Platelet Arachidonic Acid Research Articles

Guanine nucleotides inhibit agonist-stimulated arachidonic acid release in both intact and saponin-permeabilized human platelets

The effects of guanine nucleotides on arachidonic acid (AA) release were studied in intact and saponin-permeabilized human platelets. While GTP[S] itself caused a stimulation of AA release in permeabilized cells, GTP[S], GDP[S], GTP, ATP and other nucleotides inhibited AA release in response to thrombin and other agonists in intact, as well as permeabilized platelets. Inhibition of agonist-stimulated AA release by nucleotides was partially attenuated by addition of ADP, and was abolished by prior stimulation of platelets to discharge the ADP-containing dense granules. These results suggest: (i) that released ADP plays an important contributory role in agonist-stimulated platelet AA release, and (ii) that guanine nucleotides can modulate platelet activation through an extracellular action which is distinct from their effects on G-proteins.

Extract of a spice — Omum (Trachyspermum ammi)-shows antiaggregatory effects and alters arachidonic acid metabolism in human platelets

An ethereal extract of omum ( Trachyspermum ammi; Hindustani: ajwan) — a frequently consumed spice — was found to inhibit platelet aggregation induced by arachidonic acid (AA), epinephrine and collagen; in this respect it was most effective against AA-induced aggregation. Inhibition of aggregation by omum could be explained by its effect on platelet thromboxane production as suggested by the following experimental observation. (i) Omum reduced TxB 2 formation in intact platelet preparations from added arachidonate, and (ii) it reduced the formation of TxB 2 from AA-labelled platelets after stimulation with Ca 2+-ionophore A23187 by a direct action on cyclooxygenase as it did not affect the release of AA from labelled platelets. An increased formation of lipoxygenase-derived products from exogenous AA in omum-treated platelets was apparently due to redirection of AA from cyclooxygenase to the lipoxygenase pathway.

Platelet synthesis of cyclooxygenase and lipoxygenase products in type I and type II diabetes

In 24 type I and 22 type II diabetic patients without vascular complications and in 25 controls platelet thromboxane A2 (TxA2) and prostaglandin E2 (PGE 2) production (by radioimmunoassay-RIA) and 1–14C arachidonic acid (AA) metabolism (by high pressure liquid chromatography-HPLC) after thrombin stimulation were studied. Platelets both from type I and type II diabetics generated larger amounts of TxB2 (p < 0.001) and PGE 2 (p<0.005) than controls, independently of the presence of retinopathy. No significant differences in platelet AA uptake or metabolism via the cyclooxygenase (CO) route, after thrombin stimulation (5 NIH U/ml), were observed in diabetic patients: lipoxygenase metabolites were found to be slightly, but significantly decreased. A positive linear relationship (r=0.64, p < 0.001) was found between HbA-1c and TxB2 production, but not with fasting plasma glucose. These results indicate that metabolic alterations can affect platelet function independently of vascular complications. The absence of alterations in intraplatelet 1–14C AA metabolism via CO, in the presence of increased TxB2 and PGE 2 production from endogenous AA, suggests that the activation of CO is not the only possible mechanism of platelet activation and that probably an increased availability of platelet AA plays an important role in the enhanced platelet aggregation commonly found in diabetics.

Low platelet apachidonic acid in young patients with brain infarction

Fatty acid patterns of plasma and platelet lipids, platelet aggregation and thromboxane A 2(TxA 2) production were studied in young patients (n = 12) with brain infarction and in healthy controls (n = 13). Platelet arachidonic acid content was significantly reduced in the stroke patients, but in vitro platelet aggregation was similar in the two groups. A low dose of acetosalicylic acid (ASA) (100 mg) suppressed thromboxane production and normalized the platelet arachidonic acid values. The low arachidonic acid in platelets is probably due to its increased consumption, indicating platelet activation in vivo.

Abnormal platelet reactivity in diabetic microvascular disease: Is thromboxane synthesis related to platelet arachidonic acid content?

Abnormal platelet reactivity in diabetic microvascular disease: Is thromboxane synthesis related to platelet arachidonic acid content?

Evidence for a role for Na+-H+ exchange in activation of human platelets by PAF.

We have found previously that inhibitors of Na+-H+ exchange block platelet arachidonic acid release and subsequent secondary aggregation and serotonin release in response to epinephrine, ADP, and thrombin (0.004 U/ml). The present study demonstrates that the addition of ethylisopropylamiloride, an inhibitor of Na+-H+ exchange, leads to an inhibition of platelet activating factor-induced serotonin release and thromboxane B2 production in human platelets in citrated plasma. In addition, platelet activating factor-induced platelet secretion is blocked by the cyclooxygenase inhibitor indomethacin or the thromboxane antagonist SQ 29548, indicating that arachidonic acid mobilization and metabolism is required for platelet activating factor to elicit platelet activation. Our interpretation of the present findings is that platelet activating factor-induced secretion of dense granules from the human platelet requires the production of cyclooxygenase metabolites from arachidonic acid and that Na+-H+ exchange plays an important, albeit not exclusive, role in mobilization of arachidonic acid in response to platelet activating factor.

Procoagulant activity of platelet arachidonic acid metabolites.

1 Arachidonate metabolites have been extracted from indomethacin-treated human platelets after incubation with arachidonic acid. 2 After separation from platelet phospholipids, the extracts promoted the generation of large amounts of thrombin in normal plasma, but not in plasma devoid of lipoproteins. 3 The procoagulant activity was associated with a minor component of the mixture, which was active at concentrations below 10 micrograms ml-1. 4 The activity was similar to that of autoxidised arachidonic acid previously described. 5 Platelet arachidonic acid metabolites could play a role in the coagulation system.

Macrophage eicosanoid formation is stimulated by platelet arachidonic acid and prostaglandin endoperoxide transfer

The present study was designed to determine whether platelets transfer arachidonic acid or prostaglandin endoperoxide intermediates to macrophages which may be further metabolized into cyclooxygenase products. Adherent peritoneal macrophages were prepared from rats fed either a control diet or an essential fatty acid-deficient diet, and incubated with a suspension of washed rat platelets. Macrophage cyclooxygenase metabolism was inhibited by aspirin. In the presence of a thromboxane synthetase inhibitor, 7-(1-imidazolyl)heptanoic acid, immunoreactive 6-ketoprostaglandin F 1 α formation was significantly increased 3-fold. Since this increase was greater (P <00.01) than that seen with either 7-(1-imidazolyl)hepatanoic acid-treated platelets or aspirin-treated macrophages alone, these results indicate that shunting of endoperoxide from platelets to macrophages may have occurred. In further experiments, macrophages from essential fatty acid-deficient rats were substituted for normal macrophages. Essential fatty acid-deficient macrophages, depleted of arachidonic acid, produced only 2% of the amount of eicosanoids compared to macrophages from control rats. When platelets were exposed to aspirin, stimulated with thrombin, and added to essential fatty acid-deficient macrophages, significantly more immunoreactive 6-ketoprostaglandin F 1 α was formed than in the absence of platelets. This increased macrophage immunoreactive 6-ketoprostaglandin F α synthesis, therefore, must have occurred from platelet-derived arachidonic acid. These data indicate that in vitro, in the presence of an inhibition of thromboxane synthetase, prostaglandin endoperoxides, as well as arachidonic acid, may be transferred between these two cell types.

Effects of a 4-week freshwater fish (trout) diet on platelet aggregation, platelet fatty acids, serum lipids, and coagulation factors.

Eight healthy subjects consumed a diet in which all animal products were replaced by 750 g/day of freshwater trout. Platelet eicosapentaenoic acid (EPA) as a percentage of total platelet fatty acids rose from a prediet level of 0.2 +/- 0.4% to 3.4 +/- 1.6% after 4 weeks on the diet. Platelet arachidonic acid remained unchanged. Platelets became more hyperaggregable to collagen (p less than .025) but became hypoaggregable to arachidonic acid (p less than .05). The Ivy bleeding time became prolonged rising from a mean of 148 seconds before the diet to 168 seconds at 2 weeks and 202 seconds at 4 weeks. Serum lipids, coagulation profiles, and blood pressure remained unchanged. The changes induced by this diet were much less marked than changes induced by marine fish diets and diets supplemented by marine fish oil extracts, despite the fact that an equivalent amount of EPA was consumed and incorporated into platelets. These findings suggest that other substances, in addition to n-3 unsaturated fatty acids, may be responsible for the antithrombotic properties of marine fish.

Inhibition of platelet aggregation by novel triphenylethylene analogs

The present study has evaluated the effect of some newly synthesized triphenylethylene (TPE) analogs on platelet arachidonic acid metabolism and function. All compounds tested inhibited arachidonic acid induced platelet aggregation and several were superior to aspirin in their relative potency. Introduction of a carboxyl function into the alpha-ring, which should enhance binding according to proposed structural models for cyclooxygenase inhibitors, was not found to be beneficial. Increased structural rigidity, which resulted from covalent linkage of two aromatic rings in this series, did not eliminate anti-aggregatory properties.

Influence of certain compounds of plant origin on platelet aggregation and arachidonic acid metabolism in human platelets

Influence of certain compounds of plant origin on platelet aggregation and arachidonic acid metabolism in human platelets

Onion exerts antiaggregatory effects by altering arachidonic acid metabolism in platelets

In vitro effects of an oily extract of onion were examined on the metabolism of arachidonic acid (AA) in human platelets. Onion was found to reduce the formation of thromboxane and lipoxygenase products from exogenous arachidonic acid in platelets; it did not inhibit the incorporation of AA into platelet phospholipids. While not affecting the platelet phospholipase activity it did reduce the formation of thromboxane B 2 and lipoxygenase products in platelets that were prelabelled with arachidonic acid and then activated by A23187. This suggests that onion inhibits the formation of AA metabolites by exerting its effect at steps later than the liberation of AA. With concentrations of onion extract producing abolition of AA-induced aggregation, only partial inhibition of aggregation was observed with ADP- and epinephrine-induced aggregation. Onion did not inhibit A23187 induced aggregation. The results suggest that inhibition of platelet aggregation by onion is mediated largely by its effect on platelet thromboxane production.

Evidence for the mechanism by which garlic inhibits platelet aggregation

Aqueous extract of garlic inhibited aggregation induced by ADP, collagen, arachidonate (AA), epinephrine and calcium ionophore A23187 in a dose-dependent manner. In an attempt to clarify the mechanism of inhibition of aggregation, metabolism of arachidonic acid in platelets was examined in the presence of garlic extract. It was found that: (1) garlic reduced the formation of thromboxane from exogenous AA; (ii) garlic inhibited the phospholipase activity; (iii) garlic inhibited the formation of thromboxane and lipoxygenase products formed in platelets prelabelled with AA; and (iv) garlic inhibited the incorporation of arachidonate into platelet phospholipids. These effects may explain, in part, inhibition of platelet aggregation. Further, since garlic was also effective in inhibiting aggregation induced by calcium ionophore A23187 it may be suggested that the antiaggregation effect may be related to intraplatelet mobilization of calcium. Inhibition of epinephrine-induced aggregation by garlic extract may suggest that it may be inhibiting uptake of calcium into platelets thereby lowering cytosolic calcium concentrations. Thus garlic appears to be in possession of components which might exert their effects at various stages involved in the process of platelet aggregation.

Substrate for endothelial prostacyclin production in the presence of platelets exposed to collagen is derived from the platelets rather than the endothelium

Interactions between vascular endothelial cells and blood platelets have been investigated using a model microcirculation consisting of microcarrier beads colonized with human umbilical vein endothelial cells (HUVECs) and perfused with washed platelet suspensions. To simulate the effects of endothelial desquamation and exposure of subendothelium, fibrillar collagen in suspension was coinjected with the platelets. In this model, neither the passage of platelets alone nor collagen alone stimulated prostacyclin (PGI2) production by the HUVECs. Platelets activated by coinjection with collagen released thromboxane A2 (TXA2), and this was associated with the simultaneous production of PGI2 by the HUVECs. By means of double-isotope experiments with [3H]arachidonic acid (AA) incorporated into platelets and [14C]-AA into HUVECs, it was shown that all the PGI2 generated was derived from platelet AA and/or endoperoxides. This interpretation was strengthened by the finding that PGI2 production was not prevented by treatment of HUVECs with indomethacin followed by perfusion with collagen-stimulated platelets. AA metabolites in double-isotope label experiments were further characterized by reverse-phase chromatography, and it was shown that both cyclooxygenase and lipoxygenase products of the HUVECs were derived from platelet membrane lipid. Thrombin regularly produced transient PGI2 release, but showed rapid tachyphylaxis. Platelet-derived compounds including ADP, ATP, and platelet-activating factor (PAF) did not produce PGI2 release by HUVECs in this system. Thus, the transfer of AA and metabolites from collagen- stimulated platelets is likely to be the mechanism for PGI2 production in the context of minor degrees of endothelial desquamation.

Substrate for Endothelial Prostacyclin Production in the Presence of Platelets Exposed to Collagen Is Derived From the Platelets Rather Than the Endothelium

Interactions between vascular endothelial cells and blood platelets have been investigated using a model microcirculation consisting of microcarrier beads colonized with human umbilical vein endothelial cells (HUVECs) and perfused with washed platelet suspensions. To simulate the effects of endothelial desquamation and exposure of subendothelium, fibrillar collagen in suspension was coinjected with the platelets. In this model, neither the passage of platelets alone nor collagen alone stimulated prostacyclin (PGI2) production by the HUVECs. Platelets activated by coinjection with collagen released thromboxane A2 (TXA2), and this was associated with the simultaneous production of PGI2 by the HUVECs. By means of double-isotope experiments with [3H]arachidonic acid (AA) incorporated into platelets and [14C]-AA into HUVECs, it was shown that all the PGI2 generated was derived from platelet AA and/or endoperoxides. This interpretation was strengthened by the finding that PGI2 production was not prevented by treatment of HUVECs with indomethacin followed by perfusion with collagen-stimulated platelets. AA metabolites in double-isotope label experiments were further characterized by reverse-phase chromatography, and it was shown that both cyclooxygenase and lipoxygenase products of the HUVECs were derived from platelet membrane lipid. Thrombin regularly produced transient PGI2 release, but showed rapid tachyphylaxis. Platelet-derived compounds including ADP, ATP, and platelet-activating factor (PAF) did not produce PGI2 release by HUVECs in this system. Thus, the transfer of AA and metabolites from collagen-stimulated platelets is likely to be the mechanism for PGI2 production in the context of minor degrees of endothelial desquamation.

The Effect of Vegetarian Diets on Plasma Lipid and Platelet Levels

Vegetarians have an apparent diminished risk for the development of ischemic coronary heart disease. This may be secondary to dietary effects of plasma lipids and lipoproteins, but platelets, which may also play a role, have also been observed to have aberrant functions in vegetarians. We measured plasma lipid and lipoprotein levels, platelet function, platelet fatty acid levels, and platelet active prostaglandins in ten strict vegetarians (vegans), 15 lactovegetarians, and 25 age- and sex-matched omnivorous controls. The most striking observations were a highly significant rise in platelet linoleic acid concentration and a decline in platelet arachidonic acid concentration in both vegetarian subgroups as compared with omnivorous controls. Serum thromboxane and prostacyclin levels as well as results of platelet aggregation studies did not differ among the groups tested. Cholesterol levels were significantly lower in both vegetarian groups as compared with controls, but plasma high- and low-density lipoprotein levels were lower only in the vegan subgroup as compared with omnivores. If diet produces these changes in platelet fatty acid and plasma lipid levels it may contribute to the decreased risk of coronary heart disease and possibly atherosclerosis in vegetarians.

Biosynthesis of thromboxane in patients with systemic sclerosis and Raynaud's phenomenon.

Thromboxane A2, the predominant cyclo-oxygenase product of arachidonic acid in platelets, is a potent vasoconstrictor and platelet agonist. Analysis of urinary metabolites by gas chromatography and mass spectrometry is a specific non-invasive method of measuring the biosynthesis of thromboxane that avoids the problem of platelet activation ex vivo. Excretion of the major urinary thromboxane metabolite, 2,3-dinor-thromboxane B2, was significantly increased (p less than 0.001) in 10 patients (nine women) with systemic sclerosis complicated by Raynaud's phenomenon compared with healthy controls (486 (SD 88) v 162 (38) ng/g creatinine) and increased further in the patients (to 1007 (212) ng/g creatinine) during application of a cold stimulus sufficient to induce digital vasoconstriction. Consistent with an increase in platelet-vascular interactions in vivo, excretion of a prostacyclin metabolite was also significantly increased (p less than 0.005) in the patients with systemic sclerosis (248 (39) v 112 (10) ng/g creatinine) and tended to increase further on cooling. Biosynthesis of thromboxane is increased in patients with systemic sclerosis and may exacerbate digital vasospasm that such patients develop when cold. This observation and the concomitant increase in the formation of prostacyclin provide a rationale for evaluating compounds that prevent the synthesis of thromboxane A2 or inhibit its action while preserving the potential homoeostatic role of prostacyclin.

Familial essential thrombocythemia

Primary or essential thrombocythemia is rarely observed in childhood, and familial occurrence has been reported only once. In this study, essential thrombocythemia is documented in five members of both sexes from two to 62 years of age in three successive generations. The propositus had a persistent elevation of the platelet count, splenomegaly, a normal hemoglobin level, a normal white blood cell count, and abnormal platelet aggregation. Platelet arachidonic acid metabolites assayed by high-performance liquid chromatography and serum thrombopoietin levels were normal. Megakaryocytes were increased in number and size. Both mature and early immature megakaryocytes, but no atypical megakaryocytes, were identified by surface immunofluorescence. Bone marrow cultures showed normal myeloid and erythroid colony formation, and chromosome studies revealed a normal female karyotype. These findings support the concept that familial essential thrombocythemia is a myeloproliferative disorder that is transmitted by an autosomal dominant mode of inheritance, and that untreated young women and children with essential thrombocythemia have long survival.

Altered renal and platelet arachidonic acid metabolism in cirrhosis

Urinary excretion rates of prostaglandin (PG) E2, PGF2α, 6-keto-PGF1α, and thromboxane (TX) B2 were evaluated in three groups of cirrhotic patients [without ascites (group 1, 13 cases), with ascites and normal renal function (group 2, 15 cases), and with ascites and renal failure (group 3, 5 cases)]and in 14 healthy controls. All urinary arachidonate metabolites were significantly increased in group 2 patients. Patients with renal failure showed lower PGE2, PGF2α, and TXB2 values than those from group 2; PGF2α values were also lower than controls. Platelet TXA2 production during whole blood clotting was significantly reduced in all groups of patients. Administration of low-dose aspirin and sulindac, two cyclooxygenase inhibitors selectively sparing renal cyclooxygenase activity, effectively inhibited platelet TXA2 production without affecting urinary TXB2 excretion, thus ruling out platelets as a possible source of urinary TXB2. We conclude that patients with ascites and normal renal function show an overall activation of the renal PG system. Renal production of vasodilating PGE2 and PGI2 may be involved in supporting renal function in these patients. A reduced platelet synthesis of proaggregatory TXA2 also occurs in cirrhotic patients. This may play a role in the bleeding tendency of cirrhosis.

血小板Ｌｉｐｏｘｙｇｅｎａｓｅ系代謝産物による好中球活性化について

We studied effects of platelet lipoxygenase (LO) metabolites on 5-LO metabolism and degranulation of neutrophils by using 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and 12-hydroxyeicosatetraenoic acid (12-HETE) which were prepared from human platelet lysate incubated with arachidonic acid (AA) in the presence of indomethacin.When 5-LO products obtained from incubations of human neutrophils (3×107 cells/ml) with platelet activating factor (PAF) and AA were analysed by reversed-phase high-performance liquid chromatography, the addition of platelets (15-30×107 cells/ml) to the reaction mixture remarkably increased the synthesis of 5-LO products including leukotriene B4, 5-HETE and 5S, 12S-diHETE. While the 5-LO metabolism of neutrophils was similarly stimulated by cyclo-oxygenase-inhibited platelets, the synthesis of 5-LO products was significantly (p<0.001) reduced when LO-deficient platelets from patients with myeloproliferative disorders were added to the incubation mixture instead of normal platelets. The addition of 12-HPETE activated the 5-LO metabolism of neutrophils incubated with PAF and AA, but 12-HETE did not have such stimulating effects at all.Release of N-acetylglucosaminidase (NAGase) from cytochalasin B-treated neutrophils was estimated using p-nitrophenyl-2-acetamido-2-deoxy-β-D-glucopyranoside as a substrate. 12-HPETE induced not only the release of small amounts of NAGase, but also augmented the effects of N-formylmethionylleucylphenylalanine (FMLP) on the release reaction. Unlike the 5-LO metabolism, the release of NAGase from FMLP-stimulated neutrophils were increased by 12-HETE to some extent.These findings suggest that platelet LO metabolites, especially 12-HPETE, modulate neutrophil functions in some pathological states including inflammation, thrombosis and hemorrhage.