Inhibition of platelet aggregation by unsaturated fatty acids through interference with a thromboxane-mediated process

cis- and trans-unsaturated fatty acids with 18 carbon atoms (oleic, linoleic, elaidic and linolelaidic acid) inhibited aggregation of washed rabbit platelets stimulated with collagen, arachidonic acid and U46619 when in the same concentration ranges. Thrombin-induced aggregation was not affected by any of them. Saturated fatty acid (stearic acid) had no effect on this response. The inhibition is independent of the induced change in membrane fluidity, since trans-isomers could not induce the change in fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Unsaturated fatty acids, except linoleic acid, did not interfere with the formation of thromboxane B2 from exogenously added arachidonic acid. All the unsaturated fatty acids only slightly inhibited the arachidonic acid liberation by phospholipase A2 in platelet lysate. This indicates that the unsaturated fatty acids may block a process after formation of thromboxane A2 in response to collagen and arachidonic acid. The increase in phosphatidic acid formation stimulated with U46619 was inhibited dose dependently by each of the unsaturated fatty acids but that stimulated with thrombin was not affected by any of them. Phospholipase C activity measured by diacylglycerol formation in unstimulated platelet lysate was not inhibited by the fatty acids. The elevation of cytosolic free Ca2+ induced by arachidonic acid or U46619 and Ca2+ influx by collagen were inhibited almost completely at the same concentration as that which inhibited their aggregation. These data suggest that the unsaturated fatty acids were intercalated into the membrane and inhibited collagen- and arachidonic acid-induced platelet aggregation by causing a significant suppression of the thromboxane A2-mediated increase in cytosolic free Ca2+, probably due to interference with the receptor-operated Ca2+ channel.     

Inhibition of platelet aggregation and arachidonate metabolism in platelets by procyanidins

The effects of procyanidins on platelet aggregation and arachidonate metabolism in platelets were studied. Nine procyanidins were used in this investigation. Procyanidins B-2-S, EEC and C-1 significantly induced the inhibition of platelet aggregation, and the potency of inhibition was comparable with aspirin. Procyanidin B-2-S was used as a representative of procyanidins for further studies on the effect on arachidonate metabolism. In arachidonate metabolism by fatty acid cyclooxygenase pathway, B-2-S inhibited TXB2 and HHT formation by intact platelets treated with exogenous arachidonic acid. It also inhibited TXB2 formation measured by a specific radioimmunoassay when the cells were challenged with calcium ionophore A23187. In cell-free system, B-2-S inhibited both TXB2 and 12-HETE bioxynthesis in platelet microsome and cytosol, respectively. The inhibitory effect on thromboxane biosynthesis might explain the inhibitory effect of procyanidins on platelet aggregation.     

High concentrations of arachidonic acid induce platelet aggregation and serotonin release independent of prostagladin endoperoxides and thromboxane A2

We examined platelet aggregation and serotonin release, induced by less than 60 μM arachidonic acid, using washed platelet suspensions in the absense of albumin. The concentration of arachidonic acid use did not cause platelet lysis. Platelet responses induced by less than 20 μM arachidonic acid were inhibited by aspirin, whereas those induced by above 30 μM arachidonic acid were not inhibited, even by both aspirin and 5,8,11,14-eicosatetraynoic acid. Although phosphatidic acid and 1,2-diacylglcerol increased after the addition of arachidonic acid in aspirin-treated platelets, the amounts were not parallel to platelet aggregation. Oleic, linoleic and linolenic acids also induced platelet responses, while palmitic, stearic and arachidic acids did not. EDTA, dibutyryl cyclic AMP, apyrase and creatine phosphate / creatin phosphokinase brought about almost the same effects in platelet responses induced by the unsaturated fatty acids, other than arachodinic acid, as those induced by 40 μM arachodonic acid. These results suggest that the mechanism of the actions of more than 30 μM arachodinic acid on platelets is the same as that of the other unsaturated fatty acids and is independent of prostaglandin endoperoxides, thromboxane A₂ and, perhaps, phosphatidic acid and 1,2-diacylglycerol.     

Superoxide dismutase triggers activation of "primed" platelets

Superoxide dismutase (SOD) triggers activation of human platelets exposed to subthreshold concentrations of arachidonic acid and collagen. The subthreshold concentrations used are not able to activate platelets but "prime" platelets to be activated by SOD. The addition of SOD to arachidonic acid-or collagen-primed platelets induced aggregation, thromboxane A2 production, and release of [3H]serotonin. Superoxide dismutase does not have any effect on resting platelets and ADP-, thrombin-, calcium ionophore A23187-, PAF-, or U46619-stimulated platelets. Furthermore, superoxide dismutase-dependent platelet activation is fully prevented by catalase and/or aspirin, suggesting a role for H2O2 and the involvement of the cyclooxygenase pathway of arachidonic acid in such activation.     

Antiplatelet effects of chelerythrine chloride isolated from Zanthoxylum simulans

Chelerythrine chloride is an antiplatelet agent isolated from Zanthoxylum simulans. Aggregation and ATP release of washed rabbit platelets caused by ADP, arachidonic acid, PAF, collagen, ionophore A23187 and thrombin were inhibited by chelerythrine chloride. Less inhibition was observed in platelet-rich plasma. The thromboxane B2 formation of washed platelets caused by arachidonic acid, collagen, ionophore A23187 and thrombin was decreased by chelerythrine chloride. Phosphoinositides breakdown caused by collagen and PAF was completely inhibited by chelerythrine chloride, while that of thrombin was only partially suppressed. Chelerythrine chloride inhibited the intracellular calcium increase caused by arachidonic acid, PAF, collagen and thrombin in quin-2/AM-loaded platelets. The cyclic AMP level of washed platelets did not elevated by chelerythrine chloride. The antiplatelet effect of chelerythrine chloride was not dependent on the incubation time and the aggregability of platelets inhibited by chelerythrine chloride was easily recovered after sedimenting the platelets by centrifugation and then the platelet pellets were resuspended. Chelerythrine chloride did not cause any platelet lysis, since lactate dehydrogenase activity was not found in the supernatant. These data indicate that the inhibitory effect of chelerythrine chloride on rabbit platelet aggregation and release reaction is due to the inhibition on thromboxane formation and phosphoinositides breakdown.     

Biphasic effect on platelet aggregation by phospholipase a purified from Vipera russellii snake venom

A basic phospholipase A was isolated from Vipera russellii snake venom. It induced a biphasic effect on washed rabbit platelets suspended in Tyrode's solution. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The second phase was an inhibitory effect on platelet aggregation, occurring 5 min after the addition of the venom phospholipase A without stirring or after a recovery from the reversible aggregation. The aggregating phase could be inhibited by indomethacin, tetracaine, papaverine, creatine phosphate/creatine phosphokinase, mepacrine, verapamil, sodium nitroprusside, prostaglandin E1 or bovine serum albumin. The venom phospholipase A released free fatty acids from synthetic phosphatidylcholine and intact platelets. p-Bromophenacyl bromide-modified venom phospholipase A lost its phospholipase A enzymatic and platelet-aggregating activities, but protected platelets from the aggregation induced by the native enzyme. The second phase of the venom phospholipase A action showed a different degree of inhibition on platelet aggregation induced by some activators in following order: arachidonic acid greater than collagen greater than thrombin greater than ionophore A23187. The longer the incubation time or the higher the concentration of the venom phospholipase A, the more pronounced was the inhibitory effect. The venom phospholipase A did not affect the thrombin-induced release reaction which was caused by intracellular Ca2+ mobilization in the presence of EDTA, but inhibited collagen-induced release reaction which was caused by Ca2+ influx from extracellular medium. The inhibitory effect of the venom phospholipase A and also lysophosphatidylcholine or arachidonic acid could be antagonized or reversed by bovine serum albumin. It was concluded that the first stimulatory phase of the venom phospholipase A action might be due to arachidonate liberation from platelet membrane. The second phase of inhibition of platelet aggregation and the release of ATP might be due to the inhibitory action of the split products produced by this venom phospholipase A.     

Platelet desensitization by arachidonic acid is associated with the suppression of endoperoxide/thromboxane A2 binding to the membrane receptor

The inhibitory mechanism of high levels of exogenously added arachidonic acid on activation of washed human platelets was investigated. While low levels of arachidonic acid (5-10 microM) induced aggregation, ATP secretion and increase in cytoplasmic free Ca2+ concentration (first phase of activation), these platelet responses did not occur significantly at high concentrations (30-50 microM). However, much higher concentrations than 80 microM again elicited these responses (second phase). The first phase of platelet activation was inhibited by cyclooxygenase inhibitor, indomethacin, whereas the second one was independent of such treatment. Thromboxane B2 was produced dose-dependently until reaching a plateau at arachidonic acid concentrations higher than 20 microM, irrespective of the lack of aggregation and secretion at high concentrations. After that the amount of free arachidonic acid which remained unmetabolized in platelets gradually increased. High concentrations of arachidonic acid as well as other polyunsaturated fatty acids caused desensitization of platelets in response to U46619, and also depressed the specific [3H]U46619-binding to the receptor as well as other polyunsaturated fatty acids. The amount free arachidonic acid needed in platelets to suppress [3H]U46619 binding corresponded to that needed to inhibit platelet aggregation. Furthermore, arachidonic acid dose-dependently induced fluidization of lipid phase of platelet membranes as detected by 1,6-diphenyl-1,3,5-hexatriene. These results suggest that the inhibition of platelet response by high levels of arachidonic acid can be attributed to interference with endoperoxide/thromboxane A2 binding to the receptor, probably due to perturbation of the membrane lipid phase due to excess amounts of free arachidonic acid remaining in the membranes.     

Effects of free and macromolecular-bound TXA2 receptor antagonist BM 1..505 on U46619-induced platelet aggregation

The goal of this study was to synthesize a macromolecular probe of the TXA₂ receptor antagonist BM13.505 which is unable to penetrate the platelet membrane for localization and characterization of the TXA₂ receptor. The active NHS-ester of BM13.505 was synthesized and purified. It was used for covalent coupling of BM13.505 to bovine serum albumin, a macromolecular carrier. Inhibitory effects of free and macromolecular bound BM13.505 on aggregatory properties of U46619-stimulated platelets were measured and compared to TXA₂ generation in platelets, as determined by TXB₂ radioimmuno assay. No inhibitory effects of free and macromolecular-bound BM13.505 on ADP- or thrombin-induced platelet aggregation were observed. Equimolar concentrations of free or macromolecular bound BM13.505 inhibited U46619-induced platelet aggregation and TXA₂ generation with equal potency. IC₅₀-values for platelet aggregation inhibition by free and macromolecular bound BM13.505 were 64 nM and 96 nM respectively. It appears that the TXA₂ receptor ligand binding site is located close to the outer membrane surface of platelets. Interaction of macromolecular bound BM13.505 with the platelet thromboxane receptor does not depend on the availability of the free carboxyl residue in BM13.505. The method for coupling a TXA₂ receptor antagonist to a macromolecule will aid in constructing probes for the localization and characterization of the TXA₂ receptor.     

Thiopental enhances human platelet aggregation by increasing arachidonic acid release.

Conflicting results have been reported regarding the effect of thiopental on aggregation and cytosolic calcium levels in platelets. The present study attempted to clarify these phenomena. Using platelet-rich plasma or washed suspensions, platelet aggregation, thromboxane (TX) B2 formation, arachidonic acid (AA) release, and cytosolic free calcium concentrations ([Ca2+]i) were measured in the presence or absence of thiopental (30-300 microM). Platelet activation was induced by adenosine diphosphate (ADP, 0.5-15 microM), epinephrine (0.1-20 microM) arachidonic acid (0.5-1.5 mM), or (+)-9,11-epithia-11,12-methano-TXA2 (STA2, 30-500 nM). Measurements of primary aggregation were performed in the presence of indomethacin (10 microM). Low concentrations of ADP and epinephrine, which did not induce secondary aggregation in a control study, induced strong secondary aggregation in the presence of thiopental (> or = 100 microM). Thiopental (> or = 100 microM) also increased the TXB2 formation induced by ADP and epinephrine. Thiopental (300 microM) increased ADP- and epinephrine-induced 3H-AA release. Thiopental (300 microM) also augmented the ADP- and epinephrine-induced increases in [Ca2+]i in the presence of indomethacin. Thiopental appears to enhance ADP- and epinephrine-induced secondary platelet aggregation by increasing AA release during primary aggregation, possibly by the activation of phospholipase A2.     

Irreversible platelet aggregation does not depend on lipoxygenase metabolites

Previous investigations in our laboratory demonstrated the existence of an intrinsic mechanism, termed membrane modulation, capable of restoring sensitivity to aspirin treated platelets, resulting in irreversible aggregation in response to arachidonic acid (AA). The mechanism underlying correction of aspirin induced inhibition of platelet function, however, was not clear. In the present study we have evaluated the role of lipoxygenase (LO) metabolites of AA in securing irreversible aggregation of drug induced cyclooxygenase (CO) deficient platelets. Platelets treated with aspirin or Ibuprofen did not convert radiolabeled AA to thromboxane, but generated significant quantities of hydroxy acids via the LO pathway. However, drug exposed platelets, when stirred with epinephrine first and then challenged with AA, aggregated irreversibly. Eicosatetraynoic acid (ETYA 1, U53119) inhibited AA conversion by the LO pathway, whereas 5,8,11,14-eicosatetraynoic acid (ETYA 2) inhibited AA conversion by both CO and LO enzymes. Yet, at the inhibitory concentration these fatty acids failed to prevent AA induced irreversible aggregation of CO deficient, alpha adrenergic receptor stimulated platelets. Results of four studies show that the generation of LO metabolites of AA are not essential for securing irreversible aggregation of platelets.     

Inhibitory mechanism of cis-polyunsaturated fatty acids on platelet aggregation: the relation with their effects on Ca2+ mobilization, cyclic AMP levels and membrane fluidity

The in vitro inhibitory effects of cis-polyunsaturated fatty acids, linolenic (18:2 delta 9,12), alpha-linoleic (18:3 delta 9,12,15) and eicosatrienoic (20:3 delta 11,14,17) acid, on bovine platelet aggregation and their inhibitory mechanism were investigated. These fatty acids inhibited platelet aggregation induced by ADP and thrombin to similar extent. Fluorescence analyses with fura-2-loaded platelets showed that, in the concentration ranges that inhibited aggregation, they also inhibited agonist-induced increase in cytoplasmic Ca2+. According to radioimmunoassay study, addition of these fatty acids increased cyclic AMP contents in the presence of theophylline corresponded with their inhibitory effects on aggregation. These fatty acids induced a 1.6-1.8-fold increase over basal concentration of cyclic AMP in the concentration ranges that fully inhibited aggregation. On the other hand, saturated fatty acid, stearic acid, affected neither aggregation nor cyclic AMP levels. As reported previously [1985) Biochim. Biophys. Acta 818, 391), these unsaturated fatty acids induced increase in membrane fluidity in the same concentration range. These results suggest that inhibition of platelet aggregation by cis-polyunsaturated fatty acids is due to the increase in cyclic AMP levels. This increase seems to be due to stimulation of adenylate cyclase which is mediated by membrane perturbation.     

Inhibitory action of soluble elastin on thromboxane B2 formation in blood platelets

Soluble elastin, prepared from insoluble elastin by treatment with oxalic acid or elastase, was found to inhibit the formation of thromboxane B₂ both from [1-¹⁴C]arachidonic acid added to washed platelets and from [1-¹⁴C]arachidonic acid in prelabeled platelets on stimulation with thrombin. In both systems, the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) was accelerated. Oxalic acid-treated soluble elastin st 1 and 10 mg/ml inhibited the formation of thromboxane B₂ from exogenously supplied arachidonic acid 21 and 59%, respectively, and the formation of thromboxane B₂ in prelabeled platelets stimulated by thrombin 44 and 94%, respectively. These concentrations of elastin increased the formation of 12-HETE from exogenously supplied arachidonic acid about 3.4- and 7.3-times, respectively. Almost all the added arachidonic acid was converted to metabolites. In prelabeled platelets, soluble elastin at 1 and 10 mg/ml increased the formation of 12-HETE stimulated by thrombin about 1.3- and 2.8-times, respectively, and inhibited the thrombin-induced total productions of thromboxane B₂ (12-hydroxy-5,8,10-heptadecatrienoic acid (12-HETE) and free arachidonic acid by 26 and 25%, respectively. Elastase-treated digested elastin also inhibited the formation of thromboxane B₂ and stimulated the formation of 12-HETE in prelabeled platelets stimulated by thrombin. This inhibitory action of elastin was not replaced by desmosine. The level of cAMP in platelets was not affected by soluble elastin. Soluble elastin was also found to inhibit platelet aggregation induced by thrombin. However, the inhibitory action of soluble elastin on platelet aggregation cannot be explained by inhibition of thromboxane B₂ formation by the elastin.     

Effects of barbiturates on human platelet aggregation differ depending on their chemical structures

The effects of barbiturates on human platelet function are not fully understood. Since we have already revealed the effects and mechanisms of thiopental, thiamylal, and pentobarbital in platelets, the present study attempted to elucidate (i) the effects of other barbiturates on human platelet aggregation, (ii) the underlying mechanisms, and (iii) the structure-function relationship of barbiturates in platelets. Barbiturates, including amobarbital, butalbital, secobarbital, barbital, phenobarbital, metharbital, and primidone, were examined. Human platelet aggregation induced by adenosine diphosphate (ADP), epinephrine, and (+)-9,11-epithia-11,12-methano-thromboxane A2 (STA2), a thromboxane A2 analog, was measured using an 8-channel light-transmission aggregometer. The cytosolic free calcium concentration ([Ca2+]i) was measured by fluorometer using fura-2 loaded platelets. Inositol 1,4,5-trisphosphate (IP3) formation induced by STA2 was determined by a commercially available IP3 assay kit. Amobarbital, butalbital, and secobarbital suppressed ADP-, epinephrine- and STA2-induced platelet aggregation and the STA2-induced [Ca2+]i increase, even when Ca2+ influx was blocked by Ni2+. However, they did not affect STA2-induced IP3 formation. Barbital, phenobarbital, metharbital, and primidone (up to 1 mM) had no effect on ADP- and epinephrine-induced platelet aggregation. Thus, we conclude that amobarbital, butalbital, and secobarbital inhibit platelet aggregation by suppressing [Ca2+]i increase without affecting IP3 formation. However, these antiaggregatory effects may not have clinical importance, since the barbiturate concentrations used were higher than clinically relevant ones. The other tested barbiturates had no effects on platelet aggregation. The data indicate that the effects of barbiturates on platelet aggregation differ depending on their chemical structures.     

Enrichment of human platelet phospholipids with linoleic acid diminishes thromboxane release

We have investigated whether exposure of human platelets to elevated concentrations of linoleic acid, the principal dietary polyunsaturate, would influence platelet thromboxane A₂ release. Platelets were incubated with albumin-bound linoleic acid at 30°C for 24 h, with prostaglandin E₁ added to prevent aggregation. The linoleic acid supplemented platelets released, on averaged, 50% less thromboxane A₂ in response to stimulation with thrombin than corresponding control platelets. Other fatty acids were without appreciable effect. The inhibition of thrombin-stimulated thromboxane A₂ release was dependent on the time and temperature of incubation, as well as on the concentration of added linoleic acid. Supplementation increased the amount of linoleic acid in the platelet phospholipids, but the arachidonic acid content of the phospholipids was reduced. [1-¹⁴C]Linoleic acid was not converted to arachidonic acid by the platelets. Linoleic acid was released exclusively form the inositol phosphoglycerides when the enriched platelets were stimulated with thrombin. The linoleate-enriched platelets converted less [1-¹⁴C]arachidonic acid to all prostaglandin products, suggesting that the platelet cyclooxygenase was partially inhibited.     

Arachidonic acid-induced human platelet aggregation independent of cyclooxygenase and lipoxygenase

It is generally agreed that arachidonic acid (20: 4 omega 6) can stimulate platelet aggregation after conversion to prostaglandin G2 and H2 and thence to thromboxane A2. This action is prevented by cyclooxygenase inhibitors. Washed platelets were isolated on metrizamide gradient and resuspended in a Ca2+-free buffer. Their stimulation by C 20: 4 6 was followed by 14C serotonin (5HT) release, thromboxane (TX) synthesis and an increase of light transmission, not dependent on aggregation, accompanied by slight lysis (14%). The addition of extrinsic Ca2+ suppressed lysis and allowed the formation of aggregates. Under these conditions, cyclooxygenase inhibitors such as acetyl salicylic acid, indomethacin or flurbiprofen totally suppressed TX synthesis without preventing platelet aggregation or [14C]-5HT release. Other C 20 polyunsaturated fatty acids could not substitute for C 20: 4 omega 6 in inducing aggregation, and Ca2+ was found to be a prerequisite for protection of the cell against lysis as well as for aggregation in the absence or TX formation. The use of the lipoxygenase inhibitor BW 755 C did not prevent C 20: 4 omega 6-induced aggregation of aspirin-treated platelets, suggesting that the phenomenon was independent of this pathway also. The total suppression of oxidative metabolism with these inhibitors was verified by the analysis of icosanoids using glass capillary column gas chromatography. It is suggested that under these conditions, C 20: 4 omega 6-induced platelet aggregation might be due to an increased membrane permeability to Ca2+ induced by this fatty acid in the absence of oxidation.     

Influence of dietary fatty acids on membrane fluidity and activation of rat platelets

The apparent steady-state fluorescence anisotropy of DPH- or TMA-DPH-labeled washed rat platelets is strongly affected by factors that also influence the turbidity by these platelet suspensions. Sonicated preparations from platelet lipids have a low turbidity and give anisotropy values which are hardly affected by the experimental conditions. We studied the effect of four high-fat diets on membrane fluidity, lipid composition and activation tendency of washed platelets. The diets contained 50 energy% of oils with different levels of saturated and (poly)unsaturated fatty acids. Only small diet-induced differences in DPH fluorescence anisotropy were found, which were comparable for intact platelets and platelet lipids. These differences were unrelated to the degree of saturation of the dietary fatty acids. Platelets from rats fed mainly saturated fatty acids differed significantly from other diet groups in a higher unsaturation degree of phospholipids and a lower cholesterol/phospholipid ratio, but this was not detected by DPH in terms of decreased anisotropy. These platelets aggregated less than other platelets in response to thrombin or collagen. The lower response to collagen persisted in indomethacin-treated platelets activated with the thromboxane A2 mimetic U46619, indicating a different sensitivity of these platelets for thromboxane A2. We conclude that in rat platelets: (a) the overall membrane fluidity and phospholipid unsaturation degree are subject to strong homeostatic control; (b) steady-state anisotropy with DPH or TMA-DPH label is inadequate to reveal subtile changes in lipid profile; (c) changes in platelet responsiveness to thrombin and thromboxane A2, rather than (plasma) membrane fluidity, determine the effect of dietary fatty acids on platelet aggregation.     

Purification and characterization of mitochondrial thioesterase from rabbit myocardium and its inhibition by palmitoyl carnitine

The inhibitory effects of saturated fatty acids with 4 to 18 carbon atoms on ADP-induced aggregation of bovine platelets were investigated. The inhibitory effects of the acids increased with increase of their alkyl chain length up to C14. On the other hand, from C16 the inhibitory effects tended to decrease with increase of chain length, and stearic acid (C18) was not inhibitory. There was a linear relationship between the inhibitory effects and alkyl chain lengths up to C12. This linear relation and the slope of the linear regression line suggested that the inhibitory effects of the acids depended on their partition into the membrane. The fatty acids decreased the fluorescence of the surface charge probe 2-p-toluidinylnaphthalene-6-sulfonate, indicating that they increased the negative charge on the membrane surface. The relative effects of the acids on the fluorescence were consistent with their relative inhibitory effects on aggregation. These results suggest that the inhibition of platelet aggregation by saturated fatty acids is due to a change in the membrane surface charge of the platelet plasma membrane.     

Dietary n-9, n-6, and n-3 fatty acids modify linoleic acid more than arachidonic acid levels in plasma and platelet lipids and minimally affect platelet thromboxane formation in the rabbit

We have studied the effects of semisynthetic diets containing 5% by weight (12% of the energy) of either olive oil (70% oleic acid, OA) or corn oil (58% linoleic acid), or fish oil (Max EPA, containing about 30% eicosapentaenoic, EPA C 20:5 n-3, plus docosahexaenoic, DHA C 22:6 n-3, acids, and less than 2% linoleic acid), fed to male rabbits for a period of five weeks, on plasma and platelet fatty acids and platelet thromboxane formation. Aim of the study was to quantitate the absolute changes of n-6 and n-3 fatty acid levels in plasma and platelet lipid pools after dietary manipulations and to correlate the effects on eicosanoid-precursor fatty acids with those on platelet thromboxane formation. The major differences were found when comparing the group fed fish oil and depleted linoleic acid vs the other groups. The accumulation of n-3 fatty acids in various lipid classes was associated with modifications in the distribution of linoleic acid and arachidonic acid in different lipid pools. In platelets maximal incorporation of n-3 fatty acids occurred in phosphatidyl ethanolamine, which also participated in most of the total arachidonic acid reduction occurring in platelets, and linoleic acid, more than archidonic acid, was replaced by n-3 fatty acids in various phospholipids. The archidonic acid content of phosphatidyl choline was unaffected and that of phosphatidyl inositol only marginally reduced. Thromboxane formation by thrombin stimulated platelets did not differ among the three groups, and this may be related to the minimal changes of arachidonic acid in phosphatidyl choline and phosphatidyl inositol.     

Manipulation of platelet aggregation by prostaglandins and their fatty acid precursors: pharmacological basis for a therapeutic approach

Addition of the one-, two- or three- series endoperoxide to human platelet-rich plasma tend to suppress aggregation, through the action of their respective non-enzymatic breakdown products PGE1, PGD2, or PGD3 all of which elevate cyclic AMP levels. On the other hand, these stable primary products do not arise in appreciable amounts from intrinsic endoperoxides generated from either endogenous or exogenous free fatty acids. 5,8,11,14,17-Eicosapentaenoic acid (EPA) suppresses arachidonic acid (5,8,11,14-eicosatetraenoic acid) conversion by cyclooxygenase (as well as lipoxygenase) to aggregatory metabolites in platelets. Exogenously added EPA was capable of inhibiting PRP aggregation induced either by exogenous or endogenous (released by ADP or collagen) arachidonate. The hypothetical combination of an EPA-rich diet and a thromboxane synthetase inhibitor might abolish production of the pro-aggregatory species, thromboxane A2, and enhance formation of the anti-aggregatory metabolite, prostacyclin. Whereas EPA is not detectably metabolized by platelets, dihomo-gamma-linolenic acid (8,11,14-eicosatrienoic acid) is primarily converted by cyclooxygenase and thromboxane synthetase into the inactive metabolite, 12-hydroxyheptadecadienoic (HHD) acid. Pretreatment of human platelet suspensions with the thromboxane synthetase inhibitor imidazole unmasks the aggregatory property of PGH1 and DLL which was partially compromised by the PGE1 formed. The combination of the thromboxane synthetase inhibitor and an adenylate cyclase inhibitor unmasks a complete irreversible aggregation by DLL or PGH1. The basis of a dietary strategy that replaces AA with DLL must rely on the production by the platelet of an inactive metabolite (HHD) rather than thromboxane A2.     

Inhibitory mechanisms of metallothionein on platelet aggregation in in vitro and platelet plug formation in in vivo experiments

Metallothionein (MT) is a low-molecular-weight, cysteine-rich protein that contains heavy metals such as cadmium and zinc. The biological function of MT in platelets is not yet understood. Therefore, the aim of this study was to systematically examine the inhibitory mechanisms of metallothionein in platelet aggregation. In this study, metallothionein concentration-dependently (1-8 microM) inhibited platelet aggregation in human platelets stimulated by agonists. Metallothionein (4 and 8 microM) inhibited phosphoinositide breakdown in [3H]-inositol-labeled platelets, intracellular Ca+2 mobilization in Fura-2 AM-loaded platelets, and thromboxane A2 formation stimulated by collagen. In addition, metallothionein (4 and 8 microM) significantly increased the formation of cyclic GMP but not cyclic AMP in human platelets. Rapid phosphorylation of a protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by PDBu (100 nM). This phosphorylation was markedly inhibited by metallothionein (4 and 8 microM) in phosphorus-32-labeled platelets. In an in vivo thrombotic study, platelet thrombus formation was induced by irradiation of mesenteric venules in mice pretreated with fluorescein sodium. Metallothionein (6 microg/g) significantly prolonged the latency period for inducing platelet plug formation in mesenteric venules. These results indicate that the antiplatelet activity of metallothionein may involve the following pathways: (1) metallothionein may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown and thromboxane A2 formation, thereby leading to inhibition of intracellular Ca+2 mobilization; (ii) Metallothionein also activated the formation of cyclic GMP in human platelets, resulting in inhibition of platelet aggregation. The results strongly indicate that metallothionein provides protection against thromboembolism.