Studies on the mechanism of antiaggregatory effect of Moutan Cortex

The effects of Moutan Cortex and one of its major components, paeonol, on platelet aggregation and arachidonic acid (AA) metabolism in human platelets were studied. One week oral administration of water extract of Moutan Cortex [Moutan Cortex (w), 3 g/day] significantly reduced platelet aggregation and thromboxane B2 (TXB2) formation induced by collagen, epinephrine and ADP. Paeonol dose-dependently inhibited ADP and collagen induced platelet aggregation in vitro. Moutan Cortex (w) and paeonol dose-dependently inhibited the conversion of exogenous [14C]AA to [14C]heptadecatetraenoic acid [( 14C]HHT) and [14C]TXB2 by washed human platelets, while both of them increased its conversion to [14C]12-hydroxy eicosatetraenoic acid [( 14C]12-HETE). High dose of Moutan Cortex (w) inhibited the release of [14C]AA from prelabeled platelets in vitro, while paeonol did not. These results suggest that a reduction in platelet aggregation by the oral administration of Moutan Cortex might be ascribed to a decrease in thromboxane synthesis and that paeonol might play an important role in the antiaggregatory effect of Moutan Cortex because of its potent inhibitory effect on platelet aggregation and thromboxane formation.     

An analysis of inhibitory effect of cyclic GMP on arachidonic acid- or collagen-induced aggregation in rabbit platelets

Aggregation of washed rabbit platelets induced by arachidonic acid (AA) or collagen was inhibited by nitroprusside (NP) and 8-bromo cyclic GMP in a concentration-dependent manner. Although NP and 8-bromo cyclic GMP inhibited the AA-induced aggregation, these agents did not affect the conversion of exogenous AA to PG endoperoxides and TXA2 (which were observed as TXB2). On the other hand, collagen caused release of AA from phospholipids and sequential formation of TXB2 in [14C]AA prelabeled platelets. In contrast with the case in which exogenous AA was used, NP and 8-bromo cyclic GMP inhibited the collagen-induced formation of TXB2 by preventing the liberation of endogenous AA. These results indicate that cyclic GMP has at least two different inhibitory actions in platelets; one is the inhibition of AA release from phospholipids and the other is the inhibition of the action of TXA2 in platelets.     

Effects of arachidonic acid on the metabolism of eicosapentaenoic acid in washed human platelets

We examined effects of arachidonic acid (AA) on eicosapentaenoic acid (EPA) metabolism in washed human platelets. Although human platelets had been considered to metabolize scarcely EPA, a simultaneous addition of EPA and AA to washed platelet suspensions stimulated markedly EPA metabolism. In addition, the stimulatory effect was more potent over the formation of thromboxane (TX) B3 than that of 12-hydroxy-5,8,10,14,17-eicosapentaenoic acid (HEPE). The stimulation by AA can be due to AA itself and/or AA metabolites. Indomethacin decreased the stimulatory effect of AA on the HEPE formation, suggesting that cyclooxygenase product(s) of AA stimulated the HEPE formation. Among the metabolites of AA investigated, prostaglandin (PG)G2 and 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid had the stimulatory effect on both TXB2 and HEPE formations, whereas PGH2, PGD2, TXB2 and 12-hydroxy-5, 8,10,14-eicosatetraenoic acid were ineffective.     

Effects of the ratio of exogenous eicosapentaenoic acid to arachidonic acid on platelet aggregation and serotonin release

We added arachidonic acid (AA) and eicosapentaenoic acid (EPA) to washed platelet suspensions in the absence of albumin, holding the total amount of the fatty acids constant at 2 microM, and changing the ratio of EPA to AA. Platelet aggregation, serotonin release and the amount of thromboxane (TX) B2, a cyclooxygenase product synthesized from exogenous AA, decreased as the ratio was increased. The decreases were greater than the expected ones from the diminution of the amount of exogenous AA. On the other hand, 12-hydroxyeicosatetraenoic acid (HETE), a lipoxygenase product synthesized from exogenous AA, increased in the presence of EPA. Although EPA was reported to be a poor substrate for platelet cyclooxygenase, the amount of TXB3 synthesized from exogenous EPA increased markedly by the simultaneous addition of AA. These results suggest that the EPA/AA ratio-dependent decrease in platelet aggregation and serotonin release is caused at least by both the decrease in the absolute amount of AA and the inhibitory effect of EPA on AA-metabolism via the cyclooxygenase pathway. Further studies on effects of EPA-metabolites via the cyclooxygenase pathway on platelet responses will be needed.     

Persistent impairment of platelet aggregation following cessation of a short-course dietary supplementation of moderate amounts of N-3 fatty acid ethyl esters.

The duration of the effect of a short-course (1-mo twice-daily) supplementation of moderate amounts (2.28 g) of n-3 fatty acid ethyl esters (FA) on platelet lipid composition and aggregation was compared with that of olive oil (3 g/d) supplementation in 14 healthy volunteers. The FA preparation employed contained eicosapentaenoic acid (EPA) and docosahexaenoic acids (DHA) in a ratio of 1:1.4. A marked rise (p <0.05) in the plasma and platelet content of EPA and DHA, and minimal changes in the content of arachidonic acid (AA) were documented at withdrawal of the n-3 FA supplementation. EPA/AA and DHA/AA ratios in platelet phospholipids showed that the FA accumulation persisted 8-12 wks after stopping the supplementation (p <0.05). The aggregation of platelets in response to collagen or ADP, and thromboxane B2 (TXB2) formation were impaired at withdrawal. The impaired aggregation lasted 8-12 weeks (p always <0.05), whereas TXB2 formation returned to basal values 4 weeks after stopping the n-3 supplementation. No correlation was found between impaired aggregation and TXB2 formation. In contrast, the impaired sensitivity to ADP (p = 0.036) and, to a lesser extent, to collagen (p = 0.068) were related to changes in the intracellular pH (pHi) of the Na+/H+ reverse transport. No changes in platelet composition or function were observed either during or following olive oil supplementation. These results document a long-lasting impairment of platelet sensitivity to ADP and collagen; changes in the pHi values of the Na+/H+ reverse transport, and a simultaneous persistent accumulation of EPA and DHA in platelet phospholipids, after stopping a short-course dietary supplementation of moderate amounts of n-3 fatty acid ethyl esters.     

Investigation on a selective non-prostanoic thromboxane antagonist, BM 13.177, in human platelets

The mode of action of BM 13.177 (4-[2-(benzenesulfonamido)-ethyl] phenoxyacetic acid), a new anti-aggregating and anti-thrombotic agent, was studied in human washed platelets and citrated PRP. With ASA-treated platelets, BM 13.177 (0.1 - 100 microM) did not inhibit the shape change and the aggregation induced by ADP, serotonin, adrenaline, thrombin, or collagen. Therefore, BM 13.177 is neither an antagonist of ADP, serotonin, adrenaline, thrombin, or collagen nor a common pathway inhibitor like PGE1, or an inhibitor of the platelet interactions during aggregation. However, BM 13.177 (greater than or equal to 0.1 microM) produced a dose-dependent reduction of shape change, aggregation and release of [3H]serotonin induced by the stable PGH2 analogues U 46619 and U 44069 in ASA-treated platelets or ASA-treated citrated PRP. In untreated platelets, BM 13.177 inhibited platelet activation by U 46619 or U 44069 and by exogenous arachidonic acid or by endogenous arachidonic acid mobilized by hydrogen peroxide. Consequently, the ADP- and adrenaline-induced secondary aggregation and [3H]serotonin release in citrated PRP and the major effects of collagen were also inhibited. In washed platelets treated with 10 microM arachidonic acid or 100 microM hydrogen peroxide, the formation of TXB2 was not inhibited by 10 microM BM 13.177. However, the TXB2 formation after stimulation with 1,200 microM hydrogen peroxide was partially reduced by BM 13.177 to the same extent as by PGE1. This reduction may be due to the absence of a secondary release of arachidonic acid from phospholipids if the platelets were prevented from activation by BM 13.177 or PGE1. Arachidonic acid and hydrogen peroxide also induced the shape change, aggregation and release of washed platelets when thromboxane formation was inhibited by dazoxiben. Under these conditions, BM 13.177 was able to abolish the platelet response which was due to accumulating prostaglandin endoperoxides. These results show that BM 13.177 acts as a selective antagonist of TXA2 and prostaglandin endoperoxides. Its inhibitory effect on platelet function does not depend on an inhibition of either the primary release of arachidonic acid or the activities of cyclooxygenase or thromboxane synthetase.     

Influence of diamide on aggregation, cytoskeletal proteins, and arachidonic acid metabolism in human platelets

Simultaneous addition of diamide (azodicarboxylic acid-bis-dimethylamide, DIA), a SH-oxidizing agent, and collagen causes a deaggregation of otherwise irreversibly aggregating platelets. Thromboxane B2 (TXB2) and 12-HE-TE formation is inhibited depending on the concentration ratio between collagen and DIA. Thus, at 0.25 mM DIA and 20 micrograms/ml collagen neither TXB2 nor 12-HETE were measurable, but a full scale reversible aggregation is induced. Deaggregation is further attained by adding DIA to collagen-induced aggregates at a time, when maximum amplitude has been achieved. Investigation of arachidonic acid (AA) metabolites under these conditions revealed no influence of DIA on AA metabolism. Therefore, AA metabolization seems to play a minor role in collagen-induced aggregation and DIA-induced deaggregation. Polymerization of certain cytoskeletal proteins of the platelets, after addition of DIA, parallels DIA-induced deaggregation. DIA inhibits endogenous AA release, probably by interaction with platelet plasma membrane. DIA seems to inhibit the release of the alpha-granula protein thrombospondin.     

The mechanism of the inhibitory effect of protease inhibitors on platelet aggregation and cellular synthesis of prostaglandins. I. The effect on the release of arachidonic acid from phospholipids

The inhibitory effect of proteinase inhibitors on platelet aggregation was investigated. The proteinase inhibitors tested were SBTI, leupeptin and FOY (a synthetic proteinase inhibitor). Also, synthetic substrates for serine proteinases (TLME, ATEE) were tested. They completely inhibited the secondary aggregation of platelets induced by ADP or epinephrine. They also completely inhibited the platelet aggregation induced by collagen or thrombin. The aggregation induced by arachidonic acid was completely inhibited by all the proteinase inhibitors and synthetic substrates. The aggregation induced by Ca ionophore A 23187 was completely inhibited by leupeptin, FOY, TLME or ATEE but not by SBTI. It is generally accepted that platelet prostaglandin metabolism plays an important role in platelet aggregation. As the first step to elucidate the possible mechanism of the inhibitory effect of the proteinase inhibitors, their effect on the release of arachidonic acid from platelet phospholipids was investigated. The release of arachidonic acid from ¹⁴C-arachidonate incorporated gel filtered platelets (¹⁴C-AA-GFP) by thrombin or A 23187 was directly measured in the presence or absence of a proteinase inhibitor or synthetic substrate, utilizing thin layer chromatography (TLC) and a scintillation counting. The release was almost completely blocked when the aggregation of ¹⁴C-AA-GFP by thrombin or A 23187 was completely inhibited by a proteinase inhibitor or a synthetic substrate.     

Evidence for a platelet membrane defect in the myeloproliferative syndromes

Bleeding and abnormal platelet aggregation occur in patients with myeloproliferative disorders. In this study, twenty patients were examined, some sequentially, and a proportion found to have defective aggregation toward adrenaline, adenosine diphosphate (ADP), and collagen. In these seven patients, the abnormality in platelet response with defective collagen-induced [14C]serotonin release correlated with poor collagen-stimulated thromboxane B2 (TXB2) production. In contrast, five of these patients showed a normal threshold aggregation response to arachidonic acid. The combined results suggest that in these patients, there is a defect between receptor-stimulus coupling and the mobilization of arachidonic acid from membrane phospholipid.     

The mechanism of the inhibitory effect of proteinase inhibitors on platelet aggregation and cellular synthesis of prostaglandins. I. The effect on the release of arachidonic acid from phospholipids

The inhibitory effect of proteinase inhibitors on platelet aggregation was investigated. The proteinase inhibitors tested were SBTI, leupeptin and FOY (a synthetic proteinase inhibitor). Also, synthetic substrates for serine proteinases (TLME, ATEE) were tested. They completely inhibited the secondary aggregation of platelets induced by ADP or epinephrine. They also completely inhibited the platelet aggregation induced by collagen or thrombin. The aggregation induced by arachidonic acid was completely inhibited by all the proteinase inhibitors and synthetic substrates. The aggregation induced by Ca ionophore A 23187 was completely inhibited by leupeptin, FOY, TLME or ATEE but not by SBTI. It is generally accepted that platelet prostaglandin metabolism plays an important role in platelet aggregation. As the first step to elucidate the possible mechanism of the inhibitory effect of the proteinase inhibitors, their effect on the release of arachidonic acid from platelet phospholipids was investigated. The release of arachidonic acid from ¹⁴C-arachidonate incorporated gel filtered platelets (¹⁴C-AA-GFP) by thrombin or A 23187 was directly measured in the presence or absence of a proteinase inhibitor or synthetic substrate, utilizing thin layer chromatography (TLC) and a scintillation counting. The release was almost completely blocked when the aggregation of ¹⁴C-AA-GFP by thrombin or A 23187 was completely inhibited by a proteinase inhibitor or a synthetic substrate.     

The effects of a mixed fish diet on platelet function, fatty acids and serum lipids

Eight healthy subjects were fed a diet containing 1–4 g eicosapentaenoic acid (EPA) daily for 8–21 days. The EPA was derived from 300–400 g per day of sardines, pilchards, herring and/or kabeljou. Sources of arachidonic acid (AA) in the diet were reduced. At the end of the experimental period there was an increase in the ratio of EPA to AA in the platelets and a decrease in platelet aggregation to ADP, epinephrine and collagen. Aggregation to AA was not reduced. Thromboxane production in response to all four agonists was reduced. Serum total and HDL cholesterol levels fell significantly but platelet counts, LDL cholesterol and triglyceride values did not change. We conclude that even a relatively modest intake of EPA derived from a mixed fish diet together with a reduction in AA intake can alter $\text{in vitro}$ platelet function and serum lipids significantly. A long term controlled trial of a palatable mixed fish diet to assess possible antithrombotic and antiatherogenic effects is justifiable.     

Comparison of the in vitro effect of eicosapentaenoic acid (EPA)-derived lipoxygenase metabolites on human platelet function with those of arachidonic acid

Eicosapentaenoic acid (EPA) has been reported to have a potent anti-aggregatory activity and to be efficiently metabolized by 12-lipoxygenase, not by cyclooxygenase in platelets. In vitro effect of 12-lipoxygenase metabolites of EPA on platelet function was studied and compared with those of arachidonic acid (AA). The 12-lipoxygenase metabolites of AA and EPA; 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and 12-hydroperoxyeicosapentaenoic acid (12-HPEPE), and their hydroxy derivatives, 12-hydroxyeicosatetraenoic acid (12-HETE) and 12-hydroxyeicosapentaenoic acid (12-HEPE) were prepared enzymatically using human platelet lysate. These compounds were purified by high performance liquid chromatography and identified by gas chromatography mass spectrometry. 12-HPETE and 12-HPEPE inhibited dose-dependently washed human platelet aggregation and serotonin (5-HT) release induced by AA and collagen. The potency of 12-HPEPE was almost equal to that of 12-HPETE. Their hydroxy derivatives, 12-HETE and 12-HEPE were less potent. 12-hydroperoxy derivatives of AA and EPA were the most potent in inhibiting platelet aggregation and 5-HT release among 5-, 12- and 15-hydroperoxy isomers of AA and EPA. The inhibitory effects of 12-HPETE and 12-HPEPE on platelet aggregation were additive.     

Suppression by beta-lactam antibiotics of thromboxane A2 generation and arachidonic acid release in rabbit platelets in vitro

High concentrations of latamoxef and some other beta-lactam antibiotics suppressed thromboxane A2 generation as determined from the thromboxane B2 level in the in vitro aggregation of rabbit platelets in agonist-induced stimulations. In aggregation induced with a low concentration of collagen, the suppression of thromboxane B2 generation correlated well with the suppressions of aggregation and serotonin (5-HT) release; collagen produced thromboxane A2-dependent aggregation and 5-HT release as judged from the inhibitory action of indomethacin. Latamoxef also suppressed thromboxane B2 generation when platelet stimulation was induced by thrombin or platelet activating factor at concentrations at which it did not affect either aggregation or 5-HT release. However, latamoxef did not affect platelet responses including thromboxane B2 generation induced by exogenous arachidonic acid or calcium ionophore, A23187. Beta-lactam antibiotics also interfered with arachidonic acid release from the membrane phospholipids of platelets which had been prelabelled with [3H]arachidonic acid and aggregated with collagen. These results suggest that in the in vitro aggregation of platelets, beta-lactam antibiotics interfere with some of the receptor-stimulated processes which lead to arachidonic acid release from the membranes and this, in turn, suppresses thromboxane B2 generation.     

Factors affecting platelet cyclic GMP levels during aggregation induced by collagen and by arachidonic acid.

Platelet aggregation, [¹⁴C]5-HT release, malondialdehyde (MDA) production and changes in platelet cyclic GMP were measured in stirred heparinized platelet-rich plasma after addition of aggregating agents in the presence and absence of specific inhibitors. Platelet cyclic GMP levels were monitored using a simple method for isolation of cyclic [³H]GMP from platelets containing [³H]GTP labelled by preincubation with [³H]guanine: the agents studied did not affect the specific radioactivity of platelet [³H]GTP. Addition of ADP or 5-HT increased platelet cyclic [³H]GMP, this effect being greater with ADP, which also caused more marked aggregation than 5-HT. These actions of ADP were blocked by creatine phosphate with creatine phosphokinase (CP+CPK) and those of 5-HT by methysergide. CP+ CPK, but not indomethacin, inhibited the increase in cyclic [³H]GMP caused by collagen at 10 μg/ml, indicating that released ADP and not formation of prostaglandin endoperoxides was responsible for this increase. With collagen at 80 μg/ml, CP+CPK, methysergide and indomethacin, individually or in combination, did not block the increase in cyclic [³H]GMP, indicating that factors in addition to ADP, 5-HT and endoperoxides or derivatives can mediate the increase. CP+CPK, alone or with methysergide, did not affect the increase in cyclic [³H]GMP caused by 1 mM arachidonic acid, while indomethacin abolished it, indicating that in this case endoperoxide formation, but not the action of released ADP, was essential. The results provide further evidence that collagen, unlike arachidonic acid, induces the release reaction by both indomethacin-sensitive and insensitive mechanisms. CP+CPK partially inhibited both the release of [¹⁴C]5-HT and production of MDA observed with collagen or arachidonic acid, suggesting that released ADP may potentiate the release reaction by enhancing endoperoxide synthesis. ADP also potentiated indomethacin-insensitive release. In all these studies the increases in cyclic [³H]GMP levels correlated much more closely with platelet aggregation than with release of [¹⁴C]5-HT or MDA formation.     

Effect of cytoplasmic pH on platelet activation by arachidonic acid

The effects of cytoplasmic acidification by nigericin, monovalent cation ionophore, on platelet activation induced by arachidonic acid (AA) were examined. AA-induced aggregation and TXB2 formation were inhibited with decreasing cytoplasmic pH (pHc). Although TXA2 receptor agonists (STA2, U-46619)-induced aggregation of some platelet samples were suppressed by nigericin, the suppression did not correspond to the acidification of pHc. Aggregation initiated by collagen was inhibited by nigericin. The inhibition, however, was not observed in platelets treated by aspirin. These results suggest that the inhibition of AA-induced aggregation by cytoplasmic acidification is due to inhibition of the metabolism of AA to TXA2.     

Platelet adenine nucleotides and arachidonic acid metabolism in the myeloproliferative disorders

Platelet aggregation to collagen, adenosine diphosphate and arachidonic acid has been investigated in 17 patients with various myeloproliferative states. Ten patients who had abnormalities of aggregation to collagen and/or ADP were also all found to have diminished intracellular and releasable adenine nucleotides but aggregation to arachidonic acid was normal. Seven other patients who had normal aggregation responses had normal platelet adenine nucleotides. In the ten patients with abnormal platelet function platelet cyclo-oxygenase activity was normal but in two patients platelet lipoxygenase activity was reduced. Thromboxane B₂ production during collagen stimulation was found to be normal suggesting normal release of endogenous arachidonic acid. These findings suggest that the platelet defect in myeloproliferative states is due to an acquired storage pool disease.     

The effect of plasma on platelet function in hypercholesterolemic rabbits and the changes in fatty acid composition of the plasma

Rabbits were fed with 1% cholesterol-containing standard diet for 1 to 3 months. The arachidonic acid (AA)-induced aggregation of the platelet-rich plasma (PRP) of the control rabbits was accelerated by substitution of hypercholesterolemic plasma. The incorporation of C-AA into thromboxane B₂ in platelets was increased approximately 1.6 times with PRP and 1.2 times with the washed platelet suspension (WPS) in hypercholesterolemic rabbits as compared with those of the control. Analysis of the fatty acid compositions of phospholipids and total lipids of hypercholesterolemic rabbits revealed an increase in AA of platelets and plasma, and a decrease in docosahexaenoic acid (DHA) in plasma. The AA/DHA ratio of plasma increased dependently on the period of feeding with the high cholesterol diet, and the increase in the ratio was parallel with the acceleration of platelet aggregation by AA in PRP.     

Splitomicin suppresses human platelet aggregation via inhibition of cyclic AMP phosphodiesterase and intracellular Ca release

Splitomicin is derived from β-naphthol and is an inhibitor of Silent Information Regulator 2 (SIR2). Its naphthoic moiety might be responsible for its inhibitory effects on platelets. The major goal of our study was to examine possible mechanisms of action of splitomicin on platelet aggregation in order to promote development of a novel anti-platelet aggregation therapy for cardiovascular and cerebrovascular diseases. To study the inhibitory effects of splitomicin on platelet aggregation, we used washed human platelets, and monitored platelet aggregation and ATP release induced by thrombin (0.1 U/ml), collagen (2 μg/ml), arachidonic acid (AA) (0.5 mM), U46619 (2 μM) or ADP (10 μM). Splitomicin inhibited platelet aggregation induced by thrombin, collagen, AA and U46619 with a concentration dependent manner. Splitomicin increased cAMP and this effect was enhanced when splitomicin (150 μM) was combined with PGE1 (0.5 μM). It did not further increase cAMP when combined with IBMX. This data indicated that splitomicin increases cAMP by inhibiting activity of phosphodiestease. In addition, splitomicin (300 μM) attenuated intracellular Ca⁺⁺ mobilization, and production of thromboxane B2 (TXB2) in platelets that was induced by thrombin, collagen, AA or U46619. The inhibitory mechanism of splitomicin on platelet aggregation may increase cyclic AMP levels via inhibition of cyclic AMP phosphodiesterase activity and subsequent inhibition of intracellular Ca⁺⁺ mobilization, TXB2 formation and ATP release.     

Superoxide anion and hydroxyl radical release by collagen-induced platelet aggregation--role of arachidonic acid metabolism

Previous study demonstrated that platelets undergoing anoxia-reoxygenation generate superoxide anion (O2-) and hydroxyl radical (OH ) which in turn contribute to activate arachidonic acid (AA) metabolism. However it has not been clarified if oxygen free radicals (OFRs) are also generated when platelets are aggregated by common agonists. We used two probes, i.e. lucigenin and salicylic acid (SA), to measure platelet release of O2- and OH(0), respectively. Among the agonists used, such as ADP, thrombin and collagen, the release of O2- and OH was observed mainly when platelets were stimulated with collagen. Such release was inhibited in platelets pre-treated by aspirin suggesting that AA metabolism was the main source of O2- and OH(0) formation. To further analyze this relationship, O2- and OH(0) formation was measured during AA-stimulated platelet aggregation (PA); we observed that O2- and OH(0) release were dependent upon AA concentration. Furthermore, we found that the incubation of platelets with AACOCF3, a potent inhibitor of cytosolic phospholipase A2, inhibited collagen-induced platelet O2- and OH(0) release. The incubation of platelets with salicylic acid or ascorbic acid, which blunt OH and O2- respectively, inhibited both collagen-induced platelet aggregation and AA-release. This study demonstrated that collagen-induced platelet aggregation is associated with O2- and OH formation, which is dependent upon AA release and metabolism.     

Biosynthesis of prostanoids, tissue fatty acid composition and thrombotic parameters in rats fed diets enriched with docosahexaenoic (22:6n3) or eicosapentaenoic (20:5n3) acids

The objective of this experiment was to elucidate the effect(s) of eicosapentaenoic (20:5n3) vs docosahexaenoic (22:6n3) acid on prostaglandin biosynthesis and related thrombotic parameters. Diets were formulated to contain oils absent in (control) or enriched with either 20:5n3 (EPA) or 22:6n3 (DHA). The diets were fed to rats for three weeks and the following evaluated: 1) bleeding time; 2) blood viscosity; 3) platelet aggregation; 4) tissue fatty acids; 5) serum thromboxane (TXB₂), aortic prostacylin (6-keto) and 6) arachidonic acid conversion to eicosanoids by lung microsomes. There were no significant differences between treatments for bleeding time, red blood cell viscosity or platelet aggregation. In EPA fed rats 20:5n3 increased significantly in platelet and aorta phospholipids. In platelets and aorta 20:4n6 was slightly decreased in EPA and DHA animals. Platelet 22:6n3 levels were not altered by treatment, but 22:6n3 increased in the aorta of EPA and DHA fed rats. Similar changes were noted in lung and liver fatty acid composition. Serum TXB₂levels were significantly decreased only in the EPA vs control group. No differences were noted for aortic 6-keto levels or in the amount of hydroxy fatty acids, PGE, TXB₂or PGF₂ produced by lung microsomes. While fish oils have been shown to alter hematologic parameters in humans this study suggests that the rat is not similarly affected. Furthermore, it is evident that in the rat, 20:5n3 and not 22:6n3 is responsible for the alterations in platelet prostaglandin biosynthesis; however, these observations may not be directly applicable to other species.