Lovastatin decreases plasma and platelet cholesterol levels and normalizes elevated platelet fluidity and aggregation in hypercholesterolemic patients

Lovastatin Decreases Plasma and Platelet Elevated Platelet Fluidity and Aggregation E. Hochgraf,

Y. Levy, M. Aviram,

Cholesterol Levels and Normalizes in Hypercholesterolemic Patients J.G. Brook, and U. Cogan

The lipid composition of whole platelets and the fluidity of platelet membranes, as well as the sensitivity of the cell to aggregation, were studied in type IIA hypercholesterolemic human subjects before and after treatment with lovastatin. Fourteen patients with primary hypercholesterolemia having initial cholesterol levels of 383 2 52 mg/dL (mean -t standard deviation) were studied and compared with 21 control subjects having cholesterol levels of 187 f. 32 mg/dL. Lovastatin was administered orally at a starting dose of 40 mg daily. The dose was increased to 80 mg daily for eight patients who did not achieve the target cholesterol level of 200 mg/dL at 6 weeks. Serum cholesterol level was decreased by 37% following 20 weeks’ administration of the drug. The fluidity of platelet membranes expressed in terms of the fluorescence anisotropy parameter was determined using the probe 1,6-diphenyl-1,3,5-hexatriene (DPH). When compared with platelets obtained from normocholesterolemic controls, platelets from hypercholesterolemic patients had a higher molar ratio of cholesterol to phospholipids ([C/PL] 0.86 f 0.15 v 0.57 f 0.06 for controls) and of phosphatidylcholine to sphingomyelin ([PC/SM] 2.64 + 0.87 v 2.00 f 0.15 for controls), enhanced fluidity (anisotropy parameter at 37°C of 0.892 & 0.066 Y 0.977 ? 0.065 for controls), and a greater tendency to aggregate (aggregation of 84.2% f 6.3% v 78.5% & 7.6% for controls). Lovastatin administration for 20 weeks to hypercholesterolemic patients markedly normalized platelet lipid composition (C/PL, 0.58 + 0.13; PC/SM, 1.84 -C 0.60). membrane fluidity (anisotropy parameter, 0.980 1?:0.033), and aggregation (aggregation, 75.9% -+ lO.O%), suggesting that lovastatin treatment atherosclerosis. Copyright 61 1994 by W.B. Saunders Company

may attenuate

T

HE LINK BETWEEN elevated plasma cholesterol levels and atherosclerosis is well established. Platelets. which play a major role in maintaining homeostasis by participating in the coagulation process, are also actively involved in atherogenesis. 1.7 Since the platelet lacks a nucieus, its membrane cholesterol is derived from bone marrow megakaryocytes and from exchange with plasma lipoproteins.-. i 4 Various investigators have demonstrated that the platelet membrane cholesterol level is increased in hypcrcholesterolemia.s Furthermore, in vivo and in vitro studies have shown that platelet activity is enhanced in hypzrcholesterolemia; increased synthesis of thromboxane B2 (TXB#’ and increased sensitivity to aggregation have been noted.“-l’1 hfembrane lipid fluidity,* which is a measure of the dynamic state of the membrane, was shown to be an important determinant of cell function.“J2 Nonetheless, the relationship between platelet membrane composition and fluidity and the function of the cell is unknown. The drug lovastatin, a potent inhibitor of 3-hydroxy-3methylglutaryl coenzyme A reductase, the rate-limiting enqme in the biosynthesis of cholesterol, was shown to be cffec’ectivein decreasing plasma cholesterol levels in human subjects.15J“ However. no information is available as to the effect of this drug on platelet properties and function. The purpose of this study was to investigate the composition and dynamics of the platelet membrane and the function of the ceil in hypercholesterolemic subjects before and after administration of lovastatin. Membrane dynamics were assessed in terms of membrane fluidity, and the

*‘Lipid fluidity” as applied to the anisotropic bilayer membrane has been used in different senses by various investigators. A discussion of the use of the term is given.13,14 Briefly, it is used here to express the relative motional freedom of the lipid molecules or substituents thereof, combining in the one term contributions of both rate and extent of movement.

Metabolism,

Vol43,

No

1 iJanuary).

1994: pp

1 l-17

the involvement

sensitivity function.

of platelets

to aggregation

SUBJECTS

was used

in the pathogenesis

to evaluate

of

platelet

AND METHODS

Subjects Fourteen patients, men and women of a mean age of 50 -+ X years with plasma cholesterol levels of 383 t 53 mg/dL, and 21 men and women control subjects, of a mean age of 30 ? 8 years with plasma cholesterol levels of 187 ? 32 mgidl. participated in the study. Age is known to increase eicosanoid biosynthesis and platelet sensitivity to stimulating agents.” A difference in age existed between the experimental and control subjects. Nonetheless, since each patient was tested at time zero and thereafter during lovastatin administration as explained below. it follows that every patient served as his own control. Therefore. age differences between the experimental and control groups are irrelevant with respect to effects of the drug on the investigated parameters. Furthermore, the control subjects served to indicate desirable levels of the evaluated parameters, thus testing the extent to which these levels were approached upon drug treatment of the patients. All patients were previously resistant to lipid-lowering diets and drugs. None of the subjects had a disease that could affect lipid metabolism, and no patient was taking any untiaggregatory agents. The patients were maintained on a low-fat ( <30% of calories). low-cholesterol ( <300 mgid) diet.lx Lovastatin was administered orally at a starting dose of 40 mg daily. The dose was increased to 80 mg daily in eight patients who did not achieve the target cholesterol level of 200 mg/dL at 6 weeks.

From the Department of Food Engineering and Biotechnology, Technion-Israel Institute of Technology, Huijk; and the Lipid Research Unit, Rumbam Medical Center and Rappaport Institute for Research in the Medical Sciences. Haifa. Israel. Submitted February 24, 1992: accepted March 3, (993. Supported by the Technion VPR Albert Einstern Research Fund. Address reprint requests to U. Cogan. PhD, Department of Food Engineering and Biotechnology, Technion, Haifa 32000. Israel. Copyright 0 1994 by W.B. Saunders Cornpan\ 0026-049519414301-0003$03.0010

11

12

HOCHGRAF ET AL

Patients signed a written consent form, and the study was approved according to the regulations of the Helsinki Declaration. Serum

Lipid Determinations

Time-matched

samples of venous

blood were withdrawn from the participants following 14 hours of fasting. Serum cholesterol and triglyceride levels were determined enzymatically using standard kits (Sigma, St Louis, MO). High-density lipoprotein (HDL) cholesterol level was measured enzymatically using the same kit after very-low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) precipitation with dextran sulfate. LDL cholesterol level was calculated according to the commonly used equation, LDL cholesterol = total cholesterol - (triglycerides/5 + HDL cholesterol).19

Plaielet Separation For platelet studies, venous blood was taken into acid-citratedextrose solution (1.4% citric acid, 2.5% sodium citrate, and 2% dextrose) at a ratio of 9:l (vol/vol). Platelet-rich plasma (PRP) was prepared by low-speed centrifugation at 100 x g for 10 minutes at 25”C, and the remaining samples were recentrifuged at 700 x g for 10 minutes to obtain platelet-poor plasma (PPP). Platelets were counted in PRP and diluted in PPP to achieve a uniform level of 3 ? 0.3 x lo5 cells/pL. Washed platelets (WP) prepared from PRP by centrifugation at 240 x g were washed twice in 5 mmol/L HEPES buffer, pH 7.4 (140 mmol/L NaCI, 2 mmol/L KCI, 1 mmol/L MgCI:, 5 mmol/L HEPES, I2 mmol/L NaHC03, and 5.5 mmol/L glucose). Fifteen microliters acetic acid (1 mol/L) was added per milliliter platelet suspension throughout the preparatory steps to ensure acidic conditions required for resuspension.?”

Platelet Aggregation Aggregation with 3.3 kg collagen/ml PRP (Hormonchemie. Munich, Germany) was performed at 37°C in a Model PAP-4 computerized aggregometer (Bio Data, Hatboro, PA) using PPP as a reference system. Results were expressed in terms of either the extent of aggregation (% of maximal amplitude) or the lag-time (min) required to initiate the aggregation response.

Analytical Determinations For phospholipid and cholesterol analysis, platelet lipids were extracted with chloroform/methanol according to the method of Folch et al.** Phospholipids were assayed according to the method of Rouser et al.” Cholesterol was assayed according to the method of Chiamori and Henry.“3 Protein was assayed using the Bio-Rad reagent kit (Bio-Rad Laboratories, Richmond, CA).

Phosphatidylcholine to Sphingomyelin Ratio Platelet lipid extract was subjected to two-dimensional thin-layer chromatography on silica gel G plates using chloroform:methanol: ammonium hydroxide (13:5:1) for the first phase and chloroform: acetone:methanol:acetic acid:water (6:8:2:2: 1) for the second phase. Following identification with iodine, the respective stains were scraped and subjected to phosphorus analysis.*’

Fluorescence Studies Platelet membrane fluidity was assessed by steady-state fluorescence polarization measurements using 1,6-diphenyl-1,3,5hexatriene (DPH) as the fluorescent probe.Z4,Z5 DPH was incorpo-

rated at a level of lo-6 mol/L into a platelet suspension containing 50 kg protein/mL in a 5-mmol/L phosphate-buffered saline (PBS), pH 7.4, containing 0.5 mmol/L CaClz and 4 mmol/L KCI. The instrument used was a self-constructed spectrofluorometer, as previously

described.zh

Excitation

was obtained

at 365 nm, and

emission was measured through (Corning Glass Works, Corning, wavelength shorter than 410 nm.

a Corning NY), which

3-74 cut-off filter, eliminates light of

The polarization of the fluorescence was measured following 30 minutes’ incubation of the mixture at 3PC and expressed as the fluorescence anisotropy, r.

r_

Ill -11 _ (IlliIJ 111+ 2IL

- 1 (111 /I,) + 2 ’

where 111 and II are the fluorescence intensities observed through a polarizer oriented parallel and perpendicular to the direction of polarization of the exciting light. Light scattering was corrected for by subtracting the relevant components reaching the detector from a DPH-free platelet suspension. The contribution of the scattering readings was less than 2%. The anisotropy parameter [(rJ r) - 11-t was calculated using a limiting anisotropy value of 0.362 for DPH.z3 The anisotropy parameter is inversely related to the fluidity and was expressed by an Arrhenius plot of log [(rO/ r) - 11-t versus 1/T.

Statistical Analysis Data were analyzed by Student’s t test. Comparisons between patients before and after drug consumption were performed by a paired test, whereas comparisons between patients and controls were performed by unpaired t test. Values are means + standard deviations. Differences at a level of P less than .05 were considered significant. RESULTS The study comprised two types of comparisons, namely between patients and between patients and a control group. Comparisons between patients before and after lovastatin treatment indicated the extent of changes in the tested parameters, with each patient serving as his own control. Comparisons between the patients and the control group healthy young subjects-tested the extent to which drug administration restored the levels of tested parameters to the respective levels of the controls. The latter comparison may indicate whether a desirable goal was reached. Lovastatin treatment, as expected, resulted in an effective decrease (37%) of serum cholesterol levels (Fig IA). This was primarily due to a decrease of 45% and 35% in LDL and VLDL cholesterol levels, respectively, whereas HDL levels remained essentially unchanged (Fig 1B). The triglyceride level, which was 184 ? 48 mg/dL for hypercholesterolemic patients as compared with 100 ? 38 mg/dL for controls, decreased with lovastatin treatment to 120 + 25 mg/dL following 12 weeks of treatment. Under the experimental conditions, platelets from hypercholesterolemic patients exhibited a significant 7% increase in collagen-induced aggregation (P < .05) and a decrease in the lag time required for the initiation of platelet aggregation compared with platelets from the control group (Fig 2A and B). Upon lovastatin administration, the extent of aggregation decreased significantly by 11% at 6 and 12 weeks and by 9.8% at 20 weeks’ treatment, as compared with the time zero level. Likewise, drug treatment resulted in a significant increase in the lag time required for initiation of platelet aggregation (Fig 2B). Thus, lovastatin treatment acted to normalize platelet aggregation. The temperature dependence of the fluorescence anisot-

LOVASTATIN

TREATMENT

AND PLATELET PROPERTIES

B

Control

0

LOVASTATIN

6 TREATMENT

12

20

-r

Control 0 LOVASTATIN

(WEEKS)

Fa IDL

6 12 20 TREATMENT (WEEKS)

Fig I. Effect of lovastatin treatment of hypercholesterolemic subjects on tota! serum cholesterol level (A) and on lipoprotein cholesterol distribution (B). Values are means + SEM (n = 14 for experimental group and 21 for control group). The control data represent an average of time-matched samples taken from this group throughout the study. l*P < 31 relative to the time zero level. rOpy parameter [(ro/r) - I]-’ Of DPH in platelet membranes from hypercholesterolemic patients and from control subjects is illustrated in Fig 3A, and that from the membranes of hypercholesterolemic patients before lovastatin treatment and 20 weeks after administration of the drug is shown in Fig 3B. Platelet membrane fluidity was significantly higher in hypercholcsterolemic patients compared with control subjects over the temperature range of 25” to 37°C (P < .05 at 25”C, P < .Olat 37°C). Lovastatin administration resulted in a gradual decrease in membrane fluidity, after 20 weeks reaching values similar to those of the controls (Fig 3B). Thtis, the anisotropy parameter values at 37°C were 0.892 -+ 0.066 before initiation of therapy, and 0.921 + 0.066, 0.978 ? 0.060, and 0.980 t 0.033 following 6, 12, and 20 weeks of treatment, respcctivcly. The corresponding values for platelets from control subjects were 0.977 2 0.065.

The question arose as to whether lovastatin acted to decrease platelet fluidity via direct binding to the cell membrane. In vitro studies showed that incubation of lovastatin over a wide concentration range of 0 to 270 kg/mL with platelets did not affect their fluidity. The high plasma cholesterol levels of the hypercholesterolemic patients were reflected in a high platelet cholesterol to phospholipids molar ratio (C/PL) of 0.86 2 0.15 (Fig 4A). Lovastatin therapy decreased the C/PL (P < .Ol), stabilizing it at nearly 0.6, which is very close to the value of the control subjects (Fig 4A). Platelet phospholipid levels were found to be quite stable and insensitive to the plasma cholesterol concentration of the various subjects (Fig 4B). Thus, changes in the C/PL were essentially due to changes in platelet cholesterol levels. The phosphatidylcholinc to sphingomyelin ratio (PC/ SM) was higher in hypcrcholesterolemic (7.64 ? 0.X7) as 1.50/ r

**

B A l

*

T

z^ 1.25 5 i w 4

1.00

0.75

0.50 Control

0

LOVASTATIN

6 12 20 TREATMENT (WEEKS)

Control LOVASTAiN

TR:ATMEN:2

(WEE;;)

Fig 2. Effect of lovastatin treatment of hypercholesterolemic subjects on platelet aggregation. (A) Extent of aggregation; (B) lag time required to initiate aggregation. Values are means f SEM (n = 10 for experimental and 21 for control group). The control data represent an average of time-matched samples taken from this group throughout the study. lP c .05 and l*P c .Ol relative to time zero level.

14

HOCHGRAF ET AL

31°C

37%

25°C

25°C

0.20 0.15

0.10

T

T

5

31°C

37°C

0.15

: t

0.05

0.10

0 L =: 8

0.00

.J

/

/

-0.05

/ -0.10 3.20

/ 3.25

3.30 1/TX103

3.40

3.35

-0.10 3.20

3.25

3.30 l/TX103

f”K-‘)

3.40

3.35

(“K-l)

Fig 3. Arrhenius plots of the temperature dependence of the DPH fluorescence anisotropy parameter of platelet membranes derived from hypercholesterolemic patients. The fluorescence aniostropy parameter is defined as [@Jr) - 11-1, where r is the fluorescence anisotropy, and r0 is the limiting anisotropy, which equals 0.362 for DPH. (A) Before initiation of lovastatin treatment, ie, time zero (0) as compared with control subjects (M). (6) Following 20 weeks’ treatment with lovastatin (a) as compared with time zero levels (0). Values are means 2 SEM (n = 14 for experimental and 21 for control group). The control data represent an average of time-matched samples taken from this group throughout the study. lP < .05 and **P < .Ol relative to time aero levels.

compared with normocholesterolemic (2.00 2 0.15) subjects (P < .05). This ratio appeared to have decreased gradually throughout the treatment, although not to a significant extent, reaching a level of 1.84 ? 0.60 after 20 weeks of therapy (Table 1). It should be emphasized that the data obtained for the control group in the various tests represent an average of samples taken from this group throughout the whole study period. The inclusion of a control group that was not treated and had samples drawn at the same time points demonstrates that the experimental parameters had not changed during the 20-week interval and that the measured differences were due to the lovastatin treatment. The

foregoing results demonstrate that lovastatin administration to hypercholesterolemic patients acted to normalize plasma and platelet cholesterol levels, platelet fluidity, and aggregation.

DISCUSSION

A high plasma cholesterol level is known to be associated with an increased sensitivity of platelets to aggregation.27s28 The same tendency was observed in this study. Lovastatin treatment markedly decreased plasma LDL and VLDL cholesterol levels and was immediately reflected in decreased platelet aggregation. This is in line with other

1 .o q PHOSPHOLIPIDS N

Control LOVASTATIN

0

6 TREATMENT

12

20 (WEEKS)

Control LOVASTATIN

6 TREATMENT

cHoLEsT!zRoL

12

20 (WEEKS)

Fig 4. Effect of lovastatin treatment of hypercholesterolemic subjects on the C/PL (A), and on the concentration of cholesterol and phospholipids (a). Values are means f SEM (n = 14 for experimental group and 21 for control group). The control data represent an average of time-matched samples taken from this group throughout the study. l*P -c .Ol relative to time zero values.

LOVASTATIN

TREATMENT

15

AND PLATELET PROPERTIES

Table 1. Effect of Lovastatin on the PC/SM

in Platelets

Lovastatin Treatment Weeks 0

6

12

20

Control

PC/SM 2.64* 2 0.39 2.37 2 0.26 2.25 + 0.10 1.84 + 0.27 2.00 f 0.05 NOTE. Results are means k SEM; n = 5 for treatment group; n = 10 for normocholesterolemic

controls.

*Values are significantly different from those of controls at P < .05.

studies in which a decreased platelet aggregation was shown to accompany a decrease in plasma cholesterol level achieved either by plasmaphoresis10 or by treatment with the drugs clofibrate9 and synvinolin.hs7 Administration of synvinolin, another hepatic hydroxymethyl glutaryl coenzyme A reductase inhibitor, to type IIA hyperlipidemic patients resulted in a significant decrease in platelet aggregation and thromboxane formation induced by collagen and arachidonate. The disparity between the maximum hypolipidemic effect of the drug achieved at 2 weeks of treatment and the maximum antiaggregatory effect achieved at 4 to 8 weeks was explained by the investigators as evidence against a direct effect of the drug on platelets.b We did not determine the lovastatin effect at periods shorter than 6 weeks. Platelets from hypercholesterolemic patients were shown to have elevated CIPL. Platelets are not capable of de novo cholesterol synthesis, and their cholesterol is derived either from plasma lipoproteins or from synthesis in bone marrow megakaryocytes.” The increased sensitivity to aggregation of platelets derived from hypercholesterolemic patients may result from structural and dynamic changes in the plasma membrane and in the membranes surrounding the granules, and from enhanced synthesis of TXA2.29 The platelet membrane is known to contain receptors for aggregation-inducing agents such as adenosine diphosphate and thrombin. 3o Changes in the dynamic properties, ic, rluidity. of the membrane affect the activity of membranebound enzymes and the expression of receptors.31 Thus, in the case of platelets. changes in the fluidity of the plasma membrane may influence the binding of aggregationinducing agents and consequently the synthesis of eicosanoids and the cascade of processes leading to aggregation. The extent of this effect is unknown at present. The effect of fluidity changes on the expression of membrane receptors and enzymes is complex. There are cases where an increase in fluidity enhances membrane protein expression, whereas in other cases it decreases such expression.3’ The subject has been discussed by Shinitzky et aL3’ who proposed that each membrane protein requires a specific lipid fluidity to function optimally. Muller ct al3J and Malle et al35 observed increased platelet membrane fluidity in type IIB and type IV hyperlipidemic patients, but not in type IIA patients. Our results also suggest that in the case of type IIA hyperlipoproteincmla the increased sensitivity to aggregation is accompanied by increased platelet fluidity. Moreover, a decrease in platelet fluidity that took place as a result of lovastatin trcatmcnt was accompanied by a decreased tendency to

aggregation. These results are in agreement with those of Berlin et al,36 who studied the effect of dietary lipids (saturated v unsaturated) on platelet aggregation and fluidity in rabbits and found a positive correlation between adenosine diphosphate-induced platelet aggregation and platelet membrane fluidity. Furthermore. in another study, treatment of seven normolipidemic women with 250 mg aspirin daily for 1 week resulted not only in elimination of aggregation, but also in a significant decrease in platelet fluidity.37 The present results are also partly in accordance with those of Moscat et al,38 who observed that platelets from patients with severe atherosclerosis exhibited an increase in membrane fluidity and an enhanced thrombinstimulated TXB2 synthesis. However, the patients studied were not hyperlipidemic, and a decrease in the platelet C/PL was observed compared with that of the control subjects3s Shattil et al39 and Shattil and Cooper40 modulated the C/PL in the platelet membrane during in vitro studies and observed that an increase in the membrane cholesterol content was associated on one hand with increased sensitivity to aggregation and on the other hand with decreased fluidity. Evidently, the inverse relationship between the platelet CiPL and membrane fluidity observed in the in vitro studies cannot be simply compared with the present in vivo studies in which a direct relationship between the platelet C/PL and membrane fluidity was found. It is likely that in vivo, additional metabolic processes take place, which modulate the platelet membrane fluidity in a direction opposing the cholesterol effect. The possibility that additional metabolic processes may modulate platelet membrane fluidity is further supported by the study of Winocour et al,41 who observed a difference in membrane fluidity in platelets isolated from diabetic patients in the absence of membrane compositional differences. Platelets appear to possess an anomalous response of fluidity to changes in the membrane CIPL, in that an increase in this ratio resulted in increased fluidity and a decrease in the ratio was accompanied by decreased fluidity. With other cell types, primarily red blood cells.J2 and also with lymphocytes,43 an inverse relationship was observed between membrane cholesterol content and membrane fluidity. However, it should be kept in mind that the platelet possesses unique metabolic processes, and these may affect the plasma membrane fluidity in response to changes initiated by hypercholesterolemia. Furthermore, although lovastatin was not found to change platelet membrane fluidity during in vitro studies, the possibility that the active forms of liver metabolites of lovastatin, namely the hydroxy acid derivatives of the lactone form of the molecule,44 bind to platelets, thereby influencing their fluidity, should not be excluded. It should be noted that whole platelets rather than isolated membranes were used for the various experiments in the present study. Previous studies demonstrated that certain characteristics of the whole cell arc in good correlation with the same properties monitored for the plasma membrane alone. Thus, Shattil et al5 showed that in platelets derived from subjects with type IIa hypercholester-

16

HOCHGRAF ET AL

olemia and from normal controls, a good correlation between the C/PL in the whole platelet and in its plasma membrane was present. However, the granule C/PL was insensitive to plasma cholesterol levels. Marcus et al45 found that the phosphatidylcholine and sphingomyelin content of the whole platelet and that of the isolated membrane are very similar. Berlin et alI6 established a good correlation (r = 2368) between the DPH fluorescence anisotropy of the whole platelet and of the separated plasma membrane. The increase in the PC/SM of platelets in hypercholesterolemia and the apparent decrease in this ratio following lovastatin treatment may partly explain the changes in fluidity, since sphingomyelin is known to act as a membrane rigidifier.46 The change in the PC/SM may reflect alterations in cellular phospholipid biosynthesis, as well as the establishment of new conditions of equilibrium between platelet and piasma phospholipids. The complexity of alterations in platelet lipid composi-

tion and function in response to changes in plasma lipid levels was further manifested in a study in which these parameters were determined in cases of liver diseases.47 Platelets derived from such patients demonstrated increased C/PL and PC/SM and decreased aggregation. Additional studies on platelet lipids in hypercholesterolemia and during treatment with cholesterol-reducing drugs are needed to better clarify the relationship between the changes in platelet lipids and platelet fluidity and function. In conclusion, treatment with lovastatin, a novel plasma cholesterol-reducing drug, resulted in profound changes in platelet lipid composition, platelet fluidity, and sensitivity to aggregation in a manner opposing the effect of hypercholesterolemia. The levels of various parameters measured in the patients following drug administration were similar to the respective values obtained in healthy young controls. Thus, by causing these changes, lovastatin may also attenuate the involvement of platelets in the development of atherosclerosis.

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Raton, FL, CRC, 1984,pp 1-51 12. Spector A, York MA: Membrane lipid composition and cellular function. J Lipid Res 26:1015-1035,1985 13. Cogan U. Schachter D: Asymmetry of lipid dynamics in human erythrocyte membrane studied with impermeant fluorophores. Biochemistry 20:6396-6403,1981 14. Schachter D. Cogan U, Abbott RE: Asymmetry of lipid dynamics in human erythrocyte membranes studies with permeant fluorophores. Biochemistry 21:2146-2150,1982 15. Tobert JA: Efficacy and long-term adverse effect pattern of lovastatin. Am J Cardiol62:28J-335, 1988

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3 I Kowalska MA, Cierniewski CS: Microenvironment changes of human blood platelet membranes associated with fibrinogen hinding. J Membr Rio1 7557-64, 1983 31. Kinsella JE. Lokesh B, Stone RA: Dietary n-3 polyunsatur.itrd fatty acids and amelioration of cardiovascular disease: PosGble mechanisms. Am J Clin Nutr 52:1-28, 1990 3 3. Shinitzky M. Borochov H. Wilbrandt W: Lipid fluidity as a physiological regulator of membrane transport and enzyme activities in Las\en UV. Ussing HH. Weith JO (eds): Membrane Transport in Erythrocytes. Munksgaard. Copenhagen, Denmark. I <)x:1 3 1. Muller S. Ziegler 0. Donner M, et al: Rheological properties and membrane fluidity of red blood cells and platelets in primary hyprrlipoproteinemia. Atherosclerosis 83:231-237. 1990 3i. Malle E. Sattler W, Prenner E, et al: Platelet membrane Ruidity in type IIA. type IIB and type IV hyperlipoproteinemia. Atherosclerosis S7:159-167, 1991 31). Berlin E. Shapiro SG. Friedland N: Platelet membrane tluitiity and aggregation of rabbit platelets. Atherosclerosis 51:223239 IYX4 3 7. Levy Y. Hochgraf E. Aviram M. et al: A sex dependent effect of a,pirin on platelet membrane fluidity. Platelet 357-59, 1992 3,1. Moscat J, Perez P, Gavilanes FG. et al: Membrane fluidity and thromboxane synthesis in platelets from patients with severe arht~rosclerosis. Thromb Res 44:1Y7-205. 1986 3’). Shattil SJ. Anavn-Galindo R. Bennett J. et al: Platelet

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