. 1984;527:25-29. [Medline] [Order article via Infotrieve]
  20. Ekeberg Ø, Kjeldsen SE, Greenwood DT, Enger E. Correlations between psychological and physiological responses to acute flight phobia stress. Scand J Clin Lab Invest. 1990;50:671-677. [Medline] [Order article via Infotrieve]
  21. Rostrup M, Mundal HH, Westheim A, Eide I. Awareness of high blood pressure increases arterial plasma catecholamines, platelet noradrenaline and adrenergic responses to mental stress. J Hypertens. 1991;9:159-166. [Medline] [Order article via Infotrieve]
  22. Friedman M, Rosenman RH. Association of specific overt behaviour pattern with blood and cardiovascular findings. JAMA. 1959;169:1286-1296.
  23. Rosenman R, Brand R, Sholtz R, Friedman M. Multivariate prediction of coronary heart disease during 8 year followup in the Western Collaborative Group Study. Am J Cardiol. 1976;37:902-910.
  24. Clayton S, Cross MJ. The aggregation of blood platelets by cate- cholamines and thrombin. J Physiol (Lond). 1963;169:82P-83P.
  25. O'Brien JR. Some effects of adrenaline and anti-adrenaline compounds on platelets in vitro and in vivo. Nature. 1963;200:763-764. [Medline] [Order article via Infotrieve]
  26. Hjemdahl P, Larsson PT, Wallén NH. Effects of stress and ß-blockade on platelet function. Circulation. 1991;84(suppl VI):VI-44-VI-61.
  27. de Simone G, Devereux RB, Chien S, Alderman MH, Atlas SA, Laragh JH. Relation of blood viscosity to demographic and physiologic variables and to cardiovascular risk factors in apparently normal adults. Circulation. 1990;8:107-117.
  28. Erikssen G, Thaulow E, Sandvik L, Stormorken H, Erikssen J. Haematocrit: a predictor of cardiovascular mortality? J Intern Med. 1993;234:493-499. [Medline] [Order article via Infotrieve]
  29. Moan A, Nordby G, Os I, Birkeland KI, Kjeldsen SE. Relationship between hemorrheological factors and insulin sensitivity in healthy, young men. Metabolism. 1994;43:423-427. [Medline] [Order article via Infotrieve]
  30. Schmouder RL, Weder AB. Platelet sodium-proton exchange is increased in essential hypertension. J Hypertens. 1989;7:325-330. [Medline] [Order article via Infotrieve]
  31. Frojmovic MM, Milton JG, Gear AL. Platelet aggregation measured in vitro by microscopic and electronic particle counting. Methods Enzymol. 1989;169:134-149. [Medline] [Order article via Infotrieve]
  32. Egan B, Conlon ME, Campbell R, Schork N, Zweifler A, Julius S. Effects of ketanserin on blood pressure and platelet aggregation in elderly men with mild hypertension. Am J Hypertens. 1988;1(suppl):S324-S330.
  33. Gupta RK, Kjeldsen SE, Motley E, Weder AB, Zweifler AJ, Julius S. Platelet function during antihypertensive treatment with quinapril, a novel angiotensin converting enzyme inhibitor. J Cardiovasc Pharmacol. 1991;17:13-19. [Medline] [Order article via Infotrieve]
  34. Zweifler AJ, Julius S. Increased platelet catecholamine content in pheochromocytoma. N Engl J Med. 1982;306:890-894. [Abstract]
  35. Peuler JD, Johnson GA. Simultaneous single isotope radioenzymatic assay of plasma norepinephrine, epinephrine and dopamine. Life Sci. 1977;21:625-636. [Medline] [Order article via Infotrieve]
  36. Grinstein S, Goetz JD, Furuya W, Rothstein A, Gelfand EW. Amiloride-sensitive Na+-H+ exchange in platelets and leukocytes: detection by electronic cell sizing. Am J Physiol. 1984;247:C293-C298. [Abstract/Free Full Text]
  37. Grant JA, Scrutton MC. Novel alpha2-adrenoceptors primarily responsible for inducing platelet aggregation. Nature. 1979;277:659-661. [Medline] [Order article via Infotrieve]
  38. Lasch P, Jakobs KH. Agonistic and antagonistic effects of various alpha-adrenergic agonists in human platelets. Naunyn Schmiedebergs Arch Pharmacol. 1979;306:119-125. [Medline] [Order article via Infotrieve]
  39. Bylund DB. Subtypes of alpha2-adrenoceptors: pharmacological and molecular biological evidence converge. Trends Pharmacol Sci. 1988;9:356-361. [Medline] [Order article via Infotrieve]
  40. Kobilka BK, Matsui H, Kobilka TS, Yang-Feng TL, Francke U, Caron MG, Lefkowitz RJ, Regan JW. Cloning, sequencing, and expression of the gene coding for the human platelet alpha 2-adrenergic receptor. Science. 1987;238:650-656. [Abstract/Free Full Text]
  41. Regan JW, Kobilka TS, Yang-Feng TL, Caron MG, Lefkowitz RJ, Kobilka BK. Cloning and expression of a human kidney cDNA for an alpha2-adrenergic receptor subtype. Proc Natl Acad Sci U S A. 1988;85:6301-6305. [Abstract/Free Full Text]
  42. Ui M. Islet-activating protein, pertussis toxin: a probe for functions of the inhibitory guanine nucleotide regulatory component of adenylate cyclase. Trends Pharmacol Sci. 1984;5:277-279.
  43. Gilman AG. G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987;56:615-649. [Medline] [Order article via Infotrieve]
  44. Kaibuchi K, Takai Y, Ogawa Y, Kimura S, Nishizuka Y. Inhibitory action of adenosine 3',5'-monophosphate on phosphatidylinositol turnover: difference in tissue response. Biochem Biophys Res Commun. 1982;104:105-112. [Medline] [Order article via Infotrieve]
  45. Watson SP, McConnell RT, Lapetina EG. The rapid formation of inositol phosphates in human platelets by thrombin is inhibited by prostacyclin. J Biol Chem. 1984;259:13199-13203. [Abstract/Free Full Text]
  46. Pannocchia A, Hardisty RM. Cyclic AMP inhibits platelet activation independently of its effect on cytosolic free calcium. Biochem Biophys Res Commun. 1985;127:339-345. [Medline] [Order article via Infotrieve]
  47. Rink TJ, Sanchez A. Effects of prostaglandin I2 and forskolin on the secretion from platelets evoked at basal concentrations of cytoplasmic free calcium by thrombin, collagen, phorbol ester and exogenous diacylglycerol. Biochem J. 1984;222:833-836. [Medline] [Order article via Infotrieve]
  48. Haslam RJ, Davidson MM, Davies T, Lynham JA, McClenaghan MD. Regulation of blood platelet function by cyclic nucleotides. Adv Cyclic Nucleotide Res. 1978;9:533-552. [Medline] [Order article via Infotrieve]
  49. Limbird LE. Receptors linked to inhibition of adenylate cyclase: additional signalling mechanisms. FASEB J. 1988;2:2686-2695. [Abstract]
  50. Siess W, Weber PC, Lapetina EG. Activation of phospholipase C is dissociated from arachidonate metabolism during platelet shape change induced by thrombin or platelet-activating factor: epinephrine does not induce phospholipase C activation or platelet shape change. J Biol Chem. 1984;259:8286-8292. [Abstract/Free Full Text]
  51. Sweatt JD, Blair IA, Cragoe EJ, Limbird LE. Inhibitors of Na+/H+ exchange block epinephrine- and ADP-induced stimulation of human platelet phospholipase C by blockade of arachidonic acid release at a prior step. J Biol Chem. 1986;261:8660-8666. [Abstract/Free Full Text]
  52. Owen NE, Feinberg H, LeBreton GC. Epinephrine induces Ca2+ uptake in human blood platelets. Am J Physiol. 1980;239:H483-H488.
  53. Powling MJ, Hardisty RM. Potentiation by adrenaline of Ca2+ influx and mobilization in stimulated human platelets: dissociation from thromboxane generation and aggregation. Thromb Haemost. 1988;59:212-215. [Medline] [Order article via Infotrieve]
  54. Gerhardt MA, Neubig RR. Epinephrine potentiates the ADP-induced calcium response in human platelets. FASEB J. 1988;2:A398. Abstract.
  55. Erne P, Bühler FR, Affolter H, Bürgisser E. Excitatory and inhibitory modulation of intracellular free calcium in human platelets. Eur J Pharmacol. 1983;91:331-332. [Medline] [Order article via Infotrieve]
  56. Johnson PC, Ware JA, Cliveden PB, Smith M, Dvorak AM, Salzman EW. Measurement of ionized calcium in blood platelets with the photoprotein aequorin: comparison with quin2. J Biol Chem. 1985;260:2069-2076. [Abstract/Free Full Text]
  57. Sweatt JD, Connolly TM, Cragoe EJ, Limbird LE. Evidence that Na+/H+ exchange regulates receptor-mediated phospholipase A2 activation in human platelets. J Biol Chem. 1986;261:8667-8673. [Abstract/Free Full Text]
  58. Zavoico GB, Cragoe EJ, Feinstein MB. Regulation of intracellular pH in human platelets: effects of thrombin, A23187, and ionomycin and evidence for activation of Na+/H+ exchange and its inhibition by amiloride analogs. J Biol Chem. 1986;261:13160-13167. [Abstract/Free Full Text]
  59. Jelsema CL, Axelrod J. Stimulation of phospholipase A2 activity in bovine rod outer segments by the beta gamma subunits of transducin and its inhibition by the alpha subunit. Proc Natl Acad Sci U S A. 1987;84:3623-3627. [Abstract/Free Full Text]
  60. Levine SP, Towell BL, Suarez AM, Knieriem LK, Harris MM, George JN. Platelet activation and secretion associated with emotional stress. Circulation. 1985;71:1129-1134. [Abstract/Free Full Text]
  61. Larsson PT, Hjemdahl P, Olsson G, Egberg N, Hornstra G. Altered platelet function during mental stress and adrenaline infusion in humans: evidence for an increased aggregability in vivo as measured by filtragometry. Clin Sci. 1989;76:369-376. [Medline] [Order article via Infotrieve]
  62. Hornstra G, ten Hoor F. The filtragometry: a new device for measuring platelet aggregation in venous blood of man. Thromb Diathes Haemorrh. 1975;34:531-544. [Medline] [Order article via Infotrieve]
  63. Laustiola K, Kaukien S, Seppälä E, Jokela T, Vapaatalo H. Adrenaline infusion evokes increased thromboxane B2 production by platelets in healthy men: the effect of beta-adrenoceptor blockade. Eur J Clin Invest. 1986;16:473-479. [Medline] [Order article via Infotrieve]
  64. Arkel YS, Haft JI, Kreutner W, Sherwood J, Williams R. Alteration in second phase platelet aggregation associated with an emotional stressful activity. Thromb Haemost. 1977;38:552-561. [Medline] [Order article via Infotrieve]
  65. Siess W, Lorenz R, Roth P, Weber PC. Plasma catecholamines, platelet aggregation and associated thromboxane formation after physical exercise, smoking, and norepinephrine infusion. Circulation. 1982;66:44-48. [Abstract/Free Full Text]
  66. Epstein FH. Mechanisms of membrane-receptor regulation. N Engl J Med. 1984;310:1570-1579. [Medline] [Order article via Infotrieve]
  67. Hollister AS, Fitzgerald A, Nadeau JHJ, Robertson D. Acute reduction in human platelet alpha-2-adrenoreceptor affinity for agonist by endogenous and exogenous catecholamines. J Clin Invest. 1983;72:1498-1505.
  68. Zweifler AJ, Romero S. Effect of phentolamine on platelet aggregation in patients with pheochromocytoma. Thromb Res. 1975;7:505-513. [Medline] [Order article via Infotrieve]
  69. Ardlie NG, Cameron HA, Garrett J. Platelet activation by circulating levels of hormones: a possible link in coronary heart disease. Thromb Res. 1984;36:315-322. [Medline] [Order article via Infotrieve]
  70. Lande K, Kjeldsen SE, Os I, Westheim A, Hjermann I, Eide I, Gjesdal K. Increased platelet and vascular smooth muscle reactivity to low-dose adrenaline infusion in mild essential hypertension. J Hypertens. 1988;6:219-225. [Medline] [Order article via Infotrieve]
  71. Aster RH. Pooling of platelets in the spleen: role in the pathogenesis of `hypersplenic' thrombocytopenia. J Clin Invest. 1966;45:645-657.
  72. Fréden K, Lundborg P, Vilén L, Kutti J. The peripheral platelet count in response to adrenergic alpha- and beta-1-receptor stimulation. Scand J Haematol. 1978;21:427-432. [Medline] [Order article via Infotrieve]
  73. Karpatkin S. Heterogeneity of human platelets, VI: correlation of platelet function with platelet volume. Blood. 1978;51:307-316. [Free Full Text]
  74. Frojmovic M, Milton J. Human platelet size, shape and related functions in health and disease. Physiol Rev. 1982;62:185-261. [Free Full Text]
  75. Thompson CB, Eaton KA, Princiotta SM, Rushin CA, Varen CR. Size dependent platelet subpopulations: relationship of platelet volume to ultrastructure, enzymatic activity and function. Br J Haematol. 1982;50:509-519. [Medline] [Order article via Infotrieve]
  76. Peerschke EIB. Induction of human platelet fibrinogen receptors by epinephrine in the absence of released ADP. Blood. 1982;60:71-77. [Abstract/Free Full Text]
  77. Bennett JS, Vilaire G. Exposure of platelet fibrinogen-receptors by ADP and epinephrine. J Clin Invest. 1979;64:1393-1401.
  78. Plow EF, Marguerie GA. Induction of the fibrinogen receptor on human platelets by epinephrine and the combination of epinephrine and ADP. J Biol Chem. 1980;255:10971-10977. [Abstract/Free Full Text]
  79. Bonga HS, Simons ER, Brass LF, Rittenhouse SE. Activation of phospholipases A and C in human platelets exposed to epinephrine: role of glycoproteins IIb/IIa and dual role of epinephrine. Proc Natl Acad Sci U S A. 1986;83:9197-9201. [Abstract/Free Full Text]
  80. Forwell GD, Ingram GIC. The effect of adrenaline infusion on human blood coagulation. J Physiol (Lond). 1957;135:371-383.
  81. Ingram GIC. Increase in antihaemophilic globulin activity following infusion of adrenaline. J Physiol (Lond). 1961;156:217-224.
  82. Hoak JC, Spector AA, Warner ED. Effect of free fatty acids on ADP-induced platelet aggregation. Nature. 1970;228:1330-1332. [Medline] [Order article via Infotrieve]
  83. Zucker MB, Nachmias VT. Platelet activation. Arteriosclerosis. 1985;5:2-18. [Abstract/Free Full Text]
  84. Zahavi J, Kakkar VV. Beta-thromboglobulin: a specific marker of in vivo platelet release reaction. Thromb Haemost. 1980;44:23-29. [Medline] [Order article via Infotrieve]
  85. Kaplan KL, Owen J. Plasma levels of beta-thromboglobulin and platelet factor 4 as indices of platelet activation in vivo. Blood. 1981;57:199-202. [Abstract/