In vitro effects of synthetic antioxidants and vitamin E on arachidonic acid metabolism and thromboxane formation in human platelets and on platelet aggregation

The “in vitro” effects of α-tocopherol, butylhydroxytoluene (BHT) and butylhydroxyanisole (BHA) were studied on aggregation of human platelets induced by collagen and arachidonic acid (AA), on the metabolic conversion of ¹⁴C AA through the cyclooxygenase and lipoxygenase pathways and on the formation of thromboxane B₂ (TXB₂) in washed platelets after stimulation with collagen.Vitamin E completely inhibited AA induced platelet aggregation only at high concentration (mM) and after 10 minutes of preincubation, with limited effects on AA metabolism in platelets and no effect on TXB₂ formation from endogenous substrate. BHA completely inhibited platelet aggregation in the 10⁻⁶M range, gave 50% inhibition of AA metabolism in the 10⁻⁵M range and almost complete inhibition of thromboxane formation in the 10⁻⁴M range. BHT was about 100 times less active on platelet aggregation and AA metabolism. The lipoxygenase and cyclooxygenase pathways were differentially affected at low concentrations of BHA and only at concentrations greater than 5×10⁻⁵M were both pathways depressed.     

9α-homo-9,11-epoxy-5,13-prostadienoic acid analogues: Specific stable agonist (SQ 26,538) and antagonist (SQ 26,536) of the human platelet thromboxane receptor

A newly synthesized 9α-homo-9,11-epoxy-5,13-prostadienoic acid analogue, SQ 26,536, (8(R)9(S)11(R)12(S)-9α-homo-9,11-epoxy-5(Z), 13(E)-15S-hydroxyprostadienoic acid) inhibited arachidonic acid (AA)-induced platelet aggregation with an I₅₀ value of 1.7 μ . SQ 26,536 did not inhibit prostaglandin (PG) synthetase activity of bovine seminal vesicle microsomes or thromboxane (Tx) synthetase activity of lysed human blood platelets. SQ 26,536 also inhibited platelet aggregation induced by epinephrine (secondary phase), 9,11-azoPGH₂ and collagen but did not inhibit the primary phase of epinephrine-induced aggregation or ADP-induced platelet aggregation. SQ 26,538 (8(R)9(S)11(R)12(S)-9α-homo-9-, 11-epoxy-5(Z),13(E)-15R-hydroxyprostadienoic acid), a 15-epimer of SQ 26,536, induced platelet aggregation with an A₅₀ value of 2.5 μ . SQ 26,536 competitively inhibited SQ 26,538-induced platelet aggregation with a K_i value of 3 μ . Neither indomethacin, a PG synthetase inhibitor, nor SQ 80,338 (1-(3-phenyl-2-propenyl)-1H-imidazole), a Tx synthetase inhibitor, inhibited SQ 26,538- or 9,11-azoPGH₂-induced platelet aggregation. These data indicate that SQ 26,536 and SQ 26,538 are stable antagonist and agonist, respectively, of the human blood platelet thromboxane receptor.     

Effect of dipyridamole of prostaglandin generation by human platelets and vessel walls

These experiments were conducted to determine the effects of dipyridemole on human platelet aggregation, platelet thromboxane A₂ (TXA₂) and human vessel wall prostacyclin (PGI₂) generation. Dipyridamole in varying concentrations (5 to 50 μg/ml) had no direct effect on ADP-induced platelet aggregation in vitro, but it potentiated PGI₂-induced platelet aggregation inhibition at these concentrations. Dipyridamole also inhibited arachidonic acid-induced platelet TXA₂ generation at these concentrations. In continuously perfused umbilical vein segments, dipyridamole treatment resulted in stimulation of PGI₂ release determined by bioassay and by measurement of its stable metabolite 6-keto-PGF_1α. Minimum concentration of dipyridamole causing PGI₂ release was 50 μg/ml. These in vitro studies suggest that anti-thrombotic effects of dipyridamole in man are mediated mainly by potentiation of PGI₂ activity and to some extent by TXA₂ suppression. Stimulation of PGI₂ release by human vessels may not be seen in usual therapeutic concentrations.     

Inhibition of platelet aggregation by S-nitroso-cysteine via cGMP-independent mechanisms: evidence of inhibition of thromboxane A2 synthesis in human blood platelets

S-Nitroso-cysteine (SNC), a putative endothelium-derived relaxing factor, potently inhibited collagen- and arachidonic acid-induced platelet aggregation (IC₅₀=100 nM) and thromboxane A₂ (TxA₂) synthesis of human blood platelets. ODQ, a selective inhibitor of the soluble guanylyl cyclase, inhibited SNC-induced formation of cGMP but did not reverse inhibition by SNC of collagen- and arachidonic acid-induced platelet aggregation. Combination of ODQ with SQ-29548, a specific platelet TxA₂ receptor antagonist, did not modify the antiaggregatory action of SNC. Our study shows that SNC inhibits platelet aggregation by cGMP-independent mechanisms that may involve inhibition of TxA₂ synthesis in human platelets.     

OKY-1581: A selective inhibitor of thromboxane synthesis and

OKY-1581 is an effective inhibitor of thromboxane synthesis and . The generation of thromboxane B₂ (TxB₂), prostaglandin E (PGE) and prostaglandin F (PGF) was measured following clotting and during platelet aggregation induced by collagen. The presence of OKY 1581 either or caused a reduction in TxB₂ generation during clotting and platelet aggregation with a concomitant increase in PGE and PGF. The effect could be observed two hours after oral or subcutaneous administration of 5 to 100 mg per rabbit and lasted for 24 to 48 hours. The reduction in TxB₂ was not accompanied by an inhibition of clotting or platelet aggregation. OKY-1581 appears to be a suitable agent for studying the role of TxB₂ in atherosclerosis.     

A comparison of imidazole and 9,11-azoprosta-5,13-dienoic acid Two selective thromboxane synthetase inhibitors

Two selective thromboxane A₂ synthetase inhibitors, imidazole and 9,11-azoprosta-5,13-dienoic acid (azo analog I) were compared to determine their effects on the quantitative formation of thromboxane B₂ and prostaglandin E₂ accompanying human platelet aggregation. Azo analog I was at least 200 times more potent, on a molar basis, than imidazole in suppressing thromboxane B₂ formation in either platelet-rich plasma or washed platelet suspensions aggregated with arachidonic acid or prostaglandin H₂. The inhibitors differed in their effect on the aggregation response itself. Azo analog I selectively suppressed thromboxane A₂ formation with an accompanying, parallel, suppression of the platelet aggregation.Imidazole selectively suppressed thromboxane A₂ formation, but only suppressed the accompanying aggregation in platelet rich plasma, and not washed platelet suspensions. The results indicate that azo analog I functions by competitive inhibition of prostaglandin H₂ on the thromboxane synthetase, and that imidazole, while it suppresses thromboxane A₂ formation, may have an associated agonist activity that enhances platelet aggregation. The data presented support this hypothesis, and they emphasize the importance of thromboxane A₂ in arachidonate mediated platelet aggregation.     

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.     

Acetyl glycerylphosphorylcholine inhibition of prostaglandin I2-stimulated adenosine 3',5'-cyclic monophosphate levels in human platelets. Evidence for thromboxane A2 dependence

Previous studies with AGEPC (1-O-hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) stress the independence of the proaggregatory activity of AGEPC from the platelet cyclooxygenase. However, our dose response analyses in human platelet-rich plasma show distinct primary and secondary waves of aggregation in response to AGEPC. Second wave aggregation is inhibited completely by either 10 micro M indomethacin, a cyclooxygenase inhibitor, or 5.6 micro M 9,11-azoprosta-5,13-dienoic acid, a thromboxane A2 synthetase inhibitor. Simultaneous addition of AGEPC and prostaglandin I2 to platelet-rich plasma results in a marked increase in platelet cyclic AMP, which is not different from the prostaglandin I2 response alone. However, if prostaglandin I2 is added to AGEPC-stimulated platelets at a point where secondary aggregation is just beginning, AGEPC can attenuate prostaglandin I2-stimulated cyclic AMP accumulation. The inhibition by AGEPC is blocked by either cyclooxygenase or thromboxane A2 synthetase inhibitors, and radioimmunoassay of thromboxane B2 confirmed that the inhibition of prostaglandin I2-stimulated cyclic AMP accumulation is due to thromboxane A2 synthesis, and that AGEPC-stimulated secondary aggregation does not start until thromboxane A2 is synthesized. These data suggest that much of the bioactivity of AGEPC is attributable to thromboxane A2.     

Action of nonsteroidal anti-inflammatory agents on fibrinolysis and platelet aggregation

The influence of non-steroidal antiphlogistics (NSA, fluor derivatives of phenylanthranilic acid) on fibrinolysis, platelet function, prostaglandin metabolism and pharmacokinetics of indirect anticoagulants was studied in rats and rabbits in vitro and in vivo. NSA were found to shorten the euglobulin lysis time and to enlarge the lysis zones on fibrin plates. They potentiated the fibrinolytic activity of streptokinase and trypsin. Furthermore, they inhibited platelet aggregation induced by arachidonic acid in rabbits. NSA in combination with inhibitors of thromboxane synthetase potentiated inhibition of aggregation. After oral administration, NSA inhibited formation of thromboxane A2 and prostacyclin in rabbits in a dose-dependent manner. At comparatively low doses, thromboxane A2 synthesis was more effectively inhibited than prostacyclin formation. Due to pharmacokinetic interactions NSA enhanced the anticoagulant effect of indirect anticoagulants and accelerated their distribution and elimination.     

Antiplatelet Effect of Catechol Is Related to Inhibition of Cyclooxygenase,Reactive Oxygen Species,ERK/p38 Signaling and Thromboxane A2 Production

Catechol (benzenediol) is present in plant-derived products, such as vegetables, fruits, coffee, tea, wine, areca nut and cigarette smoke. Because platelet dysfunction is a risk factor of cardiovascular diseases, including stroke, atherosclerosis and myocardial infarction, the purpose of this study was to evaluate the anti-platelet and anti-inflammatory effect of catechol and its mechanisms. The effects of catechol on cyclooxygenase (COX) activity, arachidonic acid (AA)-induced aggregation, thromboxane B₂ (TXB₂) production, lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) production and extracellular signal-regulated kinase (ERK)/p38 phosphorylation were determined in rabbit platelets. In addition, its effect on IL-1β-induced prostaglandin E₂ (PGE₂) production by fibroblasts was determined. The ex vivo effect of catechol on platelet aggregation was also measured. Catechol (5-25 µM) suppressed AA-induced platelet aggregation and inhibited TXB₂ production at concentrations of 0.5–5 µM; however, it showed little cytotoxicity and did not alter U46619-induced platelet aggregation. Catechol (10–50 µM) suppressed COX-1 activity by 29–44% and COX-2 activity by 29–50%. It also inhibited IL-1β-induced PGE₂ production, but not COX-2 expression of fibroblasts. Moreover, catechol (1–10 µM) attenuated AA-induced ROS production in platelets and phorbol myristate acetate (PMA)-induced ROS production in human polymorphonuclear leukocytes. Exposure of platelets to catechol decreased AA-induced ERK and p38 phosphorylation. Finally, intravenous administration of catechol (2.5–5 µmole/mouse) attenuated ex vivo AA-induced platelet aggregation. These results suggest that catechol exhibited anti-platelet and anti-inflammatory effects, which were mediated by inhibition of COX, ROS and TXA₂ production as well as ERK/p38 phosphorylation. The anti-platelet effect of catechol was confirmed by ex vivo analysis. Exposure to catechol may affect platelet function and thus cardiovascular health.     

Mechanisms involved in the antiplatelet activity of ketamine in human platelets

The aim of this study was to systematically examine the inhibitory mechanisms of ketamine in platelet aggregation. In this study, ketamine concentration-dependently (100–350 µM) inhibited platelet aggregation both in washed human platelet suspensions and platelet-rich plasma stimulated by agonists. Ketamine inhibited phosphoinositide breakdown and intracellular Ca²⁺ mobilization in human platelets stimulated by collagen. Ketamine (200 and 350 µM) significantly inhibited thromboxane (Tx) A₂ formation stimulated by collagen. Moreover, ketamine (200 and 350 µM) increased the fluorescence of platelet membranes tagged with diphenylhexatriene. Rapid phosphorylation of a platelet protein ofMr 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12,13-dibutyrate (100 nM). This phosphorylation was markedly inhibited by ketamine (350 µM). These results indicate that the antiplatelet activity of ketamine may be involved in the following pathways. Ketamine may change platelet membrane fluidity, with a resultant influence on activation of phospholipase C, and subsequent inhibition of phosphoinositide breakdown and phosphorylation of P47, thereby leading to inhibition of intracellular Ca²⁺ mobilization and TxA₂ formation, ultimately resulting in inhibition of platelet aggregation.     

Regulatory role of cyclic adenosine 3′,5′-monophosphate on the plattelet cyclooxygenase and platelet function

Thromboxane A₂ plays and important role in arachidonic acid- and prostaglandin H₂-induced platelet aggregation. Agents that stimulate platelet adenylate cyclase (prostaglandin I₂, prostaglandin I₁, and prostaglandin E₁) and dibutyryl cyclic AMP inhibit both thromboxane A₂ formation and arachidonate-induced aggregation platelet-rich plasma. Despite complete suppression of aggregation with agents that elevate cyclic AMP, considerable thromboxane A₂ is still formed. Prostaglandin H₂-induced aggregations which bypass the cyclooxygenase regulatory step are also inhibited by agents that elevate cyclic AMP without any measurable effect on thromboxane A₂ production. These data demonstrate that cyclic AMP can inhibit platelet aggregation by a mechanism independent of its ability to suppress the cycyooxygenase enzyme. Parallel experiments with washed platelet preparations suggest that they may be an inadequate mode for studying relationship between the platelet cyclooxygenase and platelet function.     

Potentiation of anti-aggregating activity of PGI2 by 7-ethoxycarbonyl-6,8-dimethyl-4-hydroxymethyl-1(2H)-phthalazinone (EG-626) in rabbit platelets in vitro

Anti-aggregating activity of 7-ethoxycarbonyl-6,8-dimethyl-4-hydroxymethyl-1(2H)-phthalazinone (EG-626) was tested using rabbit platelets in vitro. EG-626 alone, when added before, prevented platelet aggregation induced by ADP, as did PGI₂, papaverine and dipyridamole. Spontaneous disaggregation was also accelerated when EG-626 was added after the maximal aggregation induced by ADP. EG-626 alone also inhibited platelet aggregation induced by collagen and arachidonic acid. ID₅₀s of these agents in ADP-induced aggregation were 7–9 nM for PGI₂, 223 μM for EG-626, 266 μM for papaverine and 957 μM for dipyridamole. When EG-626 was used in combination with PGI₂, a threshold dose (50 μM) of EG-626 potentiated the anti-aggregating effect of subthreshold dose (3 nM) of PGI₂ upto 100% inhibition in collagen-induced platelet aggregation. The marked potentiating effect of EG-626 was accompanied by an accumulation of cyclic AMP in the platelets. These effects might be due to inhibition of phosphodiesterase. Papaverine and dipyridamole, other phosphodiesterase inhibitors, also potentiated the anti-aggregating activity of PGI₂. The activity of papaverine, however, was one eighth of EG-626 and that of dipyridamole was much less. The most effective combination of PGI₂ and EG-626 to induce 50% inhibition was obtained with 20% of ID₅₀ of each agent, whereas that of PGI₂ and papaverine or dipyridamole was 39 or 41%, respectively.     

Selective inhibition of prostaglandin endoperoxide thromboxane isomerase by 1-carboxyalkylimidazoles

1-Carboxyalkylimidazoles inhibited the conversion of prostaglandin H₂ to thromboxane B₂ and 12L-hydroxy-5, 8, 10- heptadecatrienoic acid by a partially purified enzyme (prostaglandin endoperoxide thromboxane isomerase) from bovine platelet microsomes. The degree of the inhibition was dependent on the length of carboxyalkyl chain. 1-Carboxyheptylimidazole was the most potent inhibitor, and an almost complete inhibition was obtained at a concentration on the order of 1 μM. The inhibition, as examined with 1-carboxyheptylimidazole, was of noncompetitive type. These 1-carboxyalkylimidazoles did not affect the formation of prostaglandin H₂ from arachidonic acid. Such a selective inhibition was also demonstrated by the reaction of bovine platelet microsomes with arachidonic acid in the presence of 1-carboxyheptylimidazole, resulting in the accumulation of prostaglandin H₂ as an intermediate. Furthermore, a series of 1-alkylimidazoles with no carboxyl group also inhibited the isomerase at higher concentrations. However, the inhibition was not specific for the isomerase; namely, the prostaglandin H₂ formation from arachidonic acid was also affected.     

Thromboxane synthetase inhibitors as pharmacological tools: differential biochemical and biological effects on platelet suspensions

The comparative effects of three so called "thromboxane-synthetase-inhibitors" (imidazole, N-0164, and U-51605) on arachidonate metabolism and on platelet aggregation were studied. All three compounds blocked platelet microsomal thromboxane synthesis from prostaglandin endoperoxides without affecting platelet adenyl cyclase. Imidazole, blocked thromboxane synthesis in intact platelets either from arachidonic acid or PGH2, without affecting aggregation. U-51605 simultaneously inhibited thromboxane synthesis and platelet suspension aggregation. N-0164 inhibited aggregation probably at extracellular sites, at concentrations that did not alter arachidonate or PGH2 metabolism. High concentrations of N-0164 simultaneously inhibited PG cyclo-oxygenase and thromboxane synthetase. The lack of specificity of these compounds requires that other actions of these compound must be considered when they are used as pharmacological tools to inhibit thromboxane synthetase.     

Inhibition of ADP-induced platelet aggregation by reduced factor Xa

Treatment of blood coagulation factor Xa with insolubilized hexyl-agarose derivative of prostaglandin E1 (PGE1) results in the generation of two sulfhydryl groups in the protein molecule. The reduced factor Xa was found to be a potent inhibitor of platelet aggregation and thromboxane A2 synthesis induced by ADP. In contrast to the inhibition of thromboxane formation, the reduced factor Xa had no effect on the formation of PGE2 indicating that thromboxane synthetase might be selectively inhibited by the reduced factor Xa. Incubation with oxidized glutathione reversed the inhibitory activity of factor Xa previously exposed to the insolubilized hormone. Soluble PGE1 also reduces factor Xa, but more slowly than the insolubilized PGE1. PGE1 also exhibits reducing ability as tested with redox dyes. Reduction of factor Xa by dithiothreitol also transformed the coagulation factor into an inhibitor of platelet aggregation and thromboxane A2 formation. These experiments indicate that reduction of factor Xa leads to a reversible alteration of the molecule which inhibits platelet aggregation induced by ADP. This effect of reduced factor Xa is probably mediated through the inhibition of thromboxane A2 synthesis.     

Effects of Danshensu on Platelet Aggregation and Thrombosis: In Vivo Arteriovenous Shunt and Venous Thrombosis Models in Rats

Danshensu, a type of dihydroxyphenyl lactic acid, is one of the most abundant active phenolic acids in the dried root of Salvia miltiorrhizae (Lamiaceae)—widely used traditional Chinese medicine. The effects of danshensu on platelet aggregation and thrombus formation in rats were examined using various methods. It was found that danshensu significantly reduced thrombus weight in 2 experimental thrombosis models; dose-dependent inhibition of adenosine diphosphate (ADP) and arachidonic acid (AA)-induced platelet aggregation occurred in normal and blood stasis-induced rats; Danshensu also significantly mitigated blood viscosity, plasma viscosity and hematocrit levels. Moreover, danshensu significantly inhibited venous thrombosis-induced expression of cyclooxygenases-2 (COX-2) rather than cyclooxygenases-1(COX-1) in the venous walls, down regulated thromboxane B₂ (TXB₂) and up regulated 6-keto prostaglandin F_1α (6-keto-PGF_1α), normalizing the TXB₂/6-keto-PGF_1α ratio. In addition, danshensu did not induce gastric lesions and even had protective effects on aspirin-induced ulcer formation at doses as high as 60 mg/kg. These findings suggest that the antithrombotic and antiplatelet aggregation effects of danshensu are attributed to its highly selective inhibition of COX-2 and ability to normalize the thromboxane A₂(TXA₂)/prostacyclin(PGI₂) balance. These findings suggest that danshensu have great prospects in antithrombotic and antiplatelet therapy.     

Inhibition of prostaglandin E1-induced activation of adenylate cyclase in human blood platelet membrane

Activation of human blood platelet adenylate cyclase is initiated through the binding of prostaglandin E1 to the membrane receptors. Incubation of platelet membrane with [3H]prostaglandin E1 at pH 7.5 in the presence of 5 mM MgCl2 showed that the binding of the autacoid was rapid, reversible and highly specific. The binding was linearly proportional to the activation of adenylate cyclase. Although the membrane-bound radioligand could not be removed either by GTP or its stable analogue 5'-guanylylimido diphosphate, 150 nM cyclic AMP displaced about 40% of the bound agonist from the membrane. Scatchard analyses of the binding of the prostanoid to the membrane in the presence or absence of cyclic AMP showed that the nucleotide specifically inhibited the high-affinity binding sites without affecting the low-affinity binding sites. Incubation of the membrane with 150 mM cyclic AMP and varying amounts of prostaglandin E1 (25 nM to 1.0 microM) showed that the percent removal of the membrane-bound autacoid was similar to the percent inhibition of adenylate cyclase at each concentration of the agonist. At a concentration of 25 nM prostaglandin E1, both the binding of the agonist and the activity of adenylate cyclase were maximally inhibited by 40%. With the increase of the agonist concentration in the assay mixture, the inhibitory effects of the nucleotide gradually decreased and at a concentration of 1.0 microM prostaglandin E1 the effect of the nucleotide became negligible. These results show that cyclic AMP inhibits the activation of adenylate cyclase by low concentrations of prostaglandin E1 through the inhibition of the binding of the agonist to high-affinity binding sites.     

2-(6-carboxyhexyl)cyclopentanone hexylhydrazone: A potent inhibitor of the blood platelet cyclo-oxygenase

Previous studies have demonstrated that 13-azaprostanoic acid (13-APA) is a potent and specific antagonist of thromboxane A₂/prostaglandin H₂ (TXA₂/PGH₂) at the platelet receptor level. In the present study we evaluated the effects of a new azaprostanoid, 2-(6-carboxyhexyl) cyclopentanone hexylhydrazone (CPH), on human platelet function. This hydrazone was found to completely inhibit arachidonic acid (AA)-induced platelet aggregation at 1 uM CPH. On the other hand, CPH was not an effective inhibitor of PGH₂-induced aggregation. Furthermore, 100 uM CPH was completely ineffective in blocking platelet aggregation stimulated by adenosine diphosphate (ADP) or the stable prostaglandin endoperoxide analog U46619 (which presumably acts at the TXA₂/PGH₂ receptor). Measurement of platelet thromboxane B₂ (TXB₂) production demonstrated that the primary site-of-action of CPH is at the cyclo-oxygenase level. Thus, CPH inhibited TXB₂ formation from AA in a dose-dependent manner (0.1 uM–100 uM CPH)². In contrast, CPH blocked TXB₂ production from PGH₂ only at the highest CPH concentration tested, i.e., 100 uM. These results indicate that relative to 13-APA, addition of a second nitrogen at C₁₄ and a double bond between the 12- and 13- positions results in a loss of receptor activity but produces a high affinity for the platelet cyclo-oxygenase.     

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

It is generally agreed that arachidonic acid (20:4ω6) 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 substituted for C 20:4ω6 in inducing aggregation, and Ca²⁺ was found to be a prerequesite for protection of the cell against lysis as well as for aggregation in the absence or TX formation. The use fo the lipoxygenase inhibitor BW 755 C did not prevent C 20:4ω6-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 condition, C 20:4ω6-induced platelet aggregation might be due to an increased membrane permeability to Ca²⁺ induced by this fatty acid in the absence of oxidation.