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Platelets and aging I.—Aggregation, arachidonate metabolism and antioxidant status
THROMBOSIS RESEARCH 49; 331-342, 1988 0049-3848/88 $3.00 t .OO Printed in the USA. Copyright (c) 1988 Pergamon Press plc. All rights reserved.
PLATELETS AND AGING I-AGGREGATION, ARACHIDONATE METABOLISM AND ANTIOXIDANT STATUS.
E. VERICEL'*, M. DECHAVANNEl
M. CROSET', P. SEDIVY*, and M. LAGARDEl*.
Ph.
COURPRON3,
d'Hemobiologie, Faculti? de Medecine 1. INSERM U.63, Laboratoire Alexis Carrel, Lyon, France. Sante, Vitry-sur-Seine, France. 2. Rh8ne-Poulenc A. Charial, Francheville, 3. Maison de Sante et de Cure Medicale France. * New address: Universite de Bourgogne, CNRS U.A. 273, B.P. 138, 21004 Dijon, France. (Received 18.9.1987; Accepted in original form 22.9.1987 by Editor B. Vargaftig) (Received by Executive Editorial Office 30.11.1987) ABSTRACT Platelet functions were investigated in sixteen old (78-94 years) and eight young (25-35 years) subjects. Whole blood platelet aggregation induced by collagen was higher in the elderly. Similarly, aggregation of platelet rich plasma and plasma-free platelets induced by various agents was increased but the collagen-induced release of ATP was reduced. In agreement with the enhanced platelet the increase of thromboxane formation aggregability, (under thrombin stimulation) was also noted in platelets from elderly people. To further assess platelet and vascular function in vivo, we measured the excretion of urinary TXBB, 2,3-dinor TXB2, 6-keto-PGFla and 2,3-dinor6-keto-PGFla. The four metabolites were all increased in the elder population. In addition, a significant reduction of platelet vitamin E was observed in the elderly people, although the plasma content was normal. These results indicate numerous modifications of platelet behaviour with aging. They include the increased platelet susceptibility to aggregation, and the depletion of ATP granule content, which could reflect an activation in vivo in agreement with the enhanced urinary excretion of thromboxane and prostacyclin metabolites. We hypothesize that platelet hyperactivity associated with the enhanced oxygenated metabolism of arachidonic acid could be linked to vitamin E depletion. These changes may reveal a prethrombotic state in the elderly population. Key words:
elderly people, platelet A2, urinary prostanoids, 331
functions, thromboxane vitamin E.
PLATELET FUNCTIONS AND AGING
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INTRODUCTION Some physiological changes in cell and organ functions are associated with a high appear with aging. These modifications particularly in cardiovascular system (l), incidence of diseases, and hyperactive platelets are believed to be involved in the development of these diseases. Several studies have reported enhanced in vivo platelet bleeding activation in elderly people. They include shortened time (2), reduced platelet lifespan (3), enhanced platelet release reaction (4,5), and increased platelet sensitivity to various aggregating agents (6,7). The role of platelets in hemostasis and thrombosis has acid (AA) been well documented (8), and the arachidonic metabolism has been involved in platelet functions (9). Activation of platelets by various agents leads to the liberation of AA from membrane phospholipids which is subsequently oxygenated by the cyclooxygenase and lipoxygenase pathways. The former converts AA into cyclic prostaglandin endoperoxides, PGG2 and H2, further isomerized into thromboxane A2 (TXAB), a potent proaggregating and vasoconstricting substance (10). The latter oxygenates AA into 12-hydroperoxy-eicosatetraenoic acid which is then reduced into its 12-hydroxy derivative (12-HPETE), (12-HETE) by a glutathione-dependent peroxidase (11,12). On the other hand, AA is converted into prostacyclin of platelet aggregation (13) and (PGI2), a strong inhibitor vasodilator (14), and in vivo PGI2 biosynthesis seems to be altered with age (15). The purpose of this study was to investigate platelet function changes with special reference to AA metabolism in vitro and in vivo both in young and old people.
MATERIAL
AND
METHODS
Subjects Sixteen old (between 78-94 years) and eight young (between 25 and 35 years) subjects were investigated after selected in an old informed consent. Old subjects were carefully people's home and those having metabolic or malignant diseases Selected subjects had not taken any drug for at were excluded. least ten days before venous punction of blood. Aggregation studies * Platelet aggregation studies in whole blood. citrate was Blood containing l/10 vol. of 3.8 % trisodium diluted with an equal volume of isotonic saline. Platelet aggregation was performed using a whole-blood aggregometer 500 PA) and the change in impedance was (Chrono-Log Corp, Havertown, Collagen (Horm, Munich, FRG) was used as the recorded. aggregating agent. * Platelet aggregation studies in the presence of autologous plasma. Platelet-rich plasma (PRP) was prepared by centrifugation of blood collected onto l/10 vol. of 3.8 % trisodium citrate. Platelet aggregation was done by the turbidimetric method of Born (16). The following aggregating substances were used: ADP and
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PLATELET FUNCTIONS AND AGING
333
epinephrine (Stago, Paris, France), arachidonic acid (Sigma, St Louis, MO), platelet activating factor (PAF)(Bachem, Budendorf, Switzerland) and collagen. * Platelet aggregation studies with plasma-free platelets. Platelets were isolated from their plasma as previously described (17) and their aggregation was induced by different agents such as thrombin (Sigma, St Louis, MO), 9 methano-analogue of prostaglandin H2 (U-46619) (gift from Dr. J.E. Pike, The IJpjohn Company, Kalamazoo, MI) and calcium ionophore A 23187 (Boehringer, Mannheim, FRG). * ATP measurement. Collagen-induced release of ATP was measured by Luminescence with the luciferin-luciferase system added in PRP using the aggregometer 500. Metabolism of arachidonic acid * Platelets were stimulated with 0.1 U/ml thrombin for 4 minutes at 37°C and AA metabolites were extracted from the incubate. Hydroxy derivatives, namely 12-HETE and 12-hydroxyand quantified by heptadecatrienoic acid (HHT), were purified HPLC (18) in using 13-hydroxy-octadecadienoic acid (13-HODE) instead of 15-OH-20:3, and thromboxane B2 (TXB2) was measured by GLC (19). * Oxygenated products from AA were also measured in serum. They were extracted at pH3 with ethyl acetate and separated by tlidimensional thin-layer chromatography (TLC). In the first the mixture hexane/diethyl ether/acetic acid (60/40/l) dimension, was used to provide monohydroxy derivatives which were measured by HPLC as above. The second elution with the organic phase of the mixture ethylacetate/isooctane/acetic acid/water (100/50/20/100) allowed to separate TXB2 and 6-keto-PGFla (6KPGFla). These products were quantified by radioimmunoassay (Institut Pasteur Production, Paris, France) according to Dray et al (20). * Finally, to further assess the in vivo AA metabolism in platelets and the vascular wall, we measured the excretion of TXBZ, 2,3-dinor-TXB2 (TXBB-M), 6KPGFla and 2,3-dinorketo-PGFla (GKPGFla-M) in urine. 20 ml urine samples acidified to pH3 were passed through Sep-Pack Cl8 cartridges (Waters Associates, Milford, MA) and eluted with dichloromethane. The four metabolites contained in the extracts were separated on TLC using chloroform/methanol/acetic acid/water (90/8/l/0.8) as the eluent (21) and quantified by enzyme immunoassays (22). The yield recovery was calculated on the basis of tritiated TXB2 and 6KPGFla added prior to urine extraction. Vitamin
E determination Plasma and platelet vitamin E (gamma and alpha tocopherols) were determined by HPLC. Plasma or platelet suspension (1 volume) was diluted by water (1 volume) containing 1% ascorbic acid. Ethanol (1 volume), containing tocol (gift from Hoffmann-La Roche) used as an internal standard, was added and vitamin E was extracted by hexane (3 volumes). After centrifugation, supernatant was analysed by HPLC with a nucleosil C 18 column and tocopherols were measured according to their absorbance at 292 nm.
PLATELET FUNCTIONS AND AGING
334
selenium quantification Samples were mineralized perchloric acids mixture. After selenium was measured by atomic (Perkin-Elmer 5000).
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Plasma
by sulfuric, nitric and reduction by chlorhydric absorption spectrometry
acid,
Statistics Results are expressed as mean + S.D. and the Student's test or the Cochran's test was used to compare two groups.
t
RESULTS Platelet aggregation studies showed some age-related differences. In whole blood, aggregation induced by 2 ug/ml collagen was significantly increased in the elderly group as compared to young adults (17.1 + 4.7 vs 12.9 f 3.4 ohms, ~(0.05, n=15 and 8 respectively). In the same way, % aggregation of PRP was higher in the aged people group when induced by either arachidonic acid, collagen or epinephrine (TABLE 1). The same tendency was observed when ADP or PAF was used as the aggregating agent but the differences did not reach significance (TABLE 1).
TABLE Platelet aggregation Values (mean f S.D.)
1
in PRP when induced by various represent % of aggregation. Old
People
Young
agents.
People
ADP 8.6 ? 5.8 4.1 f 4.5 (n=16) (n=8) 0.6 nM --___--_____--___-__~-___-~~~-~~~~~~~~~~~~-_____________________ ADP 22.5 f 11.8 15.8 + 11.6 (n=16) (n=7) 1.2 nM ___-~__-~~~_----_---~-____--~_---~~_--~~~--_______--___---__---arachidonic acid 64.5 f 31.0 23.3 + 41.3 (n=16) (n=8) 300 uM __--___--~~__---~--~~-___-~~~-~~~~~-~~~~~-_---____--__--~~~_-_-~ collagen 32.0 f 31.4 1.3 f 3.7 (n=lO) (n=8) 0.25 ug/ml ______--____--_-_--_~-___--~~__~~~~--~~~~-~---____--~_--~~_---~~ PAF 33.4 a 23.3 23.0 +- 26.3 (n=16) (n=8) 190 nM _--__-______---__-__~-~----~~--~~~~-~~~~~~~---____-~___--~___-~~ epinephrine 55.6 +- 26.3 17.0 + 34.2 (n=9) (n=7) 2 IJM
N.S.
: not
significant
N.S.
N.S.
pco.02
pto.02
N.S.
pco.05
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PLATELET FUNCTIONS AND AGING
Table 2 shows the results obtained when the aggregating tests were done with plasma-free platelets. Two concentrations of acid. As shown in the agonists were used, except for arachidonic table, differences between young and old subjects were the highest when low concentrations of aggregating agents were used. TABLE
2
Aggregation of platelets isolated from their plasma when (mean f S.D.) represent % of by various agents. Values aggregation. Old
arachidonic 2 uM
acid
People
83.6 + 5.2 (n=16)
Young
induced
People
75.7 f 6.1 (n=8)
pXO.01
N.S. 84.8 f 3.7 88.7 + 5.4 thrombin (n=l6) (n=8) 15 mu/ml ____-__-____________~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~------_-_ p
U.46619 57 nM
U.46619 14 nM
48.7 f 34.0 (n=ll)
5.3 k 15.1 (n=8)
______-----_________~~~~~~~~~~~~~~~~~~~-~~~~~~~~~~~~~~~~~~~-~---A.23187 79.0 f 21.0 87.7 i: 4.4 0.25 uM (n=16) (n=8)
N.S.
p
N.S.
A.23187 6.25 nM
56.8 + 34.2 (n=16)
0.2 + 0.5 (n=7)
PAF 95 nM
56.1 f 22.0 (n=15)
45.4 + 28.2 (n=8)
N.S.
PAF 12 nM
76.0 f 23.1 (n=7)
35.2 f 22.0 (n=8)
p
p
During the aggregation of PRP, granule secretion was also appreciated by the measurement of ATP release. The release of ATP induced by collagen (0.44 pg/ml) was lower in the old people group than in young subjects (5.9 + 4.2 vs 12.5 f 5.4 nmoles ATP / lOSplatelets, p
336
PLATELET FUNCTIONS AND
AGING
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TABLE 3 Metabolism of platelet endogenous arachidonic acid under thrombin stimulation (0.1 U/ml). Results are means k S.D. nmoles/lOg platelets
Old People
Young People
TXB2
1.09 f 0.52 0.65 zt 0.22 p
Conversion of platelet endogenous AA into the main oxygenated products TXBB, HHT and 12-HETE under thrombin stimulation is reported in table 3. The lipoxygenase product tended to decrease in the elderly compared yo young adults, although not significantly (TABLE 3). In contrast, HHT tended to increase and TXB2 was enhanced significantly but the sum of the two was not significantly higher in the elderly group. The sum of the three main metabolites, which can be grossly considered to represent the endogenous AA liberated from phospholipids, was equal in both groups (TABLE 3). At the opposite, cyclooxygenase products (TXBB, HHT) were significantly depressed in serum from elderly subjects and 12-HETE tended to decrease whilst the production of 6KPGFla was two fold higher (TABLE 4). Finally, the urinary excretion of TXBB, 6KPGFla and GKPGFla-M was significantly enhanced in the elder subjects versus the young adults and the same tendency could be observed for that 0f Tx~2-M (TABLE 5).
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TABLE Oxygenated metabolites means + S.D. nmoles/ml
337
PLATELET FUNCTIONS AND AGING
4
of arachidonic
Old
acid
in serum.
Young
People
Results
are
People
0.49 2 0.18 0.28 + 0.19 (n=6) (n=16) _____________---____~~~~~~~~~~~~~~~-~~~~~~~~~-~~~~~~--~~---_----1.15 f 0.36 0.55 f 0.36 HHT TXB2
p
p
_______________________!~~~~!_____________!~~~!_-----------__-_-1.68 + 0.35 0.83 f 0.54 (n=l6) (n=6) ~---------------~~~~~~~~~~~~~~~~-~~~~~~~------------_-___________ 1.47 ?: 0.57 0.95 f 0.65 12-HETE (n=16) (n=7) ----------------~~~~~~~~~~~~~~~-~~~~~~~~~~~~~~~-------___________ 3.23 + 0.94 1.79 f 1.15 TXBBtHHTtlB-HETE (n=16) (n=6) ~---------______~~--~---~------_~~~-~--~------~~~-------______--____________________~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~__~~~~~~ 13.3 ?: 8.1 27.2 2 14.8 6-keto-PGFla (n=6) (n=16) (pmoles/ml)
TXB2+HHT
TABLE Levels of some urinary prostanoids Results are expressed in ng/mmole Old People (n=l6)
p
N.S.
pxo.02
p=o.o5
5 in the two groups of age. of creatinine (mean ?: S.D.).
Young People (n=7)
TXB2 3.1 ?: 1.2 p
Plasma and platelet vitamin E were also measured and no difference could be found in plasma gamma and alpha tocopherol contents when both groups were compared (TABLE 6). In contrast, a significant decrease of these tocopherols was observed in platelets from elderly people (TABLE 6). Selenium, another parameter of the antioxidant status, was measured in the plasma. It was signicantly lower in the elderly people (34.5 f 11.9 Ir$/ml) than in young adults (64.3 + 19.7 yg/ml).
TABLE
6
Plasma and platelet tocopherol concentrations people. Results are means f S.D. Old Plasma
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338
(nmoles/ml)
gamma-tocopherol
People (n=lO)
0.48
Young
in old
and
young
People
(n=8)
f 0. 14 ,,,%b 0.61
? 0.17
N.S.
alpha-tocopherol 10.3 f 2.8 11.2 ?: 3.5 N.S. ____________________~~~_~~~~~~-------------------------_-------Platelets (n=ll) (n=8) (nmoles/lOg platelets) gamma-tocopherol
0.05
+ 0.02
0.09
f 0.05
p
alpha-tocopherol
0.94
+ 0.21
1.41
f 0.55
p
DISCUSSION It is generally acknowledged that incidence of thrombotic complications increases with age suggesting alterations of the hemostatic balance in elderly people. In the present study, we have evaluated platelet aggregability in a group of aged subjects and another one of young adults. Various tests of aggregation indicate that platelet aggregability increases with age. Indeed, platelet aggregation studies using the classical turbidimetric method of Born (16) show that platelets from aged-people are more reactive in response to low concentrations of agonist, especially when they involve the AA metabolism in their mechanism of action. As a matter of fact, no significant difference was found in using ADP and in some way PAF acether which do not require thromboxane formation to induce platelet aggregation, at least at low reports concentrations (23). These findings agree with previous from Johnson et al (6) and Couch et al (7) indicating that platelet aggregability increased in the elderly population. We have also measured platelet aggregation in a more physiological situation such as the presence of all blood elements. The results obtained confirm the enhanced platelet aggregability in the aged individuals. We found that release of ATP induced by collagen was diminished in platelets from the elderly. This finding contrasts with the results from Kasjanovova and Balaz (24). These authors reported an elevation of the released ATP from elderly subjects. The same agonist (collagen) was used to initiate platelet release A possible explanation for reaction with similar concentrations. this discrepancy could be the age of the subjects who were only over 59 years versus over 78 years in our study. However, Zahavi et al (4) and Sie et al (5) have found an enhanced plasma B-thromboglobulin (B-TG) in old people (over 80 years). Platelet specific platelet protein to be released, factor 4 (PF4), another was also increased in plasma with age. In addition, platelets from elderly people are less dense and their mepacrin-labeled that ATP, granule content is decreased (5). We may then speculate
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like R-TG and PF4, would be discharged in the circulation due to platelet hyperactivity and that ex vivo further stimulation of those platelets would lead to lower release of their granule content. We have previously reported that exogenous AA metabolism, in particular its oxygenation via the cyclooxygenase and lipoxygenase pathways was increased in platelets from elderly the people (25). In the present study, we have investigated endogenous AA metabolism when thrombin was the stimulus. In agreement with the enhanced platelet aggregation observed, we found an increase of thromboxane B2 formation in elderly subjects. In aged rats, Giani et al (26) have also reported both increases of platelet aggregation and thromboxane formation. In we did not find such a difference in the production of contrast, 12-HETE and it was even decreased although not significantly. In study (27) showed that the accordance with these data, a previous lipoxygenase activity itself does not change with age but the production of 1X-HETE decreased subsequently to the reduction of glutathione peroxidase activity. The activity of this enzyme is selenium-dependent (28) and a recent report from Shoene et al (29) showed that platelets from selenium-deficient rats aggregated to a significantly higher rate than those from selenium-supplemented rats, and also produced more thromboxane content of platelets, but B2. We did not measure the selenium that of plasma was significantly reduced in elderly people, suggesting a mild deficiency which may be related to the alterations in the production of cyclooxygenase and lipoxygenase metabolites that we observed in our study. The increased thromboxane formation under stimulation with low concentrations of thrombin is in a good agreement with the increased TXB2 and TXBB-M in urine which may be considered to represent the basal production of the body. This also agrees with similar enhancement of 6KPGFla and GKPGFla-M excretion which has already been reported in aging (15) and believed to characterize a pre-thrombotic state with platelet activation in vivo (30). In contrast to these results observed under "basal" activation or in vitro weak activation, those obtained in serum, which results from a huge production of endogenous thrombin, revealed a decreased formation of oxygenated metabolites from AA. One possible explanation would be a reduced availability of AA from platelet phospholipids that we have found significantly depressed in these lipid compartments in the elderly (see the following (presumably providing from paper). The reason why serum 6KPGFla monocytes via PGH2 of platelet origin (31)) was increased in the elderly is not clear but cannot be taken into account in the endogenous AA availability because of its low amount. Finally, platelet but not plasma tocopherols were found significantly reduced in elderly people, in agreement with previous findings (32). This suggest a cellular hyper consumption of these antioxidants instead of a deficient state of nutritional origin. Like in experimentally deficient animals (33) and in diabetes (34), the enhanced platelet functions and oxygenated metabolism of AA might be associated with an increased "peroxide tonew of the cell closely related to a depletion of natural antioxidants, of which vitamin E is the marker in the present study.
340
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We conclude that platelet hyperactivity in the elderly, preferentially associated with the enhanced oxygenated metabolism of AA, might be linked to the antioxidant status / peroxide tone of the cell. ACKNOWLEDGEMENTS This work was for providing U 46619 gamma-tocopherol.
supported by INSERM. We thank Dr. J.E. Pike and Dr. 0. Hurstel who supplied tocol and
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12. BRYANT, R.W. and BAILEY, J.M. Altered lipoxygenase metabolism and decreased glutathione peroxidase activity in platelets from selenium-deficient rats. Biochem. Biophys. Res. Commun., 92, 268-276, 1980. 13. MONCADA, S., HIGGS, E.A. and VANE, J.R. Human arterial and venous tissues generate prostacyclin (prostaglandin X), a potent inhibitor of platelet aggregation. Lancet, 1, 18-22, 1977. 14. MONCADA, S. Biological 76, 3-31, Pharmacol.,
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The effects of vitamin N.Y. Acad. Sci., 393,
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of
PLATELETS AND AGING I-AGGREGATION, ARACHIDONATE METABOLISM AND ANTIOXIDANT STATUS.
E. VERICEL'*, M. DECHAVANNEl
M. CROSET', P. SEDIVY*, and M. LAGARDEl*.
Ph.
COURPRON3,
d'Hemobiologie, Faculti? de Medecine 1. INSERM U.63, Laboratoire Alexis Carrel, Lyon, France. Sante, Vitry-sur-Seine, France. 2. Rh8ne-Poulenc A. Charial, Francheville, 3. Maison de Sante et de Cure Medicale France. * New address: Universite de Bourgogne, CNRS U.A. 273, B.P. 138, 21004 Dijon, France. (Received 18.9.1987; Accepted in original form 22.9.1987 by Editor B. Vargaftig) (Received by Executive Editorial Office 30.11.1987) ABSTRACT Platelet functions were investigated in sixteen old (78-94 years) and eight young (25-35 years) subjects. Whole blood platelet aggregation induced by collagen was higher in the elderly. Similarly, aggregation of platelet rich plasma and plasma-free platelets induced by various agents was increased but the collagen-induced release of ATP was reduced. In agreement with the enhanced platelet the increase of thromboxane formation aggregability, (under thrombin stimulation) was also noted in platelets from elderly people. To further assess platelet and vascular function in vivo, we measured the excretion of urinary TXBB, 2,3-dinor TXB2, 6-keto-PGFla and 2,3-dinor6-keto-PGFla. The four metabolites were all increased in the elder population. In addition, a significant reduction of platelet vitamin E was observed in the elderly people, although the plasma content was normal. These results indicate numerous modifications of platelet behaviour with aging. They include the increased platelet susceptibility to aggregation, and the depletion of ATP granule content, which could reflect an activation in vivo in agreement with the enhanced urinary excretion of thromboxane and prostacyclin metabolites. We hypothesize that platelet hyperactivity associated with the enhanced oxygenated metabolism of arachidonic acid could be linked to vitamin E depletion. These changes may reveal a prethrombotic state in the elderly population. Key words:
elderly people, platelet A2, urinary prostanoids, 331
functions, thromboxane vitamin E.
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INTRODUCTION Some physiological changes in cell and organ functions are associated with a high appear with aging. These modifications particularly in cardiovascular system (l), incidence of diseases, and hyperactive platelets are believed to be involved in the development of these diseases. Several studies have reported enhanced in vivo platelet bleeding activation in elderly people. They include shortened time (2), reduced platelet lifespan (3), enhanced platelet release reaction (4,5), and increased platelet sensitivity to various aggregating agents (6,7). The role of platelets in hemostasis and thrombosis has acid (AA) been well documented (8), and the arachidonic metabolism has been involved in platelet functions (9). Activation of platelets by various agents leads to the liberation of AA from membrane phospholipids which is subsequently oxygenated by the cyclooxygenase and lipoxygenase pathways. The former converts AA into cyclic prostaglandin endoperoxides, PGG2 and H2, further isomerized into thromboxane A2 (TXAB), a potent proaggregating and vasoconstricting substance (10). The latter oxygenates AA into 12-hydroperoxy-eicosatetraenoic acid which is then reduced into its 12-hydroxy derivative (12-HPETE), (12-HETE) by a glutathione-dependent peroxidase (11,12). On the other hand, AA is converted into prostacyclin of platelet aggregation (13) and (PGI2), a strong inhibitor vasodilator (14), and in vivo PGI2 biosynthesis seems to be altered with age (15). The purpose of this study was to investigate platelet function changes with special reference to AA metabolism in vitro and in vivo both in young and old people.
MATERIAL
AND
METHODS
Subjects Sixteen old (between 78-94 years) and eight young (between 25 and 35 years) subjects were investigated after selected in an old informed consent. Old subjects were carefully people's home and those having metabolic or malignant diseases Selected subjects had not taken any drug for at were excluded. least ten days before venous punction of blood. Aggregation studies * Platelet aggregation studies in whole blood. citrate was Blood containing l/10 vol. of 3.8 % trisodium diluted with an equal volume of isotonic saline. Platelet aggregation was performed using a whole-blood aggregometer 500 PA) and the change in impedance was (Chrono-Log Corp, Havertown, Collagen (Horm, Munich, FRG) was used as the recorded. aggregating agent. * Platelet aggregation studies in the presence of autologous plasma. Platelet-rich plasma (PRP) was prepared by centrifugation of blood collected onto l/10 vol. of 3.8 % trisodium citrate. Platelet aggregation was done by the turbidimetric method of Born (16). The following aggregating substances were used: ADP and
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333
epinephrine (Stago, Paris, France), arachidonic acid (Sigma, St Louis, MO), platelet activating factor (PAF)(Bachem, Budendorf, Switzerland) and collagen. * Platelet aggregation studies with plasma-free platelets. Platelets were isolated from their plasma as previously described (17) and their aggregation was induced by different agents such as thrombin (Sigma, St Louis, MO), 9 methano-analogue of prostaglandin H2 (U-46619) (gift from Dr. J.E. Pike, The IJpjohn Company, Kalamazoo, MI) and calcium ionophore A 23187 (Boehringer, Mannheim, FRG). * ATP measurement. Collagen-induced release of ATP was measured by Luminescence with the luciferin-luciferase system added in PRP using the aggregometer 500. Metabolism of arachidonic acid * Platelets were stimulated with 0.1 U/ml thrombin for 4 minutes at 37°C and AA metabolites were extracted from the incubate. Hydroxy derivatives, namely 12-HETE and 12-hydroxyand quantified by heptadecatrienoic acid (HHT), were purified HPLC (18) in using 13-hydroxy-octadecadienoic acid (13-HODE) instead of 15-OH-20:3, and thromboxane B2 (TXB2) was measured by GLC (19). * Oxygenated products from AA were also measured in serum. They were extracted at pH3 with ethyl acetate and separated by tlidimensional thin-layer chromatography (TLC). In the first the mixture hexane/diethyl ether/acetic acid (60/40/l) dimension, was used to provide monohydroxy derivatives which were measured by HPLC as above. The second elution with the organic phase of the mixture ethylacetate/isooctane/acetic acid/water (100/50/20/100) allowed to separate TXB2 and 6-keto-PGFla (6KPGFla). These products were quantified by radioimmunoassay (Institut Pasteur Production, Paris, France) according to Dray et al (20). * Finally, to further assess the in vivo AA metabolism in platelets and the vascular wall, we measured the excretion of TXBZ, 2,3-dinor-TXB2 (TXBB-M), 6KPGFla and 2,3-dinorketo-PGFla (GKPGFla-M) in urine. 20 ml urine samples acidified to pH3 were passed through Sep-Pack Cl8 cartridges (Waters Associates, Milford, MA) and eluted with dichloromethane. The four metabolites contained in the extracts were separated on TLC using chloroform/methanol/acetic acid/water (90/8/l/0.8) as the eluent (21) and quantified by enzyme immunoassays (22). The yield recovery was calculated on the basis of tritiated TXB2 and 6KPGFla added prior to urine extraction. Vitamin
E determination Plasma and platelet vitamin E (gamma and alpha tocopherols) were determined by HPLC. Plasma or platelet suspension (1 volume) was diluted by water (1 volume) containing 1% ascorbic acid. Ethanol (1 volume), containing tocol (gift from Hoffmann-La Roche) used as an internal standard, was added and vitamin E was extracted by hexane (3 volumes). After centrifugation, supernatant was analysed by HPLC with a nucleosil C 18 column and tocopherols were measured according to their absorbance at 292 nm.
PLATELET FUNCTIONS AND AGING
334
selenium quantification Samples were mineralized perchloric acids mixture. After selenium was measured by atomic (Perkin-Elmer 5000).
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Plasma
by sulfuric, nitric and reduction by chlorhydric absorption spectrometry
acid,
Statistics Results are expressed as mean + S.D. and the Student's test or the Cochran's test was used to compare two groups.
t
RESULTS Platelet aggregation studies showed some age-related differences. In whole blood, aggregation induced by 2 ug/ml collagen was significantly increased in the elderly group as compared to young adults (17.1 + 4.7 vs 12.9 f 3.4 ohms, ~(0.05, n=15 and 8 respectively). In the same way, % aggregation of PRP was higher in the aged people group when induced by either arachidonic acid, collagen or epinephrine (TABLE 1). The same tendency was observed when ADP or PAF was used as the aggregating agent but the differences did not reach significance (TABLE 1).
TABLE Platelet aggregation Values (mean f S.D.)
1
in PRP when induced by various represent % of aggregation. Old
People
Young
agents.
People
ADP 8.6 ? 5.8 4.1 f 4.5 (n=16) (n=8) 0.6 nM --___--_____--___-__~-___-~~~-~~~~~~~~~~~~-_____________________ ADP 22.5 f 11.8 15.8 + 11.6 (n=16) (n=7) 1.2 nM ___-~__-~~~_----_---~-____--~_---~~_--~~~--_______--___---__---arachidonic acid 64.5 f 31.0 23.3 + 41.3 (n=16) (n=8) 300 uM __--___--~~__---~--~~-___-~~~-~~~~~-~~~~~-_---____--__--~~~_-_-~ collagen 32.0 f 31.4 1.3 f 3.7 (n=lO) (n=8) 0.25 ug/ml ______--____--_-_--_~-___--~~__~~~~--~~~~-~---____--~_--~~_---~~ PAF 33.4 a 23.3 23.0 +- 26.3 (n=16) (n=8) 190 nM _--__-______---__-__~-~----~~--~~~~-~~~~~~~---____-~___--~___-~~ epinephrine 55.6 +- 26.3 17.0 + 34.2 (n=9) (n=7) 2 IJM
N.S.
: not
significant
N.S.
N.S.
pco.02
pto.02
N.S.
pco.05
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PLATELET FUNCTIONS AND AGING
Table 2 shows the results obtained when the aggregating tests were done with plasma-free platelets. Two concentrations of acid. As shown in the agonists were used, except for arachidonic table, differences between young and old subjects were the highest when low concentrations of aggregating agents were used. TABLE
2
Aggregation of platelets isolated from their plasma when (mean f S.D.) represent % of by various agents. Values aggregation. Old
arachidonic 2 uM
acid
People
83.6 + 5.2 (n=16)
Young
induced
People
75.7 f 6.1 (n=8)
pXO.01
N.S. 84.8 f 3.7 88.7 + 5.4 thrombin (n=l6) (n=8) 15 mu/ml ____-__-____________~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~------_-_ p
U.46619 57 nM
U.46619 14 nM
48.7 f 34.0 (n=ll)
5.3 k 15.1 (n=8)
______-----_________~~~~~~~~~~~~~~~~~~~-~~~~~~~~~~~~~~~~~~~-~---A.23187 79.0 f 21.0 87.7 i: 4.4 0.25 uM (n=16) (n=8)
N.S.
p
N.S.
A.23187 6.25 nM
56.8 + 34.2 (n=16)
0.2 + 0.5 (n=7)
PAF 95 nM
56.1 f 22.0 (n=15)
45.4 + 28.2 (n=8)
N.S.
PAF 12 nM
76.0 f 23.1 (n=7)
35.2 f 22.0 (n=8)
p
p
During the aggregation of PRP, granule secretion was also appreciated by the measurement of ATP release. The release of ATP induced by collagen (0.44 pg/ml) was lower in the old people group than in young subjects (5.9 + 4.2 vs 12.5 f 5.4 nmoles ATP / lOSplatelets, p
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PLATELET FUNCTIONS AND
AGING
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TABLE 3 Metabolism of platelet endogenous arachidonic acid under thrombin stimulation (0.1 U/ml). Results are means k S.D. nmoles/lOg platelets
Old People
Young People
TXB2
1.09 f 0.52 0.65 zt 0.22 p
Conversion of platelet endogenous AA into the main oxygenated products TXBB, HHT and 12-HETE under thrombin stimulation is reported in table 3. The lipoxygenase product tended to decrease in the elderly compared yo young adults, although not significantly (TABLE 3). In contrast, HHT tended to increase and TXB2 was enhanced significantly but the sum of the two was not significantly higher in the elderly group. The sum of the three main metabolites, which can be grossly considered to represent the endogenous AA liberated from phospholipids, was equal in both groups (TABLE 3). At the opposite, cyclooxygenase products (TXBB, HHT) were significantly depressed in serum from elderly subjects and 12-HETE tended to decrease whilst the production of 6KPGFla was two fold higher (TABLE 4). Finally, the urinary excretion of TXBB, 6KPGFla and GKPGFla-M was significantly enhanced in the elder subjects versus the young adults and the same tendency could be observed for that 0f Tx~2-M (TABLE 5).
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TABLE Oxygenated metabolites means + S.D. nmoles/ml
337
PLATELET FUNCTIONS AND AGING
4
of arachidonic
Old
acid
in serum.
Young
People
Results
are
People
0.49 2 0.18 0.28 + 0.19 (n=6) (n=16) _____________---____~~~~~~~~~~~~~~~-~~~~~~~~~-~~~~~~--~~---_----1.15 f 0.36 0.55 f 0.36 HHT TXB2
p
p
_______________________!~~~~!_____________!~~~!_-----------__-_-1.68 + 0.35 0.83 f 0.54 (n=l6) (n=6) ~---------------~~~~~~~~~~~~~~~~-~~~~~~~------------_-___________ 1.47 ?: 0.57 0.95 f 0.65 12-HETE (n=16) (n=7) ----------------~~~~~~~~~~~~~~~-~~~~~~~~~~~~~~~-------___________ 3.23 + 0.94 1.79 f 1.15 TXBBtHHTtlB-HETE (n=16) (n=6) ~---------______~~--~---~------_~~~-~--~------~~~-------______--____________________~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~__~~~~~~ 13.3 ?: 8.1 27.2 2 14.8 6-keto-PGFla (n=6) (n=16) (pmoles/ml)
TXB2+HHT
TABLE Levels of some urinary prostanoids Results are expressed in ng/mmole Old People (n=l6)
p
N.S.
pxo.02
p=o.o5
5 in the two groups of age. of creatinine (mean ?: S.D.).
Young People (n=7)
TXB2 3.1 ?: 1.2 p
Plasma and platelet vitamin E were also measured and no difference could be found in plasma gamma and alpha tocopherol contents when both groups were compared (TABLE 6). In contrast, a significant decrease of these tocopherols was observed in platelets from elderly people (TABLE 6). Selenium, another parameter of the antioxidant status, was measured in the plasma. It was signicantly lower in the elderly people (34.5 f 11.9 Ir$/ml) than in young adults (64.3 + 19.7 yg/ml).
TABLE
6
Plasma and platelet tocopherol concentrations people. Results are means f S.D. Old Plasma
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PLATELET FUNCTIONS AND AGING
338
(nmoles/ml)
gamma-tocopherol
People (n=lO)
0.48
Young
in old
and
young
People
(n=8)
f 0. 14 ,,,%b 0.61
? 0.17
N.S.
alpha-tocopherol 10.3 f 2.8 11.2 ?: 3.5 N.S. ____________________~~~_~~~~~~-------------------------_-------Platelets (n=ll) (n=8) (nmoles/lOg platelets) gamma-tocopherol
0.05
+ 0.02
0.09
f 0.05
p
alpha-tocopherol
0.94
+ 0.21
1.41
f 0.55
p
DISCUSSION It is generally acknowledged that incidence of thrombotic complications increases with age suggesting alterations of the hemostatic balance in elderly people. In the present study, we have evaluated platelet aggregability in a group of aged subjects and another one of young adults. Various tests of aggregation indicate that platelet aggregability increases with age. Indeed, platelet aggregation studies using the classical turbidimetric method of Born (16) show that platelets from aged-people are more reactive in response to low concentrations of agonist, especially when they involve the AA metabolism in their mechanism of action. As a matter of fact, no significant difference was found in using ADP and in some way PAF acether which do not require thromboxane formation to induce platelet aggregation, at least at low reports concentrations (23). These findings agree with previous from Johnson et al (6) and Couch et al (7) indicating that platelet aggregability increased in the elderly population. We have also measured platelet aggregation in a more physiological situation such as the presence of all blood elements. The results obtained confirm the enhanced platelet aggregability in the aged individuals. We found that release of ATP induced by collagen was diminished in platelets from the elderly. This finding contrasts with the results from Kasjanovova and Balaz (24). These authors reported an elevation of the released ATP from elderly subjects. The same agonist (collagen) was used to initiate platelet release A possible explanation for reaction with similar concentrations. this discrepancy could be the age of the subjects who were only over 59 years versus over 78 years in our study. However, Zahavi et al (4) and Sie et al (5) have found an enhanced plasma B-thromboglobulin (B-TG) in old people (over 80 years). Platelet specific platelet protein to be released, factor 4 (PF4), another was also increased in plasma with age. In addition, platelets from elderly people are less dense and their mepacrin-labeled that ATP, granule content is decreased (5). We may then speculate
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like R-TG and PF4, would be discharged in the circulation due to platelet hyperactivity and that ex vivo further stimulation of those platelets would lead to lower release of their granule content. We have previously reported that exogenous AA metabolism, in particular its oxygenation via the cyclooxygenase and lipoxygenase pathways was increased in platelets from elderly the people (25). In the present study, we have investigated endogenous AA metabolism when thrombin was the stimulus. In agreement with the enhanced platelet aggregation observed, we found an increase of thromboxane B2 formation in elderly subjects. In aged rats, Giani et al (26) have also reported both increases of platelet aggregation and thromboxane formation. In we did not find such a difference in the production of contrast, 12-HETE and it was even decreased although not significantly. In study (27) showed that the accordance with these data, a previous lipoxygenase activity itself does not change with age but the production of 1X-HETE decreased subsequently to the reduction of glutathione peroxidase activity. The activity of this enzyme is selenium-dependent (28) and a recent report from Shoene et al (29) showed that platelets from selenium-deficient rats aggregated to a significantly higher rate than those from selenium-supplemented rats, and also produced more thromboxane content of platelets, but B2. We did not measure the selenium that of plasma was significantly reduced in elderly people, suggesting a mild deficiency which may be related to the alterations in the production of cyclooxygenase and lipoxygenase metabolites that we observed in our study. The increased thromboxane formation under stimulation with low concentrations of thrombin is in a good agreement with the increased TXB2 and TXBB-M in urine which may be considered to represent the basal production of the body. This also agrees with similar enhancement of 6KPGFla and GKPGFla-M excretion which has already been reported in aging (15) and believed to characterize a pre-thrombotic state with platelet activation in vivo (30). In contrast to these results observed under "basal" activation or in vitro weak activation, those obtained in serum, which results from a huge production of endogenous thrombin, revealed a decreased formation of oxygenated metabolites from AA. One possible explanation would be a reduced availability of AA from platelet phospholipids that we have found significantly depressed in these lipid compartments in the elderly (see the following (presumably providing from paper). The reason why serum 6KPGFla monocytes via PGH2 of platelet origin (31)) was increased in the elderly is not clear but cannot be taken into account in the endogenous AA availability because of its low amount. Finally, platelet but not plasma tocopherols were found significantly reduced in elderly people, in agreement with previous findings (32). This suggest a cellular hyper consumption of these antioxidants instead of a deficient state of nutritional origin. Like in experimentally deficient animals (33) and in diabetes (34), the enhanced platelet functions and oxygenated metabolism of AA might be associated with an increased "peroxide tonew of the cell closely related to a depletion of natural antioxidants, of which vitamin E is the marker in the present study.
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We conclude that platelet hyperactivity in the elderly, preferentially associated with the enhanced oxygenated metabolism of AA, might be linked to the antioxidant status / peroxide tone of the cell. ACKNOWLEDGEMENTS This work was for providing U 46619 gamma-tocopherol.
supported by INSERM. We thank Dr. J.E. Pike and Dr. 0. Hurstel who supplied tocol and
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