Arachidonate-induced Aggregation Research Articles

Monitoring aspirin therapy in children after interventional cardiac catheterization: laboratory measures, dose response, and clinical outcomes.

Very few studies have investigated dose response of aspirin and agreement of different platelet function assays in children. One hundred five children were studied at baseline and after interventional cardiac catheterization during aspirin treatment and, in cases of aspirin resistance (AR), after dose increase. Results from arachidonate-induced aggregation (AA) were compared with aggregation induced by ADP, PFA-100 closure times (CTs), urinary 11-dehydro-thromboxane B2 (urinary 11-dhTxB2) levels, and Impact-R % surface coverage. Aspirin at 2-5 mg/kg/day inhibited platelet function in a large majority. While 19 % showed bruising and mild epistaxis, no thrombotic complications were recorded. AR was detected by AA in seven children (6.7 %). After dose increase, the majority showed inhibition by aspirin. Infants had higher urinary 11-dhTxB2 baseline levels; this assay showed some correlation with AA. Both assays manifested high sensitivity and specificity for aspirin while inferior results were found for the other assays. With the PFA-100, 15.2 % of patients were found to have AR, but this corresponded to AR by AA in only one of seven children. While there was poor agreement among assays, AA and urinary 11-dhTxB2 show good specificity for the monitoring of aspirin therapy in children. Aspirin at 2-5 mg/kg inhibits platelet function; AR in children is rare and can be overcome by dose increase.

Synthesis and Biological Evaluation of Bridged Analogues of the Platelet Aggregation Inhibitor Trifenagrel

The synthesis and preliminary biological investigation of three methylene bridged heterocyclic analogues of trifenagrel, a potent inhibitor of arachidonate-induced aggregation of platelets, is reported. The synthesis of the compounds is based on the cyclocondensation reaction of a tricyclic 1,2-diketone with an appropriate aldehyde, hydrazide or a diamine to form an imidazole, a 1,2,4-triazine or a pyrazine moiety respectively. In comparison to trifenagrel, its methylene- bridged analogue had much reduced activity, while its pyrazine analogue was devoid of activity.

Aspirin Resistance in Children after Interventional Cardiac Catheterisation.

Several large trials have demonstrated the benefit of aspirin for the prevention of arterial thrombotic events in adults. Aspirin has also been increasingly used as an anti-platelet agent in children. However, very few well-designed clinical trials assessing the clinical efficacy and dosing of aspirin in children have been performed. In children, aspirin is usually administrated at a dose between 1 and 10 mg/kg/day. The term “aspirin resistance” (AR) describes the inability of aspirin to protect individuals from arterial thrombotic events (clinical AR) or the failure of aspirin to produce an expected response on laboratory measures of platelet activation and aggregation (laboratory AR). In adults, a prevalence of 5%–51% of AR had been reported, depending on the laboratory method used. A recent study using the platelet function analyser PFA-100 and levels of urinary 11-dehydrothromboxane B2 (11-dTXB2) excretion to define AR in paediatric cardiology patients, found a prevalence of 26% of laboratory AR (Heistein et al 2007). Here we studied the frequency of AR in children given aspirin at 3–5 mg/kg/d after interventional cardiac catheterization for atrial or ventricular septal defects or patent ductus arteriosus. Blood samples were taken 1–3 months after the initiation of aspirin therapy. Definition of AR was based on results from platelet aggregometry (% arachidonate-induced aggregation (AAG) of ≥ 20%) and results were compared with collagen/epinephrine closure times using the platelet function analyser PFA-100 and the levels of 11-dTXB2 excretion. As cyclooxygenase 1 (COX-1) is the primary target of aspirin and mutations could lead to a loss of functional protein, the presence of COX-1 was analyzed by western blot. Further, all children were followed clinically and side effects were monitored.Of 56 children included to date (median age: 10 y, range: 10 mo −20 y), 44 have completed the study. AR, was detected in 6 (14%) children. Of these 6 children, 3 had a normal response to aspirin (i.e. decreased AAG to <20%) after doubling the dose to 6–10 mg/kg, 1 child did not respond to a dose increase, and 2 were unavailable for further studies. Children under the age of 7 had higher urinary 11-dTXB2 levels initially compared to older children (323 vs 178 ng/mmol creatinine; p< 0.001). During aspirin therapy, 11-dTXB2 levels decreased to 38 ± 15 ng/mmol creatinine (mean ± SD). However, in 2 children, 11-dTXB2 decreased only minimally (81 and 84% of pre-aspirin level, respectively; results > mean+2SD ): Of these 2 children, one child had AR by the AAG definition even after aspirin dose increase and the other revealed AR by the PFA-100, with normal closure time, but not by AAG. While PFA-100 closure times were prolonged in 48% of children already prior to administration of aspirin, all children with AR according to AAG had prolonged closure times during aspirin-therapy. All 6 AR patients and 10 patients with a normal response to aspirin by AAG demonstrated equal amounts of COX-1 by western blot. While 6 children (14%) showed easy bruising and epistaxis during aspirin therapy, no severe bleeding or thrombotic complications were recordedIn conclusion, AR was detected in 14% of children after interventional cardiac catheterization by AAG and could be overcome in some patients by increasing the dose of aspirin. These results are in keeping with findings from adults. By the use of the PFA-100 a previous study found a prevalence of 26% of AR in children (Heistein et al 2007). However, the authors did not correlate these results to AAG. In our cohort, the assessment of AR by the PFA100 was hampered by a high rate of abnormal PFA results previous to the initiation of therapy. Although reduction of 11-dTXB2 excretion indicates an inhibition of the COX-1 pathway by aspirin therapy, 11-dTXB2 and AAG results were not always congruent.

Alteration of prostaglandin metabolism in alcoholic liver disease: its association with platelet dysfunction after chronic alcohol ingestion

Recently, hepatic microcirculation has been focused on as an important pathogenic factor in progression of alcoholic liver disease (ALD). Therefore, blood levels of several prostaglandins, which are associated with organ microcirculation, were determined in various liver diseases, including ALD. Blood thromboxane B2 (TXB2) level was significantly increased in ALD, when compared to other types of liver diseases, whereas both 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) and prostaglandin E were not changed. These consequences resulted in the imbalance of 6-keto PGF1 alpha to TXB2, which might promote platelet aggregation and blood vessel contraction. Indeed, the increase of beta-thromboglobulin and platelet factor 4 in blood was observed in ALD. Furthermore, in ALD, the rate of arachidonate-induced platelet aggregation was prominently enhanced, and malondialdehyde production in platelet, which was well correlated with blood TXB2 levels, significantly increased. Thus, the present study may indicate that, in ALD, hyper-aggregability of platelet is produced, because of the derangement of prostaglandin metabolism and platelet dysfunction.

2‐Phenyl‐3‐(quinolizidin‐1‐yl)‐5‐substituted Indoles as Platelet Antiaggregating Agents.

AbstractFor Abstract see ChemInform Abstract in Full Text.

2-Phenyl-3-(quinolizidin-1-yl)-5-substituted indoles as platelet antiaggregating agents

A set of ten 2-phenyl-3-(quinolizidin-1-yl)-5-substituted indoles was prepared through the Fischer cyclization of lupinyl- and epi-lupinylphenylketone 4-substituted phenylhydrazones.Compounds were tested for antiaggregating activity on human platelets activated by adenosine diphosphate (ADP), collagen and adrenaline. At 2.5 × 10 –4 M concentration most compounds strongly inhibited the aggregation induced by all the agonists considered and many of them still displayed good activity at 0.625 × 10 –4 M concentration. The least active ( 1c) and one of the most active ( 1d) compounds were also tested for antiaggregating activity on rabbit platelets activated by ADP, PAF and sodium arachidonate. Both the compounds were active against ADP and PAF, but only 1d inhibited the arachidonate-induced aggregation (100% at 8 × 10 –6 M concentration) and increased the bleeding time in mice. The same compounds were subjected to a general pharmacological screening and found to display several activities; of particular interest was the dose dependent reduction of serum cholesterol and heparin precipitating betalipoproteins in hypercholesterolemic mice exerted by 1c, which was still significant at the oral dose of 10 mg/kg.

Enhanced arachidonic acid and calcium metabolism in cholesteryl sulfate-enriched rat platelets

We previously found that cholesteryl sulfate (CS), a minor lipid constituent, is present in blood platelets and might influence platelet aggregation. In this study, we investigated whether CS modifies platelet arachidonic acid and calcium metabolism. In CS-loaded rat platelets, we found a potentiation of the arachidonate-induced aggregation, an increase in the thrombin-induced release of radiolabeled arachidonate preincorporated into platelet phospholipids and an increased formation of cyclooxygenase and lipoxygenase products. In addition, the calcium uptake in non-stimulated as well as in thrombin-stimulated CS-loaded platelets was enhanced. The above-mentioned effects were never observed with cholesterol, cholesteryl acetate, estrone and estrone sulfate. Although the precise determinants for the platelet CS concentration are not presently known, our findings might suggest a new role for CS as a modulator of the activity of these cells.

Antiplatelet principles from a food spice clove (Syzgium aromaticum L)

In continuation of our studies on the oil of cloves — a common kitchen spice and a drug for home medicine — we have isolated and identified two antiplatelet components, eugenol and acetyl eugenol. They inhibited arachidonate-, adrenaline- and collagen-induced platelet aggregation; they were more potent in inhibiting aggregation by the first two agonists. Their inhibitory effect was reversible. These components were antiaggregatory by a combination of at least two effects: (i) inhibition of platelet thromboxane formation, and (ii) increased formation of 12-lipoxygenase products (12-HPETE). Though the presence of plasma proteins would reduce the effective concentration of these substances due to binding, the relatively lower amounts of these components which inhibited arachidonate-induced aggregation when compared to their effects on thromboxane production was intriguing. The answer might partly lie in an increased formation of 12-HPETE facilitated by albumin which acts as a ‘conduit’ to divert free arachadonic acid (AA) from the platelet cyclooxygenase (CO) to the lipoxygenase pathway (22). Based on their IC 50 values, it was found that both eugenol and acetyl eugenol were more potent than aspirin in inhibiting platelet aggregation induced by arachidonate, adrenaline and collagen. In arachidonate-induced aggregation eugenol was on a par with indomethacin. It was found that eugenol and acetyl eugenol when used in combination potentiated inhibition of platelet aggregation induced by arachidonate, adrenaline and collagen. This effect was, however, not evident from the metabolism of AA in platelets; when used in combination the two compounds produced an additive effect.

γ-pyrone compounds. IV: Synthesis and antiplatelet effects of mono- and dioxygenated xanthones and xanthonoxypropanolamine

AbstractXanthodilol, mono- and dioxygenated xanthones, and 1,3-, 2,3-, 3,4-, 3,5-, 1,6-, 2,6-, and 3,6-dioxygenated xanthones were synthesized from benzophenone precursors by Friedel-Crafts acylation and subsequent base-catalyzed cyclization to eliminate methanol. 3-Hydroxyxanthone, xanthodilol, 2,3-dihydroxyxanthone diacetate, and 3,4-dihydroxyxanthone and its diacetate showed potent antiplatelet effects on arachidonate- and collagen-induced aggregation. 3,5-Dihydroxyxanthone and its diacetate, 1,6-dimethoxyxanthone, and 3,6-dihydroxyxanthone and its diacetate showed potent antiplatelet effects on arachidonate-induced aggregation.

Unexpected Effects of Aurin Tricarboxylic Acid on Human Platelets

Aurin tricarboxylic acid (ATA) is a potent inhibitor of ristocetin-mediated platelet agglutination and of shear-induced, von Willebrand factor (vWf)-mediated platelet aggregation, probably via inhibition of vWf interaction with glycoprotein Ib (GPIb). We examined the effects of ATA (both the sodium salt and a solution of ATA in ethanol) on platelet functions in citrated plasma (PRP) and in suspensions of washed platelets in Tyrode-albumin solution (contains 2 mM Ca2+). ATA (42-211 micrograms/ml) blocked aggregation and release of granule contents induced by thrombin (0.15 U/ml in PRP; 0.03 U/ml in platelet suspension). Responses to higher concentrations of thrombin were not inhibited. ATA also prolonged thrombin-induced clotting of fibrinogen. Since ATA had no effect on fibrinogen-induced responses of chymotrypsin-treated platelets, ATA probably acts on thrombin rather than on fibrinogen. In PRP and platelet suspensions, ATA (acid form 106 micrograms/ml; sodium salt 122 micrograms/ml) had little effect on ADP-induced platelet aggregation. The sodium salt of ATA (61-122 micrograms/ml) enhanced collagen-induced aggregation and release by platelets in citrated plasma and by washed platelets; the enhancement was extensively inhibited by aspirin. With platelet suspensions, ATA significantly enhanced aggregation and release caused by low concentrations of sodium arachidonate (15-50 microM); aggregation and release caused by higher concentrations of arachidonate were somewhat inhibited by ATA. Arachidonate-induced aggregation and release were also enhanced by ATA in PRP. ATA enhanced aggregation and release induced by the calcium ionophore A23187; aspirin had little effect on the enhancement.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormal Platelet Functions in a Patient with Abetalipoproteinemia

Studies with isolated lipoproteins and washed platelets suggest that lipoproteins may affect platelet functions. We investigated platelet-rich plasma (PRP) from a patient with abetalipoproteinemia (ABL), whose plasma lacks apo-B containing lipoproteins (VLDL, LDL and chylomicrons). ABL-PRP aggregated poorly with different agonists and failed to respond to arachidonate. Thromboxane B2 (TxB2) formation was severely impaired. After gel-filtration most of the aggregation defects persisted in agreement with reduced metabolism of endogenous arachidonate. However, arachidonate-induced aggregation and TxB2 production partially normalized. Normal platelets suspended in ABL-plasma showed similar defects in aggregation and TxB2 production but arachidonate-induced aggregation was much lower than expected on the basis of TxB2. We conclude that the abnormal platelet functions in ABL-PRP are caused by (i) an intrinsic platelet abnormality due to reduced arachidonate mobilization and (ii) a property in ABL plasma that inhibits aggregation partially by trapping the arachidonate and partially by an unidentified mechanism. The latter properties may be the result of the abnormal lipid composition of ABL-plasma.

Benzodiazepine analogues inhibit arachidonate‐induced aggregation and thromboxane synthesis in human platelets

1. Benzodiazepine analogues inhibit human platelet aggregation induced by arachidonate with an EC50 value of 0.68 microM for PK 11195, the most potent analogue used. 2. There was a highly significant correlation between the inhibition of arachidonate-induced aggregation and the affinity for the peripheral-type of benzodiazepine binding sites. 3. There was no significant correlation between the inhibition of the platelet activating factor (PAF)-induced aggregation and the binding to the peripheral-type of benzodiazepine binding sites. 4. The inhibition of platelet aggregation seems to result from the inhibition of arachidonic acid cyclo-oxygenation, since the synthesis of thromboxane and 12-hydroxy-heptadecatrienoic acid, both cyclo-oxygenase products, was reduced. 5. Our results suggest that peripheral-type of benzodiazepine binding sites on human platelets could be linked to cyclo-oxygenase.

Inhibitors of platelet aggregation from onion

Five new sulphur-containing compounds and five diastereoisomers which indicated potent inhibitory efrects for collagen-induced aggregation of human platelets in vitro as well as ADP- or arachidonate-induced aggregation were isolated from onion. All isolated compounds have a 1-(methylsulphinyl)-propyl alkyl (or alkenyl) disulphide skeleton. It was predicted that they were formed by the interaction of the onion lachrymatory factor, thiopropanal S-oxide, with alkane (or alkene) sulphenic acids produced in crushed onion.

Inhibition of platelet aggregation by idebenone and the mechanism of the inhibition

The inhibitory effect of 6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone (idebenone) on platelet aggregation was studied in rat and human platelets in vitro, and the mechanism of inhibition was examined in rat platelets. Idebenone inhibited the aggregation induced by collagen and thrombin in washed platelets, and by arachidonate and ADP in platelet-rich plasma (PRP). The inhibition was more prominent in collagen- and arachidonate-induced aggregation. In collagen-induced aggregation of human platelets, idebenone was 8-fold more potent than aspirin. In addition, idebenone inhibited prostaglandin synthesis and thromboxane B 2 production, and also increased the cyclic AMP content in platelets. However, the concentration of idebenone required to inhibit thromboxane B 2 production was much lower than that required to increase cyclic AMP. These results indicate that idebenone inhibits platelet aggregation by inhibiting thromboxane B 2 synthesis rather than by increasing cyclic AMP content.

Release of Human Epidermal Growth Factor from Platelets in Accordance with Aggregation in vitro.

We measured the intra-platelet content of human epidermal growth factor (hEGF) and beta-thromboglobulin (beta-TG) and the quantities of these released from platelets during in vitro aggregation. The intra-platelet amounts of hEGF and beta-TG in 10(8) platelets were 104.9 +/- 18.9 (Mean +/- SEM) pg and 2920.9 +/- 149.9 ng, respectively. During platelet aggregation elicited by 9, 11-epithio-11, 12-methano-thromboxane A2, a stable thromboxane A2 agonist, hEGF and beta-TG were released in amounts about 50% and 40% of the respective content in platelets. Also during arachidonate-induced aggregation, hEGF and beta-TG were released at about 60% and 50%, respectively. Various concentrations of thromboxane A2 antagonist, (9, 11), (11, 12)-di-deoxa-9, 11-dimethyl-methano-11, 12-methano-13, 14-dihydro-13-aza-14-oxo-15-cyclopentyl-16, 17, 18, 19, 20-pentanor-15-epi-thromboxane A2, suppressed both aggregation and release reactions in a dose-dependent manner. There were good correlations between the platelet aggregation rate and released beta-TG (r = 0.9368, p less than 0.01) or hEGF (r = 0.8931, p less than 0.01) and between released beta-TG and hEGF (r = 0.9385, p less than 0.01). These results suggest that hEGF is released from platelets in a similar fashion to beta-TG in vitro.

Lymphocyte aggregation in response to adrenergic stimulation

A nonanemic chronic lymphocytic leukemia patient with nearly 500,000 lymphocytes/microL underwent leukapheresis when she presented with CNS symptoms and retinal vascular engorgement. Respiratory distress developed during the cell separator run, which led us to ask whether the procedure could have changed the adhesive properties of her cells. C5a desarginine, N-f-Met-Leu-Phe, adenosine diphosphate, and collagen all failed to aggregate her lymphocytes in vitro, but arachidonic acid, excess free calcium, and 4 mumol/L epinephrine did aggregate the cells. Arachidonate-induced aggregation appeared to be a toxic phenomenon: the ED50 for aggregation was statistically indistinguishable from that for cytotoxicity, and aspirin only mildly blunted the response. In contrast, epinephrine-induced aggregation was not associated with lactic dehydrogenase release or the loss of trypan blue exclusion and was blunted by propranolol; radiopindolol-binding studies confirmed the presence of a beta-adrenergic receptor. There were approximately 3,000 receptors/cell, with no statistically significant difference between normal and chronic lymphocytic leukemia B cells or between B cells and T cells (separated by rosetting techniques). The Kd for the B cells' receptor, however, was less than that for T cells by a factor of ten (P less than .01). We conclude that B cells may aggregate when stimulated and that they--like T cells--have beta-adrenergic receptors. Adrenergically mediated changes in B cell adhesiveness may play a role in regulating lymphocyte traffic; in the rare patient with truly enormous B cell counts, we postulate that they may be an occasional cause of morbidity.

Lymphocyte Aggregation in Response to Adrenergic Stimulation

AbstractA nonanemic chronic lymphocytic leukemia patient with nearly 500,000 lymphocytes μL underwent leukapheresis when she presented with CNS symptoms and retinal vascular engorgement. Respiratory distress developed during the cell separator run, which led us to ask whether the procedure could have changed the adhesive properties of her cells. C5adeearginine, N-f-Met-Leu-Phe, adenosine diphosphate, and collagen all failed to aggregate her lymphocytes in vitro, but arachidonic acid, excess free calcium, and 4 μmol/L epinephrine did aggregate the cells. Arachidonate-induced aggregation appeared to be a toxic phenomenon: the ED50 for aggregation was statistically indistinguishable from that for cytotoxicity, and aspirin only mildly blunted the response. In contrast, epinephrine-induced aggregation was not associated with lactic dehydrogenase release or the loss of trypan blue exclusion and was blunted by propranolol; radiopindolol-binding studies confirmed the presence of a β-adrenergic receptor. There were approximately 3,000 receptors /cell, with no statistically significant difference between normal and chronic lymphocytic leukemia B cells or between B cells and T cells (separated by rosetting techniques). The Kd for the B cells’ receptor, however, was less than that for T cells by a factor of ten (P < .01). We conclude that B cells may aggregate when stimulated and that they—like T cells—have β-adrenergic receptors. Adrenergically mediated changes in B cell adhesiveness may play a role in regulating lymphocyte traffic; in the rare patient with truly enormous B cell counts, we postulate that they may be an occasional cause of morbidity.

N-Ethylmaleimide inhibits Ca 2+ influx induced by collagen or arachidonate on rabbit platelets

N-Ethylmaleimide dose dependently inhibited platelet aggregation induced by collagen or arachidonate but did not inhibit the aggregation by thrombin or ionophore A23187 within the concentrations tested. [ 3H]Arachidonate release from membrane phospholipids of the collagen-stimulated platelets was inhibited by N-ethylmaleimide in parallel with the inhibition of aggregation, but not in response to A23187. N-Ethylmaleimide prevented 45Ca 2+ influx into platelet cells from outer medium induced by collagen, and also inhibited the increase in the concentration of cytoplasmic free Ca 2+, which probably results from Ca 2+ influx, as monitored by quin2 fluorescence, under stimulation with arachidonate. The concentration of N-ethylmaleimide giving a complete inhibition of Ca 2+ influx was consistent with that required to inhibit collagen- or arachidonate-induced aggregation. Prostaglandin metabolism from arachidonate to thromboxane A 2 was not disturbed by N-ethylmaleimide, while phosphatidate formation induced by arachidonate was slightly inhibited by it at concentrations at which aggregation was completely inhibited. These data suggest that N-ethylmaleimide preferentially suppresses increase in cytoplasmic free Ca 2+ which is linked to thromboxane A 2-receptor occupation in collagen- or arachidonate-stimulated platelets, probably due to blockage of Ca 2+ influx through Ca 2+-channel protein, thereby inhibiting aggregation induced by these agonists.

Effect of OKY-046, a selective thromboxane inhibitor, on prostaglandin production and function of pregnant rat uteri and platelets

The effect of OKY-046 (ONO, Japan), a selective TX inhibitor, was studied for its effect on uterine and platelet activity. On day 21 of pregnancy, rats were injected with either 0, 1, or 5 mg/kg OKY-046 via the tail vein. One hour following injections, in vitro activity of uteri and platelets was assessed. A decrease (P<.01) in uterine TXB 2 production (measured by RIA) occurred with increasing OKY-046 dose ( 104 ± 31 vs 44 ± 6 vs 24 ± 2 ng TXB 2/g tissue/45 min). OKY-046 treatment had no effect on other prostanoids. Contractile activity was not affected by OKY-046. The amount of TXB 2 produced in platelets from OKY-046 (5 mg/Kg) treated rats was 45.5% less than that from controls (P<.001). Likewise, arachidonate-induced aggregation of platelets from OKY-046 treated rats was 46.1% less (P<.05) than that of controls. In summary, in vivo administration of OKY-046 selectively reduced uterine TXB 2 without altering other prostanoids, or affecting uterine contractions. In contrast, both platelet TXB 2 production and platelet function (aggregation) was decreased.

Vitamin E and vitamin C inhibit arachidonate-induced aggregation of human peripheral blood leukocytes in vitro.

Arachidonate induces aggregation of human polymorphonuclear (PMN) and mononuclear (MNL) blood leukocytes. This is mediated by the lipoxygenase pathway, as it is prevented by lipoxygenase inhibitors and can also be induced by leukotriene B4 (LTB4). Vitamin E and vitamin C have profound effects on the functional state of leukocytes, some of which may involve the lipoxygenase pathway. This study shows that both vitamins inhibit arachidonate-induced aggregation of PMN and MNL, in a concentration-dependent way. BW-755, previously shown to inhibit arachidonate-induced PMN and NML aggregation, was found to potentiate the inhibitory activity of both vitamins. When LTB4 was used as an aggregating agent, vitamin E markedly inhibited PMN and MNL aggregation, whereas vitamin C was ineffective. The prevention of PMN and MNL aggregation by vitamin E might account, at least partially, for the reported beneficial effects of vitamin E supplementation in some experimental syndromes characterized by leukocyte activation.