Chylomicrons from patients with Type V hyperlipoproteinemia inhibit platelet function

Atherosclerosis. 56 (1985) 157-167 Elsevier Scientific Publishers Ireland,

157 Ltd.

Chylomicrons from Patients with Type V Hyperlipoproteinemia Inhibit Platelet Function Michael Aviram, Bianca Furman

and J. Gerald Brook

The Lipid Research Unit, Rambam Medical Center and the Faculty of Medicine, Technion Israel Institute of Technology, Haija (Israel)

(Revised,

(Received 19 April, 1984) received 23 October, 1984; 15 January, (Accepted 17 January, 1985)

1985)

Summary Platelet aggregation and [‘4C]serotonin release induced by collagen and also by ADP and thrombin were significantly decreased in patients with primary Type V hyperlipoproteinemia. Platelets derived from these patients lost their hyporesponsiveness to thrombin and ADP (but not to collagen) after washings and isolation from their plasma environment. On incubation of platelets derived from normolipidemic controls with plasma derived from patients, platelet aggregation and [‘4C]serotonin release were lowered by 20% and 30%, respectively. On incubation of these platelets with 100 mg/dl of chylomicron triglyceride, a 40% reduction in both platelet aggregation and [‘4C]serotonin release was observed. The inhibition of platelet activity was positively correlated with chylomicron concentration up to a concentration of 225 mg/dl by chylomicron triglyceride. In 2 patients, bezafibrate administration (600 mg/day) resulted in marked reduction of plasma triglyceride concentration and a parallel improvement in platelet function. The platelet hyporesponsiveness in patients with Type V hyperlipoproteinemia appears to be a consequence of platelet-chylomicron interaction. This depressed platelet function may be responsible for the absence of overt atherosclerosis noted in these patients. Key words:

Chylomicrons - Platelet aggregation lipoproteinemia

- Platelet release - Type V hyper-

This research was supported by a grant from the National Council for Research and Development, Israel, and the G.S.F., Munich, F.R.G. Address reprint requests to Dr. Aviram, The Lipid Research Unit, Rambam Medical Centre, Haifa 35254, Israel. 0021-9150/85/$03.30

0 1985 Elsevier Scientific

Publishers

Ireland,

Ltd.

158

Introduction Patients with Type V hyperlipoproteinemia have exceedingly high levels of triglycerides in their plasma, due to the accumulation of both chylomicrons and very low density lipoproteins (VLDL) [l]. There appear to be both an overproduction of endogenous triglycerides and defective clearance of all triglyceride-rich lipoproteins (TRP) in these patients [2]. Since a common saturable-triglyceride-removal mechanism for both chylomicrons and VLDL has been shown in man [3], any defect in this mechanism would result in the accumulation of large amounts of both these lipoproteins. It has been suggested that exogenous triglycerides, the chylomicrons, are not associated with extensive atherogenesis [4], whereas chylomicron remnants may be extremely atherogenic [5]. Blood platelets appear to have an important role in the initiation and development of the atherogenic lesion [6]. They also interact with lipoproteins [7,8], with consequent far-reaching effects on their activity [9,10]. Low density lipoproteins (LDL) enhance platelet activation, whereas high density lipoproteins (HDL) have the opposite effect [ll]. We postulate that chylomicrons are not atherogenic, since they inhibit platelet function. In fact, a rise in exogenous triglycerides in plasma results in a decrease in platelet adhesiveness [12]. Our present study was designed to determine platelet function in patients with Type V hyperlipoproteinemia and to study the effect of the chylomicrons isolated from these patients on the various modes of function of platelets derived from normolipidemic individuals. Methods Subjects Five male patients with primary Type V hyperlipoproteinemia were studied and compared with 5 normolipidemic, healthy controls, matched for age (35-50 years) and sex. Patients with Type V hyperlipidemia were characterized by high levels of both triglycerides and cholesterol, milky plasma, a clear, creamy layer at the top of the plasma column after refrigeration for 12 h at 4°C and increased amounts of VLDL and of chylomicrons as revealed on paper electrophoresis. All blood samples were recovered after fasting 12 h. None of the patients had diabetes mellitus, and both patients and controls were free of all medication for at least 2 weeks prior to testing. Collection of blood Venous blood (75 ml) was collected through siliconized needles into plastic syringes, in 1 mM EDTA for lipoprotein separation (25 ml) and in ACD (2.5% sodium citrate, 1.4% citric acid and 2% dextrose) for platelet studies (55 ml, v/v = l/6). The pH was thus reduced to 6.9. Platelet preparation Four and a half volumes of blood were added to 0.5 volume of ACD in plastic tubes. Samples were centrifuged at 200 X g for 10 min at 23’C. Platelet-rich plasma

159

(PRP) was separated with plastic tips and the remaining samples recentrifuged at 2000 x g for 10 min at 23°C in order to prepare the platelet-poor plasma (PPP) preparation. Prior to testing platelet function, platelets were diluted to a standard concentration of 200000 platelets/p1 of plasma with PPP. Platelet aggregation Platelet aggregation was studied in a dual aggregometer (Chronolog Corp., Broomall, PA) [13]. ADP, 2.5 PM (Sigma Chemical Co., St. Louis, MO); collagen, 1 pgg/ml (Hormon Chemie, Munich, F.R.G.); and thrombin, 0.2 U/ml (Parke Davis Co., Detroit, MI), were the aggregating agents. PRP (450 ~1) was placed in a siliconized cuvette, 8 mm in diameter, and stirred (1000 rpm at 37’C) by means of a plastic bar. The cuvette was placed in the aggregometer, as was a cuvette containing the same amount of PPP as a control. Fifty ~1 of the aggregating agent were added, and the light transmittance was continuously monitored by a Heath Servo Recorder (Model EV-20B). [‘4C]Serotonin release [lo] Three ml of PRP were incubated for 30 min with 0.2 pCi/ml of [‘4C]serotonin (50.7 mCi/mmol, New England Nuclear, Boston, MA, NEC-225). After removal of the excess free [r4C]serotonin by centrifugation (1500 x g for 10 min at room temperature), the platelet pellet was resuspended in an equivalent volume of plasma or buffer. Four hundred and fifty ~1 of this preparation were placed in a siliconized cuvette in the aggregometer, and 50 ~1 of aggregating agent were added. Aggregation was performed for 5 min at 37°C. The suspension was then removed and placed into a plastic minivial containing EDTA (50 mM final concentration), placed on ice, centrifuged for 10 min at 8000 rpm in a cooled minicentrifuge, and then 250 ~1 of the supernatant was placed in vials containing 15 ml scintillation fluid. A control sample with no aggregating agent was always included. [r4C]serotonin release was calculated from the total radioactivity and that obtained from the sample and the control supernatants. Plasma /3-thromboglobulin was determined using a specific radioimmunoassay kit (Amersham). Washed platelets (WP) These were obtained by adding acetic acid (10 mM) to a PRP solution which was then centrifuged for 10 min at 1300 x g. The supernatant was removed and the pellet resuspended in an equal volume of Hepes buffer (5 mM Hepes, 137 mM sodium chloride, 2.7 mM potassium chloride, 1.2 mM magnesium chloride, 12 mM sodium bicarbonate, 0.4 mM sodium biphosphate, 0.6 mM glucose, 0.35% human albumin, pH 7.4). The WP preparation was found to be free of contaminating plasma lipoproteins by standard immunochemical and electrophoretic procedures. Incubation procedures PRP was treated with acetic acid (10 mM) before centrifugation at 2000 rpm for 10 min. The platelet pellet was resuspended either in Hepes buffer or in PPP derived from the controls or from the patients, using equivalent volumes. After incubation

160

for 30 min at 37’C, determination of platelet function was undertaken. WP were also incubated under similar conditions with Hepes buffer containing chylomicrons derived from the patients’ plasma, and platelet function was then determined. The incubation was carried out for 30 min since we have previously found that all plasma lipoproteins affect platelet function to saturation within this time [7,9-111. Isolation of chylomicrons Plasma chylomicrons were isolated by sequential flotation in a Beckman L2-65B preparative ultracentrifuge [14] at 4°C for 30 min at 20000 x g. Chylomicrons were similarly prepared from 5 normal subjects 3 h after a fatty meal consisting of 100 g of saturated fat given as cream porridge. Experimental protocol The following experiments were performed: (1) Platelet aggregation and [‘4C]serotonin release were determined in PRP derived from the patient and control groups. (2) Platelet aggregation and [‘4C]serotonin release were studied using WP derived from the patient and control groups. (3) WP were prepared from normolipedemic controls and, after resuspension in the plasma derived from either the type V phenotype patients or the controls, platelet function was determined. (4) Chylomicrons were isolated from the Type V phenotype patients and incubated with WP derived from the controls. Platelet aggregation and release were then determined. (5) Chylomicrons were suspended in Hepes buffer, in increasing concentrations, and incubated with normal WP. The effect of chylomicron concentration on platelet aggregation and release was then determined. (6) Chylomicrons and chylomicron-deficient plasma were incubated with PRP and WP derived from the controls. Platelet aggregation in response to ADP and collagen was then determined. (7) Bezafibrate (Boehringer Mannheim GmbH) at a dose of 600 mg/day was administered to the patients. In those patients who showed reduction in plasma triglycerides, platelet aggregation was determined. In addition, plasma derived from these patients was incubated with control platelets and platelet function evaluated. The plasma from 2 patients was too milky for aggregation studies, and both PRP and PPP were diluted l/4 (v/v) with saline before aggregation could be assessed. Results The platelets derived from Type V hyperlipoproteinemic patients demonstrated significantly less aggregation and [‘4C]serotonin release than those derived from the normolipidemic controls (Fig. 1). Aggregation induced by collagen was lowered by 36% and that induced by ADP, by 20%. [i4C]serotonin release was 40% less in the patient group. Plasma P-thromboglobulin levels from unstimulated plasma were significantly decreased (P < 0.01) in the patients (25 k 4.1 ng/ml) in comparison to the normal controls (34 _t 3.7 ng/ml). Patient chylomicron concentration in the PRP was 150-285 mg triglyceride/dl. In order to delete the effect of plasma factor(s), the experiment was repeated using washed-platelet preparations derived from the 2 sources (PRP and WP). Platelet aggregation and [i4C]serotonin release in WP

161

Collagen

Collagen

ADP

ADP

Fig. 1. Platelet aggregation and [‘4C]serotonin release in PRP preparation derived from primary Type V hyperlipoproteinemic patients (dashed bars) and from normal control subjects (open bars). Values are mean + SD. **P < 0.02 (patients vs controls).

induced by ADP and thrombin was similar in both normal controls and patients (Table 1). Collagen-induced platelet activation, however, was still lower in the patients in comparison to the normal group, as was found also in PRP (Table 1 and Fig. 1). Resuspension of WP derived from normal subjects with patient plasma resulted in significant depression of platelet aggregation and [t4C]serotonin release compared to platelets resuspended and incubated in control plasma. Collagen- and ADP-induced aggregation was 20% lower and the [14C]serotonin release 30% lower than the results obtained with control plasma (Fig. 2).

TABLE

1

PLATELET AGGREGATION AND [‘4C]SEROTONIN RELEASE IN WASHED PLATELETS FROM PHENOTYPE V HYPERLIPOPROTEINEMIC PATIENTS AND FROM NORMAL SUBJECTS Subjects

Platelet aggregation

(%)

Platelet [‘4C]serotonin

release (S)

Collagen

ADP

ADP

(2.5 PM)

Thrombin (0.2 U/ml)

Collagen

(1 pg/mI)

(1 &ml)

(2.5 PM)

Thrombin (0.2 U/ml)

Normal subjects 1 60 2 80 3 38 4 76 5 60 Mean *SE 63*7

12 16 29 17 25 20+2

12 71 81 88 loo 8255

40 63 69 51 45 54*5

13 11 12 11 8 11+1

73 69 88 82 62 75*5

Patients 6 7 8 9 10 Mean f SE P values

13 17 25 15 23 19+2 NS

14 89 84 80 97 85+4 NS

34 52 43 55 42 45+4 < 0.05

11 9 12 8 12 10*1 NS

70 66 74 88 62 72k5 NS

NS denotes

47 82 28 66 62 57*10 i 0.05 not significant.

162

Collagen

ADP

Collagen

ADP

Fig. 2. Effect of plasma derived from primary Type V hyperlipoproteinemic patients on normal platelet activity. Normal washed platelets (2OOOOO/~l) were incubated with either patient (dashed bars) or normal control plasma (open bars) for 30 mm at 37°C. Platelet functions were then analyzed. Values are mean * SD. **P -c 0.02 (patients vs controls).

COllagen

ADP

Thrombin

COllagOn

ADP

Thrombin

r

Fig. 3. Effect of plasma chylomicrons derived from primary Type V normal platelet function in each individual. The effect of collagen thrombin (0.2 U/ml) on normal washed platelet function was analyzed presence of ( + , dashed bars) 100 mg triglycerides/ml of chylomicrons. individual, and the horizontal line indicates the mean level of platelet *P < 0.01, **p < 0.02.

100 Chylomicrons

hyperlipoproteinemic patients on (1 &ml), ADP (2.5 PM) and without (-, open bars) or in the Each bar represents a different function. Significance ( + vs - ):

200

TG Concentration

cmg/dlr

2

Fig. 4. Effect of chylomicron concentrations on normal platelet aggregation (O-O) and on [‘4C]serotonin release (O-O). Plasma chylomicrons were separated from the plasma of primary Type V hyperlipoproteinemic patients, and incubation was carried out using normal washed platelets (200000/~1) at 37°C for 30 mm. Platelet function induced by collagen (1 pg/ml) was then analyzed in vitro. Values are means of 3 different experiments with 12% maximal variation.

163 TABLE

2

THE EFFECT OF CHYLOMICRONS AND LET AGGREGATION IN PLATELET-RICH TION

CHYLOMICRON-DEFICIENT PLASMA PLASMA AND WASHED-PLATELETS

ON PLATEPREPARA-

CDP denotes chylomicron-deficient plasma. Normal platelets (200000/~1) were incubated with either 100 mg/dl chylomicron triglyceride or 10% CDP at 37’C for 30 min, and platelet aggregation was then determined. The results represent mean * SD of 5 experiments. Platelet preparation

Aggregation

amplitude

(%)

ADP (2.5 ,uM)

Platelet-rich plasma Washed platelets

Collagen

(1 pgg/ml)

Control

+ Chylomicrons

+ CDP

Control

+ Chylomicrons

+ CDP

79 k 14 32+7

65 + 11 * 2Ok4’

74*10** 285 6’*

s5+13 76+ 3

74*10* 64+ 6+

x0*12 ** 71*5 **

* P < 0.01; **P < 0.05(significance

vs the control

experiments).

The incubation of WP derived from the controls with chylomicrons suspended in Hepes buffer at a concentration of 100 mg/dl of chylomicron triglycerides depressed platelet aggregation and [14C]serotonin release (induced by collagen) by 16% and 40%, respectively. Both aggregation and release induced by ADP were depressed by 40%, whereas the effects induced by thrombin were lowered by 10%. Figure 3 depicts the results obtained in each individual experiment. Increasing the chylomicron concentration resulted in further depression of both platelet aggregation and release (Fig. 4). The effect was linear up to 225 mg/dl of chylomicron triglycerides. The addition of chylomicrons to both normal PRP and WP preparations resulted in depression of platelet aggregation in both instances. The addition of chylomicron-deficient plasma to these preparations also lowered the aggregation response, but to a lesser extent (Table 2). Two of the 5 patients treated with bezafibrate (600 mg/day) responded with a marked fall in plasma triglyceride levels. Platelet aggregation in these patients was TABLE

3

THE EFFECT IN PATIENTS

OF SUCCESSFUL BEZAFIBRATE TREATMENT WITH TYPE V HYPERLIPOPROTEINEMIA Patient

Plasma triglyceride

ON PLATELET

Platelet aggregation

AGGREGATION

(%)

ADP (2.5 PM)

Collagen

(1 pg/ml)

(mg/dl) Before treatment

1 2

1345 1097

61 64

70 73

After treatment

1 2

281 243

76 75

85 82

Bezafibrate (600 mg/day) were then studied.

was given for 2 months,

and plasma

lipids and platelet

aggregation

in PRP

164

TABLE

4

THE EFFECT OF PLASMA CHYLOMICRONS DERIVED FROM A FATTY MEAL, ON NORMAL PLATELET AGGREGATION

NORMAL

SUBJECTS,

AFTER

The effect of collagen (1 pg/ml) and ADP (2.5 PM) on normal washed platelet aggregation was studied after 30 min of incubation of the platelets with 100 mg triglycerides/d1 of chylomicrons derived 3 h after a fatty meal. Values represent mean f SD of 5 different subjects. Platelet aggregation

ADP (2.5 PM) Collagen (1 pg/ml)

(%)

Control

+ Chylomicrons

39* 9 70*10

25+5 64*6

* *

* PiO.01.

markedly increased (Table 3) but remained depressed in the nonresponding patients. In these latter patients, plasma triglyceride levels were not reduced, and platelet aggregation induced by collagen (1 pgg/ml) was also unchanged (68%, 72% and 71% in comparison to 66%, 70% and 69%, before and after bezafibrate therapy, respectively). On incubation of the responsers’ plasma with platelets from normolipidemic controls, an inhibitory effect on aggregation was not noted. Bezafibrate administered in vitro had very little effect on platelet aggregation. Plasma chylomicrons (100 mg triglycerides/dl) derived from 5 normal subjects 3 h after a fatty meal, like the patient chylomicrons, significantly decreased platelet aggregation (Table 4) induced by either ADP or collagen.

Discussion Platelet-lipoprotein interactions appear to be of importance in determining platelet function. We have previously shown that platelets possess specific receptors for LDL [7,9], and after incubation of platelets with LDL or VLDL there is an enhancement of in-vitro platelet aggregation and [‘4C]serotonin release. Incubation with HDL results in the opposite - a depression of both aggregation and release [lO,ll]. Furthermore, platelets derived from patients with familial hypercholesterolemia exhibit increased aggregation [8], and on incubating the plasma of these patients with platelets derived from normolipidemic individuals, marked enhancement of platelet function then occurs [15-171. This report shows that the chylomicrons also interact with platelets and influence their in-vitro function. Chylomicrons derived from patients with Type V hyperlipoproteinemia decreased both the aggregation and [i4C]serotonin release of platelets obtained from normolipidemic controls (Fig. 3). In addition, these patients with Type V hyperlipoproteinemia demonstrated decreased platelet aggregation (Fig. 1). The decreased plasma P-thromboglobulin levels in the patient group represent in-vivo support for the reduced in-vitro platelet responsiveness in Type V hyperlipoproteinemic patients.

165

In both the patients with familial hypercholesterolemia and those with Type V hyperlipoproteinemia, the effect on platelet function could be a consequence of lipoprotein-platelet interactions with secondary changes in platelet membrane composition. Alternatively, some other factor or factors affecting platelet function may be operating in the plasma of these patients. An interesting difference in platelet responsiveness exists between these 2 groups of patients. Platelets derived from the patients with Type V hyperlipoproteinemia did not maintain their diminished responsiveness (except for collagen stimulation) on removing them from their environment (Fig. 1, Table I). Thus, the presence of some ‘plasma constituent or constituents, which appears to be the chylomicrons, might be responsible for the diminished platelet responsiveness. In fact, we have demonstrated an inverse relationship between chylomicron-triglyceride concentration in the platelet media and the in-vitro platelet response (Fig. 4). The greater the chylomicron concentration, the more pronounced the depression of platelet function. Platelets derived from patients with familial hypercholesterolemia behave differently, in that they maintain their hypersensitivity even after removal from their plasma environment and repeated washings [15]. This suggests that the hypercholesterolemic environment causes a more permanent effect on platelet composition than the hypertriglyceridemic environment. In the latter, the platelet hypoactivity appears to be dependent on a permanent presence of the chylomicrons. The results showing decreased platelet activity also by chylomicrons derived from normal subjects after a fatty meal (Table 4) suggest that it is the chylomicron characteristics that affect platelet activity. The chylomicrons derived from patients with Type V hyperlipoproteinemia, although circulating for a long time in the blood, affected platelet function similarly to those obtained during alimentary lipemia. Bezafibrate resulted in marked reduction of plasma triglyceride levels in 2 of our patients (Table 3). This was accompanied by a distinct improvement in platelet responsiveness, confirming in vivo the importance of the chylomicron concentration in relation to platelet function. Both lipoproteins and platelets appear to be of importance in the pathogenesis of atherosclerosis [18]. It has been suggested that Type V hyperlipoproteinemia is not associated with extensive atherogenesis, and it has been noted that patients with Type V hyperlipoproteinemia, and in the absence of diabetes mellitus, appear to be less prone to the manifestations of atherosclerosis [19]. One may speculate that the inhibition of platelet function by the chylomicrons may represent the mechanism which suppresses atherogenesis in these patients. Patients with familial hypercholesterolemia and enhanced platelet activity have accelerated atherosclerosis [8,15-171. Whereas intact chylomicrons do not appear to be related to atherogenesis, the chylomicron remnants, which are more cholesterol-rich, do demonstrate atherogenic properties [20]. It might be that the accumulation of these remnants, rather than of the intact chylomicrons, predisposes towards the atherosclerotic process. The effect of plasma lipoproteins on platelet function in normals [7,9-11,21-241 and in hyperlipoproteinemic patients [8,15-17,25-301 may be of great importance in the determination of the risk for accelerated atherosclerosis.

166

Acknowledgement

We thank Ruth Singer for typing the manuscript. References 1 Brunzell, J.D. and Bierman E.L., Chylomicronemia syndrome, Med. Clin. N. Amer., 66 (1982) 455. 2 Lewis, B., Type V hyperlipoproteinaemia. In: B. Lewis (Ed.), The Hyperlipidaemias. Blackwell Scientific, Oxford, 1976, p. 258. 3 Brunzell, J.D., Hazzard, W.R., Porte, D. and Bierman, E.L., Evidence for a common saturable-triglyceride removal mechanism for chylomicrons and very low density lipoproteins, J. Clin. Invest., 52 (1973) 1578. 4 Lippel, K., Tyroler, H., Eder, H., Gotto. A. and Vahouny, G., Relationship of hypertriglyceridemia to atherosclerosis, Atherosclerosis, 1 (1981) 406. 5 Zilversmit, D.B., Atherogenesis - A postprandial phenomenon, Circulation, 60 (1979) 473. 6 Ross, R. and Glomset, J.A., The pathogenesis of atherosclerosis, N. Engl. J. Med., 295 (1976) 369, 402. 7 Aviram, M., Brook, J.G., Lees, A.M. and Lees, R.S., Low density lipoprotein binding to human platelets - Role of charge and of specific amino acids, Biochem. Biophys. Res. Commun., 99 (1981) 308. 8 Carvalho, A.C.A., Colman, R.W. and Lees, R.S., Platelet function in hyperlipoproteinemia, N. Engl. J. Med., 290 (1974) 434. 9 Aviram, M. and Brook, J.G., Platelet interaction with high and low density lipoproteins, Atherosclerosis, 46 (1983) 259. 10 Aviram, M. and Brook, J.G., Characterization of the effect of plasma lipoproteins in platelet function in vitro, Haemostasis, 13 (1983) 344. 11 Aviram, M. and Brook, J.G., The effect of blood constituents on platelet function ~ Role of blood cells and plasma lipoproteins, Artery, 11 (1983) 297. 12 Nordoy, A., Strom, E. and Gjedal, K., The effect of alimentary hyperlipaemia and primary hypertriglyceridaemia on platelets in man, Stand. J. Haematol., 12 (1974) 329. 13 Born, G.V.R., Aggregation of blood platelets by adenosine diphosphate and its reversal, Nature (Lond.), 194 (1962) 297. and chemical composition of ultracentri14 Havel, R.J., Eder, H.S. and Bragdon, J.H., The distribution fugally separated lipoproteins in human serum, J. Clin. Invest., 34 (1955) 1345. 15 Aviram, M. and Brook, J.G., The effect of human plasma on platelet function in familial hypercholesterolemia, Thromb. Res., 26 (1982) 101. G. and Aviram, M., Platelet function and lipoprotein levels after plasma 16 Brook, J.G., Winterstein, exchange in patients with familial hypercholesterolemia, Clin. Sci., 64 (1983) 637. in hypercholes17 Viener, A., Brook, J.G. and Aviram, M., Abnormal plasma lipoprotein composition terolemic patients induces platelet activation, Europ. J. Clin. Invest., 14 (1984) 207. cholesterol esters as the major constituent of 18 Ross, A.C. and Zilversmit, D.B., Chylomicron-remnant very low density lipoproteins in plasma of cholesterol-fed rabbits, J. Lipid Res., 18 (1977) 169. D.S., Goldstein, J.L. and Brown, MS., The familial hyperlipoproteinemias. In: J.B. 19 Fredrickson, Stanbury, J.B. Wyngaarden and D.S. Fredrickson (Eds.), The Metabolic Basis of Inherited Disease, 4th edition, McGraw-Hill, New York, 1978, p. 451. chylomicrons - Mechanism of binding and uptake of 20 Fielding, C.J., Metabolism of cholesterol-rich cholesterol esters by the vascular bed of the perfused rat heart, J. Clin. Invest., 62 (1978) 141. K.R., The aggregation of isolated human platelets in the 21 Hassall, D.G., Owen, J.S. and Bruckdorfer, presence of lipoproteins and prostacyclin, B&hem. J., 216 (1983) 43. K.R., Marenah, C.B., Turner, P., Cortese, C., Miller, N.E. 22 Hassall, D.G., Forrest, L.A., Bruckdorfer, and Lewis, B., Influence of plasma lipoproteins on platelet aggregation in a normal male population, Arteriosclerosis, 3 (1983) 332. 23 Aviram, M. and Brook, J.G., Selective release from platelet granules induced by plasma lipoproteins, Biochem. Med., 32 (1984) 30.

167 24 Aviram, M., Sirtori, C.R., Colli, S., Maderna, P., Morazzoni, G. and Tremoli, E., Plasma lipoproteins affect platelet malondialdehyde and thromboxane B, production, Biochem. Med., In press. 25 Shmulevitch, A., Brook, J.G. and Aviram, M., Interaction of modified low density lipoprotein with human platelets in normal and homozygous hypercholesterolemic subjects, B&hem. J., 224 (1984) 13. 26 Baruch, Y., Brook, J.G., Eidelmans, S. and Aviram, M., Increased concentration of high density lipoprotein in plasma and decreased platelet aggregation in primary biliary cirrhosis, Atherosclerosis, 53 (1984) 151. 27 Weschler. A., Aviram, M., Levin, M., Better. O.S. and Brook, J.G., High dose of L-carnitine increases platelet aggregation and plasma triglyceride levels in uremic patients on hemodialysis, Nephron. 38 (1984) 120. 28 Aviram, M., Winterstein, G. and Brook, J.G., Differential effect of platelet inhibitors in normal and hypercholesterolemic subjects, Brit. J. Clin. Pharmacol., In press. 29 Aviram, M., Sechter, Y. and Brook, J.G., Chylomicron-like particles in severe hypertriglyceridemia, Lipids, In press. 30 Brook, J.G., Linn, S. and Aviram, M., Dietary soya lecithin decreases plasma triglyceride levels and inhibits platelet function, B&hem. Med., In press.