Combinations of low doses of unfractionated heparin and of low-molecular-weight heparin prevent experimental venous thrombosis

Synergism between low-molecular-weight heparin and low doses of unfractionated heparin (UH) enhancing anti-factor Xa activity and the release of tissue factor pathway inhibitor was observed. The aim of this study was to verify whether this association is effective in preventing experimental venous thrombosis. Seventy rats were allocated into 7 groups: the control group treated with distilled water, the H-350 group treated with UH 350 IU/kg, the E-2 group treated with enoxaparin 2 mg/kg, the H-175 group treated with UH 175 IU/kg, the E-1 group treated with enoxaparin 1 mg/kg, the H-175 + E-1 group treated with UH 175 IU/kg plus enoxaparin 1 mg/kg, and the H-100 + E-0.5 group treated with UH 100 IU/kg plus enoxaparin 0.5 mg/kg. Forty minutes after subcutaneous injection, thrombosis was induced in vena cava. Three hours later, if present, thrombi were withdrawn and weighed. Bleeding time, activated partial thromboplastin time, thrombin time (TT), and anti-factor Xa were measured at the beginning and end of the experiment. Fortyeight other animals were treated, but without inducing thrombus, and tests were performed 40 min after injection. Thrombus developed in 90.9% of control animals, 20% of the H-350 group, 22.2% of the E-2 group, 10% of the H-175 + E-1 group, and 30% of the H-100 + E-0.5 group; there was a difference between group C and the other groups. Only in the H-350 and H-175 + E-1 groups were TT and activated partial thromboplastin time prolonged in relation to control at the end of the experiment. Forty minutes after injection, TT was prolonged in the H-350 and H-175 + E-1 groups. In conclusion, combinations of low doses of low-molecular-weight heparin and low doses of UH were as effective as high doses of each one used alone in preventing thrombus development in rat vena cava. Copyright (c) 2005 S. Karger AG, Basel.

Active and exo-site inhibition of human factor Xa: Structure of des-Gla factor Xa inhibited by NAP5, a potent nematode anticoagulant protein from Ancylostoma caninum

Hookworms are hematophagous nematodes capable of growth, development and subsistence in living host systems such as humans and other mammals. Approximately one billion, or one in six, people worldwide are infected by hookworms causing gastrointestinal blood loss and iron deficiency anemia. The hematophagous hookworm Ancylostoma caninum produces a family of small, disulfide-linked protein anticoagulants (75-84 amino acid residues). One of these nematode anticoagulant proteins, NAP5, inhibits the amidolytic activity of factor Xa (fXa) with K-i = 43 pM, and is the most potent natural fXa inhibitor identified thus far. The crystal structure of NAP5 bound at the active site of gamma-carboxyglutamic acid domainless factor Xa (des-fXa) has been determined at 3.1 angstrom resolution, which indicates that Asp189 (fXa, S1 subsite) binds to Arg40 (NAP5, P1 site) in a mode similar to that of the BPTI/trypsin interaction. However, the hydroxyl group of Ser39 of NAP5 additionally forms a hydrogen bond (2.5 angstrom) with His57 NE2 of the catalytic triad, replacing the hydrogen bond of Ser195 OG to the latter in the native structure, resulting in an interaction that has not been observed before. Furthermore, the C-terminal extension of NAP5 surprisingly interacts with the fXa exosite of a symmetry-equivalent molecule forming a short intermolecular beta-strand as observed in the structure of the NAPc2/fXa complex. This indicates that NAP5 can bind to fXa at the active site, or the exosite, and to fX at the exosite. However, unlike NAPc2, NAP5 does not inhibit fVIIa of the fVIIa/TF complex. (c) 2007 Elsevier Ltd. All rights reserved.

The Vampirome: Transcriptome and proteome analysis of the principal and accessory submaxillary glands of the vampire bat Desmodus rotundus, a vector of human rabies

Vampire bats are notorious for being the sole mammals that strictly feed on fresh blood for their survival. While their saliva has been historically associated with anticoagulants, only one antihemostatic (plasminogen activator) has been molecularly and functionally characterized. Here, RNAs from both principal and accessory submaxillary (submandibular) salivary glands of Desmodus rotundus were extracted, and ~. 200. million reads were sequenced by Illumina. The principal gland was enriched with plasminogen activators with fibrinolytic properties, members of lipocalin and secretoglobin families, which bind prohemostatic prostaglandins, and endonucleases, which cleave neutrophil-derived procoagulant NETs. Anticoagulant (tissue factor pathway inhibitor, TFPI), vasodilators (PACAP and C-natriuretic peptide), and metalloproteases (ADAMTS-1) were also abundantly expressed. Members of the TSG-6 (anti-inflammatory), antigen 5/CRISP, and CCL28-like (antimicrobial) protein families were also sequenced. Apyrases (which remove platelet agonist ADP), phosphatases (which degrade procoagulant polyphosphates), and sphingomyelinase were found at lower transcriptional levels. Accessory glands were enriched with antimicrobials (lysozyme, defensin, lactotransferrin) and protease inhibitors (TIL-domain, cystatin, Kazal). Mucins, heme-oxygenase, and IgG chains were present in both glands. Proteome analysis by nano LC-MS/MS confirmed that several transcripts are expressed in the glands. The database presented herein is accessible online at http://exon.niaid.nih.gov/transcriptome/D_rotundus/Supplemental-web.xlsx. These results reveal that bat saliva emerges as a novel source of modulators of vascular biology. Biological significance: Vampire bat saliva emerges as a novel source of antihemostatics which modulate several aspects of vascular biology. © 2013.

O Wortmannin reverte a hiporreatividade de aortas à fenilefrina associada à fase final da prenhez de ratas espontaneamente hipertensas (SHR)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Peritoneal dialysis per se is a risk factor for sclerostin-associated adynamic bone disease

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effect of nωnla, an inhibitor of No-synthase, on the release of tissue-plasminogem activator from rat aorta

Alterations in the synthesis or enhanced inactivation of nitric oxide (NO) and increase in fibrin deposition in the vascular bed lead to an imbalance that can induced intravascular coagulation. NO is produced through L-arginine pathway by constitutive and inducible nitric oxide synthase (NOS). The inducible isoform can be activated by cytokines such as tumor necrosis factor alfa. We evaluated NO-induced tissue-plasminogen activator (t-PA) release from isolated aortic segments of Wistar rats measuring the fibrinolytic activity in the fibrin plate. Inhibition of NO biossynthesis with Nω-nitro-L-arginine (NωNLA) significantly attenuated the fibrinolytic activity (FA) evoked by aortic segments of this group (GII) compared to the saline group (GI). The administration of L-arginine produced restoration of FA in this group (GIII) treated with NωNLA suggesting that t-PA arising from segments of rat aorta is influenced by NO.

Vascular hyporeactivity to angiotensin II induced by Escherichia coli endotoxin is reversed by Nω-Nitro-L-Arginine, an inhibitor of nitric oxide synthase

Septic shock or sepsis is reported to be one of the major causes of death when followed by systemic infectious trauma in humans and other mammals. Its development leads to a large drop in blood pressure and a reduction in vascular responsiveness to physiological vasoconstrictors which, if not contained, can lead to death. It is proposed that this vascular response is due to the action of bacterial cell wall products released into the bloodstream by the vascular endothelium and is considered a normal response of the body's defenses against infection. A reduction in vascular reactivity to epinephrine and norepinephrine is observed under these conditions. In the present study in rats, the aim was to assess whether those effects of hypotension and hyporeactivity are also related to another endogenous vasoconstrictor, angiotensin II (AII). We evaluated the variation in the power of this vasoconstrictor over the mean arterial pressure in anesthetized rats, before and after the establishment of hypotension by Escherichia coli endotoxin (Etx). Our results show that in this model of septic shock, there is a reduction in vascular reactivity to AII and this reduction can be reversed by the inhibitor of nitric oxide synthase, Nω-Nitro-L- Arginine (NωNLA). Our results also suggest that other endogenous factors (not yet fully known) are involved in the protection of rats against septic shock, in addition to the L-arginine NO pathway.

Infliximab prevents increased systolic blood pressure and upregulates the AKT/eNOS pathway in the aorta of spontaneously hypertensive rats

High systolic blood pressure caused by endothelial dysfunction is a comorbidity of metabolic syndrome that is mediated by local inflammatory signals. Insulin-induced vasorelaxation due to endothelial nitric oxide synthase (eNOS) activation is highly dependent on the activation of the upstream insulin-stimulated serine/threonine kinase (AKT) and is severely impaired in obese, hypertensive rodents and humans. Neutralisation of circulating tumor necrosis factor-α (TNFα) with infliximab improves glucose homeostasis, but the consequences of this pharmacological strategy on systolic blood pressure and eNOS activation are unknown. To address this issue, we assessed the temporal changes in the systolic pressure of spontaneously hypertensive rats (SHR) treated with infliximab. We also assessed the activation of critical proteins that mediate insulin activity and TNFα-mediated insulin resistance in the aorta and cardiac left ventricle. Our data demonstrate that infliximab prevents the upregulation of both systolic pressure and left ventricle hypertrophy in SHR. These effects paralleled an increase in AKT/eNOS phosphorylation and a reduction in the phosphorylation of inhibitor of nuclear factor-κB (Iκβ) and c-Jun N-terminal kinase (JNK) in the aorta. Overall, our study revealed the cardiovascular benefits of infliximab in SHR. In addition, the present findings further suggested that the reduction of systolic pressure and left ventricle hypertrophy by infliximab are secondary effects to the reduction of endothelial inflammation and the recovery of AKT/eNOS pathway activation. © 2012 Elsevier B.V.

Apocynin: Chemical and biophysical properties of a NADPH oxidase inhibitor

Apocynin is the most employed inhibitor of NADPH oxidase (NOX), a multienzymatic complex capable of catalyzing the one-electron reduction of molecular oxygen to the superoxide anion. Despite controversies about its selectivity, apocynin has been used as one of the most promising drugs in experimental models of inflammatory and neurodegenerative diseases. Here, we aimed to study the chemical and biophysical properties of apocynin. The oxidation potential was determined by cyclic voltammetry (Epa = 0.76V), the hydrophobicity index was calculated (logP = 0.83) and the molar absorption coefficient was determined (ε275nm = 1.1 × 104 M-1 cm-1). Apocynin was a weak free radical scavenger (as measured using the DPPH, peroxyl radical and nitric oxide assays) when compared to protocatechuic acid, used here as a reference antioxidant. On the other hand, apocynin was more effective than protocatechuic acid as scavenger of the non-radical species hypochlorous acid. Apocynin reacted promptly with the non-radical reactive species H2O2 only in the presence of peroxidase. This finding is relevant, since it represents a new pathway for depleting H2O2 in cellular experimental models, besides the direct inhibition of NADPH oxidase. This could be relevant for its application as an inhibitor of NOX4, since this isoform produces H 2O2 and not superoxide anion. The binding parameters calculated by fluorescence quenching showed that apocynin binds to human serum albumin (HSA) with a binding affinity of 2.19 × 104 M -1. The association did not alter the secondary and tertiary structure of HSA, as verified by synchronous fluorescence and circular dichroism. The displacement of fluorescent probes suggested that apocynin binds to site I and site II of HSA. Considering the current biomedical applications of this phytochemical, the dissemination of these chemical and biophysical properties can be very helpful for scientists and physicians interested in the use of apocynin.

Triiodothyronine Increases mRNA and Protein Leptin Levels in Short Time in 3T3-L1 Adipocytes by PI3K Pathway Activation

The present study aimed to examine the effects of thyroid hormone (TH), more precisely triiodothyronine (T3), on the modulation of leptin mRNA expression and the involvement of the phosphatidyl inositol 3 kinase (PI3K) signaling pathway in adipocytes, 3T3-L1, cell culture. We examined the involvement of this pathway in mediating TH effects by treating 3T3-L1 adipocytes with physiological (P=10nM) or supraphysiological (SI=100 nM) T3 dose during one hour (short time), in the absence or the presence of PI3K inhibitor (LY294002). The absence of any treatment was considered the control group (C). RT-qPCR was used for mRNA expression analyzes. For data analyzes ANOVA complemented with Tukey's test was used at 5% significance. T3 increased leptin mRNA expression in P (2.26 ± 0.36, p< 0.001), SI (1.99 ±0.22, p< 0.01) compared to C group (1± 0.18). This increase was completely abrogated by LY294002 in P (1.31±0.05, p< 0.001) and SI (1.33±0.31, p< 0.05). Western blotting confirmed these results at protein level, indicating the PI3K pathway dependency. To examine whether leptin is directly induced by T3, we used the translation inhibitor cycloheximide (CHX). In P, the presence of CHX maintained the levels mRNA leptin, but was completely abrogated in SI (1.14±0.09, p> 0.001). These results demonstrate that the activation of the PI3K signaling pathway has a role in TH-mediated direct and indirect leptin gene expression in 3T3-L1 adipocytes. © 2013 Oliveira et al.

Alteration in cellular viability, pro-inflammatory cytokines and nitric oxide production in nephrotoxicity generation by Amphotericin B: Involvement of PKA pathway signaling

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Clonagem, expressão e purificação de domínios da proteína AtRLI2 (RNase L Inhibitor), um supressor endógeno de silenciamento por RNA de Arabidopis thaliana, visando estudos estruturais

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Obesity Preserves Myocardial Function During Blockade of the Glycolytic Pathway

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Role of protein kinase A signaling pathway in cyclosporine nephrotoxicity

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)