Rapid return of cyclo-oxygenase active platelets in dogs after a single oral dose of aspirin

A single dose of oral aspirin in human subjects inhibits the aggregation response of platelets to arachidonate and other agents for approximately one week after ingestion. In the present study we have evaluated the rate at which cyclo-oxygenase active platelets return to the circulation in humans and dogs and compared the response curves obtained to improvements in cyclo-oxygenase activity produced by the aspirin platelets. After a single dose of aspirin, dog platelet function was compromised for several days. Normal responses to arachidonate and other aggregating agents were restored six days after aspirin, and the pattern of recovery was the same for dogs and human subjects. However, cyclo-oxygenase active platelets returned to the circulation in dogs more rapidly than in humans and chemical competence was restored in both species well before correction of the defective response to aggregating agents. The delay of 1-3 days before return of significant numbers of cyclo-oxygenase active platelets most likely reflects acetylation of bone marrow megakaryocytes by the drug. More rapid return of chemically competent cells in dogs than humans probably relates to the more rapid turnover and shorter life span of canine platelets. The basis for the discrepancy in return of chemical integrity compared to functional activity after aspirin in vivo compared to simultaneous correction of chemistry and function when 10% normal platelets are added to aspirin platelets in vitro remains unresolved.     

Dose-related effects of low dose aspirin on hemostasis parameters and prostacyclin/thromboxane ratios in late pregnancy

Background: The purpose of this study was to determine which low dose of low dose aspirin (LDA) optimized the urinary prostacyclin (PGI₂)/thromboxane (TXA₂) ratio and minimized evidence of platelet aggregation during normal late pregnancy.Methods: Twelve women with uncomplicated singleton pregnancies between 28 and 34 weeks gestation participated in a randomized blinded study. Blood samples for salicylate levels were obtained pretreatment, 4 hours and 7 days after administration of placebo, 20mg, 40mg or 80mg of aspirin. Twenty-four hour urine specimens collected at the same intervals were assayed for PGI₂ and TXA₂ metabolites. In addition, bleeding time and platelet aggregation studies were performed prior to and after 7 days of LDA or placebo.Results: A dose-related increase in bleeding time occurred with 40 mg and 80 mg of LDA, but not with the 20 mg dose or placebo. Platelet aggregation studies changed progressively from a normal baseline to abnormal with an increasing dose of LDA. The ratio increased with aspirin doses as low as 20mg, with a decrease in TXA₂ metabolites but not in PGI₂ metabolites. Serum salicylate was not detectable in any sample from any patient.Conclusion: There are dose-related changes in platelet aggregation and bleeding times with progressively increasing doses of LDA. A lower dose of LDA, such as 20–40 mg per day, may be as efficacious as higher doses in the prophylaxis of pre-eclampsia in high risk populations.     

Nitroprusside differentially inhibits ADP-stimulated calcium influx and mobilization in human platelets.

1. The effect of nitroprusside on cGMP concn., cAMP concn., shape change, aggregation, intracellular free Ca2+ concn. (by quin-2 fluorescence) and Mn2+ entry (by quenching of quin-2) was investigated in human platelets incubated with 1 mM-Ca2+ or 1 mM-EGTA. 2. Nitroprusside (10 nM-10 microM) caused similar concentration-dependent increases in platelet cGMP concn. and was without effect on cAMP concn. in the presence of extracellular Ca2+ or EGTA. 3. In ADP (3-6 microM)-stimulated platelets, nitroprusside caused 50% inhibition of shape change at 0.4 microM (+Ca2+) or 1.3 microM (+EGTA), aggregation at 0.09 microM (+Ca2+) and of increased intracellular Ca2+ at 0.02 microM (+Ca2+) or 2.1 microM (+EGTA). Entry of 1 mM-Mn2+ (-Ca2+) was inhibited by 80% by 5 microM-nitroprusside. 4. In ionomycin (20-500 nM)-stimulated platelets, nitroprusside (10 nM-100 microM) did not inhibit shape change or intracellular-Ca2+-increase responses, and only partially inhibited aggregation. 5. In phorbol myristate acetate (10 nM)-stimulated platelets, neither shape change nor aggregation was inhibited by 5 microM-nitroprusside. 6. The data demonstrate that nitroprusside inhibits ADP-mediated Ca2+ influx more potently than Ca2+ mobilization. Nitroprusside appears not to influence Ca2+ efflux or sequestration and not to affect the sensitivity of the activation mechanism to intracellular Ca2+ concn. or activation of protein kinase C.     

Effects of maternal captopril treatment during late pregnancy on neonatal lung development in rats

The renin-angiotensin system (RAS) has been implicated in pulmonary hypertension and pulmonary fibrosis. In the present study, we examined the effects of maternal exposure to captopril (2.85 mg/kg/day) during late pregnancy (G13-G21) on postnatal rat lung development. Treatment with captopril during late pregnancy caused a significant decrease in ACE activity in P0 rats. Body weight decreased at P0 (p<0.001), P8 and P15 (p<0.01) in captopril-treated rats. Lung weight of P0 and P8 pups was lower in treated-animals (p<0.05). Lungs from captopril-treated animals showed impaired alveolar formation, with enlarged distal airway spaces at P8, P15 and P30. Interalveolar wall distance measured by mean linear intercept increased in treated vs. age-matched animals at P8, P15 (p<0.001) and P30 (p<0.05) resembling new bronchopulmonary dysplasia. In control animals, the proliferating cell nuclear antigen (PCNA) marker was higher at P0 and then drops gradually, while in captopril-treated animals PCNA marker remains higher at all stages studied. α-Smooth muscle actin (α-SMA), a marker of fibroblast differentiation into myofibroblasts, was higher at the tips of developing secondary septa in captopril-treated lungs at P8 and P15. The increased expression of PCNA and α-SMA in treated pups suggest that beyond the effect caused by captopril, the developing lungs have the capacity to recover once the treatment was stopped. Taking together the low weight, histomorphological changes and increased expression of cellular markers caused by ACE inhibition during late pregnancy, it appears that the RAS could be an intrinsic factor involved in secondary septa formation during lung development.     

Equivalent inhibition of in vivo platelet function by low dose and high dose aspirin treatment

In vitro platelet function was inhibited in healthy volunteers by two different doses of aspirin, as confirmed by measurement of maximum serum production of thromboxane B2 (TXB2) by platelets. 75 mg aspirin did not fully inhibit serum TXB2 production after 24 hours, whereas 300 mg aspirin did. Inhibition of platelet function in vitro was maintained by both 75 mg/day aspirin or 300 mg/alternate day aspirin. In contrast, in vivo production of TXB2, measured as urinary levels of the 11-keto-TXB2 metabolite, was inhibited similarly by both doses of aspirin throughout the study. These findings suggest that 75 mg/day aspirin may be sufficient adequately to inhibit platelet aggregation in vivo.     

Preferential utilization of endogenous arachidonate by cyclo-oxygenase in incubations of human platelets

Thromboxane B2 (TXB2) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) formed from the endogenous and exogenous arachidonate during human platelet incubation, was evaluated by selected ion monitoring (SIM). TXB2 formed from endogenous substrate accounted for about one third of the total, whereas the great part of 12-HETE derived from exogenous arachidonate. These data indicate that under the tested conditions the pool of arachidonate that acts as substrate for cyclo-oxygenase is different from the pool that acts as substrate for lipoxygenase and that the arachidonate released from phospholipids is preferentially utilized by cyclo-oxygenase.     

孕晚期孕妇肠道菌群与孕期体重增长的相关性

【背景】孕妇体重过度增长在全球范围内呈现上升趋势,对母亲和子代健康均会造成不良影响。肠道菌群与人体众多疾病相关,其有可能作为重要的调节因素影响母婴健康。【目的】研究孕晚期孕妇肠道菌群的多样性和群落组成,探讨孕期体重增长对肠道菌群的影响。【方法】收集62位孕晚期(36.82±2.19孕周)孕妇粪便样本,利用MiSeq高通量测序技术对样品中16SrRNA基因V3-V4区序列进行测序,分析不同孕期体重增长水平的孕妇肠道菌群的多样性、群落结构和物种丰度,探讨孕期体重增长对孕妇肠道菌群的影响。【结果】与正常孕妇相比,孕期体重增长过度孕妇肠道菌群多样性显著降低,群落结构变化不大;在菌群组成上,筛选出5个与孕期体重增长最为相关的菌群,分别为Alistipes spp.、Eubacterium-nodatum group spp.、Oxalobacter spp.、Raoultella spp.和Odoribacter spp.,其中Alistipesspp.不仅是相关程度最高也是丰度最高的菌群,与孕期体重增长密切相关。【结论】孕期体重增长对孕晚期孕妇肠道菌群群落结构影响显著,在孕期保持肠道菌群稳态能够促进母婴健康。     

Antibody profile of pregnant women with antiphospholipid syndrome and pregnancy outcome after treatment with low dose aspirin and low-weight-molecular heparin

The aim of the research was to show our diagnostic and therapeutic experience with antiphospholipid syndrome (APS) in pregnant women. 36 pregnant women suspect on APS were included in the study: 32 with primary antiphospholipd syndrome (PAPS) and 4 with secondary antiphospholipid syndrome (SAPS). All pregnant women received low-molecular-weight-heparin (LMWH) and low dose aspirin (LDA) therapy. Control group represented 26 women with SAPS and previous bad reproductive anamnesis. Average pregnancy lasted 37.06 +/- 0.707 weeks. LMWH and LDA therapy was successful in 97.22%. Lupus anticoagulant (LA) was found to be more frequent in PAPS group (71.87%). Anticardiolipin antibodies (aCL) were found to be more frequent in SAPS (26.66%). For three patients (3.37%), PAPS was diagnosed due to a fact that they had positive antibeta2-glycoproteinl (antibeta-GP1). To make APS diagnosis, it is of great importance to search for all antiphospholipid antibodies. LMWH and low dose of acetylsalicylic acid should be the first choice therapy.     

Mechanism of inhibition of cyclo-oxygenase in human blood platelets by carbamate insecticides.

Carbamates are a widely used class of insecticides and herbicides. They were tested for their ability to affect human blood platelet aggregation and arachidonic acid metabolism in platelets. (1) The herbicides of the carbamate type have no, or only little, influence up to a concentration of 100 microM; the carbamate insecticides, however, inhibit both aggregation and arachidonic acid metabolism in a dose- and time-dependent manner. (2) Carbaryl, the most effective compound, inhibits platelet aggregation and cyclo-oxygenase activity completely at 10 microM. The liberation of arachidonic acid from phospholipids and the lipoxygenase pathway are not affected, whereas the products of the cyclo-oxygenase pathway are drastically decreased. (3) By using [14C]carbaryl labelled in the carbamyl or in the ring moiety, it could be proved that the carbamyl residue binds covalently to platelet proteins. In contrast with acetylsalicylic acid, which acetylates only one protein, carbaryl carbamylates a multitude of platelet proteins. (4) One of the carbamylated proteins was found to be the platelet cyclo-oxygenase, indicating that carbaryl resembles in this respect acetylsalicylic acid, which is known to inhibit this enzyme specifically by acetylation.     

Nicotinic acid inhibits thromboxane synthesis in platelets

The formation of thromboxane A₂ by phospholipase A₂ in rat platelets is inhibited by nicotinic acid. The synthesis of PGE₂ and PGF_2α is increased.Nicotinic acid inhibits collagen-induced aggregation in rat platelets.     

Resveratrol inhibits polyphosphoinositide metabolism in activated platelets

The effects of resveratrol (trans-3,4′,5-trihydroxystilbene) on activation responses and the polyphosphoinositide metabolism in human blood platelets have been studied. Resveratrol partially inhibited secretory responses (liberation of dense granule nucleotides and lysosomal acid hydrolases), microparticle formation and protein phosphorylations induced by thrombin. The effects of resveratrol on phosphoinositide metabolites, phosphatidate (PtdOH), phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate (PtdIns-4(5)-P), phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P₂), phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4-P₂) and phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P₃) were monitored in blood platelets prelabelled with [³²P]P_i. Resveratrol not only inhibited the marked increase in levels of PtdOH in platelets activated by thrombin (0.1 U/ml) but it decreased the steady state levels of the other polyphosphoinositide metabolites. The distribution of ³²P in phosphoinositides in activated platelets was consistent with inhibition of CDP-DAG inositol transferase and a weak inhibition of PtdIns-4(5)-P kinase. These observations show that resveratrol has a profound effect on phospholipids, particularly on polyphosphoinositide metabolism, and may decrease the amount of PtdIns-4,5-P₂ available for signalling in these cells.     

Aspirin inhibits arachidonic acid metabolism via lipoxygenase and cyclo-oxygenase in hamster isolated lungs

The effect of aspirin on the fate of exogenous arachidonic acid (AA) was investigated in isolated perfused lungs of female hamsters. During pulmonary infusion of aspirin (10 μM, 100 μM or 1 mM) 45 nmol of ¹⁴C-AA was infused in two minutes into the pulmonary circulation. The nonrecirculating perfusion effluent was collected for 6 minutes after the beginning of the AA infusion. Arachidonate infusion increased the perfusion pressure. This pressor response was completely abolished by 1 mM aspirin. When aspirin was infused into the pulmonary circulation, the amount of radioactivity was increased in the perfused lungs and decreased dose dependently in the nonrecirculating perfusion effluent. The amount of unmetabolized free arachidonate was not changed significantly by aspirin in the perfused lungs or in the perfusion effluent. In the effluent the amounts of all arachidonate metabolites, which were extracted with ethyl acetate first at pH 7.4 and then at pH 3.5 and analysed by thin layer chromatography, were decreased quite similarly by aspirin. The formation of arachidonate metabolites was completely inhibited by 1 mM aspirin. In the perfused lung tissue the amount of ¹⁴C-AA was increased by aspirin in phospholipids and neutral lipids. The present study indicates that the metabolism of arachidonic acid is inhibited by aspirin in hamster lungs not only via cyclo-oxygenase but also via other lipoxygenases.     

Methylation variation at IGF2 differentially methylated regions and maternal folic acid use before and during pregnancy

Folic acid (FA) supplementation before and during pregnancy has been associated with decreased risk of neural tube defects although recent reports suggest it may also increase the risk of other chronic diseases. We evaluated exposure to maternal FA supplementation before and during pregnancy in relation to aberrant DNA methylation at two differentially methylated regions (DMRs) regulating insulin-like growth factor 2 (IGF2) expression in infants. Aberrant methylation at these regions has been associated with IGF2 deregulation and increased susceptibility to several chronic diseases. Using a self-administered questionnaire, we assessed FA intake before and during pregnancy in 438 pregnant women. Pyrosequencing was used to measure methylation at two IGF2 DMRs in umbilical cord blood leukocytes. Mixed models were used to determine relationships between maternal FA supplementation before or during pregnancy and DNA methylation levels at birth. Average methylation at the H19 DMR was 61.2%. Compared to infants born to women reporting no FA intake before or during pregnancy, methylation levels at the H19 DMR decreased with increasing FA intake (2.8%, p = 0.03 and 4.9%, p = 0.04, for intake before and during pregnancy, respectively). This methylation decrease was most pronounced in male infants (p = 0.01). Methylation alterations at the H19 DMR are likely an important mechanism by which FA risks and/or benefits are conferred in utero. Because stable methylation marks at DMRs regulating imprinted genes are acquired before gastrulation, they may serve as archives of early exposures with the potential to improve our understanding of developmental origins of adult disease.Key words: folic acid, epigenetics, IGF2, periconception, prenatal, exposure     

Methylation variation at IGF2 differentially methylated regions and maternal folic acid use before and during pregnancy

Folic acid (FA) supplementation before and during pregnancy has been associated with decreased risk of neural tube defects although recent reports suggest it may also increase the risk of other chronic diseases. We evaluated exposure to maternal FA supplementation before and during pregnancy in relation to aberrant DNA methylation at two differentially methylated regions (DMRs) regulating Insulin-like Growth Factor 2 (IGF2) expression in infants. Aberrant methylation at these regions has been associated with IGF2 deregulation and increased susceptibility to several chronic diseases. Using a self-administered questionnaire, we assessed FA intake before and during pregnancy in 438 pregnant women. Pyrosequencing was used to measure methylation at two IGF2 DMRs in umbilical cord blood leukocytes. Mixed models were used to determine relationships between maternal FA supplementation before or during pregnancy and DNA methylation levels at birth. Average methylation at the H19 DMR was 61.2%. Compared to infants born to women reporting no FA intake before or during pregnancy, methylation levels at the H19 DMR decreased with increasing FA intake (2.8%, p=0.03, and 4.9%, p=0.04, for intake before and during pregnancy, respectively). This methylation decrease was most pronounced in male infants (p=0.01). Methylation alterations at the H19 DMR are likely an important mechanism by which FA risks and/or benefits are conferred in utero. Because stable methylation marks at DMRs regulating imprinted genes are acquired before gastrulation, they may serve as archives of early exposures with the potential to improve our understanding of developmental origins of adult disease.     

Low dose domoic acid in neonatal rats abolishes nicotine induced conditioned place preference during late adolescence

In this study, neonatal rats were chronically exposed to low, non-convulsive doses of the kainate receptor agonist domoic acid (DOM), or saline. Later, as adolescents, all animals were tested in a nicotine-induced conditioned place preference (CPP) paradigm. As expected, a nicotine-induced CPP was evident in the adolescent control rats, but surprisingly, not in the DOM animals. This study demonstrates the importance of KA receptors in the development of normal adolescent behaviors manifested in response to the rewarding properties of nicotine.