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Cis-unsaturated fatty acids and platelet function
Prostaglandins, Leukotrienes and Essential FattyAcids (1997) 57(4 & 5), 403-410 © HarcourtBrace& Co Lid 1997
Cis.unsaturated fatty acids and platelet function M. Mutanen Department of Applied Chemistry and Microbiology (Nutrition), PO Box 27, Fin-00014 University of Helsinki, Finland
Summary The main method to study platelet function in dietary studies has been the platelet aggregation test in vitro. Even though it is well established that dietary cis-unsaturated fatty acids (FAs) modify platelet aggregation some uncertainty still exists how to interpret the in vitro results in the context of a situation in vivo. The other ways to look at platelet activation are measurements of thromboxane metabolites in urine or the concentration of 13-thromboglobulin (I3TG) released from c~-granules. Dietary fish oil or long-chain n-3 FAs lower the high basal excretion rate of thromboxane, while only a modest effect is noticed at a low basal excretion rate. Results on the effects of other cis-unsaturated FAs on urinary TXB2 metabolites are almost totally lacking. Furthermore, platelet I3TG release in vivo does not seem to be affected by changes in dietary FAs. The regulatory function of dietary FAs in platelets is extremely complex, and clearly more should be understood about the association between dietary FAs and platelet membrane FAs in connection with platelet responses to physiological stimuli and subsequent signal transduction inside the platelets. ABBREVIATIONS
AA (C20:4n-6), arachidonic acid; ,ADP, adenosine-diphosphate; ALA (C18:3n-3), ~z-linolenic acid; ]3TG, ~-thromboglobulin; cAMP, cyclic 3',5'-adenosine monophosphate; DG, 1,2-diacylglycerol; DHA (C22:6n-3), docosahexaenoic acid; DP.A (C22:5n-3), docosapentaenoic acid; EP.A (C20:5n-3), eicosapentaenoic acid; F.A, fatty acid; GP IlbIIIa, glycoprotein IIb-IIIa; IP3, inositol 1,4,5-triphosphate; LA (C18:2n-6), linoleic acid; MG, 2-monoacylglycerol; MUFA, monounsaturated fatty acid; 0_4 (C 18: l n-9), oleic acid; PC, phosphatidyl choline; PGIa, prostacyclin; PIP2, phosphatidylinositol 4,5-biphosphate; PKC, protein kinase C; PLA2, phospholipase "A2;PLC, phospholipase C; PS, phosphatidyl serine; E%, percentage of energy; P/Sratio, ratio between polyunsaturated and saturated fatty acids; SAF.A,saturated fatty acid; TXA2, thromboxane Aa; vWF, von Willebrand factor. INTRODUCTION
The physiological function of platelets is to maintain vascular integrity and arrest bleeding. On the other hand, platelets are also involved in thrombosis and probably other pathological events such as inflammation and Correspondence to: Marja Mutanen, Tel. 00 358 9 708 5203; Fax. 00 358 9 708 5269; Email: Marja.Mutanen @helsinki.fi
asthma. Platelets take part in thrombosis by adhering to the exposed subendothelial connective tissue on a site of injury. The adhesion is followed by platelet activation, which results in aggregation and secretion of regulatory compounds. Platelets are activated only if an external stimulus, interacting with platelet surface glycoproteins or glycolipids, is able to transduce its signal inside the platelet. All the stimuli finally activate the same fibrinogen receptor (GP IIb-IIIa) which changes its conformation and binds fibrinogen, vWF or fibronectin. Even though the mechanism of GP IIb-IIIa activation is still obscure, it involves signalling pathways in both directions across the plasma membrane. Thus the metabolism and F,A composition of platelet phospholipids are emphasized. -Activation of GP Iib-IIIa starts the process of platelet aggregation, which includes changes in the association of GP IIb-IIIa with the cytoskeleton. Second messengers produced by activated platelets are important regulators in this process, which finally leads to clustering of GP Ilb-IIIa molecules and fibrinogen binding to the plasma membrane.l'2 The tightly packed fibrinogen clusters enable the unactivated, newly arriving platelet to stick to them without any further prerequisites. 3 The effects of dietary F_Ason platelet function are far from pharmacological. F,Ascould thus participate in intracellular signalling pathways which regulate either the activation and binding function of GP IIb-IIIa or subse403
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quent signal transduction. The main method to study platelet activation has been the platelet aggregation test, probably due to the fact that changes in dietary FAs often change platelet responses in vitro. The question is, however, how to interpret the results in the context of a situation in vivo. Agonist-induced in vitro aggregation does not measure platelet activation per se, but the whole cascade from the activation to final platelet plug formation. To put platelet aggregation into perspective with regard to other phases of platelet activation, the speed of the early activation should be kept in mind; it takes place in less than 1 s, and even secretion may be completed within 5 s. The major biochemical events, i.e. lipid hydrolysis and protein phosphorylations, are initiated and completed within this period. The later biochemical changes are probably related to the consolidation of platelet aggregates. 3 In this review, the data obtained from dietary studies are presented in the light of current biochemical data on FAs as regulators of platelet function. However, several still obscure issues remain. At a molecular level, it is not clear how different physiological agonists mediate their effects on the activation of GP IIb-IIIa. Furthermore, it is not known if dietary FAs are able to change platelet membrane characteristics to favor GP IIb-IIIa activation. Finally, the significance of the availability and composition of free FAs inside the platelet for signalling systems to proceed is unclear. Platelet activation involves intracellular signalling processes
Protein phosphorylations are central in intracellular signal processes. In platelets, three main categories of protein phosphorylations can be distinguished: Ca2+-dependent, DG-dependent, and cAMP-dependent. One of the early responses to most physiological agonists is the PLCcatalyzed hydrolysis of membrane PIP2 to DG and IP3. Activation of PKC is a prerequisite for platelet shape change, 4,5 and the synergistic action of intracellular Ca 2+ and PKC activation is usually needed to produce platelet aggregation and release reactions. 6 PKC also causes serine/threonine phosphorylation of GP IIIa, and may thus be involved in early activation of GP IIb-IIIa. 7 PKC is a family of closely related isoenzymes, which are grouped into three classes: Ca2+-dependent, Ca 2+independent, and atypical torms. To-date, six isoenzyrnes of PKC have been identified in human platelets. 8 The basic mechanism for PKC activation includes translocation of inactive PKC from the cytosol to the plasma membrane, where DG activates it by increasing PKC affinity for Ca 2+ and PS. The optimum activity of the membranebound PKC is regulated by the level of bilayer unsaturation, especially in PC. 9 Studies with intact platelets or of
in vitro enzymatic reactions show that DG- and Ca 2+dependent activation of PKC is enhanced by cis-unsaturated FAs esterified at the sn-2-position of membrane phospholipids. Saturated and trans-unsaturated FAs are inactive. Furthermore, cfs-unsaturated FAs liberated from PC by action of PLA2 may further activate PKC by increasing its sensitivity to Ca2+.1°-12 The in vitro results with cis-unsaturated FAs, however, indicate that activation of PKC may occur in two compartments. It seems that when membrane-bound PKC is activated by DG, cis-unsaturated FA, especially AA, activates soluble PKC when both FA and enzyme are not membrane-associated. In the cytosolic fraction, cis-unsaturated FA activates predominantly Ca2+-independent isoenzymes of PKC. 13 The physiological implications of the PKC activation by cis-unsaturated FAs is not clear at present. It has been suggested that in stimulated platelets, PKC, once initially activated by PIP2 hydrolysis, may sustain its enzymatic activity even after Ca 2+ concentration returns to basal level, when both DG and cis-unsaturated FAs are available. 12 Furthermore, the evidence of relation between PKC activation and platelet desensitization TM indicates that endogenously generated free FAs and Ca 2+independent PKC may participate in negative feedback during platelet activation. The availability of DG has a pivotal role for PKC activation in membranes. Because DG is released by PLC, the activation of PLC is also crucial for the PKC activity. AA metabolites, mainly TXA2, stimulate the activation of PLC in platelets. AA is released from PI and PC by the action of PLC and PLA2, respectively, and also from DG by combined actions of DG- and MG-lipases. PLC is inhibited by cAMP, a molecule that antagonizes all the activation events in platelets. Agonists - such as PGI2 - which raise platelet cAMP level, activate cAMP-dependent protein kinases. Also 5' phosphomonoesterase which converts IP3 to IP2 and, as a consequence, inactivates the IP3 signal for Ca ~+release, inhibits platelet activation. PKC may activate 5' phosphomonoesterase and regulate by feed-back inhibition the Ca2+-pathway of platelet activation. PKC may also abrogate PLC-mediated PIP2 hydrolysis, the biochemical event which initiates its own activation. 15 Furthermore, activators of PKC have been shown to enhance PLA2-mediated release of AA in platelets. ~6 Recent data, however, revealed a G-protein-mediated mechanism for the activation of PLA2 in intact h u m a n platelets which is negatively affected by PKC. lz These varying results may be explained by the diversity of the cytosolic PLA2 enzymes, which are divided into AA selective and non-selective enzymes. The activation mechanisms of these different types of cytosolic PLAa are not clear at the moment. ~°In addition, separate groups of PKC isoenzymes are probably involved in different aspects of platelet activation. 8
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Cis-unsaturated fatty acids and platelet function
Intracellular signalling by physiological platelet agonists used in dietary intervention studies The physiological platelet agonists used frequently in studies related to dietary interventions are thrombin, collagen, ADP, and adrenaline. They all stimulate a variety of responses in the platelet including shape change, GP IIbIlia receptor exposure (aggregation), granule secretion, and liberation of AA. Intracellular signals induced by agonists include: (i) signals causing GP IIb-IIIa activation and binding of fibrinogen; and (ii) downstream signals which result in aggregation and release reactions. Neither of these pathways is fully understood but the activation of PKC is probably a necessary component shared by most, if not all, agonists in these pathways. TM Thrombin activates directly both PIand PC-specific PLC, 19 while collagen, in addition to activating PLC, strongly activates PLA2 to liberate free FAs including AA. Thrombin and collagen induced stimulations rapidly increase the turnover of PIP2 to DG and IP3, and aggregation and secretion are associated dose dependently with the accumulation of DG in platelets. 2° Platelets contain several PLC isoenzymes and PLC-[~, which is coupled to G-proteins, is activated by thrombin and TXA2, while the mechanism of action of collagen involves tyrosine phosphorylation of PLC-72 and not G-proteins. 2~,22 TXA2, produced from liberated AA, is the actual stimulus for secretion induced by low doses of collagen in addition to its ability to activate PLC. Thrombin and high-doses of collagen can activate PKC and stimulate release reaction without TXA2 productionY Recent observations indicate that the activation of PKC is not a prerequisite for phosphorylation of cytosolic PLA2 during thrombin and collagen stimulation. 24 Weak agonists may activate platelets to expose GP IIbIIIa receptors along with intracellular Ca 2+ independently on the inositol phosphate pathway. Actually, GP IIb-IIIa exposure induced by ADP is independent of the activation of PKC, even though PKC activation is required for ADP secretionY Also primary ADP-induced aggregation (no secretion or AA release) is not dependent on the formation of DG or the activation of PKCY A decrease in PIP2 is, however, an early event in the ptatelet response to ADP. The equilibrium between PIP2 and PIP may cause mobilization of some Ca2+,even though there is no phosphorylation of P40 indicating that PKC is not activated. Phosphorylation of myosin light chain (P20), which is essential for contractile activity of the platelet, is primarily dependent upon increased cytoplasmic Ca2+.ADP causes a much more rapid increase of intracellular Ca 2+than other agonistsY Platelet fatty acids There are at least two mechanisms for FAs to regulate platelet function: membrane FA composition, and free FA © Harcourt Brace & Co Ltd 1997
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liberated from membranes and intermediates of inositol phosphate pathway (PIP2, DG, PA) during stimulation. Platelet FA composition originates from the megacaryocytesY PC is the most abundant phospholipid, and nearly 14% of its FAs is AA, almost exclusively located in the sn-2 position. PE and PI have the highest percentages of AA (ca. 37°/o and 35%, respectively), but from the actual mass of FAs, PE provides almost 25% of total AA, PC over 50% and PI only 5-6o/o. EPA selectively incorporates into PC and PE and not into PI.29 The origin of free FAs upon stimulation by PLA2 is mainly PC and probably PE. AA liberated by action of PLC and DG-lipase is less than 10% of the released AA during stimulation. 3° Due to low delta-6-desaturase activity, hardly any LA is converted into AA in intact platelets. In vitro incubations, however, indicate that AA and other FAs can enter into platelet phospholipids through de novo synthesis or 'remodelling' pathways. 3~ Platelets contain an AA specific synthase probably to ensure an adequate incorporation of AA in platelet phospholipids and also to keep free AA content low inside the platelet. The significance of the de novo synthesis and 'remodelling' pathways in vivo is not clear at present. Administration of 6 g A_A/day did not affect platelet function (drop in threshold ADP) until after 10-12 days, indicating that the regeneration of platelet population was a prerequisite for the change in platelet function? 2 Similarly, 6 g EPA/day showed that even though EPA appeared in plasma phospholipids already after 4 h, it did not incorporate into platelet phopholipids until day 6.33 In human platelets, the fatty acid composition of total lipids does not reflect changes in dietary FAs very well. When a high-SAFA diet was replaced by a diet containing a high amount of MUFA, the OA level in platelet phospholipids increased only modestly34or did not change. 35,36 Concurrently, LA either increased, 36 decreased 34 or remained constantY The main feature in dietary studies with n-6 PUFA is the constant proportion of AA in platelet total phospholipids with considerably different LA intakesY -39 In tissue phospholipids A,~ appears to be resistant to dietary changes even wher~ the amount of dietary LA is lowered to 1.8 E°/o.39 The [,roportion of LA in platelet phospholipids is increased when the intake of n-6 PUFA increases. The increase of LA is very modest even with high dietary LA intakes a n ] it does not correlate with dietary LA?9 When the intake of LA is very high (> 10E%), platelet AA may slightly decreaseY ,4° Consumption of ethyl arachidonate (6 g/day) increased AA in platelet phospholipids at the expense of LA,32 while the daily intake of 350 mg AA only significantly decreased LA.41The decreases in LA or AA after diets with long-chain n-3 PUFA are compensated with increases of EPA, DPA, and DHA in platelet lipids. 42-44 Also dietary ALA increases EPA and DPA in platelet FAs45,46and corre-
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lates positively with platelet ALA and EP& but negatively with DHA. 39 FA composition of platelet total phospholipids is not very informative if the aim is to understand the effect of the FAs on intracellular signalling systems. FAs of platelet phospholipid subclasses are regulated with a high degree of specificity in vivo, which obviously has a major role during platelet activation. Furthermore, in vitro studies indicate that significant remodelling between individual phospholipid subclasses takes place when platelets are stimulated. 47 How relevant this is in vivo is not clear at present. Studies on the incorporation of cis-unsaturated FAs of vegetable origin into individual platelet phospholipids are few. OA has been found to incorporate into the PC fraction in two studies with high-OA diets 48'49 and it also increased in the PE fraction in one study. 49 Furthermore, an increase of C20:ln-9 in the PC fraction has been found after a diet high in OA.48 High-LA diets increase LA in platelet PC and particularly in PE.48-5° Sometimes high dietary LA has also been found to lead to incorporation of AA into PE but not into PC. 48-49 Long-chain n-3 PUFA supplementation increased the incorporation of EPA, DPA, and DHA into platelet PC and PE but not into PI and PS in one studyY Small but significant incorporation of EPA into PS was, however, found in another study. 51 Furthermore, during prolonged supplementation, longchain n-3 FAs have also been found in P152 even though not always. 29 Increased n-3 FAs are often compensated with decreased AA in PE and PC 29'51 but not always. 52 Sometimes LA increases with increased incorporation of n-3 FAs into PE and PC. 52 Increased EPA level in PE and PC can also be seen after high-ALA diets. 48,49 Platelet aggregation, arachidonic acid metabolism, and granule secretion The most abundant cis-unsaturated FAs in h u m a n diets are OA and LA. Quantitatively minor FAs are AA, ALA and n-3 FAs of marine origin. Not much effort has been made to understand how FAs of vegetable origin or dietary AA regulate platelet function, while h u m a n experiments with n-3 FAs of marine origin are abundant. A very general shortcoming has been a lack of information on the FA composition of individual platelet phospholipids, which is crucial in understanding the mode of combined action of an agonist and dietary FAs. In addition, little is known about the role of the baseline diet with respect to the incorporation of FAs into platelets. Low power of the study designs and a poor methodological approach are also very common and make the interpretation of the results difficult. Furthermore, evidence from animal 53 and h u m a n 32 studies suggest that a confounding factor in human studies may be the uncontrolled intake of AA.54
Thrombin has been used as an agonist in several interventions with long-chain n-3 FAs.55,56In the eight studies reported by Malle and Kostnery decreased platelet aggregation was seen in three and no change in five studies. The results are apparently independent of the total amounts of n-3 FAs (EPA 1.6-3.8 g/day and DHA 0.25-3.0 g/day) and of the ratio between EPA and DHA. Thrombin-induced aggregation was also low in farmers having low dietary SAFA vs PUFA in two studies, 37,57 while in one study no difference was found even though the difference between dietary P/S-ratio was marked (0.26 vs. 0.59). 59 However, changing the P/S-ratio from 0.32 to 0.97 for one year decreased thrombin-induced aggregation significantly. 6° In one of these studies, decreased thrombin-induced aggregation paralleled decreased plasma ratio of AA/LA.57 LA probably has a specific effect on platelet function, since increasing the ratio of LA/ALA from 2.8 to 28 and keeping the diet constant in other respects increased platelet aggregation significantly? 8 Based on studies carried out with thrombin, there is no good insight into the possible effect of dietary FAs. One reason may be the strength of thrombin as an agonist. The sensitive concentration range is very narrow, which may weaken the detection of differences between treatments. Collagen has been used in dietary interventions more frequently than thrombin. Studies concerning n-3 FAs of marine origin 55,s6 have fairly consistently shown either no effect or decreased aggregation pattern. Aggregation seems to be similarly affected by both EPA and DHA. 33 The amount of n-3 PUFA and the length of the supplementation period may increase the variation in the results. Tremoli et a144 found long-lasting impairment especially in collagen-induced platelet aggregation after n-3 PUFA supplementation. The AA content in fish otis may differ and affect the efficiency of n-3 FAs.54Variation in n-3/n-6 FA status of individual phospholipids due to the differences in background diet may also be marked. High-ALA (8-16 E%) supplementation also decreased collagen-induced aggregation when compared with baseline diets, 45,~1 while reasonable amounts of ALA from canola or rapeseed otis had no effect.34,58,~2 When the effect of ALA was compared with that of n-3 FAs from marine origin, the reduction of collagen-induced aggregation appeared about similar. 4¢~3 The lower availability of substrate FA for TXA2 production probably explains frequently found reduced collagen aggregation after n-3 FAs. This does not explain the results on cis-unsaturated FAs of vegetable origin. The increased intake of unsaturated FAs is not associated with decreased aggregation, but quite often the opposite trend is found. The underlying mechanism probably involves intracellular signals which differ between n-6 and n-3 FAs. Some 35,58,64,65 but not all34,57,59,62,63 studies show
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increased collagen sensitivity of platelets after diets with considerable amount of PUFA when compared with diets high in SAFA. Only one study shows a decreased collagen sensitivity associated with low SAFA i n t a k e s An increase in aggregation in response to collagen was found also when a high-LA diet was compared with a high-OA diet? ~ This was not confirmed by three other studies, 34,35,~2 however. In vegan platelets, the proportion of LA was higher and that of AA lower when compared with omnivore controls, but no difference in collagen-induced aggregation was found. ``° The results from ADP-induced aggregations in dietary studies vary probably most of all. Decreases, increases and 'no effects' are all common with long-chain n-3 FAsY ,56 As reviewed by Hornstra 55 only studies classified as 'poorly controlled' showed enhanced aggregation. However, in our latest partially controlled intervention study, EPA and DHA supplementation increased ADPinduced aggregation, while similar amounts of ALA had no effect during the supplementation. ALA increased, however, ADP induced aggregation during the follow-up p e r i o d s ADP-induced aggregation was not changed or it decreased in two other studies with ALA.4~,61 When purified EPA or DHA was supplemented in healthy volunteers, reduced ADP aggregation was associated only w i t h DHA. 33 When high-LA diets (P/S-ratio between 0.7 and 1.0) were compared with either high-fat diets rich in SAFA35'~8'~° or rich in ALA,58 ADP-aggregation was significantly enhanced. On the other hand, it has been postulated that with moderate dietary LA intakes (P/S-ratio between 0.6 and 0.8) LA may be associated with impaired ADP-induced aggregationF After a cereal-based low-fat diet, ADP-induced aggregation was not affected by increased LA intake. ~3 Nor was the very high LA intake of vegans (P/S-ratio 1.48) associated with a higher ADP response than in omnivores (P/S-ratio 0.34). 40 Furthermore, OA and LA rich diets similarly enhanced ADP-induced aggregation when compared with the baseline milkfat diet. 35 Taken together, the effect of dietary FAs on ADP aggregations are very inconsistent. The intracellular signals induced by ADP are the least well understood, and regulatory functions of FAs probably arise from complex interactions between amounts and types of different FAs. During platelet in vivo activation TXA2 is always formed. 2,3-dinor-TXB2 and l l-dehydro-TXB2 are two main TXA2 metabolites found in urine and measured as in vivo indices of platelet TXAa formation. Excretion of 2,3-dinor-TXB2 is probably a more sensitive marker than the urinary level of 1 1-dehydro-TXB2.68 Dietary fish oil or long-chain n-3 FAs lower the high basal excretion rate of TXB2 very dramatically, while only a modest effect is noticed at a low basal excretion rate. The underlying mechanism is the replacement of AA with EPA and DHA © Harcourt Brace & Co Ltd 1997
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in platelet phospholipids and thus a sufficiently long supplementation period is required. 33,44,52,69,7°,71The efficiency of long-chain n-3 FAs in reducing the excretion of TXA2 metabolites may be slightly improved if dietary SAFA intal~e is low. 72 Results on the effects of other cisunsaturated FAs on urinary TXB2 metabolites are almost totally lacking. There are some indications that dietary LA or AA might regulate this excretion.41,73 Another way to look at platelet activation in vivo is to measure the concentration of [~TG released from (zgranules. The plasma level of this platelet-specific protein has not changed in supplementation studies with long-chain n-3 FAs. 43'55 Urinary excretion of high molecular weight [~TG, which presumably contains intact [3TG, has been measured in three human experiments at our department. Increased intake of ALA or EPA + DHA had no effect in one study. 4~ Nor was the level affected by high OA or LA diets (Turpeinen A. M., unpublished observation) or by high stearic or elaidic acid dietsY These results indicate that dietary FAs are not powerful enough to affect platelet granule release reactions in vivo. CONCLUSIONS
Platelet activation in vivo is an extremely complex event with involvement of FAs. Platelet aggregation tests in vitro may give very little information on the relationship between dietary FAs and platelet function in vivo, unless other parameters such as individual phospholipid FA composition and measurement of intracellular signalling are also measured. Unfortunately, this has not been very common in dietary studies. In addition, the results from an early filtragometer study in vivo, comparing saturated and polyunsaturated FAs, further support the limitations of in vitro aggregation test. 74 The total amount of a given FA in the platelet is probably not crucial but merely the factor which regulates free FA levels and types in the membrane and in platelet interior. Furthermore, platelet receptor responsiveness to physiological stimuli and subsequent signal transduction may be highly dependent on membrane FA c o m p o s i t i o n , zS-z7 A n important participant in the activation of human platelets is PKC. The physiological significance of the observation that the activation of PKC depends on cis-unsaturated FAs is, however, not understood. Well-controlled dietary studies with careful measurements of different aspects of platelet activation are clearly needed to understand what kind of diet is optimal to keep platelets 'healthy'. REFERENCES
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21. Blake R. A., Schieven G. L., Watson S. P. Collagen stimulates tyrosine phosphorylation of phospholipase C-72 but not phospholipase C-71 in human platelets. FEBS Lelt 1994; 333: 212-216. 22. Daniel J. L., Dangelmaier C., Smith J. B. Evidence for a role of tyrosine phosphorylation of phospholipase C72 in collageninduced platelet cytosolic calcium mobilization. Biachem J 1994; 302: 617-622. 23. Sano K., Takai Y., YamanishiJ., Nishizuka Y. A role of calcinm-activated phospholipid-dependent protein kinase in human platelet activation. J Biol Chem 1983; 23~: 2010-2013. 24. B6rsch-Haubold A. G., Kramer R. M., Watson S. P. Cytosolic phospholipase A2 is phosphorylated in collagen- and thrombinstimulated human platelets independent of protein kinase C and mitogen-activated protein kinase. J BioI C-hem 1995; 270: 25885-25892. 25. Pulcinelli F. M., Ashby B., Gazzaniga P. P., Daniel J. L. Protein kinase C activation is not a key step in ADP-mediated exposure of flbrinogen receptors on human platelets. FEBS Lett 1995; 364: 87-90. 26. Packham M. A., Livine A-A., Ruben D. H., Rand M. L. Activation of phospholipase C and protein kinase C has little involvement in ADP-induced primary aggregation of human platelets: effects of diacylglycerols, the diacylglycerol kinase inhibitor R59022, staurosporine and okadaic acid. Biochem J 1993; 290: 849-856. 27. Rink T. J., Sage S. O. Calcium signalling in human platelets. Ann Rev Physiol 1990; 5 2 : 4 3 1 - 4 4 9 . 28. Schick B. P., Schick P. K., Chase P. R. Lipid composition of guinea pig platelets and megakaryocytes. The megacaryocytes as a probable source of platelet lipids. Biochem Biophys Acta 1981; 663: 239-248. 29. von Schacky C., Siess W., Fischer S., Weber P. C. A comparative study of eicosapentaenoic acid metabolism by human platelets in vivo and in vitro. J Lipid Res 1985; 26: 457-464. 30. Mauco G. Phospholipids: release of arachidonate for prostaglandin and thromboxane synthesis. In: Holmsen H, ed. Platelet Responses and Metabolism, Vol III: ResponseMetabolism Relationships. Florida: CRC Press, 1987:101-116. 31. Holub B. J. Altered phospholipid metabolism in thrombinstimulated human platelets. Can J Biochem Cell Biol 1984; 62: 341-351. 32. Seyberth H. W., Oelz O., Kennedy T., et al. Increased arachidonate in lipids after administration to man: effects on prostaglandin biosynthesis. Clin Pharmacol Ther 1975; 18:521-529.
33. von Schacky C., Weber P. C. Metabolism and effects on platelet function of the purified eicosapentaenoic and docosahexaenoic acids in humans. J Clin Invest 1985; 76: 2446-2450. 34. Kwon J.-S., Snook J. T., Wardlaw G. M., Hwang D. H. Effects of diets high in saturated fatty acids, canola off, or safflower oil on platelet function, thromboxane B2 formaton, and fatty acid composition of platelet phospholipids. Am J Clin Nutr 1991; 34:351-358.
35. Mutanen M., Freese R., Valsta L. M., Ahola I., Ahlstr6m A. Rapeseed oil and sunflower oil diets enhance platelet in vitro aggregation and thromboxane production in healthy men when compared with milk fat or habitual diets. Thromb Haemostas 1992; 67: 352-356. 36. Sarkkinen E. S., Agren J. J., Ahola I., Ovaskainen M-L., Uusitupa M. I. J. Fatty acid composition of serum cholesterol esters, and erythrocyte and platelet membranes as indicators of long-term adherence to fat-modified diets. A m J Clin Nutr 1994; 39: 364-370.
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37. Renaud S., Morazain R., Godsey F., et al. Nutrients, platelet function and composition in nine groups of French and British farmers. Atherosclerosis 1986; 60:37-48. 38..Agren J. J., T0rm/il/i M-L., Nenonen M. T., H~nninen O. O. Fatty acid composition of erythrocyte, platelet, and serum lipids in strict vegans. Lipids 1995; 30: 365-369. 39. Mantzioris E., James M. J., Gibson R. A., Cleland L. G. Differences exist in the relationship between dietary linoleic and cMinolenic acids and their respective long-chain metabolites. Am J Clin Nutr 1995; 61: 320-324. 40. Sanders T. A. B., Roshanai F. Platelet phospholipid fatty acid composition and function in vegans compared with age- and sex-matched omnivore controls. Eur J CEn Nutr 1992; 46:823-831.
41. Sinclair A. J., Mann N. J. Short-term diets rich in arachidonic acid influence plasma phospholipid polyunsaturated fatty acid levels and prostacyclin and thromboxane production in humans. JNutr 1996; 126:1110S-1114S. 42. Dyerberg J., Bang H. O. Haemostatic function and platelet polyunsaturated fatty acids in Eskimos. Lancet 1979; it: 433-435. 43. Schmidt E. B., Lervang H-H., Varming K., Madsen P., Dyerberg J. Long-term supplementation with n-3 fatty acids, I: effect on blood lipids, haemostasis and blood pressure. Scandy Clin Invest 1992; 52: 221-228. 44. Tremoli E., Maderna P., Marangoni F., et at. Prolonged inhibition of platelet aggregation after n-3 fatty acid ethyl ester ingestion by healthy volunteers. A m J Clin Nutr 1995; 61: 607-613. 45. Allman M. A., Pena M. M., Pang D. Supplementation with flaxseed oil versus sunflowerseed oil in healthy young men consuming a low fat diet: effects on platelet composition and function. Eur J Clin Nutr 1995; 49:169-178. 46. Freese R., Mutanen M. N-3 Alpha-linolenic acid and marine long-chain n-3 fatty acids only slighter differ in their effects on hemostatic factors in healthy subjects. Am J Clin Nutr (in press). 47. Croset M. Incorporation and turnover of eicosapentaenoic and docosahexaenoic acids in human blood platelets in vitro. Biochem J 1992, 281: 309-316. 48. Weaver B. J., Corner E. J., Bruce V. M., McDonald B. E., Holub B.J. Dietary canola off: effect on the accumulation of eicosapentaenoic acid in the alkenylacyl fraction of human platelet ethanolamine phosphoglyceride. Am J Clin Nutr 1990, 51: 594-598. 49. Chan J. K., McDonald B. E., Gerrard J. M., Bruce V. M., Weaver B.J., Holub B. J. Effect of dietary cMinolenic acid and its ratio to linoleic acid on platelet and plasma fatty acids and thrombogenesis. Lipids 1993; 28:811-817. 50. Dougherty R. M., Galli C., Ferro-Luzzi A., Iacono J. M. Lipid and phospholipid fatty acid conlposition of plasma, red blood cells, and platelets and how they are affected by dietary lipids: a study of normal subjects from Italy, Finland, and the USA. A m J Clin Nutr 1987; 45: 443-455. 51. Mori T. A., Codde J. P., Vandongen R., Berlin L. J. New findings in the fatty acid composition of individual platelet phospholipids in man after dietary fish oil supplementation. Lipids 1987; 22: 744-750. 52. Prisco D., Filippini M., Francalanci I., Paniccia R, Gensini G. F., Serneri G. G. N. Effect on n-3 fatty acid ethyl ester supplementation on fatty acid composition of a single platelet phospholipids and on platelet functions. Metabolism 1995; 44: 562-569. 53. Steel M. S., Naughton J. M., Hopkins G. W,, Sinclair A. J., O'Dea K. Arachidonic acid supplementation dose-dependently reverses the effects of a butter-enriched diet in rats. Prostaglandins LeukotEssentFattyAcids 1993; 48: 247-251.
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54. Li B., Birdwell C., Whelan J. Antithetic relationship of dietary arachidorfic acid and eicosapentaenoic acid on eicosanoid production in vivo. JLipfd Res 1994; 35: 1869-1877. 55. Hornstra G. The significance offish and fish-oil enriched food for prevention and therapy of ischaemic cardiovascular disease. In: Vergroesen A. J., Crawford M. (Eds.): The Role of Fats in Human Nutrition. 2nd ed. London: Academic Press, 1989:151-235. 56. Malle E., Kostner G. M. Effect of fish oils on lipid variables and platelet function indices. Prostaglandins Leukot Essent Fat{y Acids 1993; 49: 645-663. 57. Renaud S., Morazain R., Godsey F., et al. Platelet functions in relation to diet and serum lipids in British farmers. Br HeartJ 1981; 46: 562-570. 58. Freese R., Mutanen M., Valsta L. M., Salminen I. Comparison of the effects of two diets rich in monounsaturated fatty acids differing in their linoleic/c~dinolenic acid ratio on platelet aggregation. Thromb Haemostas 1994; 71: 73-77. 59. Renaud S., De Backr G., Thevenon C., et al. Platelet fatty acids and function in two distinct regions of Belgium: relationship to age and dietary habits. J Intern Med 1991; 229: 79-88. 60. Renaud S., Godsey F., Dnmont E., Thevenon C., Ortchanian E., Martin J. L. Influence of long-term diet modification on platelet function and composition in Moselle farmers. A m J Clin Nutr 1986; 43: 136-150. 61. Adam O., Wolfram G., Z611ner N. Vergleich der Wirkung yon Linolens~ure und Eicosapentaens~inre auf die Prostaglandinbiosynthese und Thrombozytenfunktion beim Menchen. KEn Wochenschr 1986; 64: 274-280. 62. McDonald B. E., Gerrard J. M., Bruce V. M., Corner E. J. Comparison of the effect of canola oil and sunflower oil on plasma lipids and lipoproteins and on in vivo thromboxane *42 and prostacyclin production in healthy young men. Am y Clin Nutr 1989; 50: 1382-1388. 63. Indu M., Ghafoorunissa. n-3 fatty acids in Indian diets comparison of the effects of precursor (alpha-linolenic acid) vs. product (long chain n-3 polyunsaturated fatty acids). Nutr Res 1992; 12: 569-582. 64. Mntanen M., Krusius T., R~is~nen L., Freese R., Vahtera E., Viikari J. S. A. Habitual diet, platelet function, fibrinogen and factor VII coagulant activity in young Finns. JIntern Med 1995; 237: 577-583. 65. Turpeinen A. M., Aro A., Mutanen M. Platelet function, coagulation and fibrinolysis in health subjects consuming diets rich in stearic acid or trans fatty acids. ETRO/ISSFALMeeting: Lipids Membranes and Thrombosis, July 10-13, 1996, Maastricht, The Netherlands. (abstract). 66. Sirtori C. IL, Tremoli E., Gatti E., et al. Controlled evaluation of fat intake in the Mediterranean diet: comparative activities of olive off and corn off on plasma lipids and platelets in high-risk patients. A m J Clin Nutr 1986; 44: 635-642. 67. Renaud S. Linoleic acid, platelet aggregation and myocardial infarction. Atherosderosis 1990; 80: 255-256. 68. Lorenz R. L., Uedelhoven W. M., Fischer S., Ruetzel A., Weber P. C. A critical evaluation of urinary immunoreactive thromboxane: feasibility of its determination as a potent vascular risk indicator. BBA 1989; 993: 259-265. 69. von Schacky C., Fischer S., Weber P. C. Long-term effects of dietary marine m-3 fatty acids upon plasma and cellular lipids, platelet function, and eicosanoid formation in humans. J Clin Invest 1998; 76: 1626-1631. 70. Ferretti A, Judd J. T., Taylor P. K, Nair P. P., Flanagan V. P. Ingestion of marine oil reduces excretion of 11dehydrothromboxane B:, an index of intravascular production of thromboxane A2. Prostaglandins Leukot Essent Fatty Acids 1993; 48: 305-308.
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71. Prakash C., Nelson G. J., Wu M-M., Schmidt P. C., Phillips M. A., Blair I. A. Decreased systemic throlnboxane A2 biosynthesis in normal human subjects fed a salmon-rich diet. AmJ Clfn Nutr 1994; 60: 369-373. 72. Nordoy A., Hatcher L., Goodnight S., FitzGerald G. A., Connor W. E. Effects of dietary fat content, saturated fatty acids, and fish off on eicosanoid production and haemostafic parameters in normal men. JLab Clin Med 1994; 123: 914-920. 73. Blair I. A., Prakash C., Phillips M. A., Dougherty R. M., Iacono J. M. Dietary modification of o)6 fatty acid intake and ffs effect on urinary eicosanoid exrefion. Am J Clin Nutr 1993; BT: 154-160. 74. Hornstra G., Lewis B., Chait A., Turpeinen O., Karvonen M. J., Vergroesen A. J. Influence of dietary fat on platelet function in man. Lancet 1973; i: 1155-1157.
75. Heemskerk W. M., Feijge M. A., Kester A., Hornstra G. Dietary fat modifies thromboxane A2-induced stmulation of rat platelets. Bfochem J 1991; 278: 399-404. 76. Heemskerk W. M., Feijge M. A., Simonis M. A. G., Hornstra G. Effects of dietary fatty acids on signal transducfion and membrane cholesterol content in rat platelets. BBA 1995; 12BB: 87-97. 77. Schenrlen M., Fdrchner M., Clemens M. R., Jaschonek K. Fish oil preparations rich in docosahexaenoic acid modify platelet responsiveness to prostaglandinendoperoxide/thromboxane A2 receptor agonists. Biochem Pharmaco11993; 46: 245-249.
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Cis.unsaturated fatty acids and platelet function M. Mutanen Department of Applied Chemistry and Microbiology (Nutrition), PO Box 27, Fin-00014 University of Helsinki, Finland
Summary The main method to study platelet function in dietary studies has been the platelet aggregation test in vitro. Even though it is well established that dietary cis-unsaturated fatty acids (FAs) modify platelet aggregation some uncertainty still exists how to interpret the in vitro results in the context of a situation in vivo. The other ways to look at platelet activation are measurements of thromboxane metabolites in urine or the concentration of 13-thromboglobulin (I3TG) released from c~-granules. Dietary fish oil or long-chain n-3 FAs lower the high basal excretion rate of thromboxane, while only a modest effect is noticed at a low basal excretion rate. Results on the effects of other cis-unsaturated FAs on urinary TXB2 metabolites are almost totally lacking. Furthermore, platelet I3TG release in vivo does not seem to be affected by changes in dietary FAs. The regulatory function of dietary FAs in platelets is extremely complex, and clearly more should be understood about the association between dietary FAs and platelet membrane FAs in connection with platelet responses to physiological stimuli and subsequent signal transduction inside the platelets. ABBREVIATIONS
AA (C20:4n-6), arachidonic acid; ,ADP, adenosine-diphosphate; ALA (C18:3n-3), ~z-linolenic acid; ]3TG, ~-thromboglobulin; cAMP, cyclic 3',5'-adenosine monophosphate; DG, 1,2-diacylglycerol; DHA (C22:6n-3), docosahexaenoic acid; DP.A (C22:5n-3), docosapentaenoic acid; EP.A (C20:5n-3), eicosapentaenoic acid; F.A, fatty acid; GP IlbIIIa, glycoprotein IIb-IIIa; IP3, inositol 1,4,5-triphosphate; LA (C18:2n-6), linoleic acid; MG, 2-monoacylglycerol; MUFA, monounsaturated fatty acid; 0_4 (C 18: l n-9), oleic acid; PC, phosphatidyl choline; PGIa, prostacyclin; PIP2, phosphatidylinositol 4,5-biphosphate; PKC, protein kinase C; PLA2, phospholipase "A2;PLC, phospholipase C; PS, phosphatidyl serine; E%, percentage of energy; P/Sratio, ratio between polyunsaturated and saturated fatty acids; SAF.A,saturated fatty acid; TXA2, thromboxane Aa; vWF, von Willebrand factor. INTRODUCTION
The physiological function of platelets is to maintain vascular integrity and arrest bleeding. On the other hand, platelets are also involved in thrombosis and probably other pathological events such as inflammation and Correspondence to: Marja Mutanen, Tel. 00 358 9 708 5203; Fax. 00 358 9 708 5269; Email: Marja.Mutanen @helsinki.fi
asthma. Platelets take part in thrombosis by adhering to the exposed subendothelial connective tissue on a site of injury. The adhesion is followed by platelet activation, which results in aggregation and secretion of regulatory compounds. Platelets are activated only if an external stimulus, interacting with platelet surface glycoproteins or glycolipids, is able to transduce its signal inside the platelet. All the stimuli finally activate the same fibrinogen receptor (GP IIb-IIIa) which changes its conformation and binds fibrinogen, vWF or fibronectin. Even though the mechanism of GP IIb-IIIa activation is still obscure, it involves signalling pathways in both directions across the plasma membrane. Thus the metabolism and F,A composition of platelet phospholipids are emphasized. -Activation of GP Iib-IIIa starts the process of platelet aggregation, which includes changes in the association of GP IIb-IIIa with the cytoskeleton. Second messengers produced by activated platelets are important regulators in this process, which finally leads to clustering of GP Ilb-IIIa molecules and fibrinogen binding to the plasma membrane.l'2 The tightly packed fibrinogen clusters enable the unactivated, newly arriving platelet to stick to them without any further prerequisites. 3 The effects of dietary F_Ason platelet function are far from pharmacological. F,Ascould thus participate in intracellular signalling pathways which regulate either the activation and binding function of GP IIb-IIIa or subse403
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quent signal transduction. The main method to study platelet activation has been the platelet aggregation test, probably due to the fact that changes in dietary FAs often change platelet responses in vitro. The question is, however, how to interpret the results in the context of a situation in vivo. Agonist-induced in vitro aggregation does not measure platelet activation per se, but the whole cascade from the activation to final platelet plug formation. To put platelet aggregation into perspective with regard to other phases of platelet activation, the speed of the early activation should be kept in mind; it takes place in less than 1 s, and even secretion may be completed within 5 s. The major biochemical events, i.e. lipid hydrolysis and protein phosphorylations, are initiated and completed within this period. The later biochemical changes are probably related to the consolidation of platelet aggregates. 3 In this review, the data obtained from dietary studies are presented in the light of current biochemical data on FAs as regulators of platelet function. However, several still obscure issues remain. At a molecular level, it is not clear how different physiological agonists mediate their effects on the activation of GP IIb-IIIa. Furthermore, it is not known if dietary FAs are able to change platelet membrane characteristics to favor GP IIb-IIIa activation. Finally, the significance of the availability and composition of free FAs inside the platelet for signalling systems to proceed is unclear. Platelet activation involves intracellular signalling processes
Protein phosphorylations are central in intracellular signal processes. In platelets, three main categories of protein phosphorylations can be distinguished: Ca2+-dependent, DG-dependent, and cAMP-dependent. One of the early responses to most physiological agonists is the PLCcatalyzed hydrolysis of membrane PIP2 to DG and IP3. Activation of PKC is a prerequisite for platelet shape change, 4,5 and the synergistic action of intracellular Ca 2+ and PKC activation is usually needed to produce platelet aggregation and release reactions. 6 PKC also causes serine/threonine phosphorylation of GP IIIa, and may thus be involved in early activation of GP IIb-IIIa. 7 PKC is a family of closely related isoenzymes, which are grouped into three classes: Ca2+-dependent, Ca 2+independent, and atypical torms. To-date, six isoenzyrnes of PKC have been identified in human platelets. 8 The basic mechanism for PKC activation includes translocation of inactive PKC from the cytosol to the plasma membrane, where DG activates it by increasing PKC affinity for Ca 2+ and PS. The optimum activity of the membranebound PKC is regulated by the level of bilayer unsaturation, especially in PC. 9 Studies with intact platelets or of
in vitro enzymatic reactions show that DG- and Ca 2+dependent activation of PKC is enhanced by cis-unsaturated FAs esterified at the sn-2-position of membrane phospholipids. Saturated and trans-unsaturated FAs are inactive. Furthermore, cfs-unsaturated FAs liberated from PC by action of PLA2 may further activate PKC by increasing its sensitivity to Ca2+.1°-12 The in vitro results with cis-unsaturated FAs, however, indicate that activation of PKC may occur in two compartments. It seems that when membrane-bound PKC is activated by DG, cis-unsaturated FA, especially AA, activates soluble PKC when both FA and enzyme are not membrane-associated. In the cytosolic fraction, cis-unsaturated FA activates predominantly Ca2+-independent isoenzymes of PKC. 13 The physiological implications of the PKC activation by cis-unsaturated FAs is not clear at present. It has been suggested that in stimulated platelets, PKC, once initially activated by PIP2 hydrolysis, may sustain its enzymatic activity even after Ca 2+ concentration returns to basal level, when both DG and cis-unsaturated FAs are available. 12 Furthermore, the evidence of relation between PKC activation and platelet desensitization TM indicates that endogenously generated free FAs and Ca 2+independent PKC may participate in negative feedback during platelet activation. The availability of DG has a pivotal role for PKC activation in membranes. Because DG is released by PLC, the activation of PLC is also crucial for the PKC activity. AA metabolites, mainly TXA2, stimulate the activation of PLC in platelets. AA is released from PI and PC by the action of PLC and PLA2, respectively, and also from DG by combined actions of DG- and MG-lipases. PLC is inhibited by cAMP, a molecule that antagonizes all the activation events in platelets. Agonists - such as PGI2 - which raise platelet cAMP level, activate cAMP-dependent protein kinases. Also 5' phosphomonoesterase which converts IP3 to IP2 and, as a consequence, inactivates the IP3 signal for Ca ~+release, inhibits platelet activation. PKC may activate 5' phosphomonoesterase and regulate by feed-back inhibition the Ca2+-pathway of platelet activation. PKC may also abrogate PLC-mediated PIP2 hydrolysis, the biochemical event which initiates its own activation. 15 Furthermore, activators of PKC have been shown to enhance PLA2-mediated release of AA in platelets. ~6 Recent data, however, revealed a G-protein-mediated mechanism for the activation of PLA2 in intact h u m a n platelets which is negatively affected by PKC. lz These varying results may be explained by the diversity of the cytosolic PLA2 enzymes, which are divided into AA selective and non-selective enzymes. The activation mechanisms of these different types of cytosolic PLAa are not clear at the moment. ~°In addition, separate groups of PKC isoenzymes are probably involved in different aspects of platelet activation. 8
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Intracellular signalling by physiological platelet agonists used in dietary intervention studies The physiological platelet agonists used frequently in studies related to dietary interventions are thrombin, collagen, ADP, and adrenaline. They all stimulate a variety of responses in the platelet including shape change, GP IIbIlia receptor exposure (aggregation), granule secretion, and liberation of AA. Intracellular signals induced by agonists include: (i) signals causing GP IIb-IIIa activation and binding of fibrinogen; and (ii) downstream signals which result in aggregation and release reactions. Neither of these pathways is fully understood but the activation of PKC is probably a necessary component shared by most, if not all, agonists in these pathways. TM Thrombin activates directly both PIand PC-specific PLC, 19 while collagen, in addition to activating PLC, strongly activates PLA2 to liberate free FAs including AA. Thrombin and collagen induced stimulations rapidly increase the turnover of PIP2 to DG and IP3, and aggregation and secretion are associated dose dependently with the accumulation of DG in platelets. 2° Platelets contain several PLC isoenzymes and PLC-[~, which is coupled to G-proteins, is activated by thrombin and TXA2, while the mechanism of action of collagen involves tyrosine phosphorylation of PLC-72 and not G-proteins. 2~,22 TXA2, produced from liberated AA, is the actual stimulus for secretion induced by low doses of collagen in addition to its ability to activate PLC. Thrombin and high-doses of collagen can activate PKC and stimulate release reaction without TXA2 productionY Recent observations indicate that the activation of PKC is not a prerequisite for phosphorylation of cytosolic PLA2 during thrombin and collagen stimulation. 24 Weak agonists may activate platelets to expose GP IIbIIIa receptors along with intracellular Ca 2+ independently on the inositol phosphate pathway. Actually, GP IIb-IIIa exposure induced by ADP is independent of the activation of PKC, even though PKC activation is required for ADP secretionY Also primary ADP-induced aggregation (no secretion or AA release) is not dependent on the formation of DG or the activation of PKCY A decrease in PIP2 is, however, an early event in the ptatelet response to ADP. The equilibrium between PIP2 and PIP may cause mobilization of some Ca2+,even though there is no phosphorylation of P40 indicating that PKC is not activated. Phosphorylation of myosin light chain (P20), which is essential for contractile activity of the platelet, is primarily dependent upon increased cytoplasmic Ca2+.ADP causes a much more rapid increase of intracellular Ca 2+than other agonistsY Platelet fatty acids There are at least two mechanisms for FAs to regulate platelet function: membrane FA composition, and free FA © Harcourt Brace & Co Ltd 1997
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liberated from membranes and intermediates of inositol phosphate pathway (PIP2, DG, PA) during stimulation. Platelet FA composition originates from the megacaryocytesY PC is the most abundant phospholipid, and nearly 14% of its FAs is AA, almost exclusively located in the sn-2 position. PE and PI have the highest percentages of AA (ca. 37°/o and 35%, respectively), but from the actual mass of FAs, PE provides almost 25% of total AA, PC over 50% and PI only 5-6o/o. EPA selectively incorporates into PC and PE and not into PI.29 The origin of free FAs upon stimulation by PLA2 is mainly PC and probably PE. AA liberated by action of PLC and DG-lipase is less than 10% of the released AA during stimulation. 3° Due to low delta-6-desaturase activity, hardly any LA is converted into AA in intact platelets. In vitro incubations, however, indicate that AA and other FAs can enter into platelet phospholipids through de novo synthesis or 'remodelling' pathways. 3~ Platelets contain an AA specific synthase probably to ensure an adequate incorporation of AA in platelet phospholipids and also to keep free AA content low inside the platelet. The significance of the de novo synthesis and 'remodelling' pathways in vivo is not clear at present. Administration of 6 g A_A/day did not affect platelet function (drop in threshold ADP) until after 10-12 days, indicating that the regeneration of platelet population was a prerequisite for the change in platelet function? 2 Similarly, 6 g EPA/day showed that even though EPA appeared in plasma phospholipids already after 4 h, it did not incorporate into platelet phopholipids until day 6.33 In human platelets, the fatty acid composition of total lipids does not reflect changes in dietary FAs very well. When a high-SAFA diet was replaced by a diet containing a high amount of MUFA, the OA level in platelet phospholipids increased only modestly34or did not change. 35,36 Concurrently, LA either increased, 36 decreased 34 or remained constantY The main feature in dietary studies with n-6 PUFA is the constant proportion of AA in platelet total phospholipids with considerably different LA intakesY -39 In tissue phospholipids A,~ appears to be resistant to dietary changes even wher~ the amount of dietary LA is lowered to 1.8 E°/o.39 The [,roportion of LA in platelet phospholipids is increased when the intake of n-6 PUFA increases. The increase of LA is very modest even with high dietary LA intakes a n ] it does not correlate with dietary LA?9 When the intake of LA is very high (> 10E%), platelet AA may slightly decreaseY ,4° Consumption of ethyl arachidonate (6 g/day) increased AA in platelet phospholipids at the expense of LA,32 while the daily intake of 350 mg AA only significantly decreased LA.41The decreases in LA or AA after diets with long-chain n-3 PUFA are compensated with increases of EPA, DPA, and DHA in platelet lipids. 42-44 Also dietary ALA increases EPA and DPA in platelet FAs45,46and corre-
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lates positively with platelet ALA and EP& but negatively with DHA. 39 FA composition of platelet total phospholipids is not very informative if the aim is to understand the effect of the FAs on intracellular signalling systems. FAs of platelet phospholipid subclasses are regulated with a high degree of specificity in vivo, which obviously has a major role during platelet activation. Furthermore, in vitro studies indicate that significant remodelling between individual phospholipid subclasses takes place when platelets are stimulated. 47 How relevant this is in vivo is not clear at present. Studies on the incorporation of cis-unsaturated FAs of vegetable origin into individual platelet phospholipids are few. OA has been found to incorporate into the PC fraction in two studies with high-OA diets 48'49 and it also increased in the PE fraction in one study. 49 Furthermore, an increase of C20:ln-9 in the PC fraction has been found after a diet high in OA.48 High-LA diets increase LA in platelet PC and particularly in PE.48-5° Sometimes high dietary LA has also been found to lead to incorporation of AA into PE but not into PC. 48-49 Long-chain n-3 PUFA supplementation increased the incorporation of EPA, DPA, and DHA into platelet PC and PE but not into PI and PS in one studyY Small but significant incorporation of EPA into PS was, however, found in another study. 51 Furthermore, during prolonged supplementation, longchain n-3 FAs have also been found in P152 even though not always. 29 Increased n-3 FAs are often compensated with decreased AA in PE and PC 29'51 but not always. 52 Sometimes LA increases with increased incorporation of n-3 FAs into PE and PC. 52 Increased EPA level in PE and PC can also be seen after high-ALA diets. 48,49 Platelet aggregation, arachidonic acid metabolism, and granule secretion The most abundant cis-unsaturated FAs in h u m a n diets are OA and LA. Quantitatively minor FAs are AA, ALA and n-3 FAs of marine origin. Not much effort has been made to understand how FAs of vegetable origin or dietary AA regulate platelet function, while h u m a n experiments with n-3 FAs of marine origin are abundant. A very general shortcoming has been a lack of information on the FA composition of individual platelet phospholipids, which is crucial in understanding the mode of combined action of an agonist and dietary FAs. In addition, little is known about the role of the baseline diet with respect to the incorporation of FAs into platelets. Low power of the study designs and a poor methodological approach are also very common and make the interpretation of the results difficult. Furthermore, evidence from animal 53 and h u m a n 32 studies suggest that a confounding factor in human studies may be the uncontrolled intake of AA.54
Thrombin has been used as an agonist in several interventions with long-chain n-3 FAs.55,56In the eight studies reported by Malle and Kostnery decreased platelet aggregation was seen in three and no change in five studies. The results are apparently independent of the total amounts of n-3 FAs (EPA 1.6-3.8 g/day and DHA 0.25-3.0 g/day) and of the ratio between EPA and DHA. Thrombin-induced aggregation was also low in farmers having low dietary SAFA vs PUFA in two studies, 37,57 while in one study no difference was found even though the difference between dietary P/S-ratio was marked (0.26 vs. 0.59). 59 However, changing the P/S-ratio from 0.32 to 0.97 for one year decreased thrombin-induced aggregation significantly. 6° In one of these studies, decreased thrombin-induced aggregation paralleled decreased plasma ratio of AA/LA.57 LA probably has a specific effect on platelet function, since increasing the ratio of LA/ALA from 2.8 to 28 and keeping the diet constant in other respects increased platelet aggregation significantly? 8 Based on studies carried out with thrombin, there is no good insight into the possible effect of dietary FAs. One reason may be the strength of thrombin as an agonist. The sensitive concentration range is very narrow, which may weaken the detection of differences between treatments. Collagen has been used in dietary interventions more frequently than thrombin. Studies concerning n-3 FAs of marine origin 55,s6 have fairly consistently shown either no effect or decreased aggregation pattern. Aggregation seems to be similarly affected by both EPA and DHA. 33 The amount of n-3 PUFA and the length of the supplementation period may increase the variation in the results. Tremoli et a144 found long-lasting impairment especially in collagen-induced platelet aggregation after n-3 PUFA supplementation. The AA content in fish otis may differ and affect the efficiency of n-3 FAs.54Variation in n-3/n-6 FA status of individual phospholipids due to the differences in background diet may also be marked. High-ALA (8-16 E%) supplementation also decreased collagen-induced aggregation when compared with baseline diets, 45,~1 while reasonable amounts of ALA from canola or rapeseed otis had no effect.34,58,~2 When the effect of ALA was compared with that of n-3 FAs from marine origin, the reduction of collagen-induced aggregation appeared about similar. 4¢~3 The lower availability of substrate FA for TXA2 production probably explains frequently found reduced collagen aggregation after n-3 FAs. This does not explain the results on cis-unsaturated FAs of vegetable origin. The increased intake of unsaturated FAs is not associated with decreased aggregation, but quite often the opposite trend is found. The underlying mechanism probably involves intracellular signals which differ between n-6 and n-3 FAs. Some 35,58,64,65 but not all34,57,59,62,63 studies show
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increased collagen sensitivity of platelets after diets with considerable amount of PUFA when compared with diets high in SAFA. Only one study shows a decreased collagen sensitivity associated with low SAFA i n t a k e s An increase in aggregation in response to collagen was found also when a high-LA diet was compared with a high-OA diet? ~ This was not confirmed by three other studies, 34,35,~2 however. In vegan platelets, the proportion of LA was higher and that of AA lower when compared with omnivore controls, but no difference in collagen-induced aggregation was found. ``° The results from ADP-induced aggregations in dietary studies vary probably most of all. Decreases, increases and 'no effects' are all common with long-chain n-3 FAsY ,56 As reviewed by Hornstra 55 only studies classified as 'poorly controlled' showed enhanced aggregation. However, in our latest partially controlled intervention study, EPA and DHA supplementation increased ADPinduced aggregation, while similar amounts of ALA had no effect during the supplementation. ALA increased, however, ADP induced aggregation during the follow-up p e r i o d s ADP-induced aggregation was not changed or it decreased in two other studies with ALA.4~,61 When purified EPA or DHA was supplemented in healthy volunteers, reduced ADP aggregation was associated only w i t h DHA. 33 When high-LA diets (P/S-ratio between 0.7 and 1.0) were compared with either high-fat diets rich in SAFA35'~8'~° or rich in ALA,58 ADP-aggregation was significantly enhanced. On the other hand, it has been postulated that with moderate dietary LA intakes (P/S-ratio between 0.6 and 0.8) LA may be associated with impaired ADP-induced aggregationF After a cereal-based low-fat diet, ADP-induced aggregation was not affected by increased LA intake. ~3 Nor was the very high LA intake of vegans (P/S-ratio 1.48) associated with a higher ADP response than in omnivores (P/S-ratio 0.34). 40 Furthermore, OA and LA rich diets similarly enhanced ADP-induced aggregation when compared with the baseline milkfat diet. 35 Taken together, the effect of dietary FAs on ADP aggregations are very inconsistent. The intracellular signals induced by ADP are the least well understood, and regulatory functions of FAs probably arise from complex interactions between amounts and types of different FAs. During platelet in vivo activation TXA2 is always formed. 2,3-dinor-TXB2 and l l-dehydro-TXB2 are two main TXA2 metabolites found in urine and measured as in vivo indices of platelet TXAa formation. Excretion of 2,3-dinor-TXB2 is probably a more sensitive marker than the urinary level of 1 1-dehydro-TXB2.68 Dietary fish oil or long-chain n-3 FAs lower the high basal excretion rate of TXB2 very dramatically, while only a modest effect is noticed at a low basal excretion rate. The underlying mechanism is the replacement of AA with EPA and DHA © Harcourt Brace & Co Ltd 1997
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in platelet phospholipids and thus a sufficiently long supplementation period is required. 33,44,52,69,7°,71The efficiency of long-chain n-3 FAs in reducing the excretion of TXA2 metabolites may be slightly improved if dietary SAFA intal~e is low. 72 Results on the effects of other cisunsaturated FAs on urinary TXB2 metabolites are almost totally lacking. There are some indications that dietary LA or AA might regulate this excretion.41,73 Another way to look at platelet activation in vivo is to measure the concentration of [~TG released from (zgranules. The plasma level of this platelet-specific protein has not changed in supplementation studies with long-chain n-3 FAs. 43'55 Urinary excretion of high molecular weight [~TG, which presumably contains intact [3TG, has been measured in three human experiments at our department. Increased intake of ALA or EPA + DHA had no effect in one study. 4~ Nor was the level affected by high OA or LA diets (Turpeinen A. M., unpublished observation) or by high stearic or elaidic acid dietsY These results indicate that dietary FAs are not powerful enough to affect platelet granule release reactions in vivo. CONCLUSIONS
Platelet activation in vivo is an extremely complex event with involvement of FAs. Platelet aggregation tests in vitro may give very little information on the relationship between dietary FAs and platelet function in vivo, unless other parameters such as individual phospholipid FA composition and measurement of intracellular signalling are also measured. Unfortunately, this has not been very common in dietary studies. In addition, the results from an early filtragometer study in vivo, comparing saturated and polyunsaturated FAs, further support the limitations of in vitro aggregation test. 74 The total amount of a given FA in the platelet is probably not crucial but merely the factor which regulates free FA levels and types in the membrane and in platelet interior. Furthermore, platelet receptor responsiveness to physiological stimuli and subsequent signal transduction may be highly dependent on membrane FA c o m p o s i t i o n , zS-z7 A n important participant in the activation of human platelets is PKC. The physiological significance of the observation that the activation of PKC depends on cis-unsaturated FAs is, however, not understood. Well-controlled dietary studies with careful measurements of different aspects of platelet activation are clearly needed to understand what kind of diet is optimal to keep platelets 'healthy'. REFERENCES
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