The effect of vitamin E on platelet aggregation Joseph A. C. Gomes, M.D. D. Venkatachalapathy, M.D. Jacob I. Haft, M.D., F.A.C.C. Bronx and New York, N. Y.
Since the optimistic reports by Shute and associates I in Canada there has been great interest in vitamin E as a cure or preventative for all forms of heart disease, especially t h a t due to coronary artery disease. Vitamin E has also been used in the treatment of peripheral vascular disease and intermittent claudication with conflicting results. 2-5 In experimental models, thrombosis was discovered in fetal rats dying in mothers which were vitamin E deficient,s Similarly, hyaline thrombi in capillaries in and about the cerebellum were believed to be the primary lesions in the encephalomalacia produced in chicks by a diet deficient in vitamin E. 7 In 1964 Ochsner s claimed t h a t tocopherol was of preventive value in thromboembolism because of antithrombin properties. Since platelet aggregation is intricately related to thrombosis, and since platelet aggregation occurs during the earliest stages of experimentally induced thrombosis, it seems probable t h a t agents t h a t prevent platelets sticking to each other and to the vessel wall will be more effective in preventing thrombosis than agents t h a t prevent fibrin formation. Renaud and associates 9 in 1970 showed that an increased ratio of saturated plus monounsaturated to polyunsaturated (S + M / P ) fatty acids in the platelets of patients with coronary artery disease and the platelets of rats fed a thrombogenic diet correlated with an increased propensity From the Cardiac Section of the Bronx VA Hospital and the Mount Sinai School of Medicine, New York, N. Y. Presented in part at the 47th Annual Scientific Sessions of the American Heart Association anti the National Conference on Thrombosis and Hemostasis, Nov. 20-22, 1974, Dallas, Texas. Received for publication Feb. 5, 1975. Reprint requests: Dr. Jacob I. Haft, M.D., F.A.C.C., Chief, Cardiology Department, St. Michaels Medical Center, 306 High St., Newark, N. J. 07102.
April, 1976, Vol. 91, No. 4, pp. 425-429
of platelets to aggregate in vitro. The antioxidant properties of tocopherol by which it protects the polyunsaturated fatty acids from peroxidation are widely acknowledged by most investigators. 1~ 1! H e n c e a-tocopherol could conceivably decrease the S + M / P ratio in biologic systems by preventing the peroxidation of PUFA. Hypothetically, then, a-tocopherol would bring about an increased availability of PUFA in plasma with increased binding of PUFA by the platelet or this reaction could occur de novo in the platelets leading to a decrease in the S + M / P ratio in the platelets. The decrease in the S § M / P ratio in the platelets would in turn lead to a decreased propensity of platelets to aggregate. 9 In view of this postulate, and for the reasons mentioned, the effect of a-tocopherol acetate (vitamin E) on platelet aggregation, not previously reported, was determined. Materials and methods
Platelet aggregation studies were done in 10 men 40 to 60 years of age. Group I consisted of five patients with coronary artery disease documented by coronary angiography (three patients) or by a history of a past myocardial infarction (with diagnostic electrocardiographic [ECG] changes) that had occurred at least 6 months prior to the time of study (two patients). All five patients had angina pectoris on moderate exertion, with four to six episodes per week requiring four to six tablets of nitroglycerine per week. None of the patients had congestive heart failure and none was taking any other medications. Group II consisted of five control patients with nonspecific chest pain atypical for angina and no ECG findings, negative exercise tests, no demonstrable coronary artery disease by angiography
American Heart Journal
425
Gomes, Venkatachalapathy, and Haft
Fig. 1. Grading of platelet aggregation response curves. Left panel shows 1+ platelet aggregation (partial aggregation, first phase only). Right panel: 2+ platelet aggregation (irreversible aggregation response).
CONTROL NS 5
z+
[~]:AGREG. PREd-TOCOPHEROL' ram:AGREG.POSTo/-TOCOPHEROL 2+ :2+ PLATELET AGREG. I+--I+ PLATELET AGREG. NS NS 2+
ANGINA NS
2+
5
2+
NS 2+
22 s ,,z,
::~
'+
r-
~:) 2
(~)
IO#mEPI IOpmADP I;mEPI
IAJmADP .bumADP
CONC. OF AGENTS
2+
IO)umEPl IOjurnADP bumEPl
I#mADP
I+
.IjurnADP
CONC. OF AGENTS
I:ig,2, Bar graphs comparing pre-a-tocopherol and post-e-toeophero] plate]etaggregation response curves in the control and angina groups.
(two patients), and no relief of chest pain with nitroglycerine. Platelet aggregation studies were performed by the optical density method of Born, 1~ as modified by Mustard and associatesJ 3 Venous blood was collected in siliconized tubes by a double-syringe method. Nine volumes of blood were added to one volume of 3.8 per cent sodium citrate. Samples were centrifuged at 1,000 r.p.m, for 10 minutes at room temperature and the platelet-rich plasma
426
(PRP) was removed. The platelet count of the P R P was 250,000 to 300,000 per cubic millimeter. The remaining blood was centrifuged at 2,500 r.p.m, for 20 minutes to obtain platelet-poor plasma (PPP). The P R P was placed in a cuvette of a chrono-log aggregometer and stirred at 1,200 r.p.m, at 37 ~ C, Dose-response curves were recorded on a moving strip chart recorder and were induced by adding to P R P adenosine diphosphate (ADP, Sigma Company, St. Louis, Mo.) to
April, 1976, Vol. 91, No. 4
Effect of vitamin E on platelet aggregation
achieve final concentrations of 10, 1, and 0.1 ffM, and epinephrine (adrenaline chloride, Park Davis Company, Detroit, Mich.) to achieve concentrations of 10 and 1 ~M. T h e per cent transmission of P R P was set at 90 and t h a t of P P P at 10. Aggregation studies were done in a fasting s t a t e prior to tocopherol acetate {vitamin E) and after 1,000 I.U. of tocopherol a c e t a t e (vitamin E) per day for 8 days. Serum triglycerides and cholesterol were measured before and after t o c o p h e r o l administration. Aggregation was graded as 1 + (partial aggregation, first phase only) and 2 + (irreversible aggregation or biphasic response) as shown in Fig. 1. Pre-tocopherol aggregation response curves were compared with post-tocopherol aggregation curves, with the patients serving as their own controls. Results
Prior to tocopherol there was no significant difference in the incidence of 1 + or 2 + aggregation response to the various c o n c e n t r a t i o n s of A D P or epinephrine between the control and angina groups. Similarly, after tocopherol no significant difference was found in either group when compared with pre-tocopherol aggregation response curves (Fig. 2). When all 10 patients were c o m p a r e d before and after tocopherol, no significant changes were observed (Fig. 3). Similarly no trends were observed after tocopherol in either direction (i.e., increased or decreased aggregation responses) in all 10 patients (Fig. 4). S e r u m cholesterol and triglycerides were n o r m a l in all 10 patients; namely, 210 _ 20 and 140 _-- 20 rag. per 100 ml. and did not significantly v a r y after tocopherol. All patients felt a sense of well being and no deleterious effects were observed in either the treated or the n o n t r e a t e d group. No significant difference in the frequency of anginal episodes was noted. Discussion
In the 1940's S h u t e and his colleagues' described marked i m p r o v e m e n t in an unselected series of 84 consecutive patients with angina pectoris. However, controlled studies carried out by Donegan and associates, '4 Ravin and Katz, 1~ and Makinson and associates TM showed no beneficial effects. Boyd and associates ~ and Livingston and Jones 3 reported i m p r o v e m e n t in patients with peripheral vascular disease and i n t e r m i t t e n t
American Heart Journal
r - l = AGREG. PRE (x/-TOCOPHEROL I I =AGREG. POST~'-TOCOPHEROL 2+ = 2+ PLATELET AGREG. += I + PLATELET AGREG. LLI ~1
NS 2+
'~176 1 80
CL
NS 2+
NS 2+ 2+
z
6o
z
40 I+
'%
20
C)
Z
IONmEPI
IO/JmADP INmEPI I#mADP .I}JmADP
CONC. OF AGENTS Fig. 3. Bar grhphs comparing pre-a-tocopherol and post-atocopherol platelet aggregation responses to various concentration of aggregating agents in all ten patients expressed in percentile.
claudication; however, H a m i l t o n and associates ~ reported no significant beneficial effect. Williams and associates 5 in a more recent s t u d y r e p o r t e d i m p r o v e m e n t in two-thirds of 30 patients with femeropopliteal occlusion and poor distal arteries, after taking 400 mg. of tocopherol four times a day for 3 months, as tested on an electric treadmill. Zierler and associates" and K a y and associates TM reported t h a t tocopherol p h o s p h a t e increased plasma a n t i t h r o m b i n activity. Maracci and Comera 19 found t h a t tocopherol p h o s p h a t e had a strong a n t i t h r o m b i n activity w h e r e a s tocopherol acetate had none. Similarly, Monkhouse 2~ found t h a t the a n t i t h r o m b i n effect of tocopherol phosphate in vitro was due to the phosphate ion and not to tocopherol per se. Kiffer and Olson, ~1 in their unpublished results, found no a n t i t h r o m b i n effect of tocopherol claimed by Ochsner. 8 Recently Korsan-Bengtsen and associates 22 found, in a group of p o s t m y o c a r d i a l infarction patients who were given a-tocopherol 300 mg. per day, a highly significant prolongation of the plasma clotting time after 18, 44, and 64 weeks of treatment. T h e y found no change in the platelet count nor any change in platelet adhesiveness. T o
42 7
Gomes, Venkatachalapathy, and
Haft
POST d-TOCO '2+
PRE o~-TOCO
I+
I+
POST d-TOCO 2+
PRE d-TOCO 2+.
I+
I+'
l#m ADP
lO#m ADP 2+
~-2+
I+
2+
2+
I§
I+
O"
O'
0-
POST d-TOCO
PRE (-TOCO 2+'
~.
.l#m ADP 2+
I+
I+
O
IOjum EPI
I jura EPI
Fig. 4. Linear graphs comparing pre- and post-a-tocopherol aggregation responses to various concentration of aggregating agents. With 10~m of ADP and 10~m of epinephrine there is no change pre- and post-a-tocopherol. With l~m of ADP, 0.1~m of ADP, and l~m of epinephrine some show more aggregation, others less. and some no change. No significant trends are observed in either direction. explain the observation of a prolonged c l o t t i n g time, they postulated t h a t a higher a m o u n t of highly u n s a t u r a t e d f a t t y acids was i n c o r p o r a t e d in the phospholipid fraction of the platelets together with a - t o c o p h e r o l leading to a change in the quality of the platelet m e m b r a n e , t h e r e b y causing a reduced release of p l a t e l e t f a c t o r 3. Interestingly, t h e y found no change in p l a t e l e t adhesiveness and t h e y did n o t s t u d y the effect on platelet aggregation. If our hypothesis t h a t a - t o c o p h e r o l m i g h t decrease the S + M / P ratio in the p l a t e l e t s were valid, we would expect t h a t a - t o c o p h e r o l w o u l d decrease the ability of platelets to aggregate. T h i s did not occur. I t is possible t h a t this effect m i g h t h a v e occurred a f t e r a prolonged course of t h e r a p y and t h a t 8 days was an insufficient time to result in a change of platelet activity, b u t this is unlikely. T h e r e was no significant change in p l a t e l e t function with ~-tocopherol. 23 Previous studies h a v e shown t h a t a - t o c o p h e r o l levels go up significantly in the p l a s m a and R B C , r e a c h i n g a p e a k at 10 hours24 Cross-incubation e x p e r i m e n t s w i t h p l a s m a and R B C ' s h a v e s h o w n t h a t a - t o c o p h e r o l diffuses easily f r o m p l a s m a to R B C ' s and viceversa, ~4 and, a l t h o u g h t h e r e are no d a t a on t u r n o v e r and levels of ~ - t o c o p h e r o l in t h e
428
platelets as yet, it is likely t h a t v i t a m i n E would enter platelets rapidly. I n addition, because of the n a t u r a l life span of platelets, 8 d a y s of t h e r a p y should be sufficient to effect the generation of platelets tested, even if the ~ - t o c o p h e r o l entered the platelet only as it developed. F u r t h e r m o r e , m o s t drugs t h a t affect platelet function do so within a short t i m e period a f t e r a d m i n i s t r a t i o n . I t has also been shown t h a t platelets m a y escape the antiaggregating effect of a drug (e.g., A t r o m i d ) ~ when it is given for a prolonged period of time. T h e question w h e t h e r t o c o p h e r o l is effective in the prevention or t r e a t m e n t of t h r o m b o e m b o l i s m is still open and can be solved only b y large trials. I t seems, however, t h a t if v i t a m i n E exerts a n y beneficial effect in t h e p r e v e n t i o n or t r e a t m e n t of t h r o m b o e m b o l i c p h e n o m e n a , c o r o n a r y a r t e r y disease, peripheral v a s c u l a r disease, or t h e initiation of athrogenesis it does n o t do so via an effect on platelet aggregation.
Summary Platelet aggregation studies were p e r f o r m e d in five m e n with c o r o n a r y a r t e r y disease a n d angina pectoris and five m e n with nonspecific chest pain before and after receiving 1,000 I.U. of a-tocopherol a c e t a t e orally per d a y for 8 days.
April, 1976, Vol. 91, No. 4
Effect of vitamin E on platelet aggregation
T h e r e w a s n o s i g n i f i c a n t d i f f e r e n c e in t h e p ! a t e l e t aggregation response to three concentrations of ADP and two concentrations of epinephrine between the pre- and post-vitamin E periods a m o n g t h e 10 p a t i e n t s . I f t o c o p h e r o l a c e t a t e (vitamin E) has any beneficial effect on the prevention of thromboembolism or in the treatment of angina pectoris and peripheral vascular d i s e a s e , i t is n o t v i a i n h i b i t i o n o f p l a t e l e t a g g r e g a tion.
REFERENCES
1. Shute, W. E., Shute, E. V., and Vogelsang, A.: Vitamin E in heart disease. I. The anginal syndrome, Med. Res. 160:21, 1947. 2. Boyd, A. M.. Ratcliffe, A. H.. Jepson, R. P., and James. G. W. H.: Intermittent claudication; clinical study, J Bone Joint Surg. 31-B:325, 1949. 3. Livingston, P. D., and Jones. C.: Treatment of intermittent claudication with vitamin E, Lancet 2:602, 1958. 4. Hamilton, M., Wilson, G. M., Armitage, P., and Boyde, J. T.: The treatment of intermittent claudication with vitamin E, Lancet 2:602, 1958. 5. Williams, H. T. G., Finna, D.. and Macbeth, R. A.: Alpha tocopherol in the treatment of intermittent claudication, Surg. Gynecol. Obstet. 132:662. 1971. 6. Mason, K. E.: Essays in biology in honor of Herbert M. Evans, University of California Press, 1943, p. 401. 7. Pappenheimer, A. M.. and Goettsch, M.: Cerebellar disorder in chicks, apparently of nutritional origin, J. Exp. Med. 53:11. 1931. 8. Ochsner, A.: Thromboembolism. N. Engl. J. Med. 271:211, 1964. 9. Renaud, S., Kuba, K., Goulet, C., Lemire, Y., and Allard, C.: Relationship between fatty-acid composition of platelets and platelet aggregation in rat and man, Circ. Res. 26:533, 1970. 10. Tappel, A. L.: Vitamin E as the lipid antioxidant, Vitam. Horm. 20:493, 1962.
American Heart Journal
11.' Grun, J.: Vitamin E and the biological antioxidant theory, Ann. N. Y. Acad. Sci. 203:29, 1972. 12. Born, G. V. R.: Aggregation of blood platelets by adenosine diphosphate and its reversal, Nature 194:927, 1962. 13. Mustard, J. F., Hegardt, B., Rousel, H. C., and McMillan, R. L.: Effects of adenosine nucleotides on platelet aggregation and clotting time, J. Lab. Clin. Med. 64:548, 1964. 14. Donegan, C. K., Messer, A. L., Orgain, E. S., and Ruffin, J. M.: Negative results of tocopherol therapy in cardiovascular disease, Am. J. Med. Sci. 217:294, 1949. 15. Ravin, I. S., and Katz, K. H.: Vitamin E in the treatment of angina pectoris, N. Engl. J. Med. 240:331, 1949. 16. Makinson, D. H., Oleesky, S., and Stone, R. V.: Vitamin E in angina pectoris, Lancet 1 :100, 1948. 17. Zierler, K. L., Grob, D., and Lilienthal, J. D., Jr.: On the antithrombin and antiproteolytic activity of alpha-tocopherol phosphate, Am. J. Physiol. 153:127. 1948. 18. Kay, J. H., Hutton. S. B., Jr., Weiss, G. N., and Ochsner, A.: Studies on an antithrombin. III. A plasma antithrombin test for the prediction of intravascular clotting, Surgery 28:24, 1950. 19. Maracci, M., and Comera, A.: Vitamin E and blood coagulation: Alpha-tocopherol phosphate, Third Congr. Intern. Vitamin E. 1:25, 1955. 20. Monkhouse, F. C.: The influence of inorganic salts on plasma antithrombin activity, Thromb. Diath. Haemarrh. 9:387, 1963. 21. Olson, R. E.: Vitamin E and its relation to heart disease, Circulation 46:179, 1973. 22. Korsan-Bengtsen, K.. Elmfeldt, D.. and Holm, T.. Prolonged plasma clotting time and decreased fibrinolysis after long-term treatment with alpha-tocopherol, Thromb. Diath. Haemorrh. 31:505, 1974. 23. Gomes, J. A., Venkatachalapathy, D., and Haft. J. [.: The effect of vitamin E on platelet aggregation, Circulation (abstr. 50) III-278. 1974. 24. Keyden, H. J., and Bjornson, L.: The dynamics of vitamin E transport in the human erythrocyte. Ann. N. Y. Acad. Sci. 203:127, 1972. 25. Chakrabarti, R., Fearnley, G. R., and Evans. J. F.: Effects of clofibrate on fibrinolysis, platelet stickiness, plasma fibrinogen and cholesterol, Lancet 2:1007, 1968.
429
Since the optimistic reports by Shute and associates I in Canada there has been great interest in vitamin E as a cure or preventative for all forms of heart disease, especially t h a t due to coronary artery disease. Vitamin E has also been used in the treatment of peripheral vascular disease and intermittent claudication with conflicting results. 2-5 In experimental models, thrombosis was discovered in fetal rats dying in mothers which were vitamin E deficient,s Similarly, hyaline thrombi in capillaries in and about the cerebellum were believed to be the primary lesions in the encephalomalacia produced in chicks by a diet deficient in vitamin E. 7 In 1964 Ochsner s claimed t h a t tocopherol was of preventive value in thromboembolism because of antithrombin properties. Since platelet aggregation is intricately related to thrombosis, and since platelet aggregation occurs during the earliest stages of experimentally induced thrombosis, it seems probable t h a t agents t h a t prevent platelets sticking to each other and to the vessel wall will be more effective in preventing thrombosis than agents t h a t prevent fibrin formation. Renaud and associates 9 in 1970 showed that an increased ratio of saturated plus monounsaturated to polyunsaturated (S + M / P ) fatty acids in the platelets of patients with coronary artery disease and the platelets of rats fed a thrombogenic diet correlated with an increased propensity From the Cardiac Section of the Bronx VA Hospital and the Mount Sinai School of Medicine, New York, N. Y. Presented in part at the 47th Annual Scientific Sessions of the American Heart Association anti the National Conference on Thrombosis and Hemostasis, Nov. 20-22, 1974, Dallas, Texas. Received for publication Feb. 5, 1975. Reprint requests: Dr. Jacob I. Haft, M.D., F.A.C.C., Chief, Cardiology Department, St. Michaels Medical Center, 306 High St., Newark, N. J. 07102.
April, 1976, Vol. 91, No. 4, pp. 425-429
of platelets to aggregate in vitro. The antioxidant properties of tocopherol by which it protects the polyunsaturated fatty acids from peroxidation are widely acknowledged by most investigators. 1~ 1! H e n c e a-tocopherol could conceivably decrease the S + M / P ratio in biologic systems by preventing the peroxidation of PUFA. Hypothetically, then, a-tocopherol would bring about an increased availability of PUFA in plasma with increased binding of PUFA by the platelet or this reaction could occur de novo in the platelets leading to a decrease in the S + M / P ratio in the platelets. The decrease in the S § M / P ratio in the platelets would in turn lead to a decreased propensity of platelets to aggregate. 9 In view of this postulate, and for the reasons mentioned, the effect of a-tocopherol acetate (vitamin E) on platelet aggregation, not previously reported, was determined. Materials and methods
Platelet aggregation studies were done in 10 men 40 to 60 years of age. Group I consisted of five patients with coronary artery disease documented by coronary angiography (three patients) or by a history of a past myocardial infarction (with diagnostic electrocardiographic [ECG] changes) that had occurred at least 6 months prior to the time of study (two patients). All five patients had angina pectoris on moderate exertion, with four to six episodes per week requiring four to six tablets of nitroglycerine per week. None of the patients had congestive heart failure and none was taking any other medications. Group II consisted of five control patients with nonspecific chest pain atypical for angina and no ECG findings, negative exercise tests, no demonstrable coronary artery disease by angiography
American Heart Journal
425
Gomes, Venkatachalapathy, and Haft
Fig. 1. Grading of platelet aggregation response curves. Left panel shows 1+ platelet aggregation (partial aggregation, first phase only). Right panel: 2+ platelet aggregation (irreversible aggregation response).
CONTROL NS 5
z+
[~]:AGREG. PREd-TOCOPHEROL' ram:AGREG.POSTo/-TOCOPHEROL 2+ :2+ PLATELET AGREG. I+--I+ PLATELET AGREG. NS NS 2+
ANGINA NS
2+
5
2+
NS 2+
22 s ,,z,
::~
'+
r-
~:) 2
(~)
IO#mEPI IOpmADP I;mEPI
IAJmADP .bumADP
CONC. OF AGENTS
2+
IO)umEPl IOjurnADP bumEPl
I#mADP
I+
.IjurnADP
CONC. OF AGENTS
I:ig,2, Bar graphs comparing pre-a-tocopherol and post-e-toeophero] plate]etaggregation response curves in the control and angina groups.
(two patients), and no relief of chest pain with nitroglycerine. Platelet aggregation studies were performed by the optical density method of Born, 1~ as modified by Mustard and associatesJ 3 Venous blood was collected in siliconized tubes by a double-syringe method. Nine volumes of blood were added to one volume of 3.8 per cent sodium citrate. Samples were centrifuged at 1,000 r.p.m, for 10 minutes at room temperature and the platelet-rich plasma
426
(PRP) was removed. The platelet count of the P R P was 250,000 to 300,000 per cubic millimeter. The remaining blood was centrifuged at 2,500 r.p.m, for 20 minutes to obtain platelet-poor plasma (PPP). The P R P was placed in a cuvette of a chrono-log aggregometer and stirred at 1,200 r.p.m, at 37 ~ C, Dose-response curves were recorded on a moving strip chart recorder and were induced by adding to P R P adenosine diphosphate (ADP, Sigma Company, St. Louis, Mo.) to
April, 1976, Vol. 91, No. 4
Effect of vitamin E on platelet aggregation
achieve final concentrations of 10, 1, and 0.1 ffM, and epinephrine (adrenaline chloride, Park Davis Company, Detroit, Mich.) to achieve concentrations of 10 and 1 ~M. T h e per cent transmission of P R P was set at 90 and t h a t of P P P at 10. Aggregation studies were done in a fasting s t a t e prior to tocopherol acetate {vitamin E) and after 1,000 I.U. of tocopherol a c e t a t e (vitamin E) per day for 8 days. Serum triglycerides and cholesterol were measured before and after t o c o p h e r o l administration. Aggregation was graded as 1 + (partial aggregation, first phase only) and 2 + (irreversible aggregation or biphasic response) as shown in Fig. 1. Pre-tocopherol aggregation response curves were compared with post-tocopherol aggregation curves, with the patients serving as their own controls. Results
Prior to tocopherol there was no significant difference in the incidence of 1 + or 2 + aggregation response to the various c o n c e n t r a t i o n s of A D P or epinephrine between the control and angina groups. Similarly, after tocopherol no significant difference was found in either group when compared with pre-tocopherol aggregation response curves (Fig. 2). When all 10 patients were c o m p a r e d before and after tocopherol, no significant changes were observed (Fig. 3). Similarly no trends were observed after tocopherol in either direction (i.e., increased or decreased aggregation responses) in all 10 patients (Fig. 4). S e r u m cholesterol and triglycerides were n o r m a l in all 10 patients; namely, 210 _ 20 and 140 _-- 20 rag. per 100 ml. and did not significantly v a r y after tocopherol. All patients felt a sense of well being and no deleterious effects were observed in either the treated or the n o n t r e a t e d group. No significant difference in the frequency of anginal episodes was noted. Discussion
In the 1940's S h u t e and his colleagues' described marked i m p r o v e m e n t in an unselected series of 84 consecutive patients with angina pectoris. However, controlled studies carried out by Donegan and associates, '4 Ravin and Katz, 1~ and Makinson and associates TM showed no beneficial effects. Boyd and associates ~ and Livingston and Jones 3 reported i m p r o v e m e n t in patients with peripheral vascular disease and i n t e r m i t t e n t
American Heart Journal
r - l = AGREG. PRE (x/-TOCOPHEROL I I =AGREG. POST~'-TOCOPHEROL 2+ = 2+ PLATELET AGREG. += I + PLATELET AGREG. LLI ~1
NS 2+
'~176 1 80
CL
NS 2+
NS 2+ 2+
z
6o
z
40 I+
'%
20
C)
Z
IONmEPI
IO/JmADP INmEPI I#mADP .I}JmADP
CONC. OF AGENTS Fig. 3. Bar grhphs comparing pre-a-tocopherol and post-atocopherol platelet aggregation responses to various concentration of aggregating agents in all ten patients expressed in percentile.
claudication; however, H a m i l t o n and associates ~ reported no significant beneficial effect. Williams and associates 5 in a more recent s t u d y r e p o r t e d i m p r o v e m e n t in two-thirds of 30 patients with femeropopliteal occlusion and poor distal arteries, after taking 400 mg. of tocopherol four times a day for 3 months, as tested on an electric treadmill. Zierler and associates" and K a y and associates TM reported t h a t tocopherol p h o s p h a t e increased plasma a n t i t h r o m b i n activity. Maracci and Comera 19 found t h a t tocopherol p h o s p h a t e had a strong a n t i t h r o m b i n activity w h e r e a s tocopherol acetate had none. Similarly, Monkhouse 2~ found t h a t the a n t i t h r o m b i n effect of tocopherol phosphate in vitro was due to the phosphate ion and not to tocopherol per se. Kiffer and Olson, ~1 in their unpublished results, found no a n t i t h r o m b i n effect of tocopherol claimed by Ochsner. 8 Recently Korsan-Bengtsen and associates 22 found, in a group of p o s t m y o c a r d i a l infarction patients who were given a-tocopherol 300 mg. per day, a highly significant prolongation of the plasma clotting time after 18, 44, and 64 weeks of treatment. T h e y found no change in the platelet count nor any change in platelet adhesiveness. T o
42 7
Gomes, Venkatachalapathy, and
Haft
POST d-TOCO '2+
PRE o~-TOCO
I+
I+
POST d-TOCO 2+
PRE d-TOCO 2+.
I+
I+'
l#m ADP
lO#m ADP 2+
~-2+
I+
2+
2+
I§
I+
O"
O'
0-
POST d-TOCO
PRE (-TOCO 2+'
~.
.l#m ADP 2+
I+
I+
O
IOjum EPI
I jura EPI
Fig. 4. Linear graphs comparing pre- and post-a-tocopherol aggregation responses to various concentration of aggregating agents. With 10~m of ADP and 10~m of epinephrine there is no change pre- and post-a-tocopherol. With l~m of ADP, 0.1~m of ADP, and l~m of epinephrine some show more aggregation, others less. and some no change. No significant trends are observed in either direction. explain the observation of a prolonged c l o t t i n g time, they postulated t h a t a higher a m o u n t of highly u n s a t u r a t e d f a t t y acids was i n c o r p o r a t e d in the phospholipid fraction of the platelets together with a - t o c o p h e r o l leading to a change in the quality of the platelet m e m b r a n e , t h e r e b y causing a reduced release of p l a t e l e t f a c t o r 3. Interestingly, t h e y found no change in p l a t e l e t adhesiveness and t h e y did n o t s t u d y the effect on platelet aggregation. If our hypothesis t h a t a - t o c o p h e r o l m i g h t decrease the S + M / P ratio in the p l a t e l e t s were valid, we would expect t h a t a - t o c o p h e r o l w o u l d decrease the ability of platelets to aggregate. T h i s did not occur. I t is possible t h a t this effect m i g h t h a v e occurred a f t e r a prolonged course of t h e r a p y and t h a t 8 days was an insufficient time to result in a change of platelet activity, b u t this is unlikely. T h e r e was no significant change in p l a t e l e t function with ~-tocopherol. 23 Previous studies h a v e shown t h a t a - t o c o p h e r o l levels go up significantly in the p l a s m a and R B C , r e a c h i n g a p e a k at 10 hours24 Cross-incubation e x p e r i m e n t s w i t h p l a s m a and R B C ' s h a v e s h o w n t h a t a - t o c o p h e r o l diffuses easily f r o m p l a s m a to R B C ' s and viceversa, ~4 and, a l t h o u g h t h e r e are no d a t a on t u r n o v e r and levels of ~ - t o c o p h e r o l in t h e
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platelets as yet, it is likely t h a t v i t a m i n E would enter platelets rapidly. I n addition, because of the n a t u r a l life span of platelets, 8 d a y s of t h e r a p y should be sufficient to effect the generation of platelets tested, even if the ~ - t o c o p h e r o l entered the platelet only as it developed. F u r t h e r m o r e , m o s t drugs t h a t affect platelet function do so within a short t i m e period a f t e r a d m i n i s t r a t i o n . I t has also been shown t h a t platelets m a y escape the antiaggregating effect of a drug (e.g., A t r o m i d ) ~ when it is given for a prolonged period of time. T h e question w h e t h e r t o c o p h e r o l is effective in the prevention or t r e a t m e n t of t h r o m b o e m b o l i s m is still open and can be solved only b y large trials. I t seems, however, t h a t if v i t a m i n E exerts a n y beneficial effect in t h e p r e v e n t i o n or t r e a t m e n t of t h r o m b o e m b o l i c p h e n o m e n a , c o r o n a r y a r t e r y disease, peripheral v a s c u l a r disease, or t h e initiation of athrogenesis it does n o t do so via an effect on platelet aggregation.
Summary Platelet aggregation studies were p e r f o r m e d in five m e n with c o r o n a r y a r t e r y disease a n d angina pectoris and five m e n with nonspecific chest pain before and after receiving 1,000 I.U. of a-tocopherol a c e t a t e orally per d a y for 8 days.
April, 1976, Vol. 91, No. 4
Effect of vitamin E on platelet aggregation
T h e r e w a s n o s i g n i f i c a n t d i f f e r e n c e in t h e p ! a t e l e t aggregation response to three concentrations of ADP and two concentrations of epinephrine between the pre- and post-vitamin E periods a m o n g t h e 10 p a t i e n t s . I f t o c o p h e r o l a c e t a t e (vitamin E) has any beneficial effect on the prevention of thromboembolism or in the treatment of angina pectoris and peripheral vascular d i s e a s e , i t is n o t v i a i n h i b i t i o n o f p l a t e l e t a g g r e g a tion.
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