Relationship of thromboxane generation to the aggregation of platelets from humans: effects of eicosapentaenoic acid

A non-linear relationship between the percent aggregation of human platelets and the amount of TXB2 generated requires investigators to use caution when using the data to assess antiplatelet regimens. The relationship approximates a hyperbola with a roughly linear relationship from 0 to 70% aggregation and 0 to 50 ng TXB2 per ml of platelet-rich plasma. Above these values, the amount of TXB2 produced may increase up to 500 ng per ml of platelet-rich plasma with no clear relationship to the observed platelet function of aggregation. Also, appreciable inhibition of TXB2 formation can occur at high TXB2 levels with no detectable decrease in aggregation. Thus, assessment of antiplatelet regimens using TXB2 formation alone are unlikely to be interpretable without reference to this non-linear property of platelet function. We applied this concept when evaluating a study of forty subjects with dietary supplements of 1.8 g or 2.7 g of ethyl eicosapentaenoate (20:5n-3) for four weeks. There was a moderate, but statistically significant decrease in average values for the percent aggregation (60 +/- 15 to 45 +/- 30) and thromboxane production (51 +/- 30 to 33 +/- 31 ng/ml). Although the differences in mean values were slight relative to the overall standard deviations, reductions of platelet function were clearly evident in 31 of 40 subjects when paired results were examined relative to the recognized hyperbolic relationship.     

An improved malondialdehyde assay for estimation of thromboxane synthase activity in washed human blood platelets

After stimulation of the washed human blood platelets by arachidonic acid (AA), the concurrent evaluations for formed malondialdehyde (MDA) measured by the common photometrical thiobarbituric acid (TBA) method, and for thromboxane B2 (TXB2) measured by gas chromatography, revealed that the formed MDA exceeded the amount of TXB2 on a molar base. However, MDA and TXB2 originating from thromboxane synthase activity should be produced in approximately equimolar amounts. By treatment of the stimulated platelet samples with stannous chloride it is possible to reduce all peroxidized products of AA which generate MDA otherwise during the TBA reaction and to estimate MDA and TXB2 in a ratio of nearly 1:1. The stannous chloride treatment does not destroy the MDA and does not influence the TBA reaction with MDA. Therefore the simple and quick TBA method can be used after stannous chloride treatment for estimation of thromboxane synthase activity in AA stimulated washed human platelets.     

Effects of repeated oral doses of fenflumizole on platelet aggregation and thromboxane formation in man ex vivo

Anti-platelet effects of fenflumizole, a new cyclo-oxygenase inhibitor, were studied in man ex vivo. Fenflumizole was given to male volunteers at the oral doses of 25, 50 or 100 mg per day, each dose for a period of seven days. The formation of thromboxane B2 (TXB2) during whole blood clotting, platelet aggregation induced by arachidonic acid and ADP, the formation of TXB2 during aggregation as well as serum concentration of fenflumizole were measured repeatedly during drug administration and for a fortnight after drug discontinuation. TXB2 formation during whole blood clotting was decreased dose-dependently by fenflumizole. The degree of inhibition of TXB2 formation was proportional to fenflumizole concentration in serum within each individual. The lag phase of platelet aggregation induced by arachidonic acid was prolonged and the formation of TXB2 during aggregation decreased by fenflumizole. No total inhibition of either TXB2 synthesis or platelet aggregation was caused by the fenflumizole doses used. The results show that the degree of inhibition of platelet thromboxane forming capacity by repeated doses of fenflumizole is closely related to the concentration of the drug in blood. Platelet aggregation however is less sensitive to changes in fenflumizole levels and cannot be assessed solely on the basis of cyclo-oxygenase activity.     

Plasma prostaglandin levels and circulating fuel levels in rats with diabetic ketoacidosis: effects of cyclooxygenase inhibitors and of alpha and beta adrenergic blockade

We studied the effects of two structurally unrelated inhibitors of the fatty acid cyclooxygenase and of alpha and beta adrenergic blockade on the elevated plasma levels of 13,14-dihydro-15-keto-prostaglandin (PG)E2, 6-keto-PGF1 alpha and thromboxane (TX)B2, the stable derivatives of PGE2, PGI2 (prostacyclin) and TXA2, respectively, in rats with streptozotocin-induced diabetic ketoacidosis (DKA). Meclofenamic acid and indomethacin each produced a significant decrease in the elevated plasma levels of 13,14-dihydro-15-keto-PGE2, 6-keto-PGF1 alpha and TXB2. Phentolamine significantly reduced the plasma level of TXB2 but had no effect on the elevated circulating levels of glucose, free fatty acids, total ketones, 13,14-dihydro-15-keto-PGE2 or 6-keto-PGF1 alpha. Propranolol significantly reduced the elevated circulating levels of glucose, free fatty acids and total ketones but had no effect on the levels of the three prostaglandin derivatives. The ability of meclofenamic acid and indomethacin to reduce the plasma levels of 13,14-dihydro-15-keto-PGE2, 6-keto-PGF1 alpha and TXB2 confirms that the plasma levels of these three derivatives are elevated in rats with DKA. Since abnormalities in the production of PGI2 and perhaps other cyclooxygenase derivatives may contribute to the pathogenesis of certain important hemodynamic and gastrointestinal features of DKA, cyclooxygenase inhibitors may play a role in the management of selected patients with this disorder. Alpha adrenergic activity is essential for the maintenance of the elevated plasma TXB2 level in rats with DKA. The fall in the plasma TXB2 level during alpha adrenergic blockade appears to reflect inhibition of platelet aggregation and platelet TXA2 production, but other sources of the elevated plasma TXB2 level in DKA are not excluded. Beta adrenergic activity contributes to the maintenance of elevated circulating levels of glucose, free fatty acids and total ketones in experimental DKA but not to the elevated plasma levels of the prostaglandin derivatives.     

Platelet function studies in myeloproliferative disorders

Platelet functions were studied in 64 patients with various myeloproliferative diseases. The characteristic alterations were prolonged bleeding time, decreased platelet aggregation (but normal results induced by ristomycin), elevated level of BTG, high production of MDA, increased level of TXB2 with almost normal level of 6-keto-PGF1. However, considering the bleeding time and the amount of BTG in relation to the whole blood platelet count, no differences could be detected.     

Study on the pathogenesis of pathophysiological changes of burn systemic infection

The present prospective study showed that incidence of systemic infection in severe burn patients was 30.9%. Toxic shock and multiple organ failure (MOF) developed in all patients with uncontrolled systemic infection. Both morbidity and mortality of MOF were 76.5%. In the infection group, plasma TXB(2) and TXB(2)/6-keto-PGF(1alpha) ratio increased markedly. Their changes were closely correlated with the clinical course and deterioration of systemic infection. Circulatory platelet aggregate ratio decreased significantly, while myocardiac enzyme spectrum greatly increased. Thrombi were observed in visceral tissues from patients dying of systemic infection. These suggested that TXA(2)/PGI(2). imbalance promoting microaggregate and thrombus formation may be one of the pathogenic effects of toxic shock and MOF in burn patients.     

Antithrombotic activity of garlic: its inhibition of the synthesis of thromboxane-B2 during infusion of arachidonic acid and collagen in rabbits

Rabbits were given collagen and arachidonic acid intravenously. Blood pressure, platelet counts, plasma thromboxane-B2 (TXB2) and plasma 6-keto-prostaglandin F1 alpha, (6-keto-PGF1 alpha) were determined. Both thrombogenic agents, upon infusion of a lethal dose, caused thrombocytopenia, indicative of in vivo platelet aggregation and hypotension. These changes were associated with an increase in plasma levels of TXB2 and 6-keto-PGF1 alpha measured by radioimmunoassay (RIA). Pretreatment of rabbits with an aqueous extract of garlic (500 mgkg) provided protection from thrombocytopenia and hypotension. Thromboxane-B2 synthesis was significantly reduced in animals pretreated with garlic and then injected with a lethal dose of either collagen or arachidonic acid. The amount of TXB2 synthesized in these animals was not sufficient to induce thrombocytopenia or hypotension. All animals pretreated with garlic were well protected against the effects of collagen or arachidonate infusion, and no apparent symptoms were observed in these animals. These observations indicate that garlic may be beneficial in the prevention of thrombosis.     

Inhibition of platelet aggregation and arachidonate metabolism in platelets by procyanidins

The effects of procyanidins on platelet aggregation and arachidonate metabolism in platelets were studied. Nine procyanidins were used in this investigation. Procyanidins B-2-S, EEC and C-1 significantly induced the inhibition of platelet aggregation, and the potency of inhibition was comparable with aspirin. Procyanidin B-2-S was used as a representative of procyanidins for further studies on the effect on arachidonate metabolism. In arachidonate metabolism by fatty acid cyclooxygenase pathway, B-2-S inhibited TXB2 and HHT formation by intact platelets treated with exogenous arachidonic acid. It also inhibited TXB2 formation measured by a specific radioimmunoassay when the cells were challenged with calcium ionophore A23187. In cell-free system, B-2-S inhibited both TXB2 and 12-HETE bioxynthesis in platelet microsome and cytosol, respectively. The inhibitory effect on thromboxane biosynthesis might explain the inhibitory effect of procyanidins on platelet aggregation.     

Collagen-induced platelet activation mainly involves the protein kinase C pathway.

This study analyses early biochemical events in collagen-induced platelet activation. An early metabolic event occurring during the lag phase was the activation of PtdIns(4,5)P2-specific phospholipase C. Phosphatidic acid (PtdOH) formation, phosphorylation of P43 and P20, thromboxane B2 (TXB2) synthesis and platelet secretion began after the lag phase, and were similarly time-dependent, except for TXB2 synthesis, which was delayed. Collagen induced extensive P43 phosphorylation, whereas P20 phosphorylation was weak and always lower than with thrombin. The dose-response curves of P43 phosphorylation and granule secretion were similar, and both reached a peak at 7.5 micrograms of collagen/ml, a dose which induced half-maximal PtdOH and TXB2 formation. Sphingosine, assumed to inhibit protein kinase C, inhibited P43 phosphorylation and secretion in parallel. However, sphingosine was not specific for protein kinase C, since a 15 microM concentration, which did not inhibit P43 phosphorylation, blocked TXB2 synthesis by 50%. Sphingosine did not affect PtdOH formation at all, even at 100 microM, suggesting that collagen itself induced this PtdOH formation, independently of TXB2 generation. The absence of external Ca2+ allowed the cleavage of polyphosphoinositides and the accumulation of InsP3 to occur, but impaired P43 phosphorylation, PtdOH and TXB2 formation, and secretion; these were only restored by adding 0.11 microM-Ca2+. In conclusion, stimulation of platelet membrane receptors for collagen initiates a PtdInsP2-specific phospholipase C activation, which is independent of external Ca2+, and might be the immediate receptor-linked response. A Ca2+ influx is indispensable to the triggering of subsequent platelet responses. This stimulation predominantly involves the protein kinase C pathway associated with secretion, and appears not to be mediated by TXB2, at least during its initial stage.     

Blood platelet function in patients with obliterative arteriosclerosis of the lower limbs

The specific markers of platelet activation, e.g. platelet aggregation induced with ADP, AA and PAF as well as the levels of Beta-TG, TXB2, 6-keto-PGF1 alpha and cyclic AMP in the patients suffering from obliterative arteriosclerosis of the lower limbs were measured. It was found that these patients revealed hyperfunction of blood platelets expressed in increased sensitivity of platelets to ADP and PAF, increased levels of Beta-TG and TXB2 as well as decreased levels of 6-keto-PGF1 alpha and cyclic AMP. Obtained results support the concept that atherosclerosis consists of a wide-spread functional alteration of various types of cells.     

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.     

In vitro effect of trichosanic acid, a major component of Trichosanthes japonica on platelet aggregation and arachidonic acid metabolism in human platelets

The in vitro effect of trichosanic acid (TCA; C18:3, omega-5), a major component of Trichosanthes japonica, on platelet aggregation and arachidonic acid (AA) metabolism in human platelets was studied. TCA dose-dependently suppressed platelet aggregation of platelet rich plasma and washed platelets. TCA decreased collagen (50 micrograms/ml)-stimulated production of thromboxane B2 (TXB2) and 12-hydroxyhepta-decatrienoic acid (HHT) in a dose-dependent manner, while that of 12-hydroxyeicosatetraenoic acid (12-HETE) was rather enhanced. The conversion of exogenously added [14C]AA to [14C]TXB2 and [14C]HHT in washed platelets was dose-dependently reduced by the addition of TCA, while that to [14C]12-HETE was increased. Similar observations were obtained when linolenic acid (LNA; C18:3, omega-3) was used. These results suggest that TCA may decrease TXA2 formation in platelets, probably due to the inhibition of cyclooxygenase pathway, and thereby reduce platelet aggregation.     

Antithrombotic effect of onion in streptozotocin-induced diabetic rat

In the present study, we investigated whether onion has antithrombotic effect in streptozotocin (STZ)-induced diabetic rats. In diabetic rats, serum thromboxane B(2) (TXB(2)) level was elevated compared to that in normal, and this elevation in diabetes was significantly inhibited by treatment with onion (0.5 g/ml/kg/day, i.p.) for 4 weeks. In normal rats, the serum TXB(2) level remained unaltered after the treatment with onion. To investigate in vitro effect of onion, we examined its effect on TXB(2) formation, platelet aggregation and arachidonic acid (AA)-release in platelets from diabetic and normal rats. Onion showed a significant inhibitory effect on collagen- or AA-induced TXB(2) formation with greater potency in diabetic platelets than in normal. Similarly, more potent inhibitory effects of onion in diabetes were observed in collagen- or AA-induced platelet aggregation and collagen-induced AA release response. In conclusion, these results suggest that onion can produce more beneficial antithrombotic effect in diabetes.     

Changes in urinary thromboxane level in man during cardiopulmonary bypass: effect of thromboxane on renal tubules

ONO-3708, a thromboxane A2 (TXA2) antagonist, was administered at a dose of 2 micrograms/kg/min by a double blind method as compared with inactive placebo during cardiopulmonary bypass (CPB) procedure to study the changes of thromboxane B2 (TXB2) levels in plasma and urine and N-acetyl-glucosaminidase (NAG) level in urine. TXB2 levels in plasma and urine increased significantly (P less than 0.01) during CPB in the patients given ONO-3708 (ONO-3708 group) and in those given placebo (placebo group). The plasma TXB2 level as expected from the urinary TXB2 level was higher than the measured plasma TXB2 level showing increases in TXB2 originating from the kidney. The urinary NAG level, increased significantly (P less than 0.01) during CPB the NAG level in ONO-3708 group was significantly low as compared to placebo group. The levels of TXB2 in plasma and urine in ONO-3708 group were not different from those of the patients receiving placebo, indicating that ONO-3708 does not have any effect on TXA2 production. We concluded that the elevation of urinary TXB2 level might be due to increased TXA2 production in the kidney under hypoxic condition induced by hypotension and lowered perfusion during CPB. Furthermore, the increased production of TXA2 appears to suppress the functions of the renal proximal tubules.     

Plasma lipoproteins affect platelet malondialdehyde and thromboxane B2 production

Platelet interaction with plasma lipoproteins was studied using gel-filtered platelets free of plasma constituents and purified lipoproteins. On incubation of gel-filtered platelets with plasma lipoproteins at 30 degrees C for 30 min, 100 micrograms of protein/ml of very-low as well as low-density lipoprotein caused 10% increment in platelet aggregation and [14C]serotonin release in parallel to elevation of around 15% of malondialdehyde and thromboxane B2 production. High-density lipoprotein showed the opposite effect and reduced platelet aggregation as well as thromboxane B2 synthesis by 17 and 32%, respectively. Lipoprotein-deficient plasma enhanced platelet function. Preincubation of the platelet suspension with prostacyclin did not prevent the effect of the lipoproteins on the in vitro platelet response as well as on the platelet prostaglandin pathway. Our results suggest that the formation of thromboxane B2 and malondialdehyde is influenced by plasma lipoproteins and that these, in turn, affect platelet aggregation and the release reaction. The possible significance of these results to platelet function in hyperlipidemic patients is discussed.     

Modulation of platelet aggregation by areca nut and betel leaf ingredients: roles of reactive oxygen species and cyclooxygenase

There are 2 to 6 billion betel quid (BQ) chewers in the world. Areca nut (AN), a BQ component, modulates arachidonic acid (AA) metabolism, which is crucial for platelet function. AN extract (1 and 2 mg/ml) stimulated rabbit platelet aggregation, with induction of thromboxane B2 (TXB2) production. Contrastingly, Piper betle leaf (PBL) extract inhibited AA-, collagen-, and U46619-induced platelet aggregation, and TXB2 and prostaglandin-D2 (PGD2) production. PBL extract also inhibited platelet TXB2 and PGD2 production triggered by thrombin, platelet activating factor (PAF), and adenosine diphosphate (ADP), whereas little effect on platelet aggregation was noted. Moreover, PBL is a scavenger of O2(*-) and *OH, and inhibits xanthine oxidase activity and the (*)OH-induced PUC18 DNA breaks. Deferoxamine, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and neomycin prevented AN-induced platelet aggregation and TXB2 production. Indomethacin, genistein, and PBL extract inhibited only TXB2 production, but not platelet aggregation. Catalase, superoxide dismutase, and dimethylthiourea (DMT) showed little effect on AN-induced platelet aggregation, whereas catalase and DMT inhibited the AN-induced TXB2 production. These results suggest that AN-induced platelet aggregation is associated with iron-mediated reactive oxygen species production, calcium mobilization, phospholipase C activation, and TXB2 production. PBL inhibited platelet aggregation via both its antioxidative effects and effects on TXB2 and PGD2 production. Effects of AN and PBL on platelet aggregation and AA metabolism is crucial for platelet activation in the oral mucosa and cardiovascular system in BQ chewers.     

The relationship between thromboxane and cytosolic calcium modifiers in rat platelets.

Platelet activity is controlled, in part, by cytosolic free ionized calcium concentration ([Ca++]i). Regulation of platelet thromboxane (TXB2) synthesis may be by regulation of [Ca++]i. Dietary linoleate is a regulator of TXB2 synthesis, therefore, it may act by influencing [Ca++]i. Aspirin is a regulator of TXB2 synthesis by inhibition of cyclooxygenase; ouabain and nifedipine are regulators of [Ca++]i. This study was conducted to determine whether these affectors of TXB2 synthesis and [Ca++]i cause associated responses. Male nonobese Zucker rats were fed diets supplying 30% of energy (en%) as fat. Dietary fat was a mixture of corn oil and beef tallow to provide 3.0, 4.5, 6.0, or 7.5 en% linoleic acid, with cholesterol added to provide equal cholesterol in all diets. Rats were fed for 30 days with 6 rats/diet. Isolated rat platelets were assayed for FA composition; the percentage of linoleic acid in platelet FA rose linearly with increasing dietary linoleate (r = 0.76, P less than 0.0001). Resting and thrombin-stimulated platelet [Ca++]i and TXB2 synthesis were measured in the presence or absence of extracellular calcium and aspirin, ouabain, or nifedipine. Aspirin caused reductions in both parameters; nifedipine blocked [Ca++]i, but did not affect TXB2; ouabain increased both. Changes induced by those modifiers of TXB2 and platelet [Ca++]i caused changes that were in the same direction for both. CaCl2 caused an increase in both and the [Ca++]i was correlated with the square root of the TXB2; without CaCl2 the two were negatively correlated; aspirin, ouabain, and nifedipine treatments resulted in no significant correlations. The results suggest that there is a common modifier of [Ca++]i and TXB2 synthesis.     

Effects of diltiazem on thromboxane B2 production from platelet-rich plasma and whole blood.

The present study evaluated the effects of the calcium-channel blocking agent diltiazem on platelet aggregation and on synthesis of thromboxane B2 (the stable metabolite of thromboxane A2) from platelet-rich plasma (PRP) and whole blood samples. Our results showed that diltiazem inhibits collagen- and thrombin-induced platelet aggregation and TXB2 production from PRP. Since no significant interference with conversion of arachidonate to thromboxane A2 was demonstrated, inhibition of phospholipase A2 activity may be the prevailing mechanism of the diltiazem effect. The drug demonstrated a dose-related inhibitory activity on TXB2 synthesis from whole blood samples during spontaneous clotting or following stimulation with collagen or thrombin. The present results give further evidences for an antiplatelet activity of diltiazem and support the hypothesis that inhibition of platelet function contributes to the therapeutic efficacy of this drug in the treatment of cardiovascular diseases.     

Platelet activation in patients with colorectal cancer

Aspirin may reduce the risk of colorectal neoplasia at doses similar to those recommended for the prevention of cardiovascular disease. Thus, we aimed to address whether enhanced platelet activation, as assessed by the measurement of the urinary excretion of 11-dehydro-TXB(2) (a major enzymatic metabolite of TXB(2)), occurs in patients with colorectal cancer. In 10 patients with colorectal cancer, the urinary excretion of 11-dehydro-TXB(2) was significantly higher than in 10 controls, matched for sex, age and cardiovascular risk factors [1001(205-5571) versus 409(113-984) pg/mg creatinine, respectively, median (range), P<0.05]. The administration of aspirin 50 mg daily for 5 consecutive days to colorectal cancer patients caused a cumulative inhibition of platelet cyclooxygenase (COX)-1 activity either ex vivo, as assessed by the measurement of serum TXB(2) levels, or in vivo, as assessed by urinary 11-dehydro-TXB(2) excretion. In conclusion, enhanced platelet activation occurs in colorectal cancer patients. Permanent inactivation of platelet COX-1 by low-dose aspirin might restore anti-tumor reactivity.     

Production of platelet thromboxane A2 and arterial prostacyclin I2 from hypercholesterolemic rats.

The plasma cholesterol, plasma malonaldehyde (MDA), platelet thromboxane A2 (TXA2) and vascular prostacyclin (PGI2) were measured in male Sprague-Dawley rats fed diets supplemented with cholesterol (1%) and cholic acid (0.5%). For comparisons, measurements were made in rats fed normal diets. The concentration of cholesterol in the plasma of rats had reached a maximum in 1 week of feeding experimental diets. TXA2 production from collagen and thrombin stimulated platelets was significantly decreased in animals fed experimental diets for 1 week. The production of MDA in the plasma of animals fed experimental diets for 8 weeks was significantly lower compared to the animals fed normal diets. There was a small but significant reduction in the formation of PGI2 in rats fed experimental diets for 8 weeks. These data suggest that feeding cholesterol rich diets to rats alters the platelet membrane properties differently from human and rabbit. Furthermore, cholesterol feeding to rats had some damaging effect on the arterial PGI2 synthesis.