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Inhibitory effect of alcohol on platelet functions of rats fed saturated fats
THROMBOSIS RESEARCH 22; 221-225, 1981 0049-3848/81/010221-05$02.00/O Printed in the USA. Copyright (c) 1981 Pergamon Press Ltd. All rights reserved.
BRIEF COMMUNICATION INHIBITORY EFFECT OF ALCOHOL ON PLATELET FUNCTIONS OF RATS FED SATURATED FATS
Lilian McGregor and Serge Renaud INSERM, Unit 63, 22 avenue du Doyen LIpine 69500 Bron, France (Received 14.2.1981; in revised form 28.3.1981. Accepted by Editor H. Stormorken)
INTRODUCTION The consumption of alcohol in man appears to be inversely related to the incidence of.coronary heart disease (CHD) (1). It seems that this beneficial effect of alcohol is not related to a lowering of blood lipids since alcohol is known to be hypertriglyceridemic (2). Studies in French farmers (3), confirmed in more extensive investigations in French and British farmers (4) have shown that platelet clotting activity and platelet aggregation induced by thrombin, but mostly by ADP, epinephrine and collagen, were inversely related to the alcohol intake. Platelets appear to be closely associated with CW being involved not only in thrombosis but also in early atherosclerotic lesions (5). In our studies in farmers, we have shown that platelet functions were the only blood parameter significantly correlated with the intake of sa' turated fat (4), the environmental factor the most closely assotiiatedwith CHD (6). In the present study in rats fed saturated or polyunsaturated fats, we have observed that alcohol, both in vivo and in vitro, inhibits certain platelet functions, chiefly in animals fed a saturated diet. MATERIAL AND METHODS In vivo study : a total of 39 male Sprague Dawley rats (Charles River) were fed for 24 weeks a high fat (40 X) purified diet similar to that used previously (7), rich in either saturated fats (20 X'hydrogenated palm oil, 18 X hydrogenated coconut oil with 2 2 corn oil to prevent essential fatty acid deficiency) or polyunsaturated fats (22 X corn oil) with 18 X hydrogenated coconut oil. The other dietary ingredients were the following (weight X) : casein 24, cellulose 10, glucose 20, vitamin diet fortification mixture (ICN Nutritional Biochemicals) 2, mineral mixture (Wesson) 4. In addition, half of these rats had 6 X alcohol in their drinking water for 2 months. Blood was
Key words : Alcohol, platelet functions, saturated fats. 221
222
ALCOHOL AND PLATELETFUNCTION
vo1.22, No.l/2
collected in fasted animals. Determinations of the recalcification plasma clotting time of platelet-rich plasma (PCT), platelet aggregation and plasma lipids were done as already reported (7): In vitro studies : a. - Addition to PRP. Alcohol diluted in Tyrode (pH = 7.4) was added with stirring, two minutes before performing aggregation-on platelet-rich plasma (PRP) (500,000 platelets/ul) of rats fed either laboratory chow or the same diets as in the in vivo studies. b. - Addition to blood. Alcohol diluted (0, 4, 8 or 16 X) in incomplete Tyrode was added (0.1 ml/ml) to a pool of titrated blood (15 ml/group), taken from 8 laboratory chow fed rats and stored for 15 minutes at room temperature. Then, PRP was prepared as usual (7), and the platelet count adjusted to 400,000/~1 with PPP. During the whole process, red cells were in contact with alcohol for approximately one hour before being removed from plasma. At the time of aggregation (on PRP), platelets had been in contact with alcohol for at least 2 hours. RESULTS AND DISCUSSION In vivo studies. As shown in figure 1, the PCT of saturated fat-fed rats given alcohol in their drinking water was considerably prolonged (improved) as compared to the controls. This result was not observed with the polyunsaturated fat diet. Similary, the response to thrasbin aggregation was significantly decreased (improved), only in the saturated fat fed rats. Of special interest, is that in these two tests, 6 % alcohol in drinking water completely blocked the acceleration of the PCT or the higher response (thrombin) induced by the saturated fat feeding. Concerning ADP induced aggregation, it was also significantly inhibited by alcohol drinking, but only in saturated fat-fed rats. In animals fed the saturated fat diet, plasma triglycerides were significantly increased and cholesterol was significantly decreased by alcohol drinking.
em
I
m
6% Alcohol
FIG. 1 100
Influence on various blood parameters of 50 alcohol in drinking water (6 X), for ~ ns P < 0.001 two months, in rats TMROMSlN ADP TRIGLYCERlD5S CHOLESTIWOL PC1 fed for 24 weeks COAGULATION AGGREGATION LIP-IA purified diets rich in either saturated (S) with only 2 X corn oil or polyunsaturated (P) (22 % corn oil) fats. PCT - recalcification plasma clotting time of platelet rich plasma (PRP) ; Thrombin (2.4 Units/ml PRP) ADP (3.3 pM, final concentration)
vo1.22,
No. l/2
223
ALCOHOL AND PLATELET FUNCTION
In vitro studies. As shown in figure 2, alcohol, at dosages frequently found in drinkers (2 to 4 g/l), significantly inhibited, after 2 minutes of incubation, the response to thrombin of platelets from saturated fats or laboratory chow-fed animals. By contrast, there was no significant decrease in the thrombin-induced aggregation of polyunsaturated fat-fed animals. c
I
T
FIG. 2
!O 0 mg/ml Alcohol)= 2 mg/ml !EZI 4 mg/ml
Mean 2 S.E (4 to 5 series of tests)
1
10.
T
In vitro, addition of alcohol to PRP. on thrombin and ADP (final concentration in the figure) indu5' ted platelet aggregation of rats fed either laboratory chow, saturated (S) o or polyunsaturated P(o.01 0.05 NS ADP (3.3 PM) (P) fat diets as in THROMBIN (2.4U/ml PRP) PLATELET AGGREGATION studies of fig. 1. Each test was performed in duplicate or triplicate on pooled PRP of a least 3 rats. To perform the test, Tyrode (0.1 ml) or Tyrode containing alcohol, was first added to 0.5 ml of PRP with constant stirring, and then, tow minutes later, 0.1 ml of the aggregating agent (also diluted in Tyrode) was added. I
I
I
I
I
hT T
FIG. 3 In vitro, addition of alcohol to titrated blood. Effect on thrombin and ADP induced aggregation of rats fed laboratory chow. Each test ~88 performed in duplicate on PRP containing 8 fin81 concentration of alcohol shown in the figure.
I
MO@tl+-S.E. (4to6seriesoftests)
10
AkOhOl
0 m
0 mg/ml 4 mg/ml
m I
8 mg/ml 16 mg/ml
5
C lHR(
BIN (1.6 U/ml) ADP (3.3 fl) PLATELET AGGREGATfON
No effect could be obtained by the in vitro addition of alcohol at 4 mg/ml either to PRP (fig. 2) or to blood, (fig. 3) on ADP aggregation. It was only 16 mg/ml of alcohol in blood that slightly inhibited ADP aggregation. We do not-have any explanation for this discrepancy between in vitro and in vivo results. It is once more an indication of the differences in the me&& Gs of action between thrombin and ADP induced aggregation (8).
224
ALCOHOLAND PLATELETFUNCTION
Vol.22,
No.1/2
Alcohol, as shown by the in vitro study, seems to have a direct inhibitory effect on thrombin aggregation. This effect persists solely during the time alcohol is present in blood as suggested by preliminary studies in rats drinking 6 X alcohol. Increased microaggregate formation has been found after mixing ethanol with blood from pig and rabbit in vitro, without anticoagulant (9). Thus, it was suggested that alcohol stimulates platelet aggregation, partly through an effect on red cells. In the present study in rat, there was a marked inhibitory effect of alcohol on thrombin induced aggregation either when added to PEP or to titrated blood. The discrepancy between our results and those mentioned above (9) is probably due to the difference in the techniques used. At any rate, it seems that in vivo studies might be more relevant to the human situation than the in vitro studies. In the present investigation, rats fed saturated fats and drinking alcohol for 2 months , presented a lower response to thrombin and ADP induced aggregation. In man, our epidemiological data suggested also an inhibitory effect of alcohol on ADP, epinephrine and collagen induced aggregation (3,4). It has been reported in man (IO) that the in vitro addition of ethanol to normal platelets (from subjects probably eating saturated fats) induced a lower platelet factor 3 (PF3) availability and a slight but significant retardation in the rate of thrombin induced nucleotide release. However the decrease in ADP induced primary aggregation observed in alcoholic patients, could not be reproduced by the in vitro addition of alcohol to normal platelets (10). Similary, in our present study in rat, the inhibitory effect of alcohol in drinking water on ADP induced aggregation could not be reproduced by the in vitro addition of alcohol to PEP. Consequently, it seems that alcohol, both in man and in rat, does not inhibit instantly ADP aggregation as it does with thrombin. On the other hand, the effect of alcohol on ADP, epinephrine and collagen induced aggregation persista 12 hours or more after alcohol drinking, as this was observed in fasted subjects (3,4).
e
It has been suggested that the beneficial effect of alcohol on CED in man was mediated through and increse in WL-cholesterol (11). Since, in recent that platelet functions are also years, results have accumulated indicating closely related to CED, it seems feasible to postulate that the protective effect of alcohol on CED is partly due to its inhibitory effects on platelet functions. In the present study in rat, alcohol was as efficient as polyunsaturated fat to inhibit platelet functions. Consistent with this hypothesis is our recent study showing that 6 X alcohol, in drinking water, lowers atherosclerosis and platelet hyperaggregability in rabbits fed a saturated fat diet (4). ACENOWLEDGMENTS We thank C. Benoit, E. Dumont and C. Covacho for their skilfull technical assistance. This work, supported by an INSEEMGrant (ATP 76-63), has been presented in part at the meeting of the International Society on Thrombosis and Haemostasis, London, July 1979.
vo1.22, No.112
ALCOHOL AND PLATELET FUNCTION
225
REFERENCES 1. HENNEKENS, C.H., WILLETT, W., ROSNER, B., COLE, D. and MAYRENT, S. Effects of beer, wine and liquor in coronary deaths. J. Amer. Med. Ass., 242, 1973-1974, 1979. M.C., KAGAN, A., DOYLE, J.T., HAMES, C.G., HULLEY, S.B. and ZUKEL, W.J. Alcohol and blood lipids. The cooperative lipoprotein phenotyping study. Lancet ii, 153-155, 1977.
2. CASTELLI, W.P., GORDON, T., HJORTLAND,
3. RENAUD, S., DUMONT, E., GODSEY, F., SUPPLISSON, A. and THEVENON, C. Platelet functions in relation to dietary fats in farmers from two regions of France. Thranb. Raemoet.,a, 518-531, 1978.
4. RENAUD, S., MORAEAIN, R., MCGREGOR, L. and BADDIER, F. Dietary fats on platelet functions in relation to atherosclerosis and coronary heart disease. Haemostasie,_S,234-251, 1979.
5. HARKER, L.A., ROSS, R. and GLOMSET, J. Role of platelets in thrombogenesis. Ann. N.Y. Acad. Ski.,=, 321-329, 1976. 6. KEYS, A. Coronary heart disease in seven countries. Circuktion, Suppl. 2, 41, 1-211, 1970.
7. MCGREGOR, L., MORAZAIN, R. and RENAUD, S. A comparison of the effects of dietary short and long chain saturated fatty acids on platelet functions, Inveeplatelet phospholipids, and blood coagulation in rats. Labotiatory tigation,&, 438-442, 1980.
8. PACKHAM, M.A., KINLOUGH-RATHBONE, R.L., REIMERS, H.J., SCOTT, S. and MUSTARD, J.F. Mechanisms of platelet aggregation independent of adenosine diphosphate. In: Proetaghdins in Hematology.M.J. Silver, J.B. Smith and J.J. Kocsis (Eds) Holliswood, NY: Spectrum Publication Inc., 1977, pp. 247-276. 9.
ELMER, O., GORANSSON, G., SAKTJ,M. and BENGMARK, S. Ethanol-induced microaggregate formation in pig and rabbit blood. An in vitro study. Tkromb. i%WnO6t., z, 344-350, 1977.
10. HAIJT,J.M. and COWAN, D.H. The ffect of ethanol on hemostatic properties of human blood platelets. Amer. J. Med., s, 22-33, 1974. 11. RICCI, G. and ANGELICO, F. Alcohol consumption and coronary heart disease. Lancet , 1404.1407, 1979.
BRIEF COMMUNICATION INHIBITORY EFFECT OF ALCOHOL ON PLATELET FUNCTIONS OF RATS FED SATURATED FATS
Lilian McGregor and Serge Renaud INSERM, Unit 63, 22 avenue du Doyen LIpine 69500 Bron, France (Received 14.2.1981; in revised form 28.3.1981. Accepted by Editor H. Stormorken)
INTRODUCTION The consumption of alcohol in man appears to be inversely related to the incidence of.coronary heart disease (CHD) (1). It seems that this beneficial effect of alcohol is not related to a lowering of blood lipids since alcohol is known to be hypertriglyceridemic (2). Studies in French farmers (3), confirmed in more extensive investigations in French and British farmers (4) have shown that platelet clotting activity and platelet aggregation induced by thrombin, but mostly by ADP, epinephrine and collagen, were inversely related to the alcohol intake. Platelets appear to be closely associated with CW being involved not only in thrombosis but also in early atherosclerotic lesions (5). In our studies in farmers, we have shown that platelet functions were the only blood parameter significantly correlated with the intake of sa' turated fat (4), the environmental factor the most closely assotiiatedwith CHD (6). In the present study in rats fed saturated or polyunsaturated fats, we have observed that alcohol, both in vivo and in vitro, inhibits certain platelet functions, chiefly in animals fed a saturated diet. MATERIAL AND METHODS In vivo study : a total of 39 male Sprague Dawley rats (Charles River) were fed for 24 weeks a high fat (40 X) purified diet similar to that used previously (7), rich in either saturated fats (20 X'hydrogenated palm oil, 18 X hydrogenated coconut oil with 2 2 corn oil to prevent essential fatty acid deficiency) or polyunsaturated fats (22 X corn oil) with 18 X hydrogenated coconut oil. The other dietary ingredients were the following (weight X) : casein 24, cellulose 10, glucose 20, vitamin diet fortification mixture (ICN Nutritional Biochemicals) 2, mineral mixture (Wesson) 4. In addition, half of these rats had 6 X alcohol in their drinking water for 2 months. Blood was
Key words : Alcohol, platelet functions, saturated fats. 221
222
ALCOHOL AND PLATELETFUNCTION
vo1.22, No.l/2
collected in fasted animals. Determinations of the recalcification plasma clotting time of platelet-rich plasma (PCT), platelet aggregation and plasma lipids were done as already reported (7): In vitro studies : a. - Addition to PRP. Alcohol diluted in Tyrode (pH = 7.4) was added with stirring, two minutes before performing aggregation-on platelet-rich plasma (PRP) (500,000 platelets/ul) of rats fed either laboratory chow or the same diets as in the in vivo studies. b. - Addition to blood. Alcohol diluted (0, 4, 8 or 16 X) in incomplete Tyrode was added (0.1 ml/ml) to a pool of titrated blood (15 ml/group), taken from 8 laboratory chow fed rats and stored for 15 minutes at room temperature. Then, PRP was prepared as usual (7), and the platelet count adjusted to 400,000/~1 with PPP. During the whole process, red cells were in contact with alcohol for approximately one hour before being removed from plasma. At the time of aggregation (on PRP), platelets had been in contact with alcohol for at least 2 hours. RESULTS AND DISCUSSION In vivo studies. As shown in figure 1, the PCT of saturated fat-fed rats given alcohol in their drinking water was considerably prolonged (improved) as compared to the controls. This result was not observed with the polyunsaturated fat diet. Similary, the response to thrasbin aggregation was significantly decreased (improved), only in the saturated fat fed rats. Of special interest, is that in these two tests, 6 % alcohol in drinking water completely blocked the acceleration of the PCT or the higher response (thrombin) induced by the saturated fat feeding. Concerning ADP induced aggregation, it was also significantly inhibited by alcohol drinking, but only in saturated fat-fed rats. In animals fed the saturated fat diet, plasma triglycerides were significantly increased and cholesterol was significantly decreased by alcohol drinking.
em
I
m
6% Alcohol
FIG. 1 100
Influence on various blood parameters of 50 alcohol in drinking water (6 X), for ~ ns P < 0.001 two months, in rats TMROMSlN ADP TRIGLYCERlD5S CHOLESTIWOL PC1 fed for 24 weeks COAGULATION AGGREGATION LIP-IA purified diets rich in either saturated (S) with only 2 X corn oil or polyunsaturated (P) (22 % corn oil) fats. PCT - recalcification plasma clotting time of platelet rich plasma (PRP) ; Thrombin (2.4 Units/ml PRP) ADP (3.3 pM, final concentration)
vo1.22,
No. l/2
223
ALCOHOL AND PLATELET FUNCTION
In vitro studies. As shown in figure 2, alcohol, at dosages frequently found in drinkers (2 to 4 g/l), significantly inhibited, after 2 minutes of incubation, the response to thrombin of platelets from saturated fats or laboratory chow-fed animals. By contrast, there was no significant decrease in the thrombin-induced aggregation of polyunsaturated fat-fed animals. c
I
T
FIG. 2
!O 0 mg/ml Alcohol)= 2 mg/ml !EZI 4 mg/ml
Mean 2 S.E (4 to 5 series of tests)
1
10.
T
In vitro, addition of alcohol to PRP. on thrombin and ADP (final concentration in the figure) indu5' ted platelet aggregation of rats fed either laboratory chow, saturated (S) o or polyunsaturated P(o.01 0.05 NS ADP (3.3 PM) (P) fat diets as in THROMBIN (2.4U/ml PRP) PLATELET AGGREGATION studies of fig. 1. Each test was performed in duplicate or triplicate on pooled PRP of a least 3 rats. To perform the test, Tyrode (0.1 ml) or Tyrode containing alcohol, was first added to 0.5 ml of PRP with constant stirring, and then, tow minutes later, 0.1 ml of the aggregating agent (also diluted in Tyrode) was added. I
I
I
I
I
hT T
FIG. 3 In vitro, addition of alcohol to titrated blood. Effect on thrombin and ADP induced aggregation of rats fed laboratory chow. Each test ~88 performed in duplicate on PRP containing 8 fin81 concentration of alcohol shown in the figure.
I
MO@tl+-S.E. (4to6seriesoftests)
10
AkOhOl
0 m
0 mg/ml 4 mg/ml
m I
8 mg/ml 16 mg/ml
5
C lHR(
BIN (1.6 U/ml) ADP (3.3 fl) PLATELET AGGREGATfON
No effect could be obtained by the in vitro addition of alcohol at 4 mg/ml either to PRP (fig. 2) or to blood, (fig. 3) on ADP aggregation. It was only 16 mg/ml of alcohol in blood that slightly inhibited ADP aggregation. We do not-have any explanation for this discrepancy between in vitro and in vivo results. It is once more an indication of the differences in the me&& Gs of action between thrombin and ADP induced aggregation (8).
224
ALCOHOLAND PLATELETFUNCTION
Vol.22,
No.1/2
Alcohol, as shown by the in vitro study, seems to have a direct inhibitory effect on thrombin aggregation. This effect persists solely during the time alcohol is present in blood as suggested by preliminary studies in rats drinking 6 X alcohol. Increased microaggregate formation has been found after mixing ethanol with blood from pig and rabbit in vitro, without anticoagulant (9). Thus, it was suggested that alcohol stimulates platelet aggregation, partly through an effect on red cells. In the present study in rat, there was a marked inhibitory effect of alcohol on thrombin induced aggregation either when added to PEP or to titrated blood. The discrepancy between our results and those mentioned above (9) is probably due to the difference in the techniques used. At any rate, it seems that in vivo studies might be more relevant to the human situation than the in vitro studies. In the present investigation, rats fed saturated fats and drinking alcohol for 2 months , presented a lower response to thrombin and ADP induced aggregation. In man, our epidemiological data suggested also an inhibitory effect of alcohol on ADP, epinephrine and collagen induced aggregation (3,4). It has been reported in man (IO) that the in vitro addition of ethanol to normal platelets (from subjects probably eating saturated fats) induced a lower platelet factor 3 (PF3) availability and a slight but significant retardation in the rate of thrombin induced nucleotide release. However the decrease in ADP induced primary aggregation observed in alcoholic patients, could not be reproduced by the in vitro addition of alcohol to normal platelets (10). Similary, in our present study in rat, the inhibitory effect of alcohol in drinking water on ADP induced aggregation could not be reproduced by the in vitro addition of alcohol to PEP. Consequently, it seems that alcohol, both in man and in rat, does not inhibit instantly ADP aggregation as it does with thrombin. On the other hand, the effect of alcohol on ADP, epinephrine and collagen induced aggregation persista 12 hours or more after alcohol drinking, as this was observed in fasted subjects (3,4).
e
It has been suggested that the beneficial effect of alcohol on CED in man was mediated through and increse in WL-cholesterol (11). Since, in recent that platelet functions are also years, results have accumulated indicating closely related to CED, it seems feasible to postulate that the protective effect of alcohol on CED is partly due to its inhibitory effects on platelet functions. In the present study in rat, alcohol was as efficient as polyunsaturated fat to inhibit platelet functions. Consistent with this hypothesis is our recent study showing that 6 X alcohol, in drinking water, lowers atherosclerosis and platelet hyperaggregability in rabbits fed a saturated fat diet (4). ACENOWLEDGMENTS We thank C. Benoit, E. Dumont and C. Covacho for their skilfull technical assistance. This work, supported by an INSEEMGrant (ATP 76-63), has been presented in part at the meeting of the International Society on Thrombosis and Haemostasis, London, July 1979.
vo1.22, No.112
ALCOHOL AND PLATELET FUNCTION
225
REFERENCES 1. HENNEKENS, C.H., WILLETT, W., ROSNER, B., COLE, D. and MAYRENT, S. Effects of beer, wine and liquor in coronary deaths. J. Amer. Med. Ass., 242, 1973-1974, 1979. M.C., KAGAN, A., DOYLE, J.T., HAMES, C.G., HULLEY, S.B. and ZUKEL, W.J. Alcohol and blood lipids. The cooperative lipoprotein phenotyping study. Lancet ii, 153-155, 1977.
2. CASTELLI, W.P., GORDON, T., HJORTLAND,
3. RENAUD, S., DUMONT, E., GODSEY, F., SUPPLISSON, A. and THEVENON, C. Platelet functions in relation to dietary fats in farmers from two regions of France. Thranb. Raemoet.,a, 518-531, 1978.
4. RENAUD, S., MORAEAIN, R., MCGREGOR, L. and BADDIER, F. Dietary fats on platelet functions in relation to atherosclerosis and coronary heart disease. Haemostasie,_S,234-251, 1979.
5. HARKER, L.A., ROSS, R. and GLOMSET, J. Role of platelets in thrombogenesis. Ann. N.Y. Acad. Ski.,=, 321-329, 1976. 6. KEYS, A. Coronary heart disease in seven countries. Circuktion, Suppl. 2, 41, 1-211, 1970.
7. MCGREGOR, L., MORAZAIN, R. and RENAUD, S. A comparison of the effects of dietary short and long chain saturated fatty acids on platelet functions, Inveeplatelet phospholipids, and blood coagulation in rats. Labotiatory tigation,&, 438-442, 1980.
8. PACKHAM, M.A., KINLOUGH-RATHBONE, R.L., REIMERS, H.J., SCOTT, S. and MUSTARD, J.F. Mechanisms of platelet aggregation independent of adenosine diphosphate. In: Proetaghdins in Hematology.M.J. Silver, J.B. Smith and J.J. Kocsis (Eds) Holliswood, NY: Spectrum Publication Inc., 1977, pp. 247-276. 9.
ELMER, O., GORANSSON, G., SAKTJ,M. and BENGMARK, S. Ethanol-induced microaggregate formation in pig and rabbit blood. An in vitro study. Tkromb. i%WnO6t., z, 344-350, 1977.
10. HAIJT,J.M. and COWAN, D.H. The ffect of ethanol on hemostatic properties of human blood platelets. Amer. J. Med., s, 22-33, 1974. 11. RICCI, G. and ANGELICO, F. Alcohol consumption and coronary heart disease. Lancet , 1404.1407, 1979.