A novel single‐chain enzyme complex with chain reaction properties rapidly producing thromboxane A2 and exhibiting powerful anti‐bleeding functions

Uncontrollable bleeding is still a worldwide killer. In this study, we aimed to investigate a novel approach to exhibit effective haemostatic properties, which could possibly save lives in various bleeding emergencies. According to the structure‐based enzymatic design, we have engineered a novel single‐chain hybrid enzyme complex (SCHEC), COX‐1‐10aa‐TXAS. We linked the C‐terminus of cyclooxygenase‐1 (COX‐1) to the N‐terminus of the thromboxane A₂ (TXA₂) synthase (TXAS), through a 10‐amino acid residue linker. This recombinant COX‐1‐10aa‐TXAS can effectively pass COX‐1–derived intermediate prostaglandin (PG) H₂ (PGH₂) to the active site of TXAS, resulting in an effective chain reaction property to produce the haemostatic prostanoid, TXA₂, rapidly. Advantageously, COX‐1‐10aa‐TXAS constrains the production of other pro‐bleeding prostanoids, such as prostacyclin (PGI₂) and prostaglandin E₂ (PGE₂), through reducing the common substrate, PGH₂ being passed to synthases which produce aforementioned prostanoids. Therefore, based on these multiple properties, this novel COX‐1‐10aa‐TXAS indicated a powerful anti‐bleeding ability, which could be used to treat a variety of bleeding situations and could even be useful for bleeding prone situations, including nonsteroidal anti‐inflammatory drugs (NSAIDs)‐resulted TXA₂‐deficient and PGI₂‐mediated bleeding disorders. This novel SCHEC has a great potential to be developed into a biological haemostatic agent to treat severe haemorrhage emergencies, which will prevent the complications of blood loss and save lives.     

Identification of fecal microbiome signatures associated with familial longevity and candidate metabolites for healthy aging

Gut microbiota associated with longevity plays an important role in the adaptation to damaging stimuli accumulated during the aging process. The mechanism by which the longevity-associated microbiota protects the senescent host remains unclear, while the metabolites of the gut bacteria are of particular interest. Here, an integrated analysis of untargeted metabolomics and 16S rRNA gene sequencing was used to characterize the metabolite and microbiota profiles of long-lived individuals (aged ≥90 years) in comparison to old-elderly (aged 75–89 years), young-elderly (aged 60–74 years), and young to middle-aged (aged ≤59 years) individuals. This novel study constructed both metabolite and microbiota trajectories across aging in populations from Jiaoling county (the seventh longevity town of the world) in China. We found that the long-lived group exhibited remarkably differential metabolomic signatures, highlighting the existence of metabolic heterogeneity with aging. Importantly, we also discovered that long-lived individuals from the familial longevity cohort harbored a microbiome distinguished from that of the general population. Specifically, we identified that the levels of a candidate metabolite, pinane thromboxane A2 (PTA2), which is positively associated with aging, were consistently higher in individuals with familial longevity and their younger descendants than in those of the general population. Furtherly, functional analysis revealed that PTA2 potentiated the efficiency of microglial phagocytosis of β-amyloid 40 and enhanced an anti-inflammatory phenotype, indicating a protective role of PTA2 toward host health. Collectively, our results improve the understanding of the role of the gut microbiome in longevity and may facilitate the development of strategies for healthy aging.     

阿斯匹林对大鼠在低O2高CO2下肺动脉高压的作用

目的：研究阿斯匹林(ASA)对慢性低O2高CO2性肺动脉高压的抑制作用。方法：将SD大鼠分为正常对照组,慢性低O2高CO2组,慢性低O2高CO2 阿斯匹林组。用光镜、放射免疫等方法,观察各组大鼠肺动脉平均压(mPAP)、颈动脉平均压(mCAP)、肺细小动脉显微结构、血浆和肺匀浆TXB2及6-keto-PGF1α含量的变化。结果：①低O2高CO2组mPAP比正常组显著增高,ASA组的mPAP比低O2高CO2组显著降低,3组间mCAP比较差异无显著性。②光镜下低O2高CO2组与正常组相比,肺细小动脉管壁面积／管总面积(WA／TA)和肺细小动脉中膜厚度(PAMT)均显著增高。ASA组WA／TA和PAMT显著降低。③低O2高CO2组血浆和肺匀浆TXB2、6-keto-PGF1α浓度以及TXB2／6-keto-PGF1α比正常组显著增高,而ASA组与低O2高CO2组相比显著降低。结论：ASA有抑制慢性低O2高CO2性肺动脉高压和肺血管重构的作用。     

Urinary Thromboxane B2 in Cardiac Transplant Patients as a Screening Method of Rejection

Noninvasive methods for regular monitoring of cardiac transplant patients for acute rejection are preferable to the only currently accepted method involving frequent endomyocardial biopsies. Thromboxane A₂ (TXA₂) is synthesized in large amounts by monocytes/macrophages during organ graft rejection. It enhances T-lymphocyte clonal expansion and cytotoxic function as well as upregulating the major histocompatibility class II expression on antigen presenting cells. Experimentally increased urinary excretion of TXA₂ metabolites is associated with cardiac transplant rejection. We therefore compared urinary immunoreactive thromboxane B₂ (i-TXB₂) levels to the rejection score of the endomyocardial biopsies. In addition we graded the degree of activated lymphocytes in peripheral blood. Urinary i-TXB₂ was significantly higher in patients exhibiting medium to severe rejection than in patients without rejection (1236 ± 372 vs. 526 ± 57 pg/mL). The urine i-TXB₂ (704 ± 48 pg/mL) of all patients who participated in this study, whose endomyocardial biopsy indicated rejection, was also significantly higher than in the non-rejecting group. Increased levels of urine i-TXB₂ were associated with increased biopsy scores. Circulating activated lymphocytes was also significantly increased in patients with moderate/severe rejection compared to patients with no rejection (66 ± 11 vs. 39 ± 4 per mm (3)) (p < 0.01). Further, this study shows that urine i-TXB₂ is associated with increased endomyocardial biopsy scores (acute rejection scores) and blood lymphocyte activation. Thus we conclude that urine i-TXB₂ may be of potential value as a diagnostic screening test for helping identify cardiac transplant patients undergoing acute rejection.     

Serum semaphorin 7A is associated with the risk of acute atherothrombotic stroke

Semaphorin 7A (Sema7A), a neural guidance cue, was recently identified to regulate atherosclerosis in mice. However, the clinical relevance of Sema7A with atherosclerotic diseases remains unknown. The aim of this study was to investigate the association between serum Sema7A and the risk of acute atherothrombotic stroke (AAS). We measured serum concentrations of Sema7A in 105 newly onset AAS cases and 105 age‐ and sex‐matched controls, showing that median Sema7A level in AAS cases was over three times of that in controls (5.86 vs 1.66 ng/mL). Adjusted for hypertension, body mass index, fasting blood glucose, total cholesterol, triglyceride, high‐density lipoprotein (HDL)‐cholesterol, low‐density lipoprotein (LDL)‐cholesterol, current smoking and alcohol consumption, multivariate logistic regression showed that higher Sema7A was independently associated with the odds of AAS (OR = 6.40, 95% CI: 2.88‐14.25). Each 1‐standard deviation increase in Sema7A was associated with a threefold higher odds of AAS (OR = 3.42, 95% CI: 1.84‐6.35). Importantly, adding Sema7A to a multivariate logistic model containing conventional cardiovascular risk factors improved the area under receiver operating characteristic curves from 0.831 to 0.891 for the association with AAS. In conclusion, elevated serum Sema7A is independently associated with the risk of AAS, suggesting that it may play a potential role in AAS.     

Frequency of angiotensin II type 1 receptor gene polymorphism in Turkish acute stroke patients

This study was performed in acute stroke patients in the Turkish population to determine the frequency of the A1166C polymorphism in the AT1 gene and to examine the role of this polymorphism in acute stroke development. In this study, 257 genomic DNA samples were analysed (from 206 acute stroke patients and 51 healthy individuals). Genomic DNA was prepared from peripheral blood using the salt‐extraction method. The presence of the A1166C polymorphism in the AT1 gene was determined using the polymerase chain reaction (PCR)‐restriction fragment length polymorphism (RFLP) method. PCR products were separated by 2% agarose gel electrophoresis and visualized by a charge‐coupled device (CCD) camera. In this study, the allele frequency at the A1166C position was 92% A and 8% C for control and 97% A and 3% C for patients. This difference in allele frequency between the control group and the patient group was not statistically significant. However, genotype and allele frequencies showed a significant difference (P < 0.001) in the control and the patient groups. The results of this study show no relationship between the A1166C polymorphism in the AT1 gene and acute stroke in the Turkish population.     

A new insight into subinteractomes of functional antagonists: Thromboxane (CYP5A1) and prostacyclin (CYP8A1) synthases

The current article aims to summarize all possible spectrum of protein–protein interactions for thromboxane A synthase (CYP5A1) and prostacyclin synthase (CYP8A1). These enzymes metabolize the same substrate (prostaglandin H₂) and can participate in cardiovascular, inflammatory, immune processes, and apoptosis modulation, as well as significantly influence the risk of cancers. Binary protein–protein and multiprotein complexes are of great importance in enzyme-regulating and signal-transduction pathways. However, protein partners of CYP5A1 and CYP8A1 are not yet fully identified, although both synthases are considered as prospective drug targets. At least 36 novel protein partners of CYP5A1 and CYP8A1 were revealed from different tissue types using an approach based on affinity isolation and mass spectrometry. Enrichment analysis showed that these proteins have different molecular functions: folding (refolding), unfolded protein and chaperon binding, protein transport (export/import), posttranslational modification, protein domain-specific binding, antioxidant activity, and glutathione homeostasis. A significant part of them, belonging to molecular chaperones, were common partners for CYP5A1 and CYP8A1, while other proteins were unique with the tissue-dependent distribution. New aspects of CYP5A1 and CYP8A1 interactomics and hetero-complex formation with different protein partners, including cytochrome P450s are discussed.     

蝮蛇抗栓酶对实验性动脉粥样硬化与PGI2／TXA2平衡影响的观察

通过用蝮蛇抗栓酶（Ｓｖａｔｅ）防治家兔动脉粥样硬化（ＡＳ）实验,发现Ｓｖａｔｅ能明显减轻家兔主动脉ＡＳ;同时能明显降低血液血栓素含量,能明显升高血液前列环素含量。以上血液检查结果与Ｓｖａｔｅ治疗心脑血管疾病时临床检查结果一致。说明Ｓｖａｔｅ有防治ＡＳ的作用,其机制与Ｓｖａｔｅ具有恢复前列环素／血栓素平衡的作用密切相关。     

Effect of hypoxia on in vivo thromboxane and prostacyclin levels in arterial blood of rainbow trout, Oncorhynchus mykiss (Walbaum)

Endogenous levels of TXB₂ and 6-keto PGF_1a are reported in dorsal aortic blood from rainbow trout. Acute hypoxia induced an increase in TXB₂ levels whereas 6-keto PGF_1a was unaffected. We suggest that enhanced thromboxane synthesis might have a role in microcirculation of various organs in fish hypoxia.     

Influence of whole-body gamma irradiation upon arachidonic acid metabolism in rat platelets

The effects of whole-body gamma irradiation (8.4 Gy) were studied on arachidonic acid (AA) metabolism in rats' blood platelets, from day D + 1 to day D + 10 after irradiation. AA conversion into thromboxane B2 (TxB2) increased at D + 1 and then gradually decreased to very low values from D + 7 to D + 10. This decrease in the conversion of exogenous AA into TxB2 was due to a lower AA incorporation into platelets and not to a decrease of cyclooxygenase and thromboxane-synthetase activities. AA incorporation into membrane phospholipids of blood platelets was much more decreased than AA incorporation into whole platelets; moreover, the lipid composition of the platelet membranes was markedly modified after irradiation, which must have resulted in structural and functional changes in these membranes; from these effects of whole-body gamma irradiation on platelets, the latter's membranes appeared as a major site of in vivo radiation damage in these cells.     

Modulation of the platelet thromboxane A2 and aortic prostacyclin synthesis by dietary selenium and vitamin E

Vitamin E and selenium (Se) interact synergistically as an important antioxidant defense mechanism. Se, an essential component of glutathione peroxidase (GSH-Px) and vitamin E decompose fatty acid hydroperoxides and hydrogen peroxides generated by free radical reactions. Vitamin E and GSH-Px may modulate arachidonic acid metabolism and the activity of cyclooxygenase enzymes by affecting peroxide concentration. The balance between arterial wall prostacyclin (PGI₂) production and platelet thromboxane (TX)A₂ directly influences platelet activity. In order to elucidate the differential role of dietary vitamin E and Se in aortic PGI₂ and platelet TXA₂ synthesis, 1-mo-old F344 rats were fed semipurified diets containing different levels of vitamin E (0, 30, 200 ppm) and Se (0, 0.1, 0.2 ppm) for 2 mo. Thromboxane B₂ (TXB₂) and 6-keto-PGF₁α, were measured by radioimmunoassay (RIA) after incubation of whole blood and aortic rings at 37°C for 10 and 30 min, respectively. Vitamin E deficiency reduced plasma vitamin E to 5–17% of control-fed rats, and supplementation increased it to 53% of the control-fed rats. Se supplementation in vitamin E-supplemented animals increased plasma GSH-Px by 17%, compared to vitamin E-deficient rats. Se and vitamin E supplementation did not have a similar effect on TXB₂ and PGI₂ synthesis. Se deficiency did not alter platelet TXB₂ synthesis, but significantly decreased aortic PGI₂ synthesis. It was necessary to supplement with both antioxidants in order to increase, PGI₂ synthesis. Se and vitamin E deficient groups had a higher TXB₂/PGI₂ ratio (0.17±0.08) compared to Se- and vitamin E-supplemented groups (0.03±0.01). These results confirm previous reports in humans and animals and are in accordance with epidemiological data indicating an inverse relationship between plasma Se and platelet aggregation. Thus, further suggesting that vitamin E and Se may have a specific role in controlling TXA₂ and PGI₂ synthesis.     

Selenium and selenoproteins in prostanoid metabolism and immunity

Abstract

Selenium (Se) is an essential trace element that functions in the form of the 21st amino acid, selenocysteine (Sec) in a defined set of proteins. Se deficiency is associated with pathological conditions in humans and animals, where incorporation of Sec into selenoproteins is reduced along with their expression and catalytic activity. Supplementation of Se-deficient population with Se has shown health benefits suggesting the importance of Se in physiology. An interesting paradigm to explain, in part, the health benefits of Se stems from the observations that selenoprotein-dependent modulation of inflammation and efficient resolution of inflammation relies on mechanisms involving a group of bioactive lipid mediators, prostanoids, which orchestrate a concerted action toward maintenance and restoration of homeostatic immune responses. Such an effect involves the interaction of various immune cells with these lipid mediators where cellular redox gatekeeper functions of selenoproteins further aid in not only dampening inflammation, but also initiating an effective and active resolution process. Here we have summarized the current literature on the multifaceted roles of Se/selenoproteins in the regulation of these bioactive lipid mediators and their immunomodulatory effects.     

白藜芦醇苷对血栓形成及血浆TXB2和6-keto-PGF1α水平的影响

研究了白藜芦醇苷(polydatim,PD)的抗血栓形成作用及其作用机制。采用小鼠尾静脉注射花生四烯酸(arachidonicacid,AA)、电刺激大鼠颈动脉血栓形成方法评价PD的抗血栓形成作用;运用放射免疫法测定polydatin对兔血浆血栓素B2(thromboxaneB2,TXB2)及6酮前列腺素F1α(6ketoprostaglandinF1α,6ketoPGF1α)水平的影响。结果显示PD对AA、电刺激大鼠颈动脉引起的血栓形成具有明显的对抗作用;PD亦能降低兔血浆TXB2含量并升高6KetoPGF1α水平。本实验提示PD有明显的抗血栓形成作用,其机制可能与其降低血浆TXB2含量及升高6KetoPGF1α水平密切相关。     

Comparative effects of endothelin (ET-1) and U46619 on human saphenous vein and gastroepiploic artery,sources of human autologous grafts

The effects of endothelin (ET-1) on smooth muscle contractile activity were investigated and compared in human saphenous vein and gastroepiploic artery, vessels frequently used in revascularization procedures. ET-1 contracted saphenous vein and gastroepiploic artery in a concentration-dependent manner. The peptide produced a greater maximal effect in the vein than in the artery and, in both preparations, ET-1 was less efficacious than U46619, an agent which mimics the actions of thromboxane A₂ at the thromboxane A₂/prostaglandin HZ receptor. The contractile response to ET-1 declined spontaneously at a more rapid rate in the artery than in the vein. The present data indicate that ET-1 has significant contractile activity in both vessels which are used for coronary arterial bypass surgery and suggest that although, a weaker vasoconstrictor than U46619, the peptide could induce vasospasm in both graft vessels.     

Oscillating prostacyclin and thromboxane generation by human vessels: biological and mathematical evidence for negative feedback control

The presented study investigates the time-dependent release of PGI₂ and TXA₂ by isolated human umbilical veins in vitro using the radio-immunoassay for measurement. After changing the nutritional fluid—Krebs-Henseleit solution at pH 7.4, 37°C, 95% O₂/5% CO₂—the release graph oscillates. These oscillations with time were verified by variance analysis and are very similar for both substances. This indicates one or several negative feedback mechanisms acting on the common path of synthesis from the membrane-bound phospholipids to PGH₂, which are effective in the regulation of eicosanoid biosynthesis in vitro. A mathematical function describing the observed PGI2 and TXA₂ synthesis is communicated.     

Altered prostanoid production by fibroblasts cultured from the lungs of human subjects with idiopathic pulmonary fibrosis

Background  

Prostanoids are known to participate in the process of fibrogenesis. Because lung fibroblasts produce prostanoids and are believed to play a central role in the pathogenesis of idiopathic pulmonary fibrosis (IPF), we hypothesized that fibroblasts (HF) cultured from the lungs of patients with IPF (HF-IPF) have an altered balance between profibrotic (thromboxane [TX]A₂) and antifibrotic (prostacyclin [PGI₂]) prostaglandins (PGs) when compared with normal human lung fibroblasts (HF-NL).     

Influence of a long-term zinc-deficient diet on rat platelet function and fatty acid composition

A reduced zinc intake is associated with numerous abnormalities and, in particular, with hemostasis dysfunction. In this report, we studied the effects of a long-term dietary zinc restriction on platelet function. Three groups of rats were analyzed: a zinc-deficient group (ZD) and two zinc-adequate fed groups, one pair-fed (PF) and one ad libitum fed (AL). We found that ZD diet (0.2 p.p.m.) impaired ADP-induced aggregation of washed platelet after 4 and 8 weeks of diet. Thrombin-induced aggregation was impaired in ZD rats and PF rats after 8 weeks. The thrombin-induced mobilization of radiolabeled arachidonate preincorporated into platelet phospholipids was followed as well as the subsequent formation of labeled cyclooxygenase and lipoxygenase products. Stimulated platelets of ZD rats exhibited a decreased production of cyclooxygenase and lipoxygenase products, particularly after 8 weeks of diet. Moreover, platelet thromboxane generation was decreased in the ZD group as studied using a radioimmunoassay after thrombin stimulation. In addition, we measured the total fatty acid compositions of platelet and plasma. As a whole, 20:5 (n – 3) and 22:5 (n – 3) fatty acids content were significantly increased in platelet lipids after 8 weeks. On the other hand, it is known that enrichment of these fatty acids through dietary studies, both in animal and human as well as in vitro incorporation in platelets, resulted in an inhibition of platelet function. Consequently, these changes in platelet membrane fatty acid composition may contribute to the impaired platelet aggregation observed in ZD rats.     

丙线照射所致大鼠胃粘膜易损性及其与内源性PGs和血栓素A_2的关系

本文观察了丙线照射大鼠胃粘膜的易损性及其与由源性PG_s和血检素A_2的关系。结果表明:丙线1500rad局部照射后28天,大鼠对牛磺胆酸所致胃粘膜坏死易损性明显加重,预先给予外源性PGE_2则可抑制这一现象;进一步采用放射免疫方法测定胃粘膜PG_s等的含量发现,照射后组织PGE和PGI_2含量明显降低,而血栓素A_2含量则明显升高,PGI_2/血栓素A_2,比值下降。这些结果说明,丙线照射可使大鼠胃粘膜的易损性明显加重,而内源性PGE和PGI_2含量的降低以及血栓素A_2含量的升高是照射造成易损性的主要原因之一。     

6-Keto-PGF_(1α)和TXB_2在胃粘膜适应性细胞保护中的作用

PGI_2和TXA_2均系前列腺酸的衍生物,胃粘膜细胞可不断合成和释放,具有很强的细胞保护作用。然而,关于它们与胃蛋白酶的适应性细胞保护作用的关系,尚未见报道。本文则采用放射免疫方法,测定了胃粘膜组织 PGI_2和TXA_2的代谢物 6-Keto-PGF_(1α)和TXB_2含量在不同情况下的变化。结果表明,单纯胃蛋白酶225U或胃蛋白酶150U溶于0.1NHCl或75U溶于0.2NHCl中,提前15min灌胃,均可防止由25％NaCl高渗溶液和沸水所致的胃粘膜坏死的发生,这种保护作用呈明显的量效关系。在上述三种配方灌胃后15min,胃粘膜组织PGI_2和TXA_2含量明显升高,约为对照组的2.0—2.15和1.7—2.0倍;且以PGI_2含量的增加占优势;胃蛋白酶浓度与两者含量呈现明显的量效关系。说明胃蛋白酶作为弱刺激对高渗和物理性烫伤所致的胃粘膜损伤均有保护作用,其作用机制是通过诱发内源性PG_s 的合成和释放而实现的,这一现象对解释胃粘膜的自身耐受机制,具有重要的生理意义。