Elevated endogenous thrombin potential is associated with an increased risk of a first deep venous thrombosis but not with the risk of recurrence

Measurement of the thrombin generating potential could provide a method for quantifying the composite effect of multiple risk factors. This study assessed the risk of a first as well as a recurrent venous thrombotic event associated with an increased endogenous thrombin potential (ETP). Analyses were performed in 360 patients and 404 control subjects of the Leiden Thrombophilia Study. The ETP was measured directly using a fluorogenic assay (Thrombinoscope). Individuals with an increased ETP, i.e. above 90th percentile measured in control subjects (>2109.0 nM x min) had a 1.5-fold [95% confidence interval (CI): 0.9-2.3] increased risk of a first deep venous thrombosis. The risk was more pronounced after the analysis was restricted to idiopathic thromboses, i.e. 1.7-fold (95% CI: 1.0-2.8). Overall, the hazard ratio of a recurrent thrombotic event associated with a high ETP, adjusted for age, sex and oral anticoagulant use was 1.1 (95% CI: 0.5-2.2). Thus, a high ETP was not associated with an increased relative risk of recurrent venous thrombosis. At present, the clinical relevance of the thrombin generation assay in predicting recurrent venous thrombosis remains uncertain.     

Oleanolic acid reprograms the liver to protect against hepatotoxicants,but is hepatotoxic at high doses

Oleanolic acid (OA) is a triterpenoid that exists widely in fruits, vegetables and medicinal herbs. OA is included in some dietary supplements and is used as a complementary and alternative medicine (CAM) in China, India, Asia, the USA and European countries. OA is effective in protecting against various hepatotoxicants, and one of the protective mechanisms is reprogramming the liver to activate the nuclear factor erythroid 2‐related factor 2 (Nrf2). OA derivatives, such as CDDO‐Im and CDDO‐Me, are even more potent Nrf2 activators. OA has recently been shown to also activate the Takeda G‐protein‐coupled receptor (TGR5). However, whereas a low dose of OA is hepatoprotective, higher doses and long‐term use of OA can produce liver injury, characterized by cholestasis. This paradoxical hepatotoxic effect occurs not only for OA, but also for other OA‐type triterpenoids. Dose and length of time of OA exposure differentiate the ability of OA to produce hepatoprotection vs hepatotoxicity. Hepatotoxicity produced by herbs is increasingly recognized and is of global concern. Given the appealing nature of OA in dietary supplements and its use as an alternative medicine around the world, as well as the development of OA derivatives (CDDO‐Im and CDDO‐Me) as therapeutics, it is important to understand not only that they program the liver to protect against hepatotoxic chemicals, but also how they produce hepatotoxicity.     

K(ATP) channel opening is an endogenous mechanism of protection against the no-reflow phenomenon but its function is compromised by hypercholesterolemia

OBJECTIVE: This study aimed to clarify the role of adenosine triphosphate-sensitive K(+) (K(ATP)) channels in the no-reflow phenomenon and in its extension by hypercholesterolemia. BACKGROUND: The no-reflow phenomenon is an important target of therapy in patients with acute myocardial infarction, but its mechanism remains unclear. METHODS: The left circumflex coronary artery was occluded for 30 or 60 min and reperfused in rabbit hearts in situ. The no-reflow zone, area at risk, and infarct size were determined by thioflavin-S, Evans blue, and tetrazolium staining, respectively. No-reflow zone size was expressed as a percentage of infarct size (%NR/IS). Hypercholesterolemia was induced by two weeks of cholesterol-enriched diet. RESULTS: A K(ATP) channel blocker, glibenclamide (0.3 mg/kg), increased %NR/IS after 30-min ischemia/90-min reperfusion from 33.6 +/- 1.9% to 45.9 +/- 1.6% and %NR/IS after 60-min ischemia/90-min reperfusion from 32.8 +/- 3.4% to 46.1 +/- 1.7%. However, N(G)-monomethyl-L-arginine (L-NMMA), a nitric oxide (NO) synthase inhibitor, and nicorandil, a hybrid of K(ATP) channel opener and nitrate, failed to significantly modify %NR/IS. Hypercholesterolemia increased %NR/IS to 61.6 +/- 0.6%, which was not further enlarged by glibenclamide, and delayed infarct healing during the subsequent five days of reperfusion. These effects of hypercholesterolemia were significantly suppressed by nicorandil. Neither glibenclamide, L-NMMA, nicorandil, nor hypercholesterolemia modified infarct size. CONCLUSIONS: The K(ATP) channel activation, but not NO, is a major mechanism of protection against microvascular injury, causing the no-reflow phenomenon in the heart. Suppression of K(ATP) channel opening may underlie the hypercholesterolemia-induced extension of no-reflow, which delays infarct healing.     

Thrombin-mediated activation of endogenous factor XI in plasma in the presence of physiological glycosaminoglycans occurs only with high concentrations of thrombin

The variable bleeding tendency associated with a genetic deficiency of factor XI (FXI) and the lack of bleeding disorders in individuals with a genetic deficiency of factor XII (FXII) suggest an alternative mechanism for FXI activation in vivo . Recently, thrombin has been shown to activate FXI. However, in plasma this activation has been shown to occur only with exogenous FXI and a non-physiological cofactor (sulphatides), and the occurrence of this reaction in a plasma environment has been questioned.
Using recently developed sensitive assays for FXIa–inhibitor complexes we found thrombin-mediated and FXII-independent activation of endogenous FXI in plasma in the presence of heparan sulphate, heparin, dermatan sulphate or dextran sulphate. Using heparan sulphate, which is present in the human vascular system, activation of about 1–2% of plasma FXI was observed, however, only after addition of very high amounts (500 nmol/l) of human α-thrombin to FXII-deficient plasma (at a 1 to 4 final dilution).
We conclude that endogenous FXI in plasma can be activated by thrombin in the presence of various glycosaminoglycans, including the physiological compounds heparan sulphate and dermatan sulphate, but only at very high concentrations of thrombin, corresponding to 100% prothrombin activation in undiluted plasma.     

不同时间阿司匹林预处理对大鼠局灶性脑缺血再灌注损伤的神经保护作用

目的探讨阿司匹林预处理对脑缺血再灌注损伤的保护作用机制并寻找有效预处理的产生时间及持续时间。方法将70只健康雄性SD大鼠,随机分为假手术组(n=10)、缺血再灌注对照组(n=10)和阿司匹林预处理组(n=50)。阿司匹林预处理组又分为5个亚组,每组10只,分别于预处理后即刻、3h、1、3、5d行大脑中动脉闭塞,以线栓法闭塞大脑中动脉制作脑缺血再灌注模型。局部脑缺血2h,于再灌22h观察阿司匹林对神经功能缺失评分、脑梗死体积、病变侧脑组织白细胞介素-1β(IL-1β)及肿瘤坏死因子-α(TNF-α)含量的影响。结果与缺血再灌注对照组比较,阿司匹林预处理后3h、1、3d组脑神经功能缺失评分降低,脑梗死体积缩小,IL-1β和TNF-α含量减低。结论阿司匹林预处理对随后的脑缺血再灌注损伤具有保护作用,其机制可能与减少IL-1β和TNF-α的过量表达有关,这种保护作用产生于预处理后3h,1d达峰,持续至第3d。     

The nuclear melatonin receptor RORα is a novel endogenous defender against myocardial ischemia/reperfusion injury

Circadian rhythm disruption or decrease in levels of circadian hormones such as melatonin increases ischemic heart disease risk. The nuclear melatonin receptors RORs are pivotally involved in circadian rhythm regulation and melatonin effects mediation. However, the functional roles of RORs in the heart have never been investigated and were therefore the subject of this study on myocardial ischemia/reperfusion (MI/R) injury pathogenesis. RORα and RORγ subtypes were detected in the adult mouse heart, and RORα but not RORγ was downregulated after MI/R. To determine the pathological consequence of MI/R‐induced reduction of RORα, we subjected RORα‐deficient staggerer mice and wild‐type (WT) littermates to MI/R injury, resulting in significantly increased myocardial infarct size, myocardial apoptosis and exacerbated contractile dysfunction in the former. Mechanistically, RORα deficiency promoted MI/R‐induced endoplasmic reticulum stress, mitochondrial impairments, and autophagy dysfunction. Moreover, RORα deficiency augmented MI/R‐induced oxidative/nitrative stress. Given the emerging evidence of RORα as an essential melatonin effects mediator, we further investigated the RORα roles in melatonin‐exerted cardioprotection, in particular against MI/R injury, which was significantly attenuated in RORα‐deficient mice, but negligibly affected by cardiac‐specific silencing of RORγ. Finally, to determine cell type‐specific effects of RORα, we generated mice with cardiomyocyte‐specific RORα overexpression and they were less vulnerable to MI/R injury. In summary, our study provides the first direct evidence that the nuclear melatonin receptor RORα is a novel endogenous protective receptor against MI/R injury and an important mediator of melatonin‐exerted cardioprotection; melatonin‐RORα axis signaling thus appears important in protection against ischemic heart injury.     

The endogenous thrombin potential and high levels of coagulation factor VIII, factor IX and factor XI.

High plasma concentrations of factor VIII, factor IX and factor XI have been reported as thrombosis risk factors. Using the thrombin generation test in platelet-poor plasma, it was aimed to describe the mechanism for this increased thrombosis risk. Endogenous thrombin potential was measured in platelet-poor plasma in 180 patients with a history of thromboembolism, and results were compared with those of 180 age-matched and sex-matched controls. Subjects with major hereditary and acquired thrombophilia were excluded. Plasma concentrations of the clotting factor VIII, factor IX and factor XI were significantly elevated in patients compared with controls. The mean endogenous thrombin potential was significantly higher in patients than in controls: 191.3 +/- 3.1 (95% confidence interval, 185.3-197.4) arbitrary units versus 180.8 +/- 2.6 (95% confidence interval, 175.7-185.9) arbitrary units (P = 0.009). The endogenous thrombin potential was significantly higher in patients with elevated factor IX and factor XI, but elevated factor VIII was not associated with a significant increase in endogenous thrombin potential. In conclusion, the increased thrombosis risk associated with high plasma concentrations of factor IX and factor XI may be explained by the increase in endogenous thrombin potential. However, this did not help explain the association between elevated factor VIII and thrombosis risk.     

The endogenous lectin of human platelets is an alpha-granule component

Platelets from patients with several bleeding disorders (congenital afibrinogenemia, Glanzmann's thrombasthenia, gray platelet syndrome, and Hermansky-Pudlak syndrome) were evaluated for both platelet-bound and platelet-free hemagglutination activities. Thrombin and A23187 activated afibrinogenemic, Hermansky-Pudlak, and thrombasthenic platelets had normal platelet-bound hemagglutination activity. Gray platelets activated by the same agents had deficient platelet-bound hemagglutination activity. In contrast, thrombin-activated afibrinogenemic, gray, and thrombasthenic platelets lacked platelet- free hemagglutination activity. Only thrombin-activated Hermansky- Pudlak platelets had a normal level of platelet-free hemagglutination activity. On the basis of these results and the distinguishing characteristics of the defective platelets, it is concluded that the alpha-granules are the origin of the enhanced hemagglutination activity. Furthermore, it is suggested that the insufficiency of the platelet-bound agglutinin may be the cause of the inability of gray platelets to aggregate normally in response to thrombin.     

An increase of C-reactive protein is associated with enhanced activation of endogenous fibrinolysis at baseline but an impaired endothelial fibrinolytic response after venous occlusion

OBJECTIVES: The goal of this study was to determine whether chronic inflammation of the vascular wall may be associated with an impaired activation of the fibrinolytic system. BACKGROUND: Inflammation plays an important role in the initiation and progression of atherosclerosis, and the fibrinolytic system may prevent local thrombus formation. METHODS: We included 50 patients six months after their first myocardial infarction. Plasma levels of the inflammatory marker C-reactive protein (CRP) were determined at basal conditions, and the fibrinolytic parameters tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1) were measured at basal conditions and after a standardized venous occlusion (VO) of the forearm. RESULTS: Patients with high CRP levels (> or =3 mg/l) showed a significantly higher t-PA activity at baseline compared with patients with medium (1 to 2.9 mg/l) and low (<1 mg/l) CRP levels (p <0.005). In contrast, patients with low CRP levels showed a higher increase of t-PA activity (p <0.05) and a higher reduction of PAI-1 activity during VO (p <0.05) compared with patients with medium and high CRP levels. A multivariate analysis that included cardiovascular risk factors and medical treatment showed that CRP is an independent predictor of the t-PA response after a standardized VO. CONCLUSIONS: Chronic low-grade inflammation is associated with enhanced activation of endogenous fibrinolysis at baseline but a reduced fibrinolytic response to VO. This impaired endogenous fibrinolytic capacity might be an important contributor to the increased coronary event rate associated with elevated CRP levels.     

GluR7 is an essential subunit of presynaptic kainate autoreceptors at hippocampal mossy fiber synapses

Presynaptic ionotropic glutamate receptors are emerging as key players in the regulation of synaptic transmission. Here we identify GluR7, a kainate receptor (KAR) subunit with no known function in the brain, as an essential subunit of presynaptic autoreceptors that facilitate hippocampal mossy fiber synaptic transmission. GluR7(-/-) mice display markedly reduced short- and long-term synaptic potentiation. Our data suggest that presynaptic KARs are GluR6/GluR7 heteromers that coassemble and are localized within synapses. We show that recombinant GluR6/GluR7 KARs exhibit low sensitivity to glutamate, and we provide evidence that presynaptic KARs at mossy fiber synapses are likely activated by high concentrations of glutamate. Overall, from our data, we propose a model whereby presynaptic KARs are localized in the presynaptic active zone close to release sites, display low affinity for glutamate, are likely Ca(2+)-permeable, are activated by single release events, and operate within a short time window to facilitate the subsequent release of glutamate.     

Entry of exendin-4 into brain is rapid but may be limited at high doses

OBJECTIVE: Peripherally administered exendin-4 is in clinical trials for the treatment of diabetes mellitus and obesity. Since its effects on food intake are mediated centrally, we determined the degree and type of its blood-to-brain penetration of the mouse blood-brain barrier (BBB). MEASUREMENTS AND RESULTS: High-performance liquid chromatography showed that exendin-4 was stable in blood, with most of the injected peptide reaching the brain intact. Capillary depletion studies with washout showed that the injected exendin-4 reached brain parenchyma rather than being trapped in the endothelial cells composing the BBB. Multiple-time regression analysis showed that exendin-4 crossed the BBB directly at a fast rate. The rapid brain entry of exendin-4, helped by its high lipophilicity as demonstrated by the octanol/buffer partition coefficient, was not dependent upon circumventricular organs and was not affected by food deprivation for 24 h. The simultaneous i.v. injection of high doses of unlabeled exendin-4 resulted in self-inhibition (saturation) that only became statistically significant (P<0.05) when the results of four experiments were combined; this suggests a possible limit to the amount of peripherally administered exendin-4 that can reach the brain after injection of high doses. CONCLUSION: The results indicate that exendin-4 is well conformed for exerting central effects involved in the control of obesity.     

The specificity of UV-induced mutations at an endogenous locus in mammalian cells.

We have used a rapid in vivo recombinational method to clone and completely sequence 34 UV-induced mutants at the adenine phosphoribosyltransferase (APRT) locus of Chinese hamster ovary cells. Among the mutants recovered, 26 were single base substitutions including 17 G.C----A.T transitions and a single A.T----G.C transition. Three of the 4 possible transversions accounted for the remaining 8 mutations. The G.C----T.A transversion was not recovered. Six tandem double or closely neighboring double-base substitutions, one double mutation consisting of a G.C----T.A transversion and an adjacent frameshift, as well as one single frameshift mutation were also recovered. UV-induced mutation appears to be targeted to dipyrimidine sites with only two exceptions. These include two double mutations where only one of the base substitutions occurred at a dipyrimidine site. The observed specificity of UV-light-induced mutations at the APRT locus is consistent with the argument that G.C----A.T transitions result primarily from the (6-4) pyrimidine pyrimidone lesion. A striking resemblance in the distribution of UV-induced mutants and a collection of 30 spontaneous mutants identified recently in our laboratory was noted. Both share a common strong site of multiple occurrence and a considerable degree of overlap with respect to site specificity. We speculate therefore that DNA context plays a significant role in mutation fixation in mammalian cells.     

The inter-observer reliability is very low at clinical examination of parastomal hernia

Introduction  

Parastomal hernia in patients with a permanent colostomy is common. The aim of this study was to evaluate the reliability of the diagnosis based on clinical examination and to compare this examination with the result of a specially designed questionnaire and computerised tomography (CT) scan.     

The adapter protein GRB10 is an endogenous negative regulator of insulin-like growth factor signaling

The growth factor IGF-I is critical for normal human somatic growth and development. Growth factor receptor-bound protein (Grb)10 is a protein that interacts with the IGF-I receptor and may thus regulate IGF-I-stimulated growth. However, the role of endogenous Grb10 in regulating IGF-I action is not known. The objective of this study was to determine the function of endogenous Grb10 in IGF signaling responses. Using small interfering RNA, we demonstrate that knockdown of Grb10 enhances IGF-I-mediated phosphorylation of insulin receptor substrate proteins, Akt/protein kinase B, and ERK1/2 and leads to a corresponding increase in DNA synthesis. Although IGF-I receptor autophosphorylation normally correlates with receptor signaling, we demonstrate a decrease in IGF-I-stimulated receptor phosphorylation in Grb10 knockdown cells. Pretreatment of cells with the protein-tyrosine phosphatase inhibitor pervanadate partially reverses this effect of Grb10 knockdown on receptor phosphorylation, indicating that endogenous Grb10 may block phosphatase access to the activated IGF-I receptor. Marked small interfering RNA knockdown of Grb10 does not result in increased or decreased expression of the related proteins Grb7 or Grb14. As further evidence for Grb10 functional specificity, the recently identified Grb10 interacting GYF proteins are shown to interact specifically with Grb10 and not with Grb7 or Grb14, using yeast two-hybrid assays. We conclude that Grb10 functions as a specific endogenous suppressor of IGF-I-stimulated cell signaling and DNA synthesis. Modulation of the Grb10-IGF-I receptor pathway may represent a mechanism that regulates IGF-I-responsive cell and tissue growth.     

The osmoprotective properties of urine for bacteria: the protective effect of betaine and human urine against low pH and high concentrations of electrolytes, sugars, and urea

Growth of Escherichia coli was inhibited in a defined minimal medium by high concentrations of electrolytes and sugars in direct relation to their osmotic strength. Choline, betaine, proline, and human urine increased resistance to these substances. In contrast, the toxic effect of urea was not altered directly by betaine or urine, but was reduced in the presence of other osmolytes. The osmolyte protective effect was augmented by betaine. The osmoprotective effect of betaine and urine was confirmed with 40 strains of enteric bacteria. Urine from 19 healthy subjects contained osmoprotective activity greater than that observed with betaine. A methanol extract of urine was found to be highly protective. Although betaine was present in the extract, it could not account for all the protective activity. Urine contains additional low-molecular-weight osmoprotective agents.     

A soluble form of the murine common gamma chain is present at high concentrations in vivo and suppresses cytokine signaling

The common gamma-chain (c) is a component of the receptors forIL-2, IL-4, IL-7, IL-9, and IL-15 and is essential for their signaltransduction. Western blotting and a newly established enzyme-linkedimmunosorbent assay detected substantial constitutive levels (50-250 ng/mL) of soluble c (sc) in sera of murine inbred strains. It wasdemonstrated that purified immune cells, such as T, B, and naturalkiller cells, and macrophages released this protein after activation.Transfection experiments with cDNA encoding the full-length c showedthat shedding of the transmembrane receptor led to the release ofsc. The shedding enzymes, however, appeared to be distinct fromthose cleaving other cytokine receptors because inhibitors ofmetalloproteases (eg, TAPI) did not influence sc release. In vivo,superantigen-induced stimulation of T cells enhanced sc serumconcentrations up to 10-fold within 6 hours. Because these findingsdemonstrated regulated expression of a yet unknown molecule in theimmune response, further experiments were performed to assess thepossible function(s) of sc. A physiological role of sc wasindicated by its capacity to specifically inhibit cell growth inducedby c-dependent cytokines. Mutational analysis revealed that theC-terminus and the WSKWS motif are essential for the cytokineinhibitory effect of the sc and for binding of the molecule tocytokine receptor-expressing cells. Thus, competitive displacement ofthe transmembrane c by excess sc is the most likely mechanism ofcell growth inhibition. It was implied that naturally produced sc isa negative modulator of c-dependent cytokines.     

The neuroendocrine polypeptide 7B2 is an endogenous inhibitor of prohormone convertase PC2.

The subtilisin-like prohormone convertase PC2 and the polypeptide 7B2 (an intracellularly cleaved protein of unknown function) are both selectively present in the regulated secretory pathway of neurons and endocrine cells. Here we demonstrate that intact recombinant 7B2 is a potent inhibitor of PC2 and prevents proPC2 cleavage in vitro, whereas the 7B2 cleavage product is virtually inactive. The PC2-related proteinase PC1/PC3 is not inhibited by 7B2. Furthermore, the carboxyl-terminal half of the 7B2 protein sequence is distantly related to the so-called potato inhibitor I family (which includes subtilisin inhibitors). Our findings indicate that 7B2 is a physiological inhibitor of PC2 and may provide alternative avenues for the manipulation of peptide hormone levels.