Laboratory effect on platelet activity within 24 h of the first 300-mg oral dose of aspirin given in hospital during the acute phase of ischemic cerebral events.

BACKGROUND AND PURPOSE: Aspirin is the most commonly used antiplatelet treatment during the acute phase of cerebral ischemic events. It inhibits the production of thromboxane (TX) A(2), a powerful platelet activator. Despite this protection, early ischemic recurrences are frequent and considered clinical failures of this therapy. Only a few trials have focused on the use of antiplatelet therapy during this phase, and none has described the laboratory effect of the first dose of aspirin given after an ischemic cerebral event. However, this study may help clinicians to understand the mechanisms of early recurrences, and to design new therapeutic strategies, in particular for patients already treated with a daily dose of aspirin. METHOD: We studied laboratory parameters of the first 300-mg oral dose of aspirin given within 48 h after an ischemic cerebral event. Two blood samples were taken from all of the patients: the first during the third hour following aspirin intake (T1) and the second during the twenty-fourth hour (T2). For patients already treated with a daily dose of aspirin, a supplementary sample was taken before aspirin intake (T0). Platelet reactivity was studied on the basis of serum TXB(2) levels, a metabolite of TXA(2), and light transmission aggregometry after stimulation of platelet-rich plasma by arachidonic acid and by two concentrations of collagen, i.e. 2 µg/ml (Col2), dependent on the TXA(2) pathway, and 20 µg/ml (Col20), independent of the TXA(2) pathway. Results with Col2 were related to results with Col20 (Col2/20 ratio) to limit the impact of variations induced by the effects of preanalytical conditions. RESULTS: Fifty patients were included. TXB(2) values (p < 0.001) and relative values of the Col2/20 ratio (p = 0.037) were significantly higher at T2 compared to T1. For patients already treated with aspirin, TXB(2) levels (p < 0.001) were significantly lower at T1 compared to T0, and the Col2/20 ratio tended to decrease (p = 0.096). CONCLUSION: Platelet reactivity recovers within 24 h following the first 300-mg oral dose of aspirin during the acute phase of a cerebral ischemic event as demonstrated by an increase in TXB(2) levels, and the Col2/20 ratio, at T2 compared to T1. This would favor early ischemic recurrences. However, for patients already treated with aspirin, this dose is able to decrease TXB(2) levels and to complete the inhibition of the TXA(2) pathway, which shows the utility of this prescription in this case.

The cardioprotective effects of marrubiin, a diterpenoid found in Leonotis leonurus extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Leonotis leonurus L. (Lamiaceae) is used as a traditional medicine for a variety of ailments in South Africa. The diterpene marrubiin is the major product constituent in specimens of this plant occurring in South Africa. MATERIALS AND METHODS: Marrubiin isolated from South African specimens of L. leonurus in addition to an organic extract of L. leonurus were tested in vivo, ex vivo and in vitro for their anticoagulant, antiplatelet and anti-inflammatory activities. RESULTS: Marrubiin and the organic extract suppressed coagulation, platelet aggregation and inflammatory markers. For the coagulation markers it was found that the organic extract and marrubiin significantly prolonged activated partial thromboplastin time (APTT). Fibrin and D-dimer formation were drastically decreased. These findings were observed in an ex vivo model and an obese rat model. Chemokines enhance leukocyte recruitment to inflammatory sites. TNF-α and RANTES secretion were significantly reduced by the extract and marrubiin when determined in the obese rat model relative to the controls. Calcium mobilization and TXB(2) synthesis were suppressed by the extract and marrubiin. An in vitro model was used to elucidate the antiplatelet mechanism and it was found that the extract and marrubiin inhibited platelet aggregation by inhibiting the binding of fibrinogen to glycoprotein (GP) IIb/IIIa receptor in a concentration dependent manner. CONCLUSION: The findings reflect that marrubiin largely contributes to the extract's anticoagulant, antiplatelet and anti-inflammatory effects observed.

Studies on the nature of anti-platelet aggregatory factors in the seeds of the Amazonian Herb Guarana (Paullinia cupana).

Guarana (Paullinia cupana) is a popular herb native to the Amazon Basin and used extensively in soft drinks in Brazil, other Latin American countries, and more recently in the United States. Extracts derived from the dried seeds of guarana possess strong anti-platelet aggregatory properties. In this study, an active fraction containing this activity was purified and analyzed by high-performance liquid chromatography/mass spectrometry (HPLC/MS) techniques. It was noted that this fraction contains catechins, epicatechins, and their dimers, with a small amount of caffeine. It is suggested that complexes containing caffeine and catechins (and their dimers) might be responsible for anti-platelet aggregatory activity in guarana seeds and might offer health benefits towards decreasing risk of thrombosis and cardiovascular disease.

French maritime pine bark extract Pycnogenol reduces thromboxane generation in blood from diabetic male rats.

The protective effect of Pycnogenol against cardiovascular diseases was clearly demonstrated. Nevertheless, little is known about its antithrombotic effect, especially in diabetes associated with enhanced thromboxane synthesis leading to severe vascular complications. Therefore, the main purpose of our study was to evaluate the effect of long-term Pycnogenol intake on synthesis of prothrombotic thromboxane A(2) (TXA(2)) in animal model of insulin-dependent diabetes. The levels of main plasma TXA(2) metabolite, thromboxane B(2) (TXB(2)), were assessed by enzyme-linked immunosorbent assay. Diabetes was induced in Wistar male rats by single injection of streptozotocin, resulting after 8 weeks in significant body weight reduction, increased plasma glucose concentrations, and decreased plasma C-peptide levels, compared to non-diabetic animals. There was no significant reduction of plasma glucose concentrations after Pycnogenol ingestion. It was found, however, that daily administration of either Pycnogenol (5mg/kg b.wt.) or acetylsalicylic acid (ASA, 10mg/kg b.wt.) significantly reduced plasma TXB(2) concentrations, and this inhibitory effect was higher in the latter case. Nonetheless, simultaneous administration of Pycnogenol and ASA did not improve effectiveness of ASA-mediated decrease in TXB(2) generation. The results of the present study suggest that Pycnogenol might have a beneficial antithrombotic effect when administered alone or as a supplementation of standard antiplatelet therapy in diabetic patients.

Inhibitory effect of curcumin, a food spice from turmeric, on platelet-activating factor- and arachidonic acid-mediated platelet aggregation through inhibition of thromboxane formation and Ca2+ signaling.

Curcumin, a dietary spice from turmeric, is known to be anti-inflammatory, anticarcinogenic, and antithrombotic. Here, we studied the mechanism of the antiplatelet action of curcumin. We show that curcumin inhibited platelet aggregation mediated by the platelet agonists epinephrine (200 microM), ADP (4 microM), platelet-activating factor (PAF; 800 nM), collagen (20 microg/mL), and arachidonic acid (AA: 0.75 mM). Curcumin preferentially inhibited PAF- and AA-induced aggregation (IC50; 25-20 microM), whereas much higher concentrations of curcumin were required to inhibit aggregation induced by other platelet agonists. Pretreatment of platelets with curcumin resulted in inhibition of platelet aggregation induced by calcium ionophore A-23187 (IC50; 100 microM), but curcumin up to 250 microM had no inhibitory effect on aggregation induced by the protein kinase C (PKC) activator phorbol myrsitate acetate (1 microM). Curcumin (100 microM) inhibited the A-23187-induced mobilization of intracellular Ca2+ as determined by using fura-2 acetoxymethyl ester. Curcumin also inhibited the formation of thromboxane A2 (TXA2) by platelets (IC50; 70 microM). These results suggest that the curcumin-mediated preferential inhibition of PAF- and AA-induced platelet aggregation involves inhibitory effects on TXA2 synthesis and Ca2+ signaling, but without the involvement of PKC.

Green tea extract decreases plasma malondialdehyde concentration but does not affect other indicators of oxidative stress, nitric oxide production, or hemostatic factors during a high-linoleic acid diet in healthy females.

BACKGROUND: Green tea contains polyphenolic catechins which can act as antioxidants and thus decrease the risk for cardiovascular diseases. AIM OF THE STUDY: To investigate whether green tea extract differs from placebo in its effects on markers of antioxidant status, lipid peroxidation, nitric oxide production, thromboxane production, and blood coagulation during a controlled high linoleic acid diet in healthy subjects. METHODS: Twenty healthy non-smoking females (23-50 years) participated in a 4-week controlled intervention study. The experimental diet was rich in linoleic acid (9 en%) and contained fat, protein, and carbohydrates: 27, 14, and 59 en%, respectively. In addition, the subjects ingested encapsulated green tea extract (3 g/d) or placebo mixture in a double-blind manner. Fasting blood samples and five 24-hour urines were collected before and at the end of the 4-week experimental period. Same samples were received from 10 control subjects. RESULTS: Green tea extract significantly decreased plasma malondialdehyde (MDA) concentration in comparison with the placebo treatment. The treatments did not differ in serum lipids, indicators of antioxidant status, urinary 8-isoprostaglandin F2 alpha, 2,3-dinorthromboxane B2, nitric oxide metabolites or coagulation indicators. CONCLUSIONS: We conclude that an amount of green tea extract which corresponds to 10 cups of tea per day for 4 weeks does not have specific effects on several indicators related to risk of cardiovascular diseases in comparison with placebo treatment. The relatively small but significant decrease in lipid peroxidation indicated by decreased plasma MDA was not associated with changes in markers of oxidative stress (urinary 8-isoprostaglandin F2 alpha and blood oxidized glutathione) or hemostasis.

Pro-thrombotic effects of a folic acid deficient diet in rat platelets and macrophages related to elevated homocysteine and decreased n-3 polyunsaturated fatty acids.

Folic acid deficiency represents a vitamin deficiency that may be due either to an inadequacy of the dietary supply or to an increased requirement. It leads to a number of abnormalities including hematological, neurological and cardiovascular disorders. In this study, we investigated whether folic acid deficiency would influence platelet and macrophage activities. For 6 weeks, rats were fed a test diet containing a low amount of folic acid (250 mu g/kg) by comparison with a control diet (750 mu g/kg). We found 40 and 32 percent reductions (P < 0.05) of plasma and erythrocyte folates, respectively in the tested group. Peritoneal macrophages of the folic acid deficient animals exhibited greater (20 x) tissue factor (TF) activity than in the controls. We also found that folate depletion significantly enhanced the thrombin- and ADP-induced platelet aggregation (+64 and + 13 percent, respectively). Moreover, the results of incubations with radiolabeled arachidonic acid indicated that platelets of folic acid deficient animals incorporated more labeling than controls did. When stimulated with thrombin, the mobilization of arachidonate from platelet phospholipids and its subsequent formation of cyclooxygenase and lipoxygenase metabolites were enhanced in the deficient animals. In particular, thromboxane biosynthesis was markedly increased. The analysis of the plasma fatty acid composition showed a decrease in the plasma unsaturation index related to a marked fall of long chain (n-3) fatty acids which was also observed in platelets. These data suggested the occurrence of an oxidative stress in folic acid deficient animals which was confirmed by increases in plasma lipid peroxidation products (more than +20 percent) and an enhanced susceptibility of erythrocytes to free radicals (+23 percent). Altogether these data suggested that folic acid deficiency altered the circulating and cellular fatty acid composition and thus influenced the balance of the platelet eicosanoid synthesis. In addition, total homocysteine and glutathione concentrations were highly increased in plasma from folate-depleted rats. From these results, we conclude that folate deficiency can potentiate the coagulation pathway mediated by the macrophage TF as well as the platelet activation process. It is suggested that these dysfunctions might be related to the loss of (n-3) polyunsaturated fatty acids. The latter could result from an increased lipid peroxidation triggered by the folic acid deficiency-induced hyperhomocysteinemia.

Effect of cyclosporin A on platelet aggregation and thromboxane/prostacyclin balance in a model of extrahepatic cholestasis in the rat.

Cyclosporin A (CsA), a potent immunosuppressor used in organ transplants and autoimmune diseases, is associated with adverse effects in the kidney, liver, and nervous system. This drug was recently shown to stimulate platelet aggregation, to increase thromboxane synthesis, and to decrease vascular prostacyclin synthesis. In experimental cholestasis, thrombocyte function is altered. The present study was designed to assess the effects of CsA on platelet function in extrahepatic biliary obstruction (EBO) in rats. Cyclosporin or its excipient (Cremaphore El, polyethoxylated castor oil) did not modify collagen-induced platelet aggregation. In animals with EBO, platelet aggregation decreased by 50%. The administration of CsA (5 or 10 mg/kg) or Cremaphore El increased aggregation by 163%, 253% and 123% respectively. Thromboxane production increased by 119% after Cremaphore El was administered, but was not significantly modified by CsA. Cholestasis increased thromboxane synthesis by 48.4%, whereas CsA showed a direct dose-dependent effect, and excipient had no significant effect. Excipient inhibited the vascular synthesis of 6-keto-PFG1 alpha by 67.2%, as did 5 mg/kg (56.8%) and 10 mg/kg CsA (27.6%). EBO led to a nonsignificant increase in the vascular synthesis of 6-keto-PFG1 alpha. Cremaphore EI inhibited prostacyclin synthesis by 79%; inhibition by CsA was dose-dependent (31% at 5 mg/kg, 60% at 10 mg/kg). Our findings show that cholestasis enhances the effects of CsA on platelet aggregation and thromboxane/prostacyclin balance. These results may reflect the vascular effects of CsA, which in turn may be enhanced by cholestasis.

Epoxygenase metabolites of docosahexaenoic and eicosapentaenoic acids inhibit platelet aggregation at concentrations below those affecting thromboxane synthesis.

Docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids are the major n-3 fatty acids in fish oils. When either DHA or EPA is added to platelet suspensions, aggregation and thromboxane synthesis are both suppressed. However, when DHA or EPA is provided as a dietary supplement, only platelet aggregation is impaired during the early phase of the diet. In the present study, we examined whether cytochrome P-450 epoxygenase metabolites of DHA/EPA can inhibit platelet aggregation without affecting thromboxane synthesis. Epoxide regioisomers of DHA, EPA and arachidonic acid (AA) and their corresponding diol hydrolysis products were chemically synthesized. Aggregation and thromboxane A2 formation were induced in washed-platelet suspensions by addition of AA and measured by turbidometry and radioimmunoassay. The ranges of aggregation inhibition (IC50) by the families of epoxide regioisomers derived from DHA, EPA and AA were 0.7 to 1.5, 3.2 to 5.4 and 1.0 to 4.0 microM, respectively. The IC50 values for the DHA, EPA and AA diol families ranged from 3.4 to 11.7, from 31 to 173 and from 16 to 86 microM, respectively. Hydrolysis greatly reduced the capacity of EPA and AA epoxides, but not of DHA epoxides, to inhibit platelet aggregation. The IC50 values of DHA, EPA and AA epoxide families for thromboxane synthesis ranged from 6 to 100, from 10 to 100 and from 1.7 to 9.1 microM, respectively. Thus, in contrast to AA epoxides, all the DHA and EPA epoxides inhibited platelet aggregation at concentrations below those that affected thromboxane synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Arachidonic acid metabolism in the human mast cell line HMC-1: 5-lipoxygenase gene expression and biosynthesis of thromboxane.

Metabolism of arachidonic acid was studied in the unique human mast cell line HMC-1. By HPLC and/or gas chromatography mass spectrometry (GC-MS), 19 oxygenated metabolites were identified, including monohydroxy acids, leukotrienes, prostaglandins, and thromboxane. Intact cells incubated with the calcium ionophore A23187 and arachidonic acid expressed 5-lipoxygenase activity and produced 5-hydroxyeicosatetraenoic acid (5-HETE) as the major metabolite (745 pmol/10(7) cells) followed by leukotriene (LT) C4 (245 pmol/10(7) cells) and 11-trans-LTC4 (74 pmol/10(7) cells). Low but clearly detectable levels of LTB4 were also observed. The total amounts of 5-LO products were comparable to those obtained with RBL-1 cells and corresponded to approx. 30% of the levels obtained with isolated human polymorphonuclear leukocytes. Time-course experiments revealed that HMC-1 cells contained the enzyme activities required to metabolize LTC4 into LTD4 and further into LTE4. The profile of prostanoids included, prostaglandin (PG) E2, PGF2 alpha, and PGD2, whereas 6-keto-PGF1 alpha, reflecting prostacyclin formation, could not be detected. Furthermore, we were able to unambiguously establish that HMC-1 cells could produce substantial amounts of thromboxane (TX) A2, measured as TXB2 (0.1-2.2 nmol/10(7) cells). Generation of TXA2 in such quantities, exceeding those of LTC4, suggests that mast cells may be an important source of thromboxane and points to a possible role for these cells in hemostasis and thrombosis. After approx. 10 passages in culture, 5-lipoxygenase activity in HMC-1 cells drastically declined concomitantly with changes in growth behavior and cell morphology. Analysis by Northern and Western blots revealed that loss of 5-lipoxygenase activity correlated well with a reduced 5-lipoxygenase gene expression at both a transcriptional and translational level. This loss of enzyme activity and gene expression may be related to a genetic abnormality propagated in HMC-1 cells, i.e., a 10;16 translocation, which thus involves the chromosome containing the 5-lipoxygenase gene.

Calcitonin gene-related peptide release in endotoxicosis may be mediated by prostaglandins.

Three cyclo-oxygenase inhibitors (ibuprofen, indomethacin, and high dose aspirin) and two inhibitors of thromboxane biosynthesis (imidazole and low dose aspirin) were used to evaluate the role of prostaglandins and thromboxane in the release of calcitonin gene-related peptide (CGRP) during endotoxicosis. Endotoxin (lipopolysaccharide B from Salmonella Enteritidis, 5 mg/kg, intravenously) was administered to rats lightly anesthetized with ether during injection. After 3 h, endotoxin significantly elevated plasma CGRP levels by 3-fold. Ibuprofen (50 mg/kg, subcutaneously), indomethacin (10 mg/kg, subcutaneously) and high dose aspirin (100 mg/kg, intraperitoneally (i.p.)), but not imidazole (30 mg/kg, i.p.) or low dose aspirin (15 mg/kg, i.p.), significantly blocked endotoxin-induced CGRP elevations, suggesting that a prostaglandin, but not thromboxane, served as a mediator of CGRP release during endotoxicosis. Because endotoxin-induced production of prostaglandins is greatly diminished in endotoxin-tolerant rats (following multiple exposures to low dose endotoxin), we tested whether endotoxin-induced CGRP release also becomes diminished in tolerant rats. Accumulation of plasma CGRP was greatly diminished in endotoxin-tolerant rats exposed to endotoxin (5 mg/kg, intravenously), consistent with a mediator role for prostaglandins in the CGRP release during endotoxicosis.

Influence of intravenous n-3 lipid supplementation on fatty acid profiles and lipid mediator generation in a patient with severe ulcerative colitis.

N-3 fatty acids were supplied to a 36-year-old female patient suffering from ulcerative colitis and severe steroid side-effects, in a sequence of parenteral and enteral administration. During a moderately active period of disease, 200 ml d-1 fish oil-derived lipid emulsion (eicosapentaenoic acid [EPA], 4.2 g; docosahexaenoic acid [DHA], 4.2 g) was infused for 9 days, in parallel with rapid tapering of the steroid dose. Disease activity declined rapidly, and the patient was subsequently provided with 16 fish oil capsules per day (EPA, 2.9 g; DHA, 1.9 g) for 2 months. At the end of this period of therapy, severe colitis recurred with intestinal and extraintestinal manifestations. The n-3 lipid emulsion was then used for intravenous alimentation (29 days, maximum dose 300 ml per day); during this time, marked improvement of the inflammatory bowel disease was noted. During both periods of parenteral n-3 lipid administration, total plasma EPA and DHA contents increased several-fold, surpassing that of arachidonic acid; this plasma n-3 fatty acid enrichment was only maintained to a minor extent during the intermediate period of dietary fish oil supplementation. The intravenously administered EPA-containing triglycerides were rapidly hydrolyzed, as evidenced by the appearance of substantial quantities of EPA in the plasma free fatty acid fraction. Platelet and neutrophil total membrane content of EPA and DHA as well as n-3 fatty acid/AA membrane ratios similarly increased during the periods of intravenous n-3 lipid administration and declined during oral fish oil uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of hyperbaric oxygen on prostaglandin and thromboxane synthesis in the cortex and the striatum of rat brain.

The effect of a previous exposure to hyperbaric oxygen (HBO) on the synthesis capacity of prostaglandin (PG) and thromboxane (TX) was investigated in the brain of male rats. Three groups of rats were used: 1. Neurotoxic HBO (n = 11): The rats were exposed to sixfold the atmospheric pressure (101.3 kPa), i.e., 6 absolute atmospheres (ATA), of pure O2 up to the first convulsion (6 ATA O2); 2. Mild hyperoxia (n = 10): The rats were exposed to compressed air at the same absolute pressure and for a similar time than that of the neurotoxic HBO group (here PO2 is 1.26 ATA); 3. Normoxia at atmospheric pressure (PO2 is 0.21 ATA) for control. There was no convulsion in groups 2 and 3. Decompression of the high pressure groups lasted 15 min. After decapitation, samples of the frontal cortex and the striatum were taken, weighed, washed, and then incubated in Krebs-Ringer bicarbonate for 1 h. The release of eicosanoids in the medium was determined by enzyme immunoassay. Mild hyperoxia only significantly reduced in the striatum the release of 6-keto-PGF1 alpha (1.3 +/- 2.4 vs 10.9 +/- 6.6 pg/mg wet tissue, p < 0.001; mean +/- SD) and PGE2 (3.2 +/- 2.7 vs 7.8 +/- 6.5 pg/mg wet tissue, p < 0.05), whereas TXB2 did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of fish oil supplementation in the third trimester of pregnancy on prostacyclin and thromboxane production.

OBJECTIVE: Disturbance in thromboxane and prostacyclin biosynthesis has been observed in preeclampsia. We studied whether fish oil supplementation in late pregnancy interferes with maternal and fetal production of thromboxane A2 and prostacyclin I2. STUDY DESIGN: Forty-seven women in the thirtieth week of pregnancy were randomly assigned in a ratio of 2:1:1 to receive fish oil (2.7 gm of n-3 fatty acid per day [Pikasol], or either olive oil or no oil supplementation as controls. Metabolites of thromboxane A2 and A3 and of prostacyclin I2 and I3 were quantified by mass spectrometry methods in serum and urine, respectively. Maternal serum and urine were sampled at baseline, in the thirty-third and thirty-seventh weeks of pregnancy. Fetal serum was sampled at delivery. RESULTS: At the thirty-seventh week the mean concentrations of the eicosapentaenoic-derived metabolites, thromboxane B3 and prostacyclin I3, was twofold to threefold higher (p < 0.001) in the group receiving fish oil compared with combined control groups. There were no significant effects of fish oil on the prostacyclin I2 metabolite, although there was a trend toward a reduction in thromboxane B2 in this group. In umbilical cord blood the mean concentration of thromboxane B2 was lowest in the group receiving fish oil (p = 0.03). CONCLUSIONS: Fish oil was metabolized to the eicosapentaenoic acid-derived eicosanoids thromboxane A3 and prostacyclin I3 in pregnant women. Correspondingly, analog products of arachidonic acid tended to be depressed. It remains to be established whether these biochemical effects will prove beneficial in the prevention or treatment of preeclampsia and intrauterine growth retardation.

Effect of vitamin E on acute iron load-potentiated aggregation, secretion, calcium uptake and thromboxane biosynthesis in rat platelets.

In the present study performed on rats, we investigated the influence of an in vivo acute iron load on several platelet parameters and their modification after vitamin E supplementation. Iron load was achieved by injecting iron dextran corresponding to 0.1 mg Fe3+ per kg in the gluteus muscles. Control rats were injected with an equal amount of a dextran solution. Analyses were performed 18 h after injection. By comparison with controls, in iron-injected animals, we found significant increases of: (1) serum total iron (by 110%); (2) aggregation of isolated platelets induced by low concentration of thrombin and ADP (by 350% and 120%, respectively); (3) thrombin-induced endogenous serotonin secretion (by 94%). We also studied the mobilization of radiolabeled arachidonate preincorporated into platelet phospholipids. The results indicated that the thrombin-stimulated release of arachidonate and formation of cyclooxygenase and lipoxygenase products (particularly thromboxane B2), were significantly increased. We also found in plasma an increase (by 67%) of malondialdehyde (MDA) as well as a decrease of vitamin E (by 60%). When vitamin E was injected the day before iron injection, platelet hyperactivity and thromboxane biosynthesis were reduced as well as the plasma MDA concentration. Consequently, given the key role of calcium flux in the activation processes in platelets, we also investigated the thrombin-induced Ca2+ uptake by means of radiocalcium. We found that in platelets from iron-treated rats the Ca2+ uptake amounted to 3670 +/- 201 pmol/10(9) platelets (plt) and was significantly different from controls (1680 +/- 192 pmol/10(9) plt, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of platelet activation and endothelial cell injury by polyphenolic compounds isolated from Lonicera japonica Thunb.

Effects of the polyphenolic compounds isolated from Lonicera japonica Thunb on platelet aggregation, platelet thromboxane biosynthesis and hydrogen peroxide-induced endothelial cell injury were studied. With regard to the inhibitory effect on human platelet aggregation, methyl caffeate, 3,4-di-O-caffeoylquinic acid and methyl 3,4-di-O-caffeoylquinate had a strong effect. They significantly inhibited the second wave of platelet aggregation induced by ADP. Concerning thromboxane biosynthesis triggered by calcium ionophore A23187 in platelets, methyl caffeate and methyl 3,4-di-O-caffeoylquinate had the most potent inhibitory effect. Methyl 3,4-di-O-caffeoylquinate directly inhibited the conversion of arachidonic acid to thromboxane by platelet microsomes, while methyl caffeate did not have any significant effect on thromboxane biosynthesis in platelet microsomes. In the prevention of hydrogen peroxide-induced endothelial cell injury in culture, protocatechuic acid, methyl caffeate, methyl chlorogenic acid and luteolin were significantly effective. The inhibitory effect on platelet activation and the cytoprotective effect on hydrogen peroxide-induced cell injury may explain the possible role of polyphenolic compounds isolated from Lonicera japonica Thunb in maintaining vascular homeostasis.

Rapeseed oil and sunflower oil diets enhance platelet in vitro aggregation and thromboxane production in healthy men when compared with milk fat or habitual diets.

In this highly controlled trial, 26 normolipidemic men (average age 28 years, range 18 to 60) were fed a baseline diet high in milk fat (MF) (fat 36% of energy, saturates 19%, monounsaturates 11%, polyunsaturates 4%), followed by a diet high in sunflower oil (SO) (fat 38% of energy, saturates 13%, monounsaturates 10%, polyunsaturates 13%) and another diet high in low erucic-acid rapeseed oil (RO) (fat 38% of energy, saturates 12%, monounsaturates 16%, polyunsaturates 8%). All diets were mixed natural diets with the same cholesterol contents. The baseline milk fat diet was given for 14 days and the oil diets for 24 days in a blind cross-over design. The platelet in vitro aggregation (slope %/min) induced by 1, 2 and 3 microM ADP and collagen (25 micrograms/ml PRP) was highly significantly (p less than 0.001) increased after both oil diets when compared with the results from the milk fat diet. The aggregation pattern determined by threshold collagen concentration confirmed increased collagen sensitivity of the platelets after the rapeseed oil diet (p less than 0.001). The enhancement of platelet aggregation was associated with increased in vitro platelet thromboxane production after the oil diets vs. the milk fat diet (p less than 0.05 after the sunflower oil diet and p less than 0.001 after the rapeseed oil diet).

[Kinetics of merthiolate-induced aggregation of human platelets]. / Kinetika mertioliat-indutsirovannoi agregatsii trombotsitov cheloveka.

Incubation of human platelets (in the form of platelet rich plasma or washed platelet suspension) with sodium merthiolate (ethyl mercuric salicylate inhibiting the arachidonic acid incorporation into phospholipids) induces their irreversible aggregation, which is accompanied by TxB2 synthesis. The merthiolate-induced aggregation has a lag-period of 0.5-10 min, whose magnitude is inversely correlated with the merthiolate concentration. The concentration dependencies of the rate of the merthiolate-induced and arachidonate-induced aggregation are threshold ones; the Hill coefficients are more than 30. The merthiolate-induced aggregation occurs in two phases: a slow phase which is independent of the arachidonic acid cyclooxygenase metabolism and a fast phase which is fully blocked by indomethacin. This aggregation is inhibited by PGE1 and ajoene (an inhibitor of the fibrinogen interaction with the fibrinogen receptor, GPIIb/IIIa). Quantitative and qualitative analyses of the experimental data were performed, using a model which took account of: (a) increase in the concentration of free endogenous arachidonic acid resulting from the inhibition by merthiolate of the arachidonic acid re-incorporation into phospholipids, and (b) existence of a threshold intracellular arachidonic acid concentration needed for the irreversible aggregation of platelets.

Aspirin-induced decline in prostacyclin production in patients with coronary artery disease is due to decreased endoperoxide shift. Analysis of the effects of a combination of aspirin and n-3 fatty acids on the eicosanoid profile.

BACKGROUND: It was the purpose of this study to determine the effects of the combination of aspirin (ASA) and fish oil, which is rich in n-3 polyunsaturated fatty acids, on the eicosanoid profile of patients with coronary artery disease. Specifically, we wanted to determine whether the ASA-induced reduction in prostacyclin production is due to inhibition of endothelial cell cyclooxygenase or to reduced endoperoxide shift from platelets and whether ASA negates the potentially beneficial effects of fish oil on the eicosanoid profile. METHODS AND RESULTS: Fourteen patients with clinically stable but advanced coronary artery disease received 12 g (n = 8) or 16 g (n = 6) of fish oil concentrate containing 6 or 8 g of n-3 fatty acids for 6 weeks. In addition to the fish oil, patients received increasing daily doses of ASA (50 mg, 100 mg, 325 mg, and 1,300 mg; the latter in four divided doses). Each dose was taken for 2 weeks. With fish oil supplementation, red blood cell phospholipid fatty acid content of arachidonic acid (AA) decreased and of eicosapentaenoic acid (EPA) increased so that EPA as a percent of AA increased from 2% to 26%. Serum thromboxane B2, which represents the production of TXA2 by maximally stimulated platelets, was suppressed by 38% on fish oil alone and by 97% or greater on all doses of ASA. Excretion of PGI2-M, the main urinary metabolite of PGI2 (derived from AA), fell from 50 +/- 4 ng/g of creatinine to 42 +/- 2 ng/g on fish oil alone (p = 0.02). On 50 mg of ASA per day, PGI2-M excretion was 26 +/- 2 ng/g of creatinine (p less than 0.001 versus fish oil alone). On 100 mg and 325 mg of ASA per day, PGI2-M was 24 +/- 3 ng/g and 27 +/- 3 ng/g, respectively (p V NS versus value on 50 mg per day). PGI3-M, the main urinary metabolite of PGI3 (derived from EPA), increased from 0.2 +/- 0.1 ng/g of creatinine to 4.9 +/- 0.7 ng/g on fish oil alone (p less than 0.001). In contrast with the marked ASA-induced decline in PGI2-M, PGI3-M excretion was not affected by the addition of ASA, even at the higher doses (4.6 +/- 0.7 ng/g and 4.9 +/- 0.5 ng/g on 325 mg per day and 325 mg four times daily, respectively). CONCLUSIONS: Moderate-dose (325 mg per day or less) ASA taken once daily has no effect on PGI3 production despite significantly reducing PGI2 production. This suggests that endothelial cell cyclooxygenase is minimally inhibited by such doses of ASA and that a large percent of the PGI2 produced in patients with advanced coronary artery disease derives from the transfer of prostaglandin endoperoxides from activated platelets to endothelial cells. The loss of these substrates accounts for the decrease in PGI2 with moderate-dose ASA. Thus, the ASA-induced decrease in PGI2 may in large part be an unavoidable consequence of ASA-induced platelet cyclooxygenase inhibition. ASA does not negate the potentially beneficial effects of n-3 fatty acids on the eicosanoid profile.