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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:759-764.)
© 1995 American Heart Association, Inc.

Articles

Platelet Cytosolic Ca²⁺ and Membrane Dynamics in Patients With Primary Hypercholesterolemia

Effects of Pravastatin

Kim-Hanh Le Quan Sang; Jaime Levenson; Jean-Louis Megnien; Alain Simon; Marie-Aude Devynck

From Pharmacologie, CNRS URA 1482, Faculté de Médecine Necker-Enfants Malades (K.-H.L.Q.S., M.-A.D.), and Centre de Médecine Préventive Cardiovasculaire, INSERM U28, Hôpital Broussais (J.L., J.-L.M., A.S.), Paris, France.

	Abstract

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Abstract This study was designed to evaluate the relationships between platelet cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and plasma lipids in patients with primary hypercholesterolemia. In a double-blind, placebo-controlled trial, we determined platelet [Ca²⁺]_i in the presence and virtual absence of extracellular Ca²⁺ and the effects of prolonged treatment with pravastatin, a selective inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Platelet [Ca²⁺]_i and membrane microviscosity were determined in 22 normotensive hypercholesterolemic men. Platelet [Ca²⁺]_i was observed to vary with in vivo plasma lipid characteristics: in untreated patients, [Ca²⁺]_i determined at low extracellular Ca²⁺ concentration was significantly associated with plasma triacylglycerols (P=.008) and with the total cholesterol to HDL cholesterol ratio (P=.044). Triacylglycerol levels also correlated inversely with the external Ca²⁺-dependent [Ca²⁺]_i rise. Pravastatin treatment reduced plasma total cholesterol (-20±3%), LDL cholesterol (-30±3%), triacylglycerols (-17±6%), and apoB levels (-25±4%) and simultaneously decreased platelet [Ca²⁺]_i measured in a low-Ca²⁺ medium by 14±6% (P=.03). However, [Ca²⁺]_i values remained positively correlated with the total cholesterol to HDL cholesterol ratio (P=.04). Pravastatin treatment did not induce marked changes in membrane microviscosity, although the changes in trimethylaminodiphenylhexatriene anisotropy were inversely correlated with those of HDL cholesterol. These results indicate that plasma lipids can modulate cytosolic Ca²⁺ in platelets by affecting Ca²⁺ transport pathways that are dependent and independent of Ca²⁺ influx.

Key Words: primary hypercholesterolemia • cytosolic Ca²⁺ concentration • membrane fluidity • platelets • pravastatin

	Introduction

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Hypercholesterolemia is a major risk factor for the development and progression of atherosclerosis and coronary heart disease and is associated with various alterations in cell reactivity and membrane properties. Hypercholesterolemia attenuates endothelium-derived relaxation and enhances sensitivity of the blood vessels to vasoconstrictors.^{1 2 3} In platelets, hypercholesterolemia is accompanied by hypersensitivity to various aggregating agents,^{4 5} increased cell membrane cholesterol content,^{6 7 8} and altered fatty acid composition.⁹ Stimulated platelets from hypercholesterolemic patients are also characterized by abnormally high platelet cytosolic Ca²⁺ concentrations ([Ca²⁺]_i),¹⁰ accelerated rates of phosphatidylinositol hydrolysis,⁷ and enhanced thromboxane release.¹¹ These changes may result from the altered membrane composition, which could, for example, lead to increased accessibility of various agonists to receptors or from direct effects of lipoproteins on Ca²⁺ transport pathways.

Several in vitro studies have indeed shown that lipoproteins can directly affect platelet function. LDL acts as a direct agonist on isolated platelets and enhances the aggregation response to other agonists such as thrombin, serotonin, ADP, and collagen,^{12 13 14} whereas HDL reduces thrombin and opposes LDL-induced platelet aggregation.^{15 16} It has also been demonstrated that LDL increases platelet [Ca²⁺]_i in both the presence and absence of extracellular Ca²⁺.¹⁷ This phenomenon has been attributed to Ca²⁺ release from intracellular stores and to thromboxane A₂ elevation. These effects are counteracted by HDL₃, which has been proposed to interfere with LDL binding,¹⁸ although this competition has not been observed by other investigators.¹⁴

There is, however, little information about the in vivo role of plasma lipoproteins on platelet reactivity and [Ca²⁺]_i. Plasma levels of LDL are correlated with enhanced platelet aggregation to epinephrine.¹² Decreasing the levels of plasma cholesterol, especially LDL cholesterol, in hypercholesterolemic patients leads to decreased platelet hypersensitivity.^{5 19} High plasma LDL levels do not appear to be associated with elevated [Ca²⁺]_i in unstimulated platelets from patients with essential hypertension⁸ or ischemic heart disease.¹⁰ In this latter study, however, all patients were being treated with drugs capable of attenuating a possible rise in [Ca²⁺]_i.

The purpose of this study was to evaluate, in patients with primary hypercholesterolemia, the relationships between platelet [Ca²⁺]_i and membrane microviscosity and plasma lipids and the changes therein, as induced by reduction of LDL cholesterol levels. Basal platelet [Ca²⁺]_i was therefore determined in the presence and virtual absence of extracellular Ca²⁺. Membrane dynamics was determined by using two fluorescent dyes, DPH (diphenylhexatriene) and TMA-DPH (trimethylaminodiphenylhexatriene), to probe different lipid areas of the cell membranes. The effects of pravastatin, a selective inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which reduces elevated total plasma cholesterol and LDL cholesterol levels in healthy subjects²⁰ and in patients with hypercholesterolemia,^{21 22} were evaluated in a double-blind, placebo-controlled trial.

	Methods

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Patient Selection
Twenty-two male subjects with type II hypercholesterolemia, aged 35 to 63 years, participated in the study. Informed consent and approval of the Hospital Ethics Committee were obtained. These patients were selected on the basis of a plasma total cholesterol value higher than 6.5 mmol/L and triacylglycerol levels not exceeding 2 mmol/L. Exclusion criteria were hypertension; diabetes; renal, hepatic, or metabolic diseases; coronary heart disease; and clinical evidence of advanced atherosclerotic lesions of the abdominal aorta, cervical arteries, or lower-limb arteries, which was confirmed by ultrasonic examination of carotid, abdominal aortic, and femoral arteries.²³ Patients who were receiving treatment with drugs capable of modifying lipid metabolism or platelet handling were also excluded.

Study Design
The study was started with a 6-week placebo period during which lipid-lowering drugs were withdrawn and dietary stabilization was established. For the duration of the trial, all patients were instructed to adhere to a low-fat, low-cholesterol diet. They were advised to maintain their usual lifestyles and physical exercise levels. At the end of this preselection period, patients with plasma total cholesterol levels higher than 5.2 mmol/L were randomized to active treatment with pravastatin (two tablets of 20 mg each) or to placebo (two tablets) once a day for 12 weeks.

Plasma lipids and platelet parameters were determined at baseline, ie, before randomization, and at the end of the 12-week treatment period.

Blood Lipid Determinations
Blood samples were obtained between 9 and 10 AM after a 12-hour overnight fast. Total serum cholesterol and triacylglycerol values were measured by an enzymatic method.²⁴ HDL cholesterol concentration was determined in serum after precipitation of VLDL and LDL by phosphotungstic acid. LDL cholesterol was calculated as total cholesterol-HDL cholesterol-triacylglycerols/2.2.

Blood Sampling and Preparation of Platelet-Rich Plasma
Venous blood was collected in tubes containing 1/10 volume anticoagulant (2.73% citric acid, 4.48% trisodium citrate, and 2% glucose). Platelet-rich plasma was obtained by centrifugation at a maximum acceleration of 530g for 5 minutes at 20°C. All platelet measurements were performed within 3 hours of blood sampling.

Measurement of Platelet [Ca²⁺]_i
Platelet [Ca²⁺]_i was determined as described previously.²⁵ A characteristic of quin 2 is that it establishes a relatively strong Ca²⁺ buffer inside the cell. It is inappropriate to use quin 2 to follow rapid [Ca²⁺]_i changes, but it constitutes a valid indicator of steady-state [Ca²⁺]_i values.

Platelets were loaded with 20 µmol/L quin 2-AM (Sigma Chemical Co) for 30 minutes at 37°C in the presence of plasma. Under these conditions, the intracellular dye concentration did not exceed 0.5 mmol/L. This procedure also minimized the accumulation of unwanted hydrolysis compounds that, at high concentrations, may alter cell functions. Platelets were washed by centrifugation at 270g for 10 minutes at 20°C and resuspended at a density of 1x10⁸/mL in a medium containing (in mmol/L) NaCl 145, KCl 5, MgSO₄ 1, Na₂HPO₄ 0.5, glucose 5, and HEPES 10, pH 7.4, at 37°C, to which 10^-3 mol/L Ca²⁺ was added or not (the low-Ca²⁺ medium contained about 10^-6 mol/L Ca²⁺).

Fluorescence intensities were measured at 37°C with excitation and emission wavelengths of 339 and 490 nm, respectively, in quartz microcuvettes with short optical pathways (2.5 and 2.5 mm). Fluorescence signals were measured in duplicate, and [Ca²⁺]_i was calculated as previously described.²⁵ The intra-assay and inter-assay coefficients of variation were 5% (n=7) and 4% (n=12), respectively.

In preliminary experiments that were performed with platelets from seven unselected subjects, [Ca²⁺]_i was found to increase linearly with external Ca²⁺ concentration (r=.99, P=.0001 for seven external Ca²⁺ concentrations ranging from 70 to 1 mmol/L; y=10^-8[7.3+8800x] mol/L).

Measurement of Platelet Membrane Microviscosity
Platelet-rich plasma was diluted five times with the aforementioned medium containing 10^-3 mol/L Ca²⁺, centrifuged at 270g for 15 minutes at 20°C, and resuspended in the same medium. The density of washed platelets was adjusted by Rayleigh scattering to 3x10⁷ cells/mL. Under these conditions and with the microcuvettes that were used (2.5x2.5-mm optical pathways), the depolarization due to light scattering was minimized. Stock solutions of TMA-DPH and DPH (Molecular Probes) prepared in dimethylformamide at a concentration of 10^-2 mol/L were diluted extemporaneously and added to platelet suspensions at final concentrations of 5 and 2x10^-7 mol/L, respectively. Fluorescence intensities were measured after 6 and 10 minutes at 37°C for TMA-DPH and DPH as described previously.²⁶ Fluorescence anisotropy is defined as

where G is the correction ratio, I_//H/I_H. The subscripts H and V refer to horizontally and vertically polarized excitation light, and the subscripts // and refer to components of the emitted light that are parallel and perpendicular, respectively, to the direction of polarization of the excitation beam.

The intra-assay and the inter-assay coefficients of variation were 0.5% (n=26) and 1% (n=4) for TMA-DPH anisotropy and 0.3% and 0.5% for DPH anisotropy, respectively.

Statistical Analysis
Results are expressed as mean±SEM. Comparisons within groups (before and after treatment) and between groups (at baseline and after 3 months of treatment) were performed by ANOVA. Correlations between plasma lipids and platelet parameters were calculated by linear regression. Stepwise multiple regression analysis was performed to confirm the results of univariate comparisons.

	Results

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The biological characteristics of the hypercholesterolemic subjects are given in the Table. In these 22 subjects, platelet [Ca²⁺]_i did not significantly vary with age, body mass index, or blood pressure. [Ca²⁺]_i was, however, associated with some plasma lipid components. In univariate analysis, platelet [Ca²⁺]_i determined in the low-Ca²⁺ medium was correlated positively with the ratio of total plasma cholesterol to HDL cholesterol and with plasma triacylglycerols (Fig 1) and negatively with apoA-I levels (r=-.453, P<.05). These relations were not observed when platelet [Ca²⁺]_i was measured in a medium containing 10^-3 mol/L external Ca²⁺, in which [Ca²⁺]_i tended to vary positively with total plasma cholesterol, LDL cholesterol, and apoB levels (P=.146, .117, and .152, respectively). The difference between [Ca²⁺]_i determined at these two external Ca²⁺ concentrations, which reflects the influence of Ca²⁺ influx, was associated with plasma triacylglycerols only (r=-.444, P=.038). Individual values of membrane dynamics were not related to plasma lipids or platelet [Ca²⁺]_i.

View this table: [in this window] [in a new window]   Table 1. Characteristics of the Patients

View larger version (25K): [in this window] [in a new window]   Figure 1. Scattergrams of individual platelet cytosolic [Ca2+] values determined in the presence of low-Ca2+ medium and plasma lipids. Top, the relation with triacylglycerol levels in untreated hypercholesterolemic patients (, n=22; r=.546, P=.008, y=48+23x). Values after 3 months on pravastatin (, n=10; r=.476, P=0.16) are given for information. Bottom, the relation with total cholesterol to HDL cholesterol ratios in untreated hypercholesterolemic patients (, n=22; r=.432, P=.044, y=50+5x) and after 3 months on pravastatin (, n=10; r=.644, P=.041, y=38+7x).

The interrelations among plasma lipid fractions raise the problem of identifying those factors responsible for their associations with platelet [Ca²⁺]_i. The role of triacylglycerols was confirmed by stepwise regression analysis with [Ca²⁺]_i, as determined in the presence of low or normal external Ca²⁺, and their difference as dependent variables and all plasma lipid values, as detected by univariate analysis to be possible modulators (P<.15), as independent variables. Only plasma triacylglycerol levels were significantly associated with platelet [Ca²⁺]_i in the low-Ca²⁺ medium (F=7.25, P=.02), with its rise dependent on external Ca²⁺ concentration (F=5.01, P=.04).

To investigate whether the aforementioned relations were really due to plasma lipid levels, the patients were randomly distributed into two groups that received either placebo or pravastatin treatment for 12 weeks. These two groups were similar with respect to age, sex ratio, body mass index, arterial blood pressure, plasma lipids, platelet [Ca²⁺]_i, and membrane microviscosity (Table).

Placebo treatment did not modify body mass index, arterial blood pressure, or plasma lipids, except for small decreases in total plasma cholesterol and LDL cholesterol (Table). No change was observed in platelet [Ca²⁺]_i irrespective of the external Ca²⁺ concentration (Fig 2), in their difference, or in DPH or TMA-DPH anisotropies (Table).

View larger version (21K): [in this window] [in a new window]   Figure 2. Semilog plot showing platelet cytosolic [Ca2+] as a function of external Ca2+ concentration in untreated subjects at baseline (,) and after placebo () or pravastatin () treatment for 3 months.

On the contrary, pravastatin treatment reduced plasma total cholesterol (-20±3%), LDL cholesterol (-30±3%), and apoB levels (-25±4%) (Table). Pravastatin also reduced triacylglycerols (-17±6%), tended to increase plasma HDL cholesterol (10±4%), and decreased the ratio of plasma total cholesterol to HDL cholesterol and of apoB to apoA-I by 22±4% for each. Blood pressure was not changed. Platelet [Ca²⁺]_i, as measured in the low-Ca²⁺ medium, decreased by 14±6% (-12.6±4.9 nmol/L, P=.03). A similar but nonsignificant decrease was observed when [Ca²⁺]_i was measured in 10^-3 mol/L Ca²⁺ medium (-11.2±6.8 nmol/L, P=.14) (Fig 2). The difference between platelet [Ca²⁺]_i measured in the two media, which reflects the influence of external Ca²⁺, also remained unchanged, as was membrane dynamics (Table).

In pravastatin-treated subjects, platelet [Ca²⁺]_i, as determined in the low-Ca²⁺ medium, was correlated positively with the total plasma cholesterol to HDL cholesterol ratio (Fig 1), similar to that observed in the untreated group. This relation was weakened when [Ca²⁺]_i was calculated at constant plasma triacylglycerol values (r'=.544, P=.12).

In the placebo and pravastatin groups, considered separately or together, individual changes in platelet TMA-DPH anisotropy were inversely correlated with those in HDL cholesterol (Fig 3). These relations were unchanged when changes in plasma triacylglycerol levels were considered as possible confounders (r'>.680 for each).

View larger version (21K): [in this window] [in a new window]   Figure 3. Scattergrams of individual changes from baseline values in plasma HDL cholesterol and platelet trimethylaminodiphenylhexatriene anisotropy in subjects on placebo (, n=12; r=-.680, P=.015, y=1.1-31.3x) or pravastatin treatment (, n=10; r=-0.698, P=.025, y=5.0-46.9x).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study was designed to evaluate the influence of hypercholesterolemia on platelet [Ca²⁺]_i and membrane dynamics. Platelets do not synthesize cholesterol, and its incorporation into the cellular membrane depends on circulating lipoprotein levels. This may affect the activity of Ca²⁺ transport pathways either specifically or more generally through changes in membrane microviscosity. This study has demonstrated that basal platelet [Ca²⁺]_i varies with in vivo plasma lipid characteristics. Plasma triacylglycerol levels and the total cholesterol to HDL cholesterol ratio were positively associated with platelet [Ca²⁺]_i values in the absence of a significant Ca²⁺ influx, whereas LDL levels tended to be associated with [Ca²⁺]_i in the presence of a physiological extracellular Ca²⁺ concentration. These findings are supported by pravastatin-induced reductions in platelet [Ca²⁺]_i.

In a low-Ca²⁺ medium, the participation of Ca²⁺ influx is minimal and platelet [Ca²⁺]_i is low; it increases as a function of external Ca²⁺ to reach values about twice as high at physiological Ca²⁺ concentration. The inverse relation between plasma triacylglycerols and the platelet Ca²⁺ influx–dependent [Ca²⁺]_i rise suggests that triacylglycerols, although minor components of biological membranes, also participate in the control of Ca²⁺ influx or that their effects can be attenuated by the membrane-stabilizing effect of extracellular Ca²⁺. The tendency of platelet [Ca²⁺]_i to rise with LDL cholesterol level agrees with our previous observation that the [Ca²⁺]_i in platelets from untreated hypertensive patients was positively correlated with plasma cholesterol, even at constant arterial pressure.²⁷

An unexpected result of this study was the HDL-independent relation between plasma triacylglycerol level and platelet [Ca²⁺]_i. We are not aware of previous reports describing a link between physiological levels of plasma triacylglycerols and platelet [Ca²⁺]_i handling. Our findings raise the possibility that plasma triacylglycerols may modulate membrane properties either directly via their incorporation into the phospholipid bilayer²⁸ or indirectly via the associated alterations in membrane composition^{8 29} and membrane dynamics.^{8 26 30} Even if triacylglycerol incorporation into the membrane bilayer is limited,²⁸ it may directly or indirectly affect Ca²⁺ transport pathways, the activities of which are very sensitive to their lipid environment. Further studies are obviously required to determine the respective importance of these various possibilities.

This association of plasma triacylglycerols with platelet [Ca²⁺]_i handling resembles those previously observed for Na⁺ and K⁺ transport^{31 32 33} and further reinforces the suggestion that the neutral lipid domains formed in the cell membranes by triacylglycerols can have functional roles.^{28 34}

The moderate decreases in plasma total cholesterol and LDL cholesterol in the placebo group, in which hypercholesterolemic patients received no active treatment, were probably due to dietary restriction, ie, low intakes of cholesterol and saturated fat. The observed decreases were not accompanied by changes in platelet [Ca²⁺]_i or membrane dynamics.

In contrast, pravastatin treatment markedly reduced plasma total cholesterol, LDL cholesterol, apoB and, to a lesser extent, plasma triacylglycerol levels. Pravastatin also tended to increase HDL cholesterol, which is considered to be a protective factor against atherosclerosis and coronary heart disease.³⁵ The lipid-lowering effects of pravastatin in the hypercholesterolemic patients agree with previous reports in both magnitude and pattern^{21 22 36} and are comparable with those of other HMG-CoA reductase inhibitors, such as simvastatin or lovastatin.^{37 38 39} Improvement in the plasma lipid profile under pravastatin treatment was accompanied by a significant decrease in platelet [Ca²⁺]_i, as determined in a medium with low external Ca²⁺, thus confirming the observed relation in untreated subjects. This finding suggests that the lipid-lowering effects of pravastatin have increased the efficacy of Ca²⁺ extrusion or storage mechanisms without major alteration of the transmembrane calcium influx. Considering the hypothesis that high triacylglycerol levels modify Ca²⁺ storage and extrusion efficacy, it is also tempting to propose that such a mechanism may participate in the exclusive relationship between plasma triacylglycerol levels and the extent of coronary calcifications that has been recently described in asymptomatic hypercholesterolemic men.²³

If plasma triacylglycerol levels indeed modulate cell Ca²⁺ handling, then reduction of the former by dietary n-3 fatty acids should also reduce [Ca²⁺]_i. The reduction of platelet [Ca²⁺]_i in hypertensive patients by a diet rich in eicosapentaenoic acid⁴⁰ is compatible with this proposal, but its interpretation is complicated by the simultaneous lowering of blood pressure and by possible direct effects of fatty acids on Ca²⁺ transport mechanisms.

Another result of the present study is the absence of a clear relationship between plasma lipids and platelet membrane microviscosity. In contrast with in vitro cholesterol enrichment, which causes a marked increase in membrane microviscosity, the mean values of TMA-DPH and DPH anisotropies in platelets from hypercholesterolemic, normotriglyceridemic patients did not differ from those of normolipidemic subjects. Furthermore, although platelet cholesterol content decreases with pravastatin treatment,⁴¹ TMA-DPH and DPH anisotropies were unchanged. This outcome, however, does not preclude a subtle role for plasma lipids as modulators of membrane dynamics, because the treatment-induced changes in TMA-DPH anisotropy were inversely related to those of HDL cholesterol.

HMG-CoA reductase inhibitors may also have effects independent of their ability to inhibit endogenous cholesterol synthesis. This class of agents has been reported to lower cytosolic pH by reducing Na⁺/H⁺ exchange and modifying the buffering capacity in cultured cells.⁴² Such acidification, if it occurs in platelets, is probably not responsible for the observed reduction in [Ca²⁺]_i, because experimental acidification does not reduce platelet [Ca²⁺]_i.⁴³

The present study indicates that in patients with primary hypercholesterolemia, the lipid-lowering effect of pravastatin is associated with a decreased platelet [Ca²⁺]_i in the absence of a Ca²⁺ influx, without marked changes in membrane microviscosity. It points out the unexpected influence of plasma triacylglycerol levels on cell Ca²⁺ handling.

	Acknowledgments

 
This study was partially supported by a research grant from Bristol-Myers Squibb. The kind participation of Muriel Del Pino in the practical organization of this study is highly appreciated.

	Footnotes

 
Reprint requests to M.-A. Devynck, PhD, Pharmacology, CNRS URA 1482, Necker Medical School, 156 rue de Vaugirard, F 75015-Paris, France.

Received May 31, 1994; accepted February 28, 1995.

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