Analysis of platelets in nonhuman primates:: II. Effects of varying dietary fatty acid ratios on platelet ultrastructure and function

EXPERIMENTAL AND MOLECULAR PATHOLOGY 35, 394-404 (1981)

Analysis of Platelets in Nonhuman Primates:

II. Effects of Varying Dietary Fatty Acid Ratios on Platelet Ultrastructure and Function

JOHN C. LEWIS, RICHARD W. ST. CLAIR, AND MELANIE S. WHITE

Department of Pathology. Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27103

Received April 27, 1981, and in revised form June 24, 1981

The effects of varying dietary fatty acid composition on in vitro aggregation and ultrastructure of African green monkey platelets are reported. Platelets from animals maintained on either a control diet (30% calories from fat; polyunsaturated:saturated:monounsaturated = 1:2.3:2.7) or a diet approximating that suggested by the Senate Select Commit- tee on Nutrition and Human Needs (P:S:M = 1: 1: 1) aggregated maximally with 20 pM ADP to a level of 65%. With lesser amounts of ADP, the maximum aggregation decreased propor- tionally to a low of 25% when using 3 @I ADP. This aggregation pattern was significantly altered when the animals were switched to a safflower oil-enriched diet which provided more polyunsaturated fatty acid (PUFA) (P:S:M = 2: 1: 1). With the high PUFA diet, platelet aggregation using ADP increased to a range of 45-85% depending upon the concentration of aggregating agent used. Thrombin-induced aggregation was unaltered by dietary change. The ADP hypersensitivity in platelets with the PUFA diet paralleled a significant drop in plasma HDL. Concomitantly, large lipid-like droplets were consistently observed in the cytoplasm of platelets obtained during the high-PUFA diet period. Platelet aggregation and ultrastructure and plasma HDL levels returned to normal with resumption of the control diet. The platelet hypersensitivity to ADP aggregation and the cytoplasmic lipid were again observed when the animals were returned to a PUFA diet containing 40% calories from fat. Results substantiate the suggestion that under certain conditions dietary PUFAs alter circulating platelets. The biochemical mechanism for the alterations remains to be elucidated.

INTRODUCTION

An increased tendency toward thrombosis has long been recognized as one of the factors associated with patterns of dietary fat consumption (1,2). This propensity, which in part is mediated through alterations in platelet function, both aggregation and procoagulant activity, has been most frequently correlated with high saturated fat intake (1,3). Conversely, diets which are relatively low in saturated fats and high in polyunsaturated fats have been linked to reductions in platelet aggregability and a decreased incidence of ischemic heart disease (4). Such observations are significant since platelet hypersensitivity has been identified in many clinical conditions including venous thromboembolism, myocardial infarc- tion, peripheral vascular disease, and hypertension (5). Recent evidence suggests that excessive polyunsaturated fat may, as well as saturated fat, increase the probability of intraarterial thrombosis. Ginglewski and Moncada have suggested that this detrimental effect of polyunsaturated fatty acids results from the rapid oxidation of polyunsaturated fatty acids to the lipid peroxides, prostaglandins (2). In light of the current national trend to decrease saturated fat intake while at the same time increase consumption of polyunsaturated fats, it seemed reasonable to explore the effects on platelets of short-term alterations in dietary fatty acid ratios. We report a study, using African green monkeys (Cercopithecus aethiops), of the effects of three different fatty acid ratios and two levels of dietary fat on plasma

394

0014-4800/81/060394-11$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

DIETARY FATTY ACIDS ALTER PLATELET ULTRASTRUCTURE 395

lipids and platelets. These diets were patterned after the recommendations of the Senate Select Committee on Nutrition and Human Needs (McGovern Commis- sion) (6). Our results clearly document changes in platelet aggregation in the animals while they were receiving diets relatively enriched in polyunsaturated fatty acids.

MATERIALS AND METHODS

Dietary Conditions

Ten male African green monkeys (C. aethiops), ranging in age from 6 to 9 years, were used in this study. The animals were maintained in the animal resource facility at Bowman Gray School of Medicine where they were fed a semipurified diet consisting primarily of a wheat flour, dry milk solid base with lactalbumin, and casein protein supplements (Table I). Each of the diets was further supplemented with fats of known composition (Table II). The diets, shown in Table I, were fed sequentially to all 10 animals for periods of 2 months each. Plasma lipids were determined after 4, 6, and 8 weeks of consuming each diet. Since there was no significant difference in these three periods, we averaged the values obtained at the above three periods to provide a single value for plasma lipid concentrations with each dietary fat. The diets containing 30% of calories from fat were fed for the first 8 months followed by a second 8-month period in which diets with 40% of calories from fat were fed. Each 8-month phase began and ended with the same control diet containing the fatty acid composition of a typical North American diet. The diet with a polyunsaturated to saturated to monounsaturated (P:S:M) fatty acid composition of 1: 1: 1 will subsequently be referred to as the McGovern diet since it was recommended by the Senate Select Committee (6). The diet with a 2:l:l P:S:M ratio will be referred to as the high polyunsatu-

TABLE I Basic Diet Composition

30% calories from fat 40% calories from fat

Ingredient Diet number: 1 2 3 4 5 6 (Ii9 Fatty acid ratio” 1:2.3:2.7 1:l:l 2:l:l 1:2.3:2.7 1:l:l 2:l:l

Wheat flour Casein Lactalbumin Sucrose Dextrin Applesause Salt mixture Vitamin mixture Alphacel Fat

Butter Lard

Safflower oil (18: 1) Safflower oil (182) Cholesterol

35.0 8.0 8.0 5.0

12.61 2.64 2.64 2.64 4.5 4.0 2.5

10.0

1.8 2.45 2.45 2.45 7.68 5.64 2.64 10.43 7.66 3.59 1.68 0 0 1.14 0 0 0.84 4.56 7.56 2.28 6.19 10.27

47.59 49.73 52.88 43.42 46.33 50.60

Nore. All diets had the basic composition shown for diet 1, the value shown for diets 2-6 are those which differ from diet 1.

(I Polyunsaturated:saturated:monounsaturated.

396 LEWIS, ST. CLAIR, AND WHITE

TABLE II Fatty Acid Compositions of Fats Used in the Various Diets

Percentage of total fatty acids

<14:0” 14:o 16:O 16:l 180 18:l 182 Unidentified

Butter 2.3 12.3 37.4 1.9 13.0 29.5 1.3 2.3 Lard - 0.7 25.8 3.3 14.7 45.9 8.0 1.6 Safflower oil - - 5.0 0.2 2.3 81.9 9.8 0.8

(18:l)” Safflower oil - - 8.0 1.3 2.6 12.7 74.8 0.6

(18:2)”

(1 No. of carbon atoms:double bonds.

rated fat diet (PUFA diet). The dietary cholesterol content was the same for all diets in that they contained 0.15 mg cholesteroYkca1 which is similar to approximately 300 mg cholesterol/day for human beings. The fats used in each of the diets were strictly controlled with the same source and lot number of fat used throughout the course of the study. Each of the fats was analyzed prior to use (Table II) to establish fatty acid composition. Two of the fats shown in Table II as safflower oil (l&l) and safflower oil (l&2) were specially provided by PVO International (Richmond, Calif.). Table III summarizes the overall experimental design. The lipid analyses performed did not include positional isomers; however, it is as- sumed that trans isomers are not present in significant amounts in the fats used in our study.

Plasma Lipid Analysis

The animals were sedated with ketamine (5 mg/kg) and blood was collected by venipuncture for determination of total plasma cholesterol and triglycerides. This procedure was repeated at biweekly intervals. At the end of each 2-month dietary period these assays were expanded to include plasma HDL determination and platelet studies. All lipid analyses were done in the Bowman Gray School of Medicine Lipid Analytic Laboratory, under strict quality control as monitored by the lipid standardization program of the Center for Disease Control. For lipid

TABLE III Overall Experimental Design

Months of study Diet Platelet studies

at end of month

Phase I 30% calories from fat

o-2

3-4

5-6

7-8

Phase II 40% calories from fat

9-11 II-12 13-14 IS- 16

North American control P:S:M = 1:2.3:2.7

McGovern P:S:M = 1:l:l

High polyunsaturated P:S:M = 2:1:1

North American control

North American control 10 McGovern 12 High polyunsaturated 14 North American control 16

2

4

6

8


analysis the animals were fasted overnight. Total plasma cholesterol and triglyceride concentrations were determined by the method of Rush et al. (7). High- density lipoprotein (HDL) cholesterol concentrations were determined by the heparin and maganese precipitation procedure (8). Because of the low concentrations of VLDL in this species we were able to estimate low-density lipoprotein cholesterol as the difference between total plasma cholesterol and HDL cholesterol .

Platelet Studies

Isolation and preparation of platelet-rich plasma for aggregation and electron microscopy was carried out as previously described (9). Prior to venipuncture, the animals were sedated using 15 mg/kg of ketamine. Blood was then drawn into plastic syringes and immediately mixed with freshly prepared 3.8% sodium citrate pH 7.4 (9 vol of blood with 1 vol of citrate). Platelet-rich plasma (PRP) was obtained by centrifugation of the anticoagulated blood at 125g for 10 min at room temperature in plastic tubes. Platelet aggregation was done by the turbidometric method of Born (10) in a Chronolog 330 Aggregometer equipped with a linear recorder (Chronolog Corporation, Havertown, Pa). For these studies adenosine diphosphate (ADP) (Sigma Chemical, St. Louis, MO.) at final concentrations in the cuvette of 3,5, 10, and 20 pM and thrombin (sodium salt, Sigma Chemical, St. Louis, MO.) at concentrations of 1, 3, and 5 units/ml were used. At all dietary stages select aggregation samples and corresponding PRP were processed for electron microscopy as previously reported (9).

RESULTS

The mean total plasma cholesterol concentrations for the animals while consuming the control diet with a fatty acid composition similar to that of the present North American diet (P:S:M = 1:2.3:2.7) ranged from 154 to 182 mg/dl (Table IV). This TPC level remained essentially unchanged during Phase 1(30% calories from fat) when the animals were switched from the control diet to one which approxi- mates the fatty acid composition recommended by the McGovern Commission. When the diet with 40% of calories from fat was fed (Phase II, Table III) there was a markedly lower plasma cholesterol concentration with the 1:l:l diet. It is doubtful that the elevated cholesterol (182 + 15 mg/dl) found at the beginning of this phase is significant, since a return to this high plasma cholesterol concentration did not occur when the control diet was again fed at the end of this diet phase. There was a consistent and significant reduction in total plasma cholesterol concentration when the animals were given the PUFA diet. This reduction in TPC, observed when either 30 or 40% of calories were derived from fat, was primarily due to changes in plasma HDL. Plasma triglyceride concentrations were not significantly altered by any of the diets, and body weight remained constant throughout the course of the experiment (Table III).

Dramatic changes in platelet aggregation were also observed when the animals were given the PUFA diet. Upon stimulation with ADP or thrombin rapid aggregation of the platelets occurred. The degree of aggregation with all diets was dependent upon concentration of the aggregating agent (Figs. 1 and 2), and irreversible aggregation was obtained when using either 20 fl ADP (Fig. 1) or 3 units/ml of thrombin (Fig. 2). Platelets within the irreversible aggregates were closely apposed and cells at the periphery of the aggregates had undergone significant


TABLE IV Plasma Lipids in African Green Monkeys Maintained on Diets of Various Fatty Acid Composition

Fatty acid composition of diet (P:S:M)

1:2.3:2.7 I:l:l 2:l:l (control) (McGovern) (High Poly.)

30% calories from fat and 0.15 mg chol/kcal Total cholesterol (mg/dl) 162 + 12 158 ‘- 14 150 f 12 HDL cholesterol (mg/dl) 79 2 6 79 2 5 70 2 3 LDL cholesterol (mg/dl) 84 + 8 792 11 81 ? 11 Triglyceride (mg/dl) 26 k 3 26 2 3 19 T 2 Body weight (kg) 4.68 T 0.11 4.75 i 0.10 4.50 i 0.12

1~2.312.7 (control)

164 * 14 77 f 5 87 c 12 22 2 3

4.64 2 0.15

40% calories from fat and 0.15 mg chol/kcal Total cholesterol (mg/dl) 182 ? 15 152 * 10 143 ” 4 154 r 13 HDL cholesterol (mg/dl) 90 f 5 86 k 6 74 k 4 82 + 5 LDL cholesterol (mg/dl) 93 f 14 67 + 7 69 t 9 73 2 9 Triglyceride (mg/dl) 18 f 1 22 k 3 24 2 4 25 k 3 Body weight (kg) 4.50 f 0.12 4.74 + 0.16 4.59 ” 0.15 4.66 -t 0.14

Nore. Results are the ,? + SEM from determinations made after 4, 6, and 8 weeks of each diet period for the 10 animals in the study. The mean of these three measurements was calculated for each animal and represents the average response of that animal to each diet. Statistics were done using this average response for the 10 animals of the study. P:S:M = polyunsaturated fat (18:2)/ saturated fat (16:O + 18:0)/mononusaturated fat (18: 1).

release (Fig. 3). When aggregated with lower concentrations of either agent, the cells had undergone shape change, but degranulation was minimal and the platelets were loosely associated within the aggregates (Fig. 4). This aggregation response pattern was consistently observed throughout the experiment, but as already pointed out, significant changes were observed with the diet containing high polyunsaturated fatty acids. Under these conditions, as shown in Fig. 5,

- . s - ._*_ LA.

JJM

0 I 2 3 4 5

Time ( min.)

FIG. 1. A series of aggregation curves showing the in vifro response of African green monkey platelets to different concentrations of adenosine diphosphate (ADP). When stimulated with 3, 5, or 10 @I ADP, the platelets undergo rapid aggregation which is maximal after 60-90 sec. The aggregation phase is followed by disaggregation over a period of 2-5 min. Treatment with 20 pMADP typically results in irreversible aggregation.


too r 90 -

60 -

70 -

60 -

I unit I

units

0. I 2

Time (min.)

FIG. 2. Two aggregation curves showing the response of African green platelets to thrombin treatment. Reversible aggregation, typically obtained with l-2 units/ml of thrombin, is overcome with thrombin concentrations of 3 units or more.

maximum platelet aggregation with ADP ranged from 45 to 85% depending upon the concentration of ADP used. This was in contrast to a range of 25-65% obtained when the animals were on either the North American control diet or the McGovern diet. The exaggerated response of platelets to ADP when the animals were fed the PUFA diet was observed when either 30 or 40% of calories were derived from fat, and in each of these cases the platelet response to ADP returned to baseline levels when the animals were returned to the North American control diet. Thrombin aggregation remained unchanged throughout the course of the

FIG. 3. An electron micrograph illustrating the tigl _ _ _ with 20 @4 ADP. Approximately 50% of the cells within these aggregates have degranulated-xil00.


FIG. 4. A platelet aggregate representative of the type which disaggregates. Most of the platelets have undergone shape change with pseudopod extension. However, the cells, most of which retain their granules, are only loosely associated within the aggregate. Compare with Fig. 3. x4100.

ADP Concentroiion

n 3um 0 5um 0 IOum

t l 20 urn

90 t

i I 2 I I I 2 I Polyunsaturated 2.3 I I 2.3 2.3 I I 2.3 Saturated 2.7 I I 2.7 2.7 I I 2.7 Monounsoturated

30% Fat 40% Fat

FIG. 5. A series of graphs illustrating the maximum aggregation obtained with ADP treatment of platelets from the animals at different dietary stages. Each curve represents a different ADP concentration and the graph points correspond to diets of various fatty acid composition, or percentage of fat as shown on the abscissa. Note that with either 30 or 40% calories from fat an exaggerated aggregation response was found when the fatty acid composition (P:S:M) was 2:l:l. This hypersensitivity to ADP aggregation was lost when the animals were returned to the control diet (1:2.3:2.7) for 2 months.


FIG. 6. Electron micrograph illustrating the morphology of unstimulated platelets from animals while on the high polyunsaturated diet (P:S:M = 21: 1). Under these conditions, the resting cells frequently had large cytoplasmic projections containing lipid droplets (arrows). ~20,300.

experiment. Ultrastructurally, the effect of the PUFA diet on platelet function (ADP aggregation) could not be correlated with apparent changes in platelet size or organelle distribution. The PUFA diet cells did differ from the North American control diet cells and the McGovern diet cells through the prevalence in PRP of platelets having one or more pseudopods and osmiphilic droplets within the cytoplasm (Fig. 6). These lipid-like droplets had diameters in the range 3500-4500 A, were often located within terminal regions of the pseudopods in nonaggregated cells, and could be identified in platelets subsequent to aggregation.

DISCUSSION Platelet hypersensitivity, long recognized as a clinically significant ramification

of excessive animal fat consumption, has primarily been correlated with long- chain fatty acids (1,3 - 5). Data substantiating this correlation, obtained from epi- demiological studies (1) and experiments involving dietary manipulation (3,5), have related platelet function and the propensity toward thrombosis to dietary levels of myristic (14:0), palmitic (16:0) and stearic (18:0) acids with the longer- chain stearic acid having the greatest correlation (1). Modification of dietary fat by lowering the relative amount of saturated fats while increasing the proportion of polyunsaturated fats has generally been associated with a decreased propensity toward thrombosis and a reduction in platelet sensitivity. Along these lines Homstra et al. (4) have, in a study involving more than 130 human volunteers, reported significant reductions in platelet aggregation times and tangents to the


aggregation slopes when individuals consuming a diet high in polyunsaturated fatty acids (P:S = 1.6) were compared to those whose diet resembled the normal Finnish diet (P:S = 0.25). Similar effects of diet modifications have been reported by Jakubowski and Ardlie (11) who studied the same group of individuals under two dietary regimens, one in which butter provided 30% of dietary calories and the other in which a commercially available polyunsaturated margarine (P:S > 3: 1) was substituted for the butter. In addition to a reduction in circulating platelet aggregates, significant reductions in whole-blood platelet counts and heparin neutraliz- ing activity were associated with consumption of polyunsaturated fatty acids. The time frame for manifestation of these fatty acid effects has not been determined; however, in an experiment designed to evaluate acute effects of saturated and unsaturated fatty acids O’Brien et al. (12) were able to detect changes in heparin-thrombin clotting time, platelet count, and platelet volume within a few hours of fat ingestion. These investigators reported that saturated fats shortened the heparin-thrombin clotting time whereas unsaturated fats had an opposite effect. Interestingly, O’Brien et al. (12) also found that both saturated and unsaturated fats caused a reduction in platelet number and an increase in platelet volume. These latter findings, suggestive of increased platelet consumption, impli- cate unsaturated as well as saturated fatty acids in the dietary enhancement of thrombosis. The suggestion that unsaturated fatty acids may adversely influence platelets has been further substantiated by the recent studies of Honour et al. (13). These investigators reported finding an increased sensitivity of ADP for thrombus formation in rabbits when either polyunsaturated or saturated fat diets were given to the animals. In addition to the platelets becoming hypersensitive to ADP, significant reductions in total plasma cholesterol levels occurred when the animals were given the polyunsaturated diets. The results of our present experiments are consistent with those of Honour et al. (13) in that we have documented a 30% increase in maximum ADP-induced platelet aggregation in all animals when given a diet enriched with polyunsaturated fatty acids (P:S = 2.0). In our experiments the hypersensitive state was found when either 30 or 40% of calories were derived from fat, and in both situations the hypertensive condition was lost when the animals were returned to the control diet (P:S = 0.43). This latter point is particularly significant since, as recently expressed by O’Brien (14), experimental repro- ducibility is a major concern in studies designed to evaluate dietary lipids and platelet function.

The biochemical mechanism by which the dietary polyunsaturated fatty acids have altered platelet sensitivity in the present study is unknown; however, several possibilities exist to explain this phenomenon. One obvious explanation for our results is the effect of elevated dietary polyunsaturated fatty acids on plasma lipids. Consistent with the finding of Honour (13), triglycerides remained unchanged throughout the course of our experiments. Total plasma cholesterol was, however, slightly lower while the animals were given the high polyunsaturated fatty acid diet, with 75% of this decrease due to a drop in HDL cholesterol levels. This is a potentially undesirable effect of polyunsaturated fatty acids, since low- ered plasma HDL levels have been correlated with increased platelet aggregation in women on oral contraceptives (IS), and since elevated HDL levels have been linked to a reduction in maximal platelet aggregation in hyperlipoproteinemic monkeys (16). It is‘important to note that a direct relationship between plasma lipoproteins and platelet sensitivity is not fully substantiated for Miller and Nor-


day (17) reported that incubation in vitro of platelet-rich plasma with increasing concentrations of very low density, low-density, or high-density lipoproteins in amounts sufficient to raise the concentration two- to sevenfold had no effect on the subsequent aggregation of platelets by ADP, 1-epinephrine, or collagen. Addition of HDL did in the study of Miller and Nordoy (17) cause a rise in platelet factor 3 activity suggesting some direct platelet response.

Conceivably, the increased ADP sensitivity is the result of direct alterations in platelet lipids in response to dietary PUFA. Such alteration could bring about changes in membrane lipid fluidity (18) and prostaglandin synthesis (2). We did not in the present experiments evaluate platelet lipid composition, however, Berlin et al. (19) have recently demonstrated significant increases in linoleic acid and arachidonic acid in rabbit platelet phospholipids when the animals were fed corn oil as opposed to cocoa butter or milk fat. These changes in rabbit platelets which extended previously reported studies in rats, are pertinent since the relative per- centages of saturated and polyunsaturated fatty acid in the phospholipids of platelet membranes correlated to alterations in membrane fluidity as determined by fluorescence depolarization (19). Since ADP aggregation is a surface event involving the reversible exposure of fibrinogen binding sites, alterations in plasma membrane fluidity similar to that reported by Berlin et al. (19) could explain the hypersensitivity to low-concentration ADP aggregation. The question of altered prostaglandin metabolism has been briefly reviewed by Gryglewski and Moncada (2) who pointed out several potential biochemical mechanisms by which polyunsaturated fatty acids participate in the process of intraarterial thrombosis. Poten- tially applicable to the results of our study is the proposed rapid conversion in the liver of dietary linoleic acid to arachidonic acid. This liver-derived arachidonate could be incorporated into plasma and membrane phospholipids, which in the platelet would become part of the phospholipid reservoir for the generation of thromboxane A, (TXA), a potent aggregator. No evidence to our knowledge is available to substantiate conversion in African green monkeys of excess dietary linoleic acid to arachidonic acid, nor is there information available with respect to changes in platelet arachidonate. However, since linoleate is the major fatty acid in our high polyunsaturated fatty acid diet, platelet hypersensitivity (particularly under conditions involving the release reaction and irreversible aggregation) mediated through enhanced TXAz synthesis is most intriguing.

Finally, our study includes ultrastructural observations of both resting platelets and platelets aggregated by varying concentrations of ADP. One of the interesting observations from these studies is the identification in resting and aggregated platelets of lipid droplets when the animals were maintained on a diet enriched in polyunsaturated fatty acids. Similar observations were reported by Hovig and Grottum who used both scanning and transmission electron microscopy to de- scribe “lipid particles” in human platelets following infusion of a soybean lipid emulsion (20). In our study the lipid inclusions appear to be a direct result of the dietary polyunsaturated fatty acids since they were rarely observed with either the control or McGovern diets. Similarly, inclusions of this type were not reported in a comparative morphometric analysis of platelets from six different species of nonhuman primates (9). The presence of these lipid inclusions gives credence to the suggestion that dietary polyunsaturated fatty acids alter the lipid composition of African green monkey platelets.


ACKNOWLEDGMENTS

The authors are grateful to Mr. Tilman Prater and Mr. Richard Taylor for electron microscopy technical support, to Ms. Nina Ann Stokes for lipid analysis, and to Mrs. Bobbie Lindsay for her assistance in the preparation of this manuscript. This work was supported by a grant from the National Dairy Council and NIH Grant HL-14164 (SCOR).

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2. GRYGLEWSKI, R. J., and MONCADA, S. (1979). Polyunsaturated fatty acids and thrombosis. Eur. J. Clin. Invesr. 9, l-2.

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11. JAKUBOWSKI, J. A. and ARDLIE, N. G. (1978). Modification of human platelet function by a diet enriched in a saturated or polyunsaturated fat. Afherosclerosis 31, 335-344.

12. O’BRIEN, J. R., ETHERINGTON, M. D., and JAMIESON, S. (1976). Acute platelet changes after large meals of saturated and unsaturated fats. Lancer 1, 878-880.

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14. O’BRIEN, J. R. (1980). Lipids, platelets, and atherosclerosis. Lancer 1, 981-982. 15. BIERSENBAUM, M. L., FLEISCHMAN, A. I., STIER, A., WATSON, P., SOMOL, H., NASO, A. M.,

and BINDER, M. (1979). Increased platelet aggregation and decreased high-density lipoprotein cholesterol in women on oral contraceptives. Amer. J. Obster. Gvnecol. 134, 638-641.

16. DEMPSEY, G. L., and WAGNER, W. D. (1979). Altered platelet function in hyperlipoproteinemic monkeys. Fed. Proc. 38, (Abstract), 1207.

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18. SHATTIL, S. J., and COOPER, R. A. (1978). Role of membrane lipid composition organization, and fluidity in human platelet function. Prog. Hernosfasis Thromb. 4, 59-86.

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20. HOVIG, T., and GROTTUM, K. A. (1973). Lipid infusions in man. Thrornb. Diath. Hue/now. 29, 450-459.

Documents

Analysis of platelets in nonhuman primates:: II. Effects of varying dietary fatty acid ratios on platelet ultrastructure and function