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Platelet aggregates in hypercholesterolemic rhesus monkeys
Vol. 7, pp. 91';-9Zi, 1975
-rHRo~IBoSI~RESEARCH Prinred
in
the United
Pergamon
States
Press,
Inc.
PLATELET AGGREGATES IN HYPERCHOLESTEROLEXIC RHESUS XONKEYS
Kenneth K. Wu, PIarkL. Armstrong, John C. Hoak and srjorie B. )tegan From the Atherosclerosis Research Center and the Divisions of HematologyOncology and Cardiology, Department of Medicine, University of Iowa College of ??edicine, Iowa City, Iowa 52242
in revised form 7.11.197j. (Received 24.9.197j; Accepted by Editor K.SI. Brikhous) ABSTRACT The short-term and long-term effects of induced hypercholesterolemia on platelet aggregates were determined in rhesus monkeys by a platelet countratio technique. Twenty control animals had normal values. Seven of the controls were then made hypercholesterolemic by an atherogenic diet containing 1.2% cholesterol, and aggregates were determined between three and nine weeks when the average serum cholesterol was essentially constant at 540 + 47 mgfdl. Platelet aggregate values were unchanged from those found in the control state after three weeks of hypercholesterolemia, but increased aggregates were found at six weeks and even more aggregates at nine weeks. After 40 weeks of hypercholesterolemia, however, normal values were found despite an average serum cholesterol of 622 + 41 mg/dl. The prevalence of increased platelet aggregates in long-term hypercholesterolemia was studied in fifty monkeys fed the same atherogenic diet for 28 months. Normal values were found in all but eight animals (16%); within this subgroup that had increased aggregates, five were given a cholesterolfree diet. Serum cholesterol fell toward control values; decreased aggregates were found at three weeks and normal values at six weeks. The data indicate that experimental hypercholesterolemia (preponderantly hyperbetalipoproteinemia) provided an essential background for the occurrence of increased platelet aggregates, but that hypercholesterolemia per se did not account for this phenomenon. The nature of other responsible factors, perhaps related to arterial injury, remains to be clarified.
INTRODUCTION Although the pathogenic relationship between atherosclerosis and thrombosis remains uncertain, it is increasingly clear from experimental studies that platelets may play an important role in initiating thrombotic complications of atherosclerosis (1).
Platelet hyperfunction has been found in patients with myocardial infarction
(2,3), type II hyperlipidemia (4) and diabetes mellitus (5). rll?
One of the key
Vo1.7,No.6
PLATELETS:HYPERCHOLESTEROLEMI4
918
questions remaining to be answered is the effect of atherogenic diets on platelet function.
Since rhesus monkeys fed atherogenic diet develop atherosclerosis similar
to human disease (6), this experimental model was used to study the effect of diet on platelet aggregates at the various stages of atherosclerosis and to study the relationship between serum cholesterol and platelet aggregates.
METHODS Seventy adult male rhesus monkeys were studied.
All animals were fed a semi-
purified diet that was cholesterol-free and contained 4% fat for baseline studies of plasma lipids (7).
Fifty monkeys were then given a semi-purified atherogenic
diet that contained 1.2% cholesterol and 40% of total calories from egg yolk fat for 28 months (8); the other 20 continued on the baseline diet to serve as controls. To study the short-term effect of atherogenic diet, 7 of the 20 control animals were then fed atherogenic diet and the blood samples for platelet aggregate and serum cholesterol studies were collected prior to, and 3, 6, 9 and 40 weeks after the initiation of the diet.
To study the effect of regression diet on the platelet
aggregates, 5 of the 50 hypercholesterolemic monkeys with increased platelet aggregates were fed a regression diet which was cholesterol-free and contained approximately 20% fat (9). The method for the determination of platelet aggregates was reported previously"(l0).
In brief, free-flowing venous blood (0.5 ml) was drawn directly into
2 separate polypropylene syringes, one containing 2 ml of buffered formalin-EDTA solution and the other 2 ml of buffered EDTA solution.
After incubation at room
temperature for 15 minutes, both samples were thoroughly mixed and centrifuged to prepare platelet-rich plasma (PRP).
Platelet counts were then performed on the PRP
samples with a Model B Coulter Counter. platelet count in the formalin-EDTA
The result was expressed as the ratio of
PRP over that in the EDTA PRP.
This method was
based upon the idea that platelet aggregates, when present, would be fixed by formalin and centrifuged out.
Therefore, the platelet count in the formalin-EDTA PRP
sample would be decreased, resulting in a iower ratio.
The validity of this method
was confirmed by in vivo studies in which thrombin was infused into rabbits and by in vitro studies in which ADP was added to blood obtained from normal subjects. The serum cholesterol was determined by the method of Abel1 et al (11).
RESULTS The mean platelet aggregate ratio of 50 hypercholesterolemic monkeys fed atherogenic diet for 28 months was 0.89 It_S.E.M. 0.02 which was not significantly different from the value of 20 weight-matched control monkeys (see Table 1).
919
TABLE 1 PLATELET AGGREGATES IN HYPERCHOLESTEROLE?lICRHESUS MONKEYS
Total Number
Body Weight (kg)
Serum Cholesterol (mg/dl)
Platelet Aggregate Ratio
Hypercholesterolemic
50
7.68 + 1.09
637.60 + 16.49
0.89 + 0.02
Control
20
7.41 + 1.55
126.77 + -
0.94 + 0.02
Group
3.00
The normal range of the platelet aggregate ratios (mean + 2 S.D.) as derived from the study of 20 control monkeys was 0.80 or higher.
The platelet aggregate ratios
in 8 of the 50 study monkeys were abnormally low, indicative of increased aggregates in these monkeys (FIG.1).
The body weight and cholesterol concentrations of
those 8 monkeys were not deviated from the rest of the study group.
As shown in
FIG. 1, the correlation between the platelet aggregate ratio and serum cholesterol concentration in the study monkeys was poor with a correlation coefficient of -0.129.
___
0
‘2
lc_~--_--_---
-----e-m-
cl ??
G to-
4i%
$
9
3 $,
0.9-
.
.
” .
aF
$.*
0.
. . .__--_-__-_ ~~~___~~_~~_~~~~~~r~~~ ??
??
.* ’
=? 0.7-
r=-0.129
'4
$
0.6 -
2
0.5 -
. ??
?l 0 \o a
.
.
?? .*
??
Atherosclerotic
6 Contra/
.
0.4O4
I 200 Serum
I 400 Cholesterol
??
I 600
I 800 (mg/dl)
FIG. 1 Platelet aggregate ratio and serum cholesterol concentration in 50 hypercholesterolemic and 20 control monkeys. The area between the two dotted lines represents the range of normal platelet aggregate ratio (mean + 2 S.D.). Seven control monkeys were made hypercholesterolemic by an arherogenic diet and the platelet aggregates were studied sequentially.
Baseline studies prior to
920
PLATEL.FTS:HYPERCHOLESTEROLEMIA
the feeding with atherogenic diet revealed a mean platelet aggregate ratio of 0.98 2 0.06 and cholesterol concentration of 123.14 mg/dl + 3.17.
Despite a marked
increase in cholesterol concentrations 3 weeks after the diet had started, the platelet aggregate values remained unchanged.
However, the aggregate ratios be-
came decreased significantly (0.72 + 0.06) at 6 weeks, and continued to drop (0.59 -+ 0.08) at 9 weeks, indicative of increasing platelet aggregate formation (FIG. 2).
600
9
-
1.0
cl
P
B
bQ.e
P
ws z4 Q.
0.8 * 0.6
. * Pc:O.Ol
:‘i-/ c
0
3
6
9%
Weeks FIG. 2 Effect of atherogenic diet on platelet aggregates. The platelet aggregate ratio dropped significantly (P < 0.01). at 6 and 9 weeks but returned to normal at 40 weeks. The change of platelet aggregate ratios in response to the atherogenic diet was not uniform.
The aggregate values became markedly abnormal in 5 monkeys and remained
normal in 2 monkeys.
While cholesterol concentrations stayed elevated, the platelet
aggregate ratios in all monkeys had returned to normal (mean 0.97 + 0.03) at 40 weeks. To evaluate the effect of regression diet on the platelet aggregate formation, 5 of the 50 chronic hypercholesterolemic monkeys with increased platelet aggregates were fed a regression diet.
At 3 weeks, the serum cholesterol concentrations re-
turned to normal in all monkeys.
On the other hand, the platelet aggregate values
started to rise at 3 weeks and became normalized at 6 weeks and 40 weeks in all 5
921
PWTELETS:KYPERCHOLESTEROLE>lIA
VOl,;,SO.6
animals (FIG. 3).
600 2 Q_l ;; 5 Q 2 5% $3 B cr,
400
200
-
$1
\
-
\
\
\
\
-
\
\
\
\f $-_
-f___~ iP
??
I
* 0
PdO.01
3
6
‘/-ijO
Weeks FIG. 3 Effect of regression diet on platelet aggregates. The serum cholesterol levels returned to normal range and the platelet aggregate values began to rise at 3 weeks and reached normal levels at six weeks and thereafter. DISCUSSION In our laboratories, we have been interested in studying the effects of high cholesterol diet on platelet function. study is quite obvious becaze
The selection of rhesus monkeys in this
the morphological and lipid changes of diet-induced
atherosclerosis in rhesus monkeys have been well established (12).
In addition,
platelet studies can be much better controlled with rhesus monkeys than with humans In human studies, the interpretation of platelet functions in usually hampered by factors that may enhance or inhibit platelet functions (13).
Cigarette smoking
(14), drugs and concomitant diseases are only a few common examples.
These ob-
stacles can be completely avoided in monkeys. That the platelet aggregates were increased only at an early phase but not at a chronic stage of diet-induced atherosclerosis is of interest.
The occurrence
of increased platelet aggregates at early stages of atherosclerosis is probably not due to increase in serum cholesterol since it remained elevated throughout the
vo1.7,~0.6
PLATELETS:HYPERCHOLESTEROLEMIA
922
course of study.
Therefore, other factors may be involved.
From previous studies
(6), it has been shown that deposition of fatty streaks in the endothelium might start as early as 9 weeks after the initiation of high cholesterol diet in rhesus monkeys.
The disruption of the endothelium may render the availability of subendo-
thelial tissues to which circulating platelets adhere (15-17). the vessel wall, platelets undergo reversible aggregation.
After adherence to
Loosely bound platelet
aggregates may presumably become detached from the vessel wall and circulate in the blood stream.
Cn the other hand, the platelet aggregates may be induced by
free fatty acids (18,19) or possibly other lipoptrotein fractions which may be present in the serum of these animals.
It has also been suggested that hyper-
cholesterolemic diets which are atherogenic have toxic effects on vessel walls; the angiotoxicity was not due to cholesterol per se but was related to alcohol-soluble substances (20).
Despite the continuation of the same diet and the persistent ele-
vation of serum cholesterol in these animals, platelet aggregates were found to return to baseline values at 40 weeks.
These results seem to suggest that, whatever
the platelet aggregate-inducing factors may be at an earlier phase, they have disappeared at this stage. The presence of increased platelet aggregates at an early stage of atherosclerosis may have important implications in human atherosclerosis.
Several
careful post mortem studies have shown that intravascular platelet aggregates could be found in the coronary arteries of patients with sudden death (21) or in the cerebral arteries of patients who died after the sudden onset of a stroke (22). The intravascular platelet aggregates were thought to be responsible for the organ infarction and the sudden death because no obvious atheromata could be detected. Although the mechanisms of the platelet aggregate formation in these patients are not elucidated, it is possible that this may represent a human counterpart of our present experimental study.
The platelet aggregates may be formed because of the
damage to the endothelium by early atherosclerosis, which may not be detected by morphological studies.
By the same token, early atherosclerosis-related platelet
aggregates may be important in patients with myocardial infarction who were found to have normal coronary angiography (23). Although the platelet aggregate value was normal in most chronic atherosclerotic animals, it was abnormal in 8 monkeys.
The body weight, and the cholesterol
concentration in this subgroup of animals were not different from those of the remaining animals.
This finding suggests that even in chronic atherosclerosis,
interaction between the endothelium and platelets may be important in causing increased platelet aggregates in some animals.
Five of these animals subsequently
fed a regression diet had not only the cholesterol but also the platelet aggregate values normalized within 6 weeks.
The normalization of platelet aggregates may be
VOl.7,?;0.6
PLATELETS:HYPERCHOLESTEROLE~lI~
923
related to the regression of vascular:lesions rather than serum cholesterol concentrations alone (9). The preliminary data suggest that the early endothelial damage and the subsequent interaction between the subendothelial tissues and platelets may play an important role in causing increased platelet aggregates and thrombotic complications at an early stage of atherosclerosis.
Although experimental hypercholesterol-
emia (preponderantly hyperbetalipoproteinemia) provides an essential background for the occurrence of increased platelet aggregates, hypercholesterolemia per se does not account for this phenomenon.
The nature of other responsible factors, perhaps
related to arterial injury, remains to be clarified.
REFERENCES 1.
POOLE, J.C.F. Thrombosis and atherosclerosis. In: Recent Advances in Thrombosis. L. Poller (Ed.). Edinburgh and London, ChurchillandLivingstone. 1973, p. 59.
2.
MLJRPHY,E.A., MUSTARD J.F. Coagulation tests and platelet economy in atherosclerotic and control subjects. Circulation. 25, 114, 1962.
3.
,Q'BRIEN,J.R., HEYWOOD, J.B., HEADY, J.A. The quantitation of platelet aggregation induced by four compounds: a study in relation to myocardial infarction. Thromb. Diath. Haemorrh. 16, 752, 1966.
4.
CARVALHO, A.C.A., COLMAN, R.W., LEES, R.S. Platelet function in hyperlipoproteinemia. New Eng. J. Med. 290, 434, 1974.
5.
KWAAN, H.C., COLWELL, J.A., CRUZ, S., SUQANWELA, N., DOBBIE, J.G. Increased platelet aggregation in diabetes mellitus. J. Lab. Clin. Med. 80, 236, 1972.
6.
WISSLER, R.W. Recent progress in studies of experimental primate atherosclerosis. Proc. Biochem. Pharmacol. 4, 378, 1968.
7.
ARMSTRONG, M.S., CONNOR, W.E., WARNER, E.D. Xanthomatosis in rhesus monkeys fed a hypercholesterolemic diet. Arch. Path. 84, 227, 1967.
8.
ARMSTRONG, M.S., CONNOR, W.E., WARNER, E.D. Tissue cholesterol concentration in the hypercholesterolemic rhesus monkey. Arch. Path. 87, 87, 1969.
9.
ARMSTRONG, M.S., WARNER, E-D., CONNOR, W.E. Regression of coronary atheromatosi: in rhesus monkeys. Circ. Res. 27, 59, 1970.
10.
WU, K.K., HOAK, J.C. A new method for the quantitative detection of platelet aggregates in patients with arterial insufficiency. Lancet. 2, 924, 1974.
11.
ABELL, L-L., LEVY, B.B., BRODIE, B.B., KENDALL, F.E. Simplified method for the estimation of total cholesterol in serum and demonstration of its specificity. J. Biol. Chem. 195, 352, 1952.
12.
ARMSTRONG, M.S., WARNER, E.D., Morphology and distribution of diet-induced atherosclerosis in rhesus monkeys. &ch. Path. 92, 395, 1971.
PLATELETS:HYPERCHOLESTEROLEMlX
924
Vo1.7,No.6
13.
MUSTARD, J-F., PACKHAM, M.A. Factors influencing platelet functions: adhesion, release and aggregation. Pharmacol. Rev, 22, 97, 1969.
14.
HAWKINS, R.I., Smoking, platelets and thrombosis. Nature. 236, 450, 1972.
15.
BIZZOZERO, J. Ueber einen neuen formbestandteil des bluter and dessen rolle bei der thrombose und der blutergerinnung. Fichows Arch. Pathol. Anat. 90,
26, 1882. 16.
FRENCH, J.E., MACFARLAND, R.G., SANDERS, A.G. The structure of hemostatic plugs and experimental thrombi in small arteries. Brit. J. Exp. Path. 45, 467, 1964.
17.
HONOUR, A.J., PICKERING, G.W., SHEPPARD, B.L. Ultrastructure and behaviour of platelet thrombi in injured arteries. Brit. J. Exp. Path. 52, 482, 1971.
18.
CONNOR, W.E., HOAK, J.C., WARNER, E.D. Massive thrombosis produced by fatty acid infusion. J. Clin. Invest. 42, 860, 1963.
19.
HOAK, J.C., WARNER, E.D., CONNOR, W.E. Platelets, fatty acid and thrombosis. Circ. Res. 20, 11, 1967.
20.
IMAI, H. Ultrastructural one day bioassay of angiotoxicity: possible prediction ofatherogenicity. (Abst.) Fed. Proc. 33, 623, 1974.
21.
HAEREM, J.W. Sudden coronary death: theoccurrence of platelet aggregates in the epicardial arteries of man. Atherosclerosis. 14, 417, 1971.
22.
JORGENSEN, L. The role of platelet embolism from crumbling emboli and of platelet aggregates arising in flowing blood. In: Thrombosis, S. Sherry, K.M. Brinkhous, E. Genton, 3-M. Stengle (Ed.) Washington, D.C., National Academy of Science. 1969, p. 506.
23.
KHAN, A.H., HAYWOCD, L.J. Myocardial infarction in nine patients with radiologically patent coronary arteries. -_ New Eng. J. Med. 291, 427, 1974.
-rHRo~IBoSI~RESEARCH Prinred
in
the United
Pergamon
States
Press,
Inc.
PLATELET AGGREGATES IN HYPERCHOLESTEROLEXIC RHESUS XONKEYS
Kenneth K. Wu, PIarkL. Armstrong, John C. Hoak and srjorie B. )tegan From the Atherosclerosis Research Center and the Divisions of HematologyOncology and Cardiology, Department of Medicine, University of Iowa College of ??edicine, Iowa City, Iowa 52242
in revised form 7.11.197j. (Received 24.9.197j; Accepted by Editor K.SI. Brikhous) ABSTRACT The short-term and long-term effects of induced hypercholesterolemia on platelet aggregates were determined in rhesus monkeys by a platelet countratio technique. Twenty control animals had normal values. Seven of the controls were then made hypercholesterolemic by an atherogenic diet containing 1.2% cholesterol, and aggregates were determined between three and nine weeks when the average serum cholesterol was essentially constant at 540 + 47 mgfdl. Platelet aggregate values were unchanged from those found in the control state after three weeks of hypercholesterolemia, but increased aggregates were found at six weeks and even more aggregates at nine weeks. After 40 weeks of hypercholesterolemia, however, normal values were found despite an average serum cholesterol of 622 + 41 mg/dl. The prevalence of increased platelet aggregates in long-term hypercholesterolemia was studied in fifty monkeys fed the same atherogenic diet for 28 months. Normal values were found in all but eight animals (16%); within this subgroup that had increased aggregates, five were given a cholesterolfree diet. Serum cholesterol fell toward control values; decreased aggregates were found at three weeks and normal values at six weeks. The data indicate that experimental hypercholesterolemia (preponderantly hyperbetalipoproteinemia) provided an essential background for the occurrence of increased platelet aggregates, but that hypercholesterolemia per se did not account for this phenomenon. The nature of other responsible factors, perhaps related to arterial injury, remains to be clarified.
INTRODUCTION Although the pathogenic relationship between atherosclerosis and thrombosis remains uncertain, it is increasingly clear from experimental studies that platelets may play an important role in initiating thrombotic complications of atherosclerosis (1).
Platelet hyperfunction has been found in patients with myocardial infarction
(2,3), type II hyperlipidemia (4) and diabetes mellitus (5). rll?
One of the key
Vo1.7,No.6
PLATELETS:HYPERCHOLESTEROLEMI4
918
questions remaining to be answered is the effect of atherogenic diets on platelet function.
Since rhesus monkeys fed atherogenic diet develop atherosclerosis similar
to human disease (6), this experimental model was used to study the effect of diet on platelet aggregates at the various stages of atherosclerosis and to study the relationship between serum cholesterol and platelet aggregates.
METHODS Seventy adult male rhesus monkeys were studied.
All animals were fed a semi-
purified diet that was cholesterol-free and contained 4% fat for baseline studies of plasma lipids (7).
Fifty monkeys were then given a semi-purified atherogenic
diet that contained 1.2% cholesterol and 40% of total calories from egg yolk fat for 28 months (8); the other 20 continued on the baseline diet to serve as controls. To study the short-term effect of atherogenic diet, 7 of the 20 control animals were then fed atherogenic diet and the blood samples for platelet aggregate and serum cholesterol studies were collected prior to, and 3, 6, 9 and 40 weeks after the initiation of the diet.
To study the effect of regression diet on the platelet
aggregates, 5 of the 50 hypercholesterolemic monkeys with increased platelet aggregates were fed a regression diet which was cholesterol-free and contained approximately 20% fat (9). The method for the determination of platelet aggregates was reported previously"(l0).
In brief, free-flowing venous blood (0.5 ml) was drawn directly into
2 separate polypropylene syringes, one containing 2 ml of buffered formalin-EDTA solution and the other 2 ml of buffered EDTA solution.
After incubation at room
temperature for 15 minutes, both samples were thoroughly mixed and centrifuged to prepare platelet-rich plasma (PRP).
Platelet counts were then performed on the PRP
samples with a Model B Coulter Counter. platelet count in the formalin-EDTA
The result was expressed as the ratio of
PRP over that in the EDTA PRP.
This method was
based upon the idea that platelet aggregates, when present, would be fixed by formalin and centrifuged out.
Therefore, the platelet count in the formalin-EDTA PRP
sample would be decreased, resulting in a iower ratio.
The validity of this method
was confirmed by in vivo studies in which thrombin was infused into rabbits and by in vitro studies in which ADP was added to blood obtained from normal subjects. The serum cholesterol was determined by the method of Abel1 et al (11).
RESULTS The mean platelet aggregate ratio of 50 hypercholesterolemic monkeys fed atherogenic diet for 28 months was 0.89 It_S.E.M. 0.02 which was not significantly different from the value of 20 weight-matched control monkeys (see Table 1).
919
TABLE 1 PLATELET AGGREGATES IN HYPERCHOLESTEROLE?lICRHESUS MONKEYS
Total Number
Body Weight (kg)
Serum Cholesterol (mg/dl)
Platelet Aggregate Ratio
Hypercholesterolemic
50
7.68 + 1.09
637.60 + 16.49
0.89 + 0.02
Control
20
7.41 + 1.55
126.77 + -
0.94 + 0.02
Group
3.00
The normal range of the platelet aggregate ratios (mean + 2 S.D.) as derived from the study of 20 control monkeys was 0.80 or higher.
The platelet aggregate ratios
in 8 of the 50 study monkeys were abnormally low, indicative of increased aggregates in these monkeys (FIG.1).
The body weight and cholesterol concentrations of
those 8 monkeys were not deviated from the rest of the study group.
As shown in
FIG. 1, the correlation between the platelet aggregate ratio and serum cholesterol concentration in the study monkeys was poor with a correlation coefficient of -0.129.
___
0
‘2
lc_~--_--_---
-----e-m-
cl ??
G to-
4i%
$
9
3 $,
0.9-
.
.
” .
aF
$.*
0.
. . .__--_-__-_ ~~~___~~_~~_~~~~~~r~~~ ??
??
.* ’
=? 0.7-
r=-0.129
'4
$
0.6 -
2
0.5 -
. ??
?l 0 \o a
.
.
?? .*
??
Atherosclerotic
6 Contra/
.
0.4O4
I 200 Serum
I 400 Cholesterol
??
I 600
I 800 (mg/dl)
FIG. 1 Platelet aggregate ratio and serum cholesterol concentration in 50 hypercholesterolemic and 20 control monkeys. The area between the two dotted lines represents the range of normal platelet aggregate ratio (mean + 2 S.D.). Seven control monkeys were made hypercholesterolemic by an arherogenic diet and the platelet aggregates were studied sequentially.
Baseline studies prior to
920
PLATEL.FTS:HYPERCHOLESTEROLEMIA
the feeding with atherogenic diet revealed a mean platelet aggregate ratio of 0.98 2 0.06 and cholesterol concentration of 123.14 mg/dl + 3.17.
Despite a marked
increase in cholesterol concentrations 3 weeks after the diet had started, the platelet aggregate values remained unchanged.
However, the aggregate ratios be-
came decreased significantly (0.72 + 0.06) at 6 weeks, and continued to drop (0.59 -+ 0.08) at 9 weeks, indicative of increasing platelet aggregate formation (FIG. 2).
600
9
-
1.0
cl
P
B
bQ.e
P
ws z4 Q.
0.8 * 0.6
. * Pc:O.Ol
:‘i-/ c
0
3
6
9%
Weeks FIG. 2 Effect of atherogenic diet on platelet aggregates. The platelet aggregate ratio dropped significantly (P < 0.01). at 6 and 9 weeks but returned to normal at 40 weeks. The change of platelet aggregate ratios in response to the atherogenic diet was not uniform.
The aggregate values became markedly abnormal in 5 monkeys and remained
normal in 2 monkeys.
While cholesterol concentrations stayed elevated, the platelet
aggregate ratios in all monkeys had returned to normal (mean 0.97 + 0.03) at 40 weeks. To evaluate the effect of regression diet on the platelet aggregate formation, 5 of the 50 chronic hypercholesterolemic monkeys with increased platelet aggregates were fed a regression diet.
At 3 weeks, the serum cholesterol concentrations re-
turned to normal in all monkeys.
On the other hand, the platelet aggregate values
started to rise at 3 weeks and became normalized at 6 weeks and 40 weeks in all 5
921
PWTELETS:KYPERCHOLESTEROLE>lIA
VOl,;,SO.6
animals (FIG. 3).
600 2 Q_l ;; 5 Q 2 5% $3 B cr,
400
200
-
$1
\
-
\
\
\
\
-
\
\
\
\f $-_
-f___~ iP
??
I
* 0
PdO.01
3
6
‘/-ijO
Weeks FIG. 3 Effect of regression diet on platelet aggregates. The serum cholesterol levels returned to normal range and the platelet aggregate values began to rise at 3 weeks and reached normal levels at six weeks and thereafter. DISCUSSION In our laboratories, we have been interested in studying the effects of high cholesterol diet on platelet function. study is quite obvious becaze
The selection of rhesus monkeys in this
the morphological and lipid changes of diet-induced
atherosclerosis in rhesus monkeys have been well established (12).
In addition,
platelet studies can be much better controlled with rhesus monkeys than with humans In human studies, the interpretation of platelet functions in usually hampered by factors that may enhance or inhibit platelet functions (13).
Cigarette smoking
(14), drugs and concomitant diseases are only a few common examples.
These ob-
stacles can be completely avoided in monkeys. That the platelet aggregates were increased only at an early phase but not at a chronic stage of diet-induced atherosclerosis is of interest.
The occurrence
of increased platelet aggregates at early stages of atherosclerosis is probably not due to increase in serum cholesterol since it remained elevated throughout the
vo1.7,~0.6
PLATELETS:HYPERCHOLESTEROLEMIA
922
course of study.
Therefore, other factors may be involved.
From previous studies
(6), it has been shown that deposition of fatty streaks in the endothelium might start as early as 9 weeks after the initiation of high cholesterol diet in rhesus monkeys.
The disruption of the endothelium may render the availability of subendo-
thelial tissues to which circulating platelets adhere (15-17). the vessel wall, platelets undergo reversible aggregation.
After adherence to
Loosely bound platelet
aggregates may presumably become detached from the vessel wall and circulate in the blood stream.
Cn the other hand, the platelet aggregates may be induced by
free fatty acids (18,19) or possibly other lipoptrotein fractions which may be present in the serum of these animals.
It has also been suggested that hyper-
cholesterolemic diets which are atherogenic have toxic effects on vessel walls; the angiotoxicity was not due to cholesterol per se but was related to alcohol-soluble substances (20).
Despite the continuation of the same diet and the persistent ele-
vation of serum cholesterol in these animals, platelet aggregates were found to return to baseline values at 40 weeks.
These results seem to suggest that, whatever
the platelet aggregate-inducing factors may be at an earlier phase, they have disappeared at this stage. The presence of increased platelet aggregates at an early stage of atherosclerosis may have important implications in human atherosclerosis.
Several
careful post mortem studies have shown that intravascular platelet aggregates could be found in the coronary arteries of patients with sudden death (21) or in the cerebral arteries of patients who died after the sudden onset of a stroke (22). The intravascular platelet aggregates were thought to be responsible for the organ infarction and the sudden death because no obvious atheromata could be detected. Although the mechanisms of the platelet aggregate formation in these patients are not elucidated, it is possible that this may represent a human counterpart of our present experimental study.
The platelet aggregates may be formed because of the
damage to the endothelium by early atherosclerosis, which may not be detected by morphological studies.
By the same token, early atherosclerosis-related platelet
aggregates may be important in patients with myocardial infarction who were found to have normal coronary angiography (23). Although the platelet aggregate value was normal in most chronic atherosclerotic animals, it was abnormal in 8 monkeys.
The body weight, and the cholesterol
concentration in this subgroup of animals were not different from those of the remaining animals.
This finding suggests that even in chronic atherosclerosis,
interaction between the endothelium and platelets may be important in causing increased platelet aggregates in some animals.
Five of these animals subsequently
fed a regression diet had not only the cholesterol but also the platelet aggregate values normalized within 6 weeks.
The normalization of platelet aggregates may be
VOl.7,?;0.6
PLATELETS:HYPERCHOLESTEROLE~lI~
923
related to the regression of vascular:lesions rather than serum cholesterol concentrations alone (9). The preliminary data suggest that the early endothelial damage and the subsequent interaction between the subendothelial tissues and platelets may play an important role in causing increased platelet aggregates and thrombotic complications at an early stage of atherosclerosis.
Although experimental hypercholesterol-
emia (preponderantly hyperbetalipoproteinemia) provides an essential background for the occurrence of increased platelet aggregates, hypercholesterolemia per se does not account for this phenomenon.
The nature of other responsible factors, perhaps
related to arterial injury, remains to be clarified.
REFERENCES 1.
POOLE, J.C.F. Thrombosis and atherosclerosis. In: Recent Advances in Thrombosis. L. Poller (Ed.). Edinburgh and London, ChurchillandLivingstone. 1973, p. 59.
2.
MLJRPHY,E.A., MUSTARD J.F. Coagulation tests and platelet economy in atherosclerotic and control subjects. Circulation. 25, 114, 1962.
3.
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