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Effects of dietary arachidonate deficiency on the aggregation of cat platelets
Camp. Biochem. Physiol. Vol. 78C, No. I, pp. 123-126, Printed in Great Britain
0306.4492/84 $3.00 + 0.00 1984 Pergamon Press Ltd
1984 c
EFFECTS OF DIETARY ARACHIDONATE DEFICIENCY ON THE AGGREGATION OF CAT PLATELETS Department
MARNIE L. MACDONALD, QUINTON R. ROGERS and JAMES G. MORRIS* of Physiological Sciences, School of Veterinary Medicine, and *Department of Animal Sciences, University of California, Davis, CA 95616, U.S.A. (Received 8
September
1983)
Abstract-l. Because the cat lacks the ability to synthesize a significant amount of arachidonate from linoleate in the liver, arachidonate is a dietary essential. We studied the effects of essential fatty acid deficiency on the aggregation of cat platelets. 2. Cat platelets aggregated in response to ADP, collagen, arachidonate and epinephrine. 3. Aggregation was impaired in platelets from cats fed arachidonate-deficient diets. 4. Including a source of hnoleate in the arachidonate-deficient 5. Arachidonate is required in the diet of the cat for normal
INTRODUCTION In some species, including the human and the rat, the type of dietary fat affects the tendency to thrombosis (Hornstra, 1975). The effect of dietary fat on thrombosis may be a result of differences in platelet aggregation as a result of changes in the fatty acid composition of platelet phospholipids. Arachidonate (5,8,11,1420:4), present in platelet phospholipids, is released when platelets are stimulated to aggregate (Silver et al., 1978). Free arachidonate is then converted to eicosanoids, including thromboxane A,, which is a potent aggregatory substance (Hamberg et al., 1975). Platelets from essential fatty acid (EFA)-deficient rats synthesize very little thromboxane A, (Vincent and Zijlstra, 1978), presumably because levels of arachidonate in platelet phospholipids are low. McGregor and Renaud (1977) observed that platelets from EFA-deficient rats were less responsive to ADP and more responsive to arachidonate than were platelets from rats fed a diet adequate in linoleate. Hornstra (1975) reported that EFA deficiency decreased the response of rat platelets to collagen, but not to ADP. Galli et al. (1981) found that low levels of arachidonate in phospholipids of rabbit platelets were associated with a higher threshold for the aggregation of platelets in response to arachidonate, and a decrease in the synthesis of thromboxane B, from exogenous arachidonate. The domestic cat (Fe& catus) is different from non-carnivorous mammals because it lacks the ability to synthesize a significant amount of arachidonate from linoleate in the liver (Hassam et al., 1977). As a result, arachidonate is a dietary essential for the cat (Rivers et al., 1975). In cats fed diets lacking arachidonate, the level of arachidonate is only 1% in plasma lipids and 6”/, in erythrocyte lipids, whether linoleate is provided in the diet or not (MacDonald et al., 1983). In cats fed commercial canned cat foods, which contain arachidonate, the level of arachidonate in plasma and erythrocyte lipids is 15525% of the total fatty acids by weight (Rivers et al., 1975; Sinclair et al., 1979).
diet did not improve platelet aggregation.
aggregation.
Marcinkiewicz et al. (1978) showed that the aggregation of cat platelets in response to collagen, ADP and arachidonate was blocked by 2-isopropyl-3nicotinylindole, an inhibitor of thromboxane synthesis. This suggests that thromboxane synthesis is an important step in the aggregation of cat platelets, as it is in platelets from other mammals. Because of the importance of arachidonate in phospholipids as a source of free arachidonate for thromboxane synthesis during aggregation, we proposed that platelets from cats fed arachidonate-deficient diets would be unable to aggregate normally. The results of this study showed that cat platelets aggregate in response to ADP, collagen, arachidonate and epinephrine, and that aggregation of platelets from cats fed diets deficient in arachidonate is impaired.
METHODS Specific pathogen-free cats were fed either a mixture of proprietary cat foods (control diet) or one of two purified diets containing either 35% (by weight) hydrogenated beef tallow (EFA-deficient diet) or 5% safflower seed oil plus 30% hydrogenated beef tallow (SSO diet). Both the EFAD and SSO diets lacked arachidonate. The level of linoleate was 6.77; of dietary energy in the SSO diet and 0.3% of dietary energy in the EFAD diet. The remainder of the EFAD and SSO diets consisted of casein, carbohydrates, vitamins and minerals, as described previously (MacDonald et al., 1983). Groups of 4-6 cats were fed each diet for approximately 2 years, beginning at 3 months of age. Blood (9 ml) was collected from the jugular vein of unanesthetized cats. Blood samples were drawn into 12ml plastic syringes containing 1 ml of 1.9% sodium citrate. (A higher concentration of sodium citrate (3.8%) completely inhibited platelet aggregation.) Samples were immediately centrifuged at room temperature at 18Og for 10min. The platelet-rich plasma (PRP) was collected and the remaining sample was centrifuged at 15OOg for 15 min to obtain platelet-poor plasma (PPP). Despite careful handling, aggregation occurred spontaneously in 23% of the samples from cats fed the control diet, 33% of the samples from cats fed the EFAD diet, and 16% of the samples from cats fed the SSO diet. Platelets in whole blood and PRP were counted by phase-contrast microscopy after a I: 100 dilution in I”/, 123
124
MARNIE L. MACDONALD Table
I. Ekts
of diet on the aggregation
et ul.
of cat platelets in response to arachiodonate,
ADP and collagen*
Diet
Platelet Platelet
count count
Response Maximum Lag (min) Threshold
in whole bloodt in PRPt
Control
EFAD
SSO
Pooled SEM
353,500” 482,800
472.195” 603,000”
182,140b 436,000”
29,854 45,770
to arachldonate aggregation (p,) with ImM arachidonate concentration (mM)
Response to ADP Maximum aggregation Percentage aggregation First phase Second phase
(“/,) to 0.4 PM ADP:
Response to collagen Maximum aggregation (%) Lag (min) at 200pg/ml collagen
87.9” I .87” 0.28”
75.6h 2.66b 0.20”h
79.6”b 2.31ab O.lgh
2.4 0.14 0.02
82.7”
14.1”
85.4”
2.8 2.5 4.9
19.5”
13.9”
21.3”
80.6
70.0”f
95.9”
87.3”
84.2“
84.2”
1.66”
‘Means not sharing a common
superscript are significantly tNumber of platelets per ~1. IDifferent from SSO value at 0.05 < P < 0.10. $DitTerent from control value at 0.05 < P < 0.10.
ammonium oxalate. Dilution of PRP with PPP was avoided because it caused platelets to aggregate spontaneously. However, the average platelet counts in PRP were not significantly different among groups (Table 1). Aggregation was measured in a Lumi-Aggregometer (Chrono-Log Corp., Havertown, Pennsylvania, U.S.A.) at 37°C with 0.45 ml of PRP and 0.05 ml of the reagent. Maximum aggregation was measured with the following final concentrations of reagents: ADP, 20 p M; arachidonate, 1 mM; collagen, 200 pg/ml. ADP, collagen and epinephrine reagents were obtained from American Dade, Miami, Florida, U.S.A. Stock solutions were prepared fresh daily. Sodium aracludonate (99% pure, Sigma Chemical Co., St Louis, Missouri, U.S.A.) was kept frozen until use. Dilutions of the stock solutions and of sodium arachidonate were made with O.SS”A NaCI. The response of platelets to epinephrine was tested 1 hr after blood collection. The thresh-
old concentration of arachidonate was defined as the min-
2.32” different,
P < 0.05,
imum concentration which caused maximal aggregation within 3 min. For the statistical analysis of aggregation parameters, Duncan’s multiple range test was used to detect significant
differences
among
groups
(Nie ef al., 1975).
RESULTS
Typical responses of cat platelets to ADP, arachidonate, collagen and epinephrine are shown in Fig. 1. The response to ADP varied among cats, but was similar to that obtained by MacMillan and Sim (1970) and Marcinkiewicz et al. (1978). Secondary aggregation occured with concentrations of ADP between 0.25 and 2 PM. Irreversible aggregation occured even when the extent of first-phase aggregation was only 5%. This confirms the marked ARACHIDONATE
ADP (/A41
30-
2.4 0.24
2.90”s
(mM)
LV
40XI60. 700Ogo-
%T
COLLAGEN
EPINEPHRINE
@g/ml)
(pM)
IO20 10
3040. 5or 60. \
70t3090L
’ 0
I
1 2
3
4
0
I
2
3
4
MINUTES Fig. I. Aggregation of cat platelets in oitro in response to ADP, collagen, arachidond’te or epinephrine. Final concentrations of reagents in platelet-rich plasma are shown next to each tracing. Reagents were added at time 0. The y0 transmittance of light is shown on the ordinate.
Arachidonate deficiency and cat platelet tendency of cat platelets to self-propagated aggregation, perhaps as a result of the release of relatively large amounts of storage compounds, including ADP (MacMillan and Sim, 1970) and 5-hydroxytryptamine (Paasonen, 1968). Aggregation also occurred in response to sodium arachidonate at concentrations between 0.15 and IOmM. However, the response to different concentrations was unlike that observed with other mammalian platelets. In cat platelets, lowering the concentration of arachidonate increased the time to maximum aggregation, but did not affect the extent of aggregation (Fig. 1). In other species, such as the rabbit, there is no lag in arachidonate-induced aggregation, and the extent of aggregation is lower with lower concentrations of arachidonate (Vargaftig and Zirinis, 1973). The response to collagen was similar to the response to arachidonate. Platelets aggregated in response to a low concentration of collagen (2 pg/ml) but there was a long lag of 5 min. MacMillan and Sim (1970) reported that cat platelets aggregated in response to 0.01&l PM epinephrine, but no data was given. Tschopp (1970) reported that epinephrine did not cause aggregation, although preincubation with epinephrine increased the extent response to of aggregation in S-hydroxytryptamine. In our studies, cat platelets aggregated in response to 10 PM epinephrine. There was no primary phase of aggregation, and the lag was long. The lag in response to epinephrine appeared to be directly proportional to the platelet count in PRP, and was only 2 min in one sample with l,OOO,OOO platelets/pi. The long lag may explain why aggregation was not observed in the study by Tschopp (1970). Also, in the study by Tschopp, blood was collected from cats anesthetized with pentobarbitone, which may depress the reactivity of platelets (McKenzie et al., 1972). A response to epinephrine also occurs with human platelets, but not with platelets from other mammalian species (Sinakos and Caen, 1967). The characteristics of aggregation of platelets from cats fed the EFAD, SSO or control diets are shown in Table 1. The platelet count in whole blood of cats fed the SSO diet was significantly lower than that of cats fed the control or EFAD diets. Two cats fed the SSO diet were thrombocytopenic, with platelet counts consistently less than 100,000. The corresponding PRP samples failed to aggregate because of low platelet counts. We studied the response to ADP of platelets from normal cats and found that at least 100,000 platelets/PI were necessary for maximal aggregation (Fig. 2). The responses of platelets to arachidonate were affected by arachidonate deficiency. The maximum aggregation in response to all concentrations of arachidonate was lower and the lag in response to 1 mM arachidonate was longer in the EFAD and SSO groups than in the control group. Thus, platelets from the arachidonate-deficient cats were unable to respond to arachidonate as well as platelets from cats fed the control diet. However, the threshold concentration of arachidonate was lower for platelets from cats fed the EFAD or SSO diets than for platelets from cats fed the control diet, so that platelets from the deficient cats responded more quickly than those
125
aggregation
from the control cats when arachidonate was limiting. These results would be consistent with an adaptation of the platelets to low concentrations of which arachidonate occur in circulating arachidonate-deficient cats. The maximum aggregation in response to ADP was lower in platelets from cats fed the EFAD diet than in platelets from cats fed the SSO or control diets (0.05 < P < 0.10). In addition, in platelets from cats fed the EFAD or SSO diets, the extent of aggregation was less and the lag was longer in response to collagen than in platelets from cats fed the control diet (0.05 < P < 0.10). There was no apparent difference among groups in the response to epinephrine (data not shown). DISCUSSION
In cats, arachidonate deficiency caused a decrease in the ability of platelets to aggregate in response to arachidonate. The responses to collagen and ADP were also slightly lower in platelets from cats fed the arachidonate-deficient diets than in those from cats fed a control diet. Platelet aggregation was not better in the SSO group than in the EFAD group. This is consistent with the negligible conversion of linoleate to arachidonate in cat liver, and supports the idea that arachidonate is specifically required for normal platelet function. Many workers have observed that the ingestion of n3-polyunsaturated fatty acids, present in marine oils, affects hemostatic parameters, including platelet aggregation, in humans and rats (Dyerberg and Jorgensen, 1982). Black et al. (1979) studied experimental cerebral infarction in cats, and observed that the extent ofinfarction and of damage was less in cats fed a supplement of menhaden oil than in unsupplemented cats. In our studies, some cats were fed diets containing 0.2% tuna oil, which provided 22:6n3 (MacDonald et al., 1983). In two cats which were studied, there were no apparent differences in any of the parameters of aggregation compared to cats fed the SSO diet (data not shown). Some authors have noted that changes in thrombotic tendency can occur without accompanying changes in platelet aggregation (Hornstra, 1975; loo-
20 IO-
100
200
PLATELET
300
COUNT
400
IN PRP
500
600
700
(XI&/J)
Fig. 2. Effect of platelet count in platelet-rich plasma on the maximum aggregation of cat platelets in response to 20 pM ADP.
126
MARN~EL. MACDONALD et al.
Dyerberg and Jorgensen, 1982). We did not examine other aspects of hemostasis in cats fed different diets, but the thrombocytopenia which occurred in two out of six cats fed the SSO diet would certainly contribute to an increased bleeding time. The thrombocytopenia appeared to be a result of arachidonate deficiency. Other investigators have observed a decreased capillary resistance in EFA-deficient rats (Kramer and Levine, 1953) which could have been a result of thrombocytopenia. Aithough arachidonate deficiency impaired the aggregation of cat platelets, the effect was relatively small. Not all agents which cause platelet aggregation were tested, and the responses of cat platelets to other agents may be affected more by arachidonate deficiency than were the responses to collagen, arachidonate and ADP. Nevertheless, cats apparently require dietary arachidonate for normal platelet aggregation. Linoleate does not meet the requjrement for arachidonate because the delta-4 desaturase, which is required for the conversion of linoleate to arachidonate, is nearly absent from cat liver (Hassam et al., 1977). Paradoxically, the low activity of this enzyme may protect the cat against some of the deleterious effects of linoleate deficiency on platelet function. Most EFA-deficient mammals synthesize an eicosatrienoic acid (5,8,1 l-20:3) from oieic acid (9-18: 1) in the liver. This triene is synthesized during EFA deficiency because of low levels of linoleate, which competes with oleate for binding to the delta-6 desaturase (Brenner, 1974). Lagarde et al. (1983) showed that 5,&l l-20:3 increases the responsiveness of human platelets to several reagents. The large increase in the level of 5,8,I l-20:3 in platelets may explain some of the effects of linoleate deficiency on platelet function in mammals other than the cat. Since the first enzyme in the synthesis of 5,8,1 l-20:3 is the delta-6 desaturase, the linoleate-deficient cat produces only a small amount of this fatty acid (MacDonald et at., 1983). If 5.8,11-20:3 is responsible for some of the abnormalities in platelet aggregation during linoleate deficiency, the cat would be less affected by linoleate deficiency than are other mammals. Further studies are needed to elucidate the roles of endogenous arachidonate and other fatty acids in the regulation of platelet aggregation in various species.
through the cyclooxygenase and lipoxygenase in platelets and vascular tissue. Lipicis 16, 165-172. Hamberg M., Svensson J. and Samuelsson B. (1975) Thromboxanes: a new group of biologically active compounds derived from prostagIandin endoperoxides. Proc. Natn. Acad. Sci. 72, 2994-2998. Hassam A. G., Rivers J. P. W. and Crawford M. A. (1977) The failure of the cat to desaturate linoleic acid: its nutritional imolications. Nutr. Metab. 21. 321-328. Hornstra G. (1975) Specific effects of types of dietary fat on arterial thrombosis. In The Role of Fats in Human Nutrition (Edited by Vergroesen A. J.), pp. 303-330. Academic Press, New York. Kramer J. and Levine V. E. (1953f Influence of fats and fattv acids on the caoillaries. J. Nutr. 50. 149-160. Lagarhe M., Burtin M.1 Sprecher H., Dechavanne M. and (1983) effect of Renaud S. Potentiating 5,8,11-eicosatrienoic acid on human platelet aggregation.
REFERENCES
Sinclair A. J., McLean J. G. and Monger E. A. (1979) Metabolism of linoleic acid in the cat. LiDi& 14.932-936. Tschopp T. B. (1970) Aggregation of cat blateleis in vitro.
Lipids 18, 291-294.
MacDonald M. L., Rogers Q. R. and Morris J. G. (1983) Role of linoleate as an essential fattv acid for the cat, independent of arachidonate synthesis. J. Nutr. 113, 1422-1433. MacMillan D. C. and Sim A. K. (1970) A comparative study of platelet aggregation in man and laboratory animals. Thromb. Dinth. haemorrh. 24, 385-394. Marcinkiewicz E., Grodzinska L. and Gryglewski R. J. (1978) Platelet aggregation and thromboxane A, formation in cat platelet-rich plasma. Pharmac. Res. Commun. 10, l--12.
McGregor L. and Renaud S. (1977) Influence of dietary linoleic acid level on coagulation, aggregation and fatty acid composition of rats. Nutr. Metab. 21 (Suppl. I), 192-196. McKenzie F. N., Svensjo E. and Arfors K.-E. (1972) EfIect of sodium ~ntobarbital anesthesia on platelet behavior in vitro and in aivo. Microvasc. Res. 4, 42-50. Nie N. H., Hull C. H., Jenkins J. G., Steinbrenner K. and Bent D. H. (1975) Statistical Package for the Social Sciences. 2nd edn. DV.422433. McGraw-Hill. New York. Paasonen, ‘M. K. (id88) Platelet S-hydroxytryptamine as a model in pharmacology. Annl Med. exp. Biol. Fenn. 46, 416-422. Rivers J. P. W., Sinclair A. J. and Crawford M. A. (1975) Inability of the cat to desaturate essential fatty acids. Nature, Land. 258, 171-173. Silver M. J., Bill T. K. and Smith J. B. (1978) Platelets and prostaglandins: the key role of platelet phospholipase A, activity. In Platelets, a Multidisciplinary Approach. (Edited by de Gaetano G. and Garattini S.), pp. 213-225. Raven Press, New York. Sinakos Z. and Caen J. P. (1967) Platelet aggregation in mammalians (human, rat, rabbit, guinea-pig, horse, dog) a comparative study. Thromb. Diath. haemorrh. 17, 99-111.
Black K. L., Culp B., Madison D., Randall 0. S. and Lands W. E. M. (19791The urotective effects of dietary fish oil on focal ‘ceret;ral infarction. Pros&gland. Med. 3, 257-268. Brenner R. R. (1974) The oxidative desaturation of unsaturated fatty acids in animals. Molec. cell. Biochem. 3, 4 l-52.
Dyerbern J. and Jorgensen K. A. (1982) Marine oils and ~throm~ogenesis. F&g. Lipid Res. 21, 255-269. Galli C., Agradi E., Petroni A. and Tremoii E. (1981) Differential effects of dietary fatty acids on the accumulation of arachidonic acid and its metabohc converslon
Thr~~b. Qiath. ha~m~~h.-~,
60 l-620. _
Vareaftirr B. 8. and Zirinis P. (1973) Platelet aggregation in&& by arachidonic acid is‘accompanied b;rel&e of potential inflammatory mediators distinct from PGE, and PGF,. Nature, New Biol. 244, 114-116. Vincent J. E. and Zijlstra F. J. (1978) Formation by phospholipase A, of prostaglandins and endoperoxides in platelets of normal and essential fatty acid deficient rats. In Adances in Prost~glandin and Thromboxane Research (Edited by Galli C.), Vol. 3, pp. 143-146. Raven Press, New York.
0306.4492/84 $3.00 + 0.00 1984 Pergamon Press Ltd
1984 c
EFFECTS OF DIETARY ARACHIDONATE DEFICIENCY ON THE AGGREGATION OF CAT PLATELETS Department
MARNIE L. MACDONALD, QUINTON R. ROGERS and JAMES G. MORRIS* of Physiological Sciences, School of Veterinary Medicine, and *Department of Animal Sciences, University of California, Davis, CA 95616, U.S.A. (Received 8
September
1983)
Abstract-l. Because the cat lacks the ability to synthesize a significant amount of arachidonate from linoleate in the liver, arachidonate is a dietary essential. We studied the effects of essential fatty acid deficiency on the aggregation of cat platelets. 2. Cat platelets aggregated in response to ADP, collagen, arachidonate and epinephrine. 3. Aggregation was impaired in platelets from cats fed arachidonate-deficient diets. 4. Including a source of hnoleate in the arachidonate-deficient 5. Arachidonate is required in the diet of the cat for normal
INTRODUCTION In some species, including the human and the rat, the type of dietary fat affects the tendency to thrombosis (Hornstra, 1975). The effect of dietary fat on thrombosis may be a result of differences in platelet aggregation as a result of changes in the fatty acid composition of platelet phospholipids. Arachidonate (5,8,11,1420:4), present in platelet phospholipids, is released when platelets are stimulated to aggregate (Silver et al., 1978). Free arachidonate is then converted to eicosanoids, including thromboxane A,, which is a potent aggregatory substance (Hamberg et al., 1975). Platelets from essential fatty acid (EFA)-deficient rats synthesize very little thromboxane A, (Vincent and Zijlstra, 1978), presumably because levels of arachidonate in platelet phospholipids are low. McGregor and Renaud (1977) observed that platelets from EFA-deficient rats were less responsive to ADP and more responsive to arachidonate than were platelets from rats fed a diet adequate in linoleate. Hornstra (1975) reported that EFA deficiency decreased the response of rat platelets to collagen, but not to ADP. Galli et al. (1981) found that low levels of arachidonate in phospholipids of rabbit platelets were associated with a higher threshold for the aggregation of platelets in response to arachidonate, and a decrease in the synthesis of thromboxane B, from exogenous arachidonate. The domestic cat (Fe& catus) is different from non-carnivorous mammals because it lacks the ability to synthesize a significant amount of arachidonate from linoleate in the liver (Hassam et al., 1977). As a result, arachidonate is a dietary essential for the cat (Rivers et al., 1975). In cats fed diets lacking arachidonate, the level of arachidonate is only 1% in plasma lipids and 6”/, in erythrocyte lipids, whether linoleate is provided in the diet or not (MacDonald et al., 1983). In cats fed commercial canned cat foods, which contain arachidonate, the level of arachidonate in plasma and erythrocyte lipids is 15525% of the total fatty acids by weight (Rivers et al., 1975; Sinclair et al., 1979).
diet did not improve platelet aggregation.
aggregation.
Marcinkiewicz et al. (1978) showed that the aggregation of cat platelets in response to collagen, ADP and arachidonate was blocked by 2-isopropyl-3nicotinylindole, an inhibitor of thromboxane synthesis. This suggests that thromboxane synthesis is an important step in the aggregation of cat platelets, as it is in platelets from other mammals. Because of the importance of arachidonate in phospholipids as a source of free arachidonate for thromboxane synthesis during aggregation, we proposed that platelets from cats fed arachidonate-deficient diets would be unable to aggregate normally. The results of this study showed that cat platelets aggregate in response to ADP, collagen, arachidonate and epinephrine, and that aggregation of platelets from cats fed diets deficient in arachidonate is impaired.
METHODS Specific pathogen-free cats were fed either a mixture of proprietary cat foods (control diet) or one of two purified diets containing either 35% (by weight) hydrogenated beef tallow (EFA-deficient diet) or 5% safflower seed oil plus 30% hydrogenated beef tallow (SSO diet). Both the EFAD and SSO diets lacked arachidonate. The level of linoleate was 6.77; of dietary energy in the SSO diet and 0.3% of dietary energy in the EFAD diet. The remainder of the EFAD and SSO diets consisted of casein, carbohydrates, vitamins and minerals, as described previously (MacDonald et al., 1983). Groups of 4-6 cats were fed each diet for approximately 2 years, beginning at 3 months of age. Blood (9 ml) was collected from the jugular vein of unanesthetized cats. Blood samples were drawn into 12ml plastic syringes containing 1 ml of 1.9% sodium citrate. (A higher concentration of sodium citrate (3.8%) completely inhibited platelet aggregation.) Samples were immediately centrifuged at room temperature at 18Og for 10min. The platelet-rich plasma (PRP) was collected and the remaining sample was centrifuged at 15OOg for 15 min to obtain platelet-poor plasma (PPP). Despite careful handling, aggregation occurred spontaneously in 23% of the samples from cats fed the control diet, 33% of the samples from cats fed the EFAD diet, and 16% of the samples from cats fed the SSO diet. Platelets in whole blood and PRP were counted by phase-contrast microscopy after a I: 100 dilution in I”/, 123
124
MARNIE L. MACDONALD Table
I. Ekts
of diet on the aggregation
et ul.
of cat platelets in response to arachiodonate,
ADP and collagen*
Diet
Platelet Platelet
count count
Response Maximum Lag (min) Threshold
in whole bloodt in PRPt
Control
EFAD
SSO
Pooled SEM
353,500” 482,800
472.195” 603,000”
182,140b 436,000”
29,854 45,770
to arachldonate aggregation (p,) with ImM arachidonate concentration (mM)
Response to ADP Maximum aggregation Percentage aggregation First phase Second phase
(“/,) to 0.4 PM ADP:
Response to collagen Maximum aggregation (%) Lag (min) at 200pg/ml collagen
87.9” I .87” 0.28”
75.6h 2.66b 0.20”h
79.6”b 2.31ab O.lgh
2.4 0.14 0.02
82.7”
14.1”
85.4”
2.8 2.5 4.9
19.5”
13.9”
21.3”
80.6
70.0”f
95.9”
87.3”
84.2“
84.2”
1.66”
‘Means not sharing a common
superscript are significantly tNumber of platelets per ~1. IDifferent from SSO value at 0.05 < P < 0.10. $DitTerent from control value at 0.05 < P < 0.10.
ammonium oxalate. Dilution of PRP with PPP was avoided because it caused platelets to aggregate spontaneously. However, the average platelet counts in PRP were not significantly different among groups (Table 1). Aggregation was measured in a Lumi-Aggregometer (Chrono-Log Corp., Havertown, Pennsylvania, U.S.A.) at 37°C with 0.45 ml of PRP and 0.05 ml of the reagent. Maximum aggregation was measured with the following final concentrations of reagents: ADP, 20 p M; arachidonate, 1 mM; collagen, 200 pg/ml. ADP, collagen and epinephrine reagents were obtained from American Dade, Miami, Florida, U.S.A. Stock solutions were prepared fresh daily. Sodium aracludonate (99% pure, Sigma Chemical Co., St Louis, Missouri, U.S.A.) was kept frozen until use. Dilutions of the stock solutions and of sodium arachidonate were made with O.SS”A NaCI. The response of platelets to epinephrine was tested 1 hr after blood collection. The thresh-
old concentration of arachidonate was defined as the min-
2.32” different,
P < 0.05,
imum concentration which caused maximal aggregation within 3 min. For the statistical analysis of aggregation parameters, Duncan’s multiple range test was used to detect significant
differences
among
groups
(Nie ef al., 1975).
RESULTS
Typical responses of cat platelets to ADP, arachidonate, collagen and epinephrine are shown in Fig. 1. The response to ADP varied among cats, but was similar to that obtained by MacMillan and Sim (1970) and Marcinkiewicz et al. (1978). Secondary aggregation occured with concentrations of ADP between 0.25 and 2 PM. Irreversible aggregation occured even when the extent of first-phase aggregation was only 5%. This confirms the marked ARACHIDONATE
ADP (/A41
30-
2.4 0.24
2.90”s
(mM)
LV
40XI60. 700Ogo-
%T
COLLAGEN
EPINEPHRINE
@g/ml)
(pM)
IO20 10
3040. 5or 60. \
70t3090L
’ 0
I
1 2
3
4
0
I
2
3
4
MINUTES Fig. I. Aggregation of cat platelets in oitro in response to ADP, collagen, arachidond’te or epinephrine. Final concentrations of reagents in platelet-rich plasma are shown next to each tracing. Reagents were added at time 0. The y0 transmittance of light is shown on the ordinate.
Arachidonate deficiency and cat platelet tendency of cat platelets to self-propagated aggregation, perhaps as a result of the release of relatively large amounts of storage compounds, including ADP (MacMillan and Sim, 1970) and 5-hydroxytryptamine (Paasonen, 1968). Aggregation also occurred in response to sodium arachidonate at concentrations between 0.15 and IOmM. However, the response to different concentrations was unlike that observed with other mammalian platelets. In cat platelets, lowering the concentration of arachidonate increased the time to maximum aggregation, but did not affect the extent of aggregation (Fig. 1). In other species, such as the rabbit, there is no lag in arachidonate-induced aggregation, and the extent of aggregation is lower with lower concentrations of arachidonate (Vargaftig and Zirinis, 1973). The response to collagen was similar to the response to arachidonate. Platelets aggregated in response to a low concentration of collagen (2 pg/ml) but there was a long lag of 5 min. MacMillan and Sim (1970) reported that cat platelets aggregated in response to 0.01&l PM epinephrine, but no data was given. Tschopp (1970) reported that epinephrine did not cause aggregation, although preincubation with epinephrine increased the extent response to of aggregation in S-hydroxytryptamine. In our studies, cat platelets aggregated in response to 10 PM epinephrine. There was no primary phase of aggregation, and the lag was long. The lag in response to epinephrine appeared to be directly proportional to the platelet count in PRP, and was only 2 min in one sample with l,OOO,OOO platelets/pi. The long lag may explain why aggregation was not observed in the study by Tschopp (1970). Also, in the study by Tschopp, blood was collected from cats anesthetized with pentobarbitone, which may depress the reactivity of platelets (McKenzie et al., 1972). A response to epinephrine also occurs with human platelets, but not with platelets from other mammalian species (Sinakos and Caen, 1967). The characteristics of aggregation of platelets from cats fed the EFAD, SSO or control diets are shown in Table 1. The platelet count in whole blood of cats fed the SSO diet was significantly lower than that of cats fed the control or EFAD diets. Two cats fed the SSO diet were thrombocytopenic, with platelet counts consistently less than 100,000. The corresponding PRP samples failed to aggregate because of low platelet counts. We studied the response to ADP of platelets from normal cats and found that at least 100,000 platelets/PI were necessary for maximal aggregation (Fig. 2). The responses of platelets to arachidonate were affected by arachidonate deficiency. The maximum aggregation in response to all concentrations of arachidonate was lower and the lag in response to 1 mM arachidonate was longer in the EFAD and SSO groups than in the control group. Thus, platelets from the arachidonate-deficient cats were unable to respond to arachidonate as well as platelets from cats fed the control diet. However, the threshold concentration of arachidonate was lower for platelets from cats fed the EFAD or SSO diets than for platelets from cats fed the control diet, so that platelets from the deficient cats responded more quickly than those
125
aggregation
from the control cats when arachidonate was limiting. These results would be consistent with an adaptation of the platelets to low concentrations of which arachidonate occur in circulating arachidonate-deficient cats. The maximum aggregation in response to ADP was lower in platelets from cats fed the EFAD diet than in platelets from cats fed the SSO or control diets (0.05 < P < 0.10). In addition, in platelets from cats fed the EFAD or SSO diets, the extent of aggregation was less and the lag was longer in response to collagen than in platelets from cats fed the control diet (0.05 < P < 0.10). There was no apparent difference among groups in the response to epinephrine (data not shown). DISCUSSION
In cats, arachidonate deficiency caused a decrease in the ability of platelets to aggregate in response to arachidonate. The responses to collagen and ADP were also slightly lower in platelets from cats fed the arachidonate-deficient diets than in those from cats fed a control diet. Platelet aggregation was not better in the SSO group than in the EFAD group. This is consistent with the negligible conversion of linoleate to arachidonate in cat liver, and supports the idea that arachidonate is specifically required for normal platelet function. Many workers have observed that the ingestion of n3-polyunsaturated fatty acids, present in marine oils, affects hemostatic parameters, including platelet aggregation, in humans and rats (Dyerberg and Jorgensen, 1982). Black et al. (1979) studied experimental cerebral infarction in cats, and observed that the extent ofinfarction and of damage was less in cats fed a supplement of menhaden oil than in unsupplemented cats. In our studies, some cats were fed diets containing 0.2% tuna oil, which provided 22:6n3 (MacDonald et al., 1983). In two cats which were studied, there were no apparent differences in any of the parameters of aggregation compared to cats fed the SSO diet (data not shown). Some authors have noted that changes in thrombotic tendency can occur without accompanying changes in platelet aggregation (Hornstra, 1975; loo-
20 IO-
100
200
PLATELET
300
COUNT
400
IN PRP
500
600
700
(XI&/J)
Fig. 2. Effect of platelet count in platelet-rich plasma on the maximum aggregation of cat platelets in response to 20 pM ADP.
126
MARN~EL. MACDONALD et al.
Dyerberg and Jorgensen, 1982). We did not examine other aspects of hemostasis in cats fed different diets, but the thrombocytopenia which occurred in two out of six cats fed the SSO diet would certainly contribute to an increased bleeding time. The thrombocytopenia appeared to be a result of arachidonate deficiency. Other investigators have observed a decreased capillary resistance in EFA-deficient rats (Kramer and Levine, 1953) which could have been a result of thrombocytopenia. Aithough arachidonate deficiency impaired the aggregation of cat platelets, the effect was relatively small. Not all agents which cause platelet aggregation were tested, and the responses of cat platelets to other agents may be affected more by arachidonate deficiency than were the responses to collagen, arachidonate and ADP. Nevertheless, cats apparently require dietary arachidonate for normal platelet aggregation. Linoleate does not meet the requjrement for arachidonate because the delta-4 desaturase, which is required for the conversion of linoleate to arachidonate, is nearly absent from cat liver (Hassam et al., 1977). Paradoxically, the low activity of this enzyme may protect the cat against some of the deleterious effects of linoleate deficiency on platelet function. Most EFA-deficient mammals synthesize an eicosatrienoic acid (5,8,1 l-20:3) from oieic acid (9-18: 1) in the liver. This triene is synthesized during EFA deficiency because of low levels of linoleate, which competes with oleate for binding to the delta-6 desaturase (Brenner, 1974). Lagarde et al. (1983) showed that 5,&l l-20:3 increases the responsiveness of human platelets to several reagents. The large increase in the level of 5,8,I l-20:3 in platelets may explain some of the effects of linoleate deficiency on platelet function in mammals other than the cat. Since the first enzyme in the synthesis of 5,8,1 l-20:3 is the delta-6 desaturase, the linoleate-deficient cat produces only a small amount of this fatty acid (MacDonald et at., 1983). If 5.8,11-20:3 is responsible for some of the abnormalities in platelet aggregation during linoleate deficiency, the cat would be less affected by linoleate deficiency than are other mammals. Further studies are needed to elucidate the roles of endogenous arachidonate and other fatty acids in the regulation of platelet aggregation in various species.
through the cyclooxygenase and lipoxygenase in platelets and vascular tissue. Lipicis 16, 165-172. Hamberg M., Svensson J. and Samuelsson B. (1975) Thromboxanes: a new group of biologically active compounds derived from prostagIandin endoperoxides. Proc. Natn. Acad. Sci. 72, 2994-2998. Hassam A. G., Rivers J. P. W. and Crawford M. A. (1977) The failure of the cat to desaturate linoleic acid: its nutritional imolications. Nutr. Metab. 21. 321-328. Hornstra G. (1975) Specific effects of types of dietary fat on arterial thrombosis. In The Role of Fats in Human Nutrition (Edited by Vergroesen A. J.), pp. 303-330. Academic Press, New York. Kramer J. and Levine V. E. (1953f Influence of fats and fattv acids on the caoillaries. J. Nutr. 50. 149-160. Lagarhe M., Burtin M.1 Sprecher H., Dechavanne M. and (1983) effect of Renaud S. Potentiating 5,8,11-eicosatrienoic acid on human platelet aggregation.
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Thr~~b. Qiath. ha~m~~h.-~,
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Vareaftirr B. 8. and Zirinis P. (1973) Platelet aggregation in&& by arachidonic acid is‘accompanied b;rel&e of potential inflammatory mediators distinct from PGE, and PGF,. Nature, New Biol. 244, 114-116. Vincent J. E. and Zijlstra F. J. (1978) Formation by phospholipase A, of prostaglandins and endoperoxides in platelets of normal and essential fatty acid deficient rats. In Adances in Prost~glandin and Thromboxane Research (Edited by Galli C.), Vol. 3, pp. 143-146. Raven Press, New York.