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.     

Stimulation of eicosapentaenoic acid metabolism in washed human platelets by 12-hydroperoxyeicosatetraenoic acid

Washed human platelets were not able to convert eicosapentaenoic acid (EPA) to thromboxane B3 (TXB3) and 12-hydroxyeicosapentaenoic acid (AA) to washed human platelets induced conversion of EPA to TXB3 and 12-HEPE. Esculetin, a specific inhibitor of 12-lipoxygenase, prevented the effect of AA, but cyclooxygenase inhibitor did not. The conversion of AA to TXB2 was not affected by the same dose of esculetin. These data suggest that products of AA formed by 12-lipoxygenase in human platelets have stimulatory effects on EPA metabolism. When AA was preincubated with washed human platelets, its effect on EPA conversion was reduced, suggesting that a labile product of AA formed by 12-lipoxygenase is involved in the facilitation of EPA metabolism. Addition of 12-hydroperoxyeicosatetraenoic acid directly to washed human platelets caused dose-dependent synthesis of TXB3 and 12-HEPE, while addition of 12-hydroxyeicosatetraenoic acid had no effect. Thus, 12-hydroperoxyeicosatetraenoic acid formed from AA promotes the metabolism of EPA in washed human platelets.     

Trans n-3 eicosapentaenoic and docosahexaenoic acid isomers exhibit different inhibitory effects on arachidonic acid metabolism in human platelets compared to the respective cis fatty acids

N-3 trans geometrical isomers of 20:5 n-3 and 22:6 n-3 were isolated from rats fed heated linseed oil. The ability of these acids to inhibit 20:4 n-6 metabolism by human platelets was examined. The concentrations required to inhibit 50% of platelet aggregation (IC50) induced by 2.5 microM 20:4 n-6 were higher for the 20:5 delta 17t isomer compared to all cis 20:5 n-3; means 29.2 and 7.6 microM, respectively (P less than 0.05). There were no significant differences in IC50 between 22:6 delta 19t and all cis 22:6 n-3; means 4.3 and 5.6 microM, respectively (P greater than 0.05). Inhibition of action of cyclooxygenase on 20:4 n-6 was similar for 20:5 delta 17t and 20:5 n-3 when examined at their IC50s, but comparison at equal concentrations indicated that 20:5 n-3 was a significantly better inhibitor (P less than 0.05). The ability to inhibit platelet aggregation was paralleled by cyclooxygenase inhibition as determined by thromboxane B2 and 12-hydroxyheptadecatrienoic acid formation. 22:6 delta 19t appeared to inhibit cyclooxygenase more completely than 22:6 n-3, examined at their IC50s or at similar concentrations (P less than 0.05). Isomers of 20:5 n-3 and 22:6 n-3 having an n-3 cis or trans bond appear to have similar modes of action, although levels required for effectiveness are different for the C20 acids.     

Accumulation of thromboxane B2 within periovulatory ovine follicles: Relationship to adhesion of platelets to endothelium

Levels of thromboxane (TX) B₂ (the stable metabolite of TXA₂) were quantified by radioimmunoassay in periovulatory ovine follicles. Follicles were obtained before, and at 8, 16, 24 and 32 h after the onset of the preovulatory surge in secretion of luteinizing hormone (the ovulatory event culminates at approximately 24 h). Unruptured follicles were segregated into tissue and fluid components. A portion of the wall of each follicle was processed for examination by high resolution light and transmission electron microscopy. Concentrations of TXB₂ in homogenates of the follicular wall, and in fluid of follicles that had not yet ruptured, were dramatically elevated at 24 h. Aggregates of platelets (the suspected source of TXA₂) were adhered to endothelial cells at this time; in some cases intra and extravascular clotting was apparent. It is suggested that platelets and(or) TXA₂ might contribute to periovulatory processes in the ewe.     

The effect of adrenalectomy on the aggregation of rat blood platelets and the endogenous formation of thromboxane B2 and 12-hydroxy-5,8,10,14-eicosatetraenoic acid

Rat platelets were isolated and labelled with [1-14C] arachidonic acid. After aggregation thromboxane B2, 12-hydroxy 5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-eicosatetraenoic acid (12-HETE) were the main metabolites formed. A comparison was made between several properties of the platelets of adrenalectomized and sham operated rats. There was no difference in collagen-induced aggregation. The amount of 12-HETE and the sum of TxB2 and 12-HETE formed from endogenous arachidonic acid after aggregation was higher in the first group.     

Changes in phosphatidylinositol and phosphatidic acid in stimulated human neutrophils: Relationship to calcium mobilization,aggregation and superoxide radical generation

Human neutrophils aggregate and release mediators of inflammation, such as active oxygen species and lysosomal enzymes, when exposed to the chemoattractant, fMet-Leu-Phe, or the tumor promotor, phorbol myristate acetate. In order to ‘stage’ events which may lead to such neutrophil responses, we determined the temporal relationship between stimulus-induced changes in the endogenous phospholipids phosphatidylinositol (PI) and phosphatidic acid, the mobilization of calcium, and the onset of aggregation and generation of superoxide anion during the initial 2 min of cell activation. Within 5 s after addition of fMet-Leu-Phe (10^?7 M) neutrophils accumulated phosphatidic acid and the levels of PI decreased, as determined by two-dimensional thin-layer chromatography and phosphorus determinations. By 5 s, phosphatidic acid levels rose approximately 3.5-fold and at 15 s the loss of PI exceeded the quantity of phosphatidic acid generated. In response to phorbol myristate acetate (1 μg/ml), however, changes in PI or phosphatidic acid were not observed until after 60 s. Accumulation of phosphatidic acid in fMet-Leu-Phe-stimulated cells was not inhibited by chelation of extracellular calcium. Neutrophils exposed to either fMet-Leu-Phe or phorbol myristate acetate also showed rapid decrements in fluorescence of cell-associated chlorotetracycline (used as an indirect probe of mobilization of intracellular membrane-associated calcium) and took up ⁴⁵Ca²⁺ from the extracellular medium (under 60 s). The results indicate that changes in calcium mobilization, together with the alterations in phospholipid metabolism (under 5 s) anteceded aggregation and the generation of O^?₂ (10–15 s) induced by fMet-Leu-Phe. In contrast, when neutrophils were exposed to phorbol myristate acetate, changes in PI and phosphatidic acid (over 60 s) were observed after the mobilization of calcium (under 5 s) and the onset of O^?₂ generation and aggregation (30–35 s).     

The differences in 12-hydroxyeicosatetraenoic acid and thromboxane B2 release from guinea pig platelets.

Anti-12(S)-hydroxyeicosatetraenoic acid (12-HETE)-antibody and anti-thromboxane B2 (TXB2)-antibody were generated and applied to the radioimmunoassay. The detection limit for 12-HETE was 16 pg. The cross-reactivities of anti-12-HETE-antibody were 4.6% for 15-HETE, 0.18% for 5-HETE and below 0.15% for leukotrienes and prostaglandins (PGs). 12-HETE and TXB2 released from guinea pig platelets were measured by radioimmunoassay. Platelet activating factor (PAF) at 10(-9) M induced the aggregation of platelets, the releases of immunoreactive-12-HETE (1.8 +/- 1.2 ng/10(8) platelets, mean +/- S.D.) and immunoreactive-TXB2 (18.5 +/- 17.3 ng/10(8) platelets). Collagen at 1 microgram/ml also evoked platelet aggregation, the releases of immunoreactive-12-HETE (2.7 +/- 1.1 ng/10(8) platelets) and immunoreactive-TXB2 (11.8 +/- 4.6 ng/10(8) platelets). By the stimulation with these compounds, TXB2 was produced in a greater amount than 12-HETE from guinea pig platelets. Although 10(-7) M and 10(-6) M U46619, a TXA2 mimetic, caused platelet aggregation, arachidonic acid metabolites were not released. These data suggest the presence of different mechanisms of platelet activation depending on each stimulus.     

Relationship of the effects of nigericin on the aggregation and cytoplasmic pH of bovine platelets in the presence of different cations

The effect of nigericin on aggregation of bovine platelets was investigated in media containing the chloride salts of various alkali metal cations of quaternary ammonium cations. In medium with K+, which has the highest permeability with the ionophore among the cations tested, nigericin slightly enhanced both ADP- and thrombin-induced aggregation. In medium with Na+, nigericin scarcely affected ADP-induced aggregation, and slightly inhibited thrombin-induced aggregation. In media with Cs+, choline and tetramethylammonium, it inhibited the aggregations induced by both ADP and thrombin. Measurement of the cytoplasmic pH with the fluorescent probe 2',7'-bis(carboxyethyl)5,6-carboxyfluorescein showed that nigericin increased the intracellular pH in K+ medium and caused its stable decrease (of about 0.6) in Cs+, choline and tetramethylammonium media, but caused only a small transient decrease in medium with Na+. These results suggest that the effects of nigericin on platelet aggregation are mainly due to its effects on the cytoplasmic pH. This conclusion is supported by the findings that the effects on platelet aggregation of other types of ionophore tested were also proportional to their effects on the cytoplasmic pH.     

Antithrombotic effects of peroxynitrite: Inhibition and reversal of aggregation in human platelets

The inhibition of platelet aggregation by peroxynitrite, a reactive oxygen species derived from the interaction of nitric oxide (NO) and superoxide, was examined in platelet-rich plasma. In this report, we have used a preparation of peroxynitrite that was free of H₂0₂ and MnO₂. As such, peroxynitrite dose-dependently (50–200 μA) inhibited aggregation of human platelets stimulated by ADP (5 μM), collagen (0.5 μg), thrombin (0.5 UlmL) and U46619 (1 PM). In addition, peroxynitrite reversed platelet aggregation induced by collagen, ADP, and thrombin. Peroxynitrite, preincubated with platelet-poor plasma or albumin (7%) for 30 min, did not alter the inhibition of platelet aggregation. This suggested that the inhibitory action of peroxynitrite may be due to nitrosylation of proteins, which by themselves possess activity, rather than conversion to NO or NO donors. Furthermore, we show that peroxynitrite increased the cGMP level only at 200 μM concentrations, further suggesting that the action of peroxynitrite was not completely due to its conversion to NO or NO donors.     

Lipoproteins from normolipidemic and dyslipidemic subjects modify the thromboxane A2 generation by platelets in clotting human blood

The study was performed to investigate the influence of lipoproteins (LP) on the thromboxane (TX) A₂ formation capacity of platelets in clotting whole blood in vitro. The different lipoprotein fractions VLDL, LDL, HDL₂ and HDL₃ were isolated from blood of normo- or dyslipidemic volunteers by ultracentrifugation. These lipoproteins were incubated in blood with different levels of serum total cholesterol (TC) taken from normolipidemics (TC < 200 mg/dl), moderate hypercholesterolemics (TC: 200–250 mg/dl) or subjects with high cholesterol level (TC > 250 mg/dl), respectively. The amount of serum TXA₂ formed within 60 min at 37°C was measured by enzyme immunoassay. The results obtained show that the efficacy of separate LP fractions to influence the TXA₂ production depends not only on the type of LP fraction but also on the source of plasma used for isolation of LP and on the cholesterol level in the blood for incubation: LDL taken from normolipidemics or moderate hyperlipidemics inhibited the TXA₂ formation in blood from normolipidemics (P < 0.02, respectively), but enhanced it in blood from persons with moderate hypercholesterolemia (P < 0.05). LDL from hyperlipidemics enhanced TXA₂ production in blood from hyperlipidemics (P < 0.05). The HDL₂ fractions inhibited the TXA₂ formation in blood from normo- and hypercholesterolemics (P < 0.02, resp.), but there was no effect of HDL₂ in clotting blood from persons with moderate hypercholesterolemia. All HDL₃ fractions tested inhibited the TXA₂ formation in all types of blood used for clotting (P < 0.02, resp.), probably due to their great cholesterol accepting capacity.     

Relative effects of flurbiprofen on platelet 12-hydroxy-eicosatetraenoic acid and thromboxane A2 production: influence on collagen-induced platelet aggregation and adhesion

Flurbiprofen has been shown to inhibit cyclo-oxygenase metabolism of arachidonic acid to thromboxane A2 (TxA2), resulting in the inhibition of platelet aggregation. Recently, our laboratory reported that the "irreversible" phase of platelet aggregation and adhesion were regulated, in part, by the lipoxygenase metabolism of arachidonic acid to 12-hydroxy-eicosatetraenoic acid (12-HETE) in platelets, and that selective inhibition of one enzyme i.e. either cyclo-oxygenase or lipoxygenase, resulted in paradoxical effects on the metabolism of arachidonic acid and platelet response related to the other pathway. Therefore, we performed experiments to assess the relative effects of flurbiprofen on TxA2 and 12-HETE synthesis, and on collagen-induced platelet aggregation and platelet adhesion to collagen-coated surfaces. "Irreversible" collagen-induced platelet aggregation was only partially inhibited by pre-incubation with 1 x 10(-6) M flurbiprofen, while TxA2 production was elevated and 12-HETE production was maximally inhibited in these platelets. At this concentration of flurbiprofen (1 x 10(-6)M), collagen-induced platelet adhesion was also reduced by 50%. At higher concentrations of flurbiprofen, both platelet aggregation and adhesion were further reduced, with a corresponding inhibition of TxA2 production. Thus it appears that the lipoxygenase pathway of arachidonic acid metabolism in platelets is not only inhibited by flurbiprofen, but is more sensitive to inhibition by flurbiprofen than the cyclo-oxygenase pathway. This differential effect of flurbiprofen on arachidonic acid metabolism in the platelet is related to differential effects on platelet function.     

A comparative study of eicosapentaenoic acid metabolism by human platelets in vivo and in vitro

During long-term dietary n-3 fatty acid supplementation, eicosapentaenoic acid (EPA) is not incorporated into phosphatidylinositol or -serine of human platelets in vivo and is not detectable in phosphatidic acid upon stimulation with thrombin. However, EPA is released from platelet phospholipids and metabolized to thromboxane B3 (TXB3). In contrast, in vitro, platelets incorporate [14C]EPA into phosphatidylinositol, whether they contain endogenous EPA in their cellular lipids or not. Following platelet stimulation, [14C]EPA appears in phosphatidic acid, as free fatty acid, and is transformed to TXB3. We conclude that the fatty acid compositions of platelet phospholipid subclasses are regulated with a high degree of specificity in vivo. Qualitative differences exist between in vivo and in vitro uptake of EPA into platelet phospholipid subclasses. After in vivo incorporation, EPA is released by action of a phospholipase A2.     

Experimental atherosclerosis in rabbits: platelet aggregation, thromboxane A2 generation and anti-aggregatory potency of prostacyclin.

Experimental atherosclerosis in rabbits was associated with increased aggregation of their platelets to arachidonic acid, and with increased generation of thromboxane A2 by their platelet-rich plasma. A heightened susceptibility of platelets to the anti-aggregatory action of prostacyclin against the ADP-induced aggregation was also observed. It is concluded that in advance atherosclerosis the platelet system is hypersensitive to biologically active metabolites of arachidonic acid.     

Predominant inhibition of ganodermic acid S on the thromboxane A2-dependent pathway in human platelets response to collagen

Ganodermic acid S (GAS), a membrane acting agent, exerts multiple effects on human platelet function (C.N. Wang et al. (1991) Biochem. J. 277, 189-197). The study reported how GAS affected the response of human gel-filtered platelets (GFP) to collagen. The agent inhibited cell aggregation by prolonging lag and shape change periods and decreasing the initial cell aggregation rate. However, the inhibitory efficiency was less than its inhibition on GFP response to U46619, a thromboxane (TX) A2 mimetic. In the agent-effect on biochemical events, GAS effectively inhibited Ca2+ mobilization, phosphorylation of myosin light chain, dense granule secretion and TXB2 generation. The inhibitions might originate from blocking Ca2+ mobilization of the TXA2-dependent pathway. GAS partially decreased the phosphorylation of most phosphotyrosine proteins from early activation to the integrin alphaIIbbeta3-regulated steps. The agent did not affect the phosphorylation of three proteins at the steps regulated by integrin alphaIIbbeta3. The results suggest that GAS inhibits the collagen response predominantly on the TXA2-dependent signaling, and the tyrosine kinase-dependent pathway in collagen response plays a major role in aggregation.     

Re-evaluation of the effects of neuropeptide Y on aggregation of human platelets.

Several studies have shown that platelets are a major source of circulating neuropeptide Y (NPY) immunoreactivity in rats, but the effects of this vasoconstrictor peptide on platelets are not well-defined. Recently, it was reported that porcine NPY was an inhibitor of in vitro human platelet aggregation induced by epinephrine, an observation which would have important implications regarding platelet-vascular interactions during states involving platelet activation and thrombosis. Thus, we undertook the present studies, in an attempt to confirm the earlier report, and to extend those observations to human NPY. In contrast to the recent report, we found no inhibitory effect of either human or porcine NPY on epinephrine- or collagen-induced aggregation of human platelets from normal subjects. Likewise, specific NPY Y-1 and Y-2 agonists had no direct or indirect action on platelet aggregation. Finally, the effect of human NPY on intraplatelet cAMP was measured. The peptide had no effect on either basal or iloprost-stimulated cAMP levels. We hypothesize that the role of NPY in the platelet-vascular interaction is in promoting vasoconstriction associated with platelet aggregation, and does not include inhibition of further thrombosis.     

Evaluation of the effects of several zoanthamine-type alkaloids on the aggregation of human platelets

Ten zoanthamine-type alkaloids from two marine zoanthids belonging to the Zoanthus genus (Zoanthus nymphaeus and Zoanthus sp.) along with one semisynthetic derivative were evaluated for their antiplatelet activities on human platelet aggregation induced by several stimulating agents. 11-Hydroxyzoanthamine (11) and a synthetic derivative of norzoanthamine (16) showed strong inhibition against thrombin-, collagen- and arachidonic acid-induced aggregation, zoanthenol (15) displayed a selective inhibitory activity induced by collagen, while zoanthaminone (10) behaved as a potent aggregant agent. These evaluations allowed us to deduce several structure-activity relationships and suggest some mechanisms of action for this type of compounds.     

Inhibition of oxidative-stress-induced platelet aggregation by androgen at physiological levels via its receptor is associated with the reduction of thromboxane A2 release from platelets

BACKGROUND: Platelets play a crucial role in the development of arterial thrombosis and other pathophysiologies leading to clinical ischemic events. Defective regulation of platelet activation/aggregation is a predominant cause for arterial thrombosis. The purposes of our study are to assess the effect of androgen at physiological concentration via its receptor on oxidative-stress-induced platelet aggregation and to further elucidate the possible mechanism. METHODS AND RESULTS: Plasma dihydrotestosterone (DHT) was determined by ELISA using a commercially available kit. Platelet aggregometer was used to measure platelet aggregation. The contents of thromboxane B(2) (TXB(2)) were assayed with radio-immunoassay. Our results showed that addition of DHT (2 nM) significantly inhibited platelet aggregation induced by hydrogen peroxide (H(2)O(2)) (10 mM, 25 mM) in PRP diluted with Tyrode's buffer. Moreover, H(2)O(2)-induced platelet aggregation decreased in sham-operated rats. However, H(2)O(2)-induced platelet aggregation significantly increased in castrated rats. Replacement of DHT inhibited H(2)O(2)-induced platelet aggregation in castrated rats. After PRP was pretreated with flutamide, H(2)O(2)-induced platelet aggregation increased in castrated rats again. Presence of DHT (2 nM) obviously inhibited H(2)O(2)-induced thromboxane A(2) (TXA(2)) release in castrated rats. Pretreatment of DHT and flutamide increased H(2)O(2)-stimulated TXA(2) release from platelet in castrated rats again. Castration caused a significant reduction in plasma testosterone and DHT levels, whereas DHT replaced at a dose of 0.25 mg/rat restored the circulating DHT to physiological levels, without being altered by treatment with flutamide. The plasma TXB(2) increased in castrated rats as compared with that in sham-operated rats. Replacement with DHT reduced plasma TXB(2) contents in castrated rats. However, flutamide supplementation increased plasma contents of TXB(2) in castrated rats again. CONCLUSION: Androgen at physiological doses via its receptor inhibits oxidative-stress-induced platelet aggregation, which is associated with the reduction of TXA(2) release from platelets.