Platelet qadrenergic receptors in hypercholesterolemia: Relationship between binding studies and epinephrine-induced platelet aggregation

Background: Platelets isolated from patients with hypercholesterolemia are more sensitive in vitro to various aggregating agents, inchuiing epinephrine, than those isolated from normocholesterolemic subjects. Increased platelet reactivity is one mechanism that may explain the enhanced risk of thromboembolism in hypercholesterolemia. This study assessed whether platelet hyperreactivity to epinephrine in hypercholesterol- emiais associated with higher cu,-adrenergic receptor density or a%inity for epinephrine. Methods: Platelet aggregation and binding studies, with use of [ 3H]yohimbine as ligand, were performed on platelets isolated from 30 patients with type IIa hypercholesterolemia and 23 control subjects. Results: Platelet aggregation in response to epinephrine was significantly higher in patients with hyper- cholesterolemia than in control subjects. A statistically significant higher cx,-adrenergic receptor density was observed in a subgroup of 13 patients with hypercholesterolemia than in 13 sex- and age-matched control subjects (280 + 61 and 230 f 49 finol/mg protein respectively; p < 0.03), but no difference was observed in receptor affinity for the ligand. In these subgroups plasma total and levels of low-density lipoprotein (LDL) cholesterol were inversely correlated with platelet aggregation but directly correlated with platelet receptor density. G~~lu.&n: Platelet o,-adrenergic receptor density is increased in hypercholesterolemia and directly correlates with plasma total and levels of LDL cholesterol, providing at least a partial explanation for the enhanced platelet response to epinephrine that is observed in hypercholesterolemia. (Clin Pharmacol Ther 1997;61:684- 91.)

Damiano Baldassarre, PhD, Nadia Mores, MD, Susanna Colli, PhD, Franc0 Pazzucconi, MD, Cesare R Sirtori, MD, PhD, and Elena Tremoli, PhD Milan and Rome, Italy

Type IIa hypercholesterolemia is accompanied by an increased risk of atherosclerosis and its acute thromboembolic complications.‘” Experimental and clinical evidence supports a link between hyper- lipidemia and the functional and biochemical alter- ations of platelets,4,5 as indicated by the greater in vitro sensitivity of platelets from patients with hy- percholesterolemia to a variety of aggregating

From the Enrica Grossi Paoletti Center, Institute of Pharmaco- logical Sciences, University of Milan, Milan, and the Institute of Pharmacology, Catholic University Rome, Rome.

Received for publication Oct. 11, 1996; accepted Jan. 24, 1997. Reprint requests: Elena Tremoh, PhD, Institute of Pharmacolog-

ical Sciences, University of Milan, Via Balzaretti, 9, 20129, Milan, Italy.

Copyright 0 1997 by Mosby-Year Book, Inc. 0009-9236/97/$5.00 + 0 13/l/80688

684

agents. 6-9 Moreover, in hypercholesterolemia, in- creased urinary excretion of the eicosanoid metab- olite 11-dehydrothromboxane B2 has been reported, which indicates that platelet activation occurs also in viv0.l’

Biochemical studies have shown that low-density lipoproteins (LDL) enhance the platelet response in the absence11T12 and in presence of agonists (thrombin, adenosine diphosphate, epinephrine, collagen) 13-15 through a direct interaction with the glycoprotein IIb-IIIa complex that results in an in- creased binding of platelets to the physiologic ligand fibrinogen. 16-‘* Changes of platelet membrane com- position, that is, increased cholesterol content or the arachidonic acid content of phospholipids,‘9-21 have been held responsible for the altered platelet reac- tivity.

CLINICAL P HARMACOLOGY &THERAPEUTICS VOLUME 61. NUMBER6 Baldassarre et al. 685

Other results show that the greater sensitivity of platelets from patients with type IIa hypercholester- olemia is associated with the patients’ reduced re- sponse to inhibitors of platelet aggregation.22,23 Platelets of such patients are less sensitive to the antiaggregatory effects of prostaglandin D, and ilo- prost, a stable prostacyclin derivative, and this phe- nomenon is ascribed to a reduction in the number of platelet-binding sites for antiaggregatory eico- sanoids23 or to a limited access of the ligand to the receptor site, consequent on the altered membrane lipid composition.24

Increased plasma cholesterol levels have been as- sociated with an increased sympathetic nervous sys- tem reactivity, as indicated by the elevated norepi- nephrine levels in plasma and in platelets of patients with hypercholesterolemia.25 This increase may in turn contribute to the platelet hyperaggregability observed in this metabolic disorder.

In an attempt to investigate the biochemical as- pects underlying the altered platelet response in hypercholesterolemia, we compared the platelet a,-adrenergic receptor status of patients with hypercholesterolemia with that of normolipidemic subjects. The relationships among platelet ct2- adrenergic receptor density, platelet sensitivity to epinephrine, and plasma cholesterol levels were also investigated.

METHODS Subjects and blood collection. Thirty patients with

type IIa hypercholesterolemia (19 men and 11 wom- en; age range, 19 to 69 years) were selected among those attending the E. Grossi Paoletti Center for the Study of Metabolic Diseases and were classified ac- cording to the World Health Organization criteria.26 The classification into type IIa was based on plasma total cholesterol and triglyceride levels and on li- poprotein fractionation. The patients were free of established atherosclerotic lesions (no angina, clau- dication, or cerebrovascular ischemia) and had no clinical evidence of arterial occlusive disease (e.g., decreased vascular pulses or bruits). Subjects with hypertension or diabetes were not included in the study. None of the patients had been taking hypo- lipidemic drugs in the 2 months before the study. All patients were on a standard low-lipid diet (approx- imate composition: 26% fat, 22% proteins, and 52% carbohydrates; P/S ratio, 0.43)27 for at least 2 months before the blood collection. The results of the platelet studies were compared with those ob- tained from samples of 23 normolipidemic individ-

uals (14 men and nine women; age range, 22 to 63 years) who were selected among relatives of patients and among the medical staff of the hospital. The control subjects had dietary habits comparable to those of patients, Both patients and control subjects had been instructed to avoid any drug that would affect platelet function for at least 15 days before blood sampling. In addition, control subjects were also instructed to avoid alcohol and fatty meals at least for 1 week before the blood sampling. The protocol of the study was approved by the ethical committee of the E. Grossi Paoletti Center, and informed consent was obtained from all the patients.

In the whole group there were 15 smokers (nine patients with hypercholesterolemia and six volun- teers) who were asked to refrain from smoking in the 12 hours before the blood collection. Venous blood was taken after an overnight fast between 8:30 and 9:30 AM from the forearm antecubital vein after the subjects rested for 15 minutes in the supine position.

Measurement of plasma lipids and lipoproteins. Plasma was obtained from blood anticoagulated with sodium ethylenediaminetetraacetic acid (EDTA; 1 mg/ml). Total cholesterol and triglycerides were mea- sured by enzyme methods.28,29 High-density lipopro- tein (HDL) cholesterol was measured after selective precipitation of apoliprotein B-containing lipopro- teins with dextran-sulfate, and magnesium chloride.30 Plasma LDL cholesterol was calculated by Friede- wald’s formula.3l

Platelet isolation. For platelet aggregation studies, platelet-rich plasma was prepared by centrifugation of blood anticoagulated with 3.8% sodium citrate, 1:9 (vol/vol), at 150g for 18 minutes. For binding studies, platelets were isolated from platelet-rich plasma according to Yamaguchi.32 In brief, blood samples (40 ml) were collected into acid citrate/ dextrose, 1.5:8.5 (vol/vol). The platelet pellet was obtained by centrifugation of platelet-rich plasma at 450g for 15 minutes and resuspended in 1 ml Tyrode-Hepes (140 mmol/L sodium chloride, 2.7 mmol/L potassium chloride, 12 mmol/L sodium bi- carbonate, 0.4 mmol/L monobasic sodium phos- phate, 0.2 mmoliL magnesium chloride, and 5 mmol/L Hepes, pH 7.35) that contained ethylene- glycoltetraacetic acid (7 mmol/L) and prostaglandin E, (1 kmol/L). Samples were centrifuged (10,OOOg for 1 minute) and finally resuspended in Tyrode- Hepes. Washed platelet samples were immediately frozen at -80” C and binding studies of 02- adrenergic receptors were performed on platelet

686 Baldassarre et al. CLINICAL PHARMA COLOGY &THERAPEUTICS

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membranes not more than 20 days after the prepa- ration of samples.33

Plutelet aggregation studies. Platelet aggregation was studied with use of an Elvi Logos aggregome- ter (Elvi Logos, Milan, Italy) as described previ- 0us1y.~ Transmittance was continuously moni- tored by an Omniscribe recorder (Elvi Logos, Milan, Italy). Epinephrine (Angelini S.p.a. Italy) was used as the aggregating agent. Dilutions in saline solution were freshly made from a stock solution (1 mg/ml).

For each subject the threshold aggregatory con- centration for biphasic platelet aggregation and the agonist concentration giving 50% aggregation (AC,,) were determined at 5 minutes.34 In addition, AC,, values were also calculated at 1 minute after the addition of the stimulus. The choice of these times at which to calculate AC,, was based on the consideration that the first wave of aggregation in response to epinephrine reflects the binding of the agonist to the a,-adrenergic receptors,35 whereas the AC+,, 5 minutes after the addition of the agonist represents the second wave. All aggregation exper- iments were performed by a single operator.

cu,-Adrenergic receptor binding assay. Binding stud- ies were performed on platelet membranes with use of [3H]yohimbine (specific activity, 88.4 Ci/mmol; New England Nuclear Corporation), as described previously.36 In brief, platelets were resuspended in ice-cold lysis buffer (5 mmol/L Tris-hydrochloric acid, 5 mmol/L Na,EDTA, pH 8), incubated for 40 minutes at 4” C, homogenized by a Teflon pestle, and centrifuged at 30,OOOg for 10 minutes.

The crude membrane pellet was washed twice, resuspended in 50 mmol/L Tris- hydrochloric acid, pH 7, and incubated at 25” C for 30 minutes with increasing [3H]yohimbine concentrations (0.5 to 20 nmol/L). The suspension was filtered through glass fiber filters (GF/C Filters, Whatman Laboratory Products, Inc., Clifton N.J.) and rinsed with ice-cold 50 mmol/L Tris- hydrochloric acid, pH 7. The filters were counted by liquid scintillation spectrometry.

Specific binding was defined as total minus non- specific binding, the latter being the binding dis- placed by 10 pmol/L phentolamine. Maximum bind- ing capacity (B,,, in femtomoles per milligram of protein) and dissociation constants (&, in nano- moles per liter) for the ol,-adrenergic receptors were determined from linear Scatchard plots. The protein concentration was determined by the method of Lowry.37

Statistical analysis. Means and SD values were used as descriptive measures of normally distributed variables. Group comparisons were made by use of the unpaired Student t test for all variables except for triglycerides, which were analyzed by the Mann- Whitney U test. Comparisons of groups matched for age and sex were performed by the paired Student t test (triglycerides after logarithmic transformation). Correlations were performed by use of parametric methods for normally distributed variables (Pearson correlation); when this condition was not satisfied, nonparametric methods (Spearman correlation) were used. Multiple stepwise regression analysis was used to determine the relative importance in pre- dicting B,, values of each variable considered [to- tal cholesterol, HDL cholesterol, LDL cholesterol, log(triglycerides), age, uric acid, and blood glucose].

RESULTS The selected group of patients with hypercholester- olemia differed from control subjects in total and LDL cholesterol (total cholesterol, 296 ?Z 34 versus 196 + 24 mg/dl; LDL, 214 2 33 versus 121 & 22 mg/dl for patients and control subjects; bothp < 0.0001). In addition, although patients were not hypertriglyceride- mic, their triglyceride levels were signilicantly higher than those of control subjects (131 + 36 versus 95 + 36 mg/dl; p < 0.0001). Other variables (age, HDL cho- lesterol, uric acid, and blood glucose) were similar in the two groups of subjects.

As expected,8 platelets from patients with hy- percholesterolemia required significantly lower amounts of epinephrine to undergo irreversible ag- gregation than platelets from control subjects (threshold aggregatory concentrations, 0.4 ? 0.41 versus 1.2 2 1.27 bg/ml;p = 0.007). Moreover, AC,, values for epinephrine calculated at 1 and 5 minutes after the addition of the stimulus were significantly lower in the patients with hypercholesterolemia than in control subjects (0.24 -+ 0.16 versus 0.49 t 0.49 and 0.36 5 0.33 versus 0.88 ? 1.02 &ml respectively; both p < O.Ol), which confirmed that platelets from patients with hypercholesterolemia respond more to epinephrine than those from con- trol subjects. In this respect, threshold aggregatory concentration and A&, were found to be highly correlated (r = 0.97 and r = 0.93 for AC,a at 1 and 5 minutes, respectively; bothp < 0.0001).

In studies of binding to [3H]yohimbine, there was no difference in B,, and I& between platelets from patients and those from control subjects (B,,, 259 rt 70.2 versus 265 + 69.5 fmol/mg protein, p >

CLINICAL PHARh4A COLOGY &THERAPEUTICS VOLUME 61, NUMBER 6 Baldassume et al. 687

Table I. Characteristics of the age- and sex-matched patients with hypercholesterolemia and control subjects Control Patients with p Value subjects hypercholesterolemia (between)

n 13 13 - Men/women 914 914 - Smokers (%) 2 (15.4) 5 (38.5) 0.37 Age 64 44.8 + 14.0 44.8 -c 14.5 0.82 LDL-C (mg/dl) 126.8 + 23.8 206.7 -c 29.4 0.0001 Total cholesterol (mg/dl) 202.0 5 24.2 285.5 -c 27.7 0.0001 Triglycerides (mg/dl) 100.1 ? 33.8 124.2 ? 28.8 0.062 HDL-C (mg/dl) 55.1 -c 11.4 53.9 t 11.4 0.78 Uric acid (mg/dl) 3.8 It 1.0 3.8 5 0.8 0.89 Blood glucose (mg/dl) 91.8 +: 13.4 93.6 2 12.9 0.72

Results are expressed as mean values k SD. HDL-C, High-density lipoprotein cholesterol; LDLC, low-density lipoprotein cholesterol. Statistical significance was determined by the paired Student t test (triglycerides after logarithmic transformation).

0.05; I(d, 4.06 + 1.42 versus 3.67 + 1.33 nmol/L,p > 0.05 in patients and control subjects, respectively).

Interestingly, we found a significant negative cor- relation between B,, and epinephrine AC,, at 1 and 5 minutes (r = -0.4 and r = -0.31, respectively; both p < 0.05), whereas the correlation between B and threshold aggregatory concentration was atmtye borderline of the statistical significance (r = -0.24; p = 0.08). No correlation was found between a,-adrenergic receptor density and lipid variables.

Although B,,, &, and platelet aggregation were similar in men and women in this study, previous reports indicate that age38,39 and se2s influence platelet ol,-adrenergic receptor density. Therefore 13 patients with hypercholesterolemia and 13 con- trol subjects matched for age and sex were selected from the whole group (Table I). These were repre- sentative of the overall groups of patients and con- trol subjects. In this subgroup, threshold aggrega- tory concentrations and AC,, values for epinephrine were again significantly lower in patients with hyper- cholesterolemia (Fig. 1). However, at variance with the observations in the whole group, B,, was sig- nificantly higher in the patients than in the matched control subjects (Fig. 2, A). No difference in I& values was found (Fig. 2, B).

As observed in the whole group, a significant inverse correlation between platelet aggregation (AC,, values and threshold aggregatory concentra- tion) and B,, was shown (Fig. 3). The levels of significance of these correlations were higher than those previously calculated (r = -0.63,~ < 0.001 for AC,, evaluated at 5 minutes; r = -0.42,~ < 0.05 for AC,, evaluated at 1 minute; and r = -0.5,~ < 0.01 for threshold aggregatory concentration). In addi-

tion, a significant inverse correlation between AC,, for epinephrine and plasma total cholesterol was detected (r = -0.36 and r = -0.39 for AC5c evalu- ated at 1 and 5 minutes, respectively; p < 0.05 for both). A nearly identical regression curve was ob- tained with use of LDL cholesterol instead of total cholesterol (r = -0.39 and r = -0.38 for AC,, evaluated at 1 and 5 minutes, respectively; p < 0.05 for both). Interestingly, in this group of subjects matched for age and sex, B,, was positively corre- lated with plasma total cholesterol and LDL choles- terol (Fig. 4). When multiple stepwise regression analysis was performed on this group, plasma total cholesterol level was the only independent predictor of B,, (l3 = 0.67, 3 = 0.301,~ = 0.003).

DISCUSSION The results indicate that the platelet hyperaggrega- bility to epinephrine observed in hypercholesterol- emia is, at least in part, related to an increased density of a,-adrenergic receptors on platelet mem- branes. Data obtained in the whole group of subjects enrolled in the study confirm and extend previous findings that indicated that platelets from patients with type IIa hypercholesterolemia are more sensi- tive to the in vitro effects of epinephrine than plate- lets from normal subjects.8 This conclusion is drawn both from the threshold concentration of epineph- rine necessary to bring about irreversible aggrega- tion and from the concentrations of epinephrine necessary to induce a 50% aggregation.

Few previous studies have examined the relation- ship between density or affinity of cqadrenergic receptors and functional alterations of platelets. A direct relationship between binding sites and

688 Baldassarre et al.

2,

=1-6 E s =I.2 E ‘C c f .8 3

.4

0 rl ACso (1 min) AC60 (5 min) TAC

CLINICAL PHARhiACOLOGY &THERAPEUTICS

TUNE 1997

Fig. 1. Platelet aggregation induced by epinephrine in control subjects (open bars) and in patients with hypercholesterolemia matched for age and sex (diagonally hatched bars). Platelet aggregation is represented as AC,, values calculated at 1 and 5 minutes from the addition of the stimulus and as threshold aggregatory concentration (TAC) for epinephrine. Values are mean 2 SEM. *p < 0.05 versus control subjects.

300- * 4-

g 28D 3.8.

h F 280. > g 3.2.

; 240. Y ;;’ 2.8,

v E 220. 2.4.

200- 2- C HC C HC

Fig. 2. [3H]Yohimbine maximal binding capacity (Bmax) and dissociation constant (KJ in platelet lysates from patients with hypercholesterolemia (HC) and in age- and sex-matched subjects with normal cholesterol levels (C). Values are mean 2 SEM. *p < 0.05 versus control subjects.

epinephrine-induced aggregation has been sug- the whole group was considered, a significantly gested in some pathologic conditions other than higher receptor density was observed in patients hypercholesterolemia, such as essential thrombocy- with hypercholesterolemia when two subgroups themia4’ or unstable angina.41 matched for age and sex were selected. The need

Although in our study no difference in the num- for careful patient selection by age and sex to ber or affinity of cy,-adrenergic receptors was appreciate differences in or,-adrenergic receptor found between patients and control subjects when density between groups indicates that appropriate

CLINICAL PHARMACOLOGY & THERAPEUTICS VOLUME 61, NUMBER 6 Baldassarre et al. 689

%I0 200 300 400

A B max (fmollmg)

07 100 200 300 400

B max (fmollmg)

r = -0.6 p * 0.0’

0 0 0

*

“T O

7 0 0

200 300 400

B max (fmollmg)

Fig. 3. Correlation of B,, and epinephrine-induced ag- gregation in the studied subjects. A, B,, versus AC,, evaluated 5 minutes after addition of the stimulus. B, B,, versus AC,, evaluated 1 minute after addition of the stimulus. C, B,, versus threshold aggregatory concentra- tion for epinephrine.

r =0.66 0 . p< 0.01

loo+ 50 150 250 350

A TC (mgldl)

r= 0.49 400. . 0 p < 0.06

6 0

s 30@

E

2 fJ 200.

0

1001 50 100 150 200 250 3 0

B LDL (mgldl)

Fig. 4. Correlation between B,, and total cholesterol (TC; A) and low-density lipoprotein (LDL) cholesterol (B) in 13 patients with hypercholesterolemia (solid circles) and 13 age- and sex-matched patients with normal cho- lesterol levels (open circles).

selection is of particular relevance in this type of studies.

The correlation of AC,, for epinephrine with oz- adrenergic receptor density, coupled with the obser- vation of identical & values in patients with hyper- cholesterolemia and control subjects, suggests that the higher platelet reactivity to epinephrine ob- served in patients is caused by an increased density of platelet qadrenergic receptors rather than to any structural or functional changes in them. Step- wise multiple regression analysis showed that plasma total cholesterol was the only independent predictor of a,-adrenergic receptor density, and we therefore hypothesized that cholesterol can directly affect platelet a,-adrenergic receptor density.

The mechanism(s) responsible for the increased

690 Baldassarre et al. CLINICAL PHARMA COLOGY &THERAPEUTICS

JUNE 1997

number of or,-adrenergic receptor in platelets from patients with hypercholesterolemia was not directly addressed in this study. Possible mechanisms in- clude changes in megakaryocyte maturation that re- sult in an increased platelet membrane area and as a consequence in an altered platelet survival. As far as this latter hypothesis is concerned, it has been shown that platelets from patients with hypercholes- terolemia have shortened survival rates.42 However, these findings have not been confirmed by others.43

The higher ol,-adrenergic receptor density de- scribed in hypercholesterolemia, coupled with the increased sympathetic activity observed in this con- dition,25 may provide an explanation for the en- hanced thrombotic risk in this clinical condition. In this regard it is important to underline that a rela- tion between platelet function and the complica- tions of coronary disease has been documented4 and that the circadian variation of ischemic events (silent ischemic episodes and myocardial infarction) with a peak incidence in the morning hours after awakening45-47 parallels enhanced platelet aggrega- bility and plasma catecholamine levels.48

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