ITRAQ-Based Proteomics Analysis of Acute Lung Injury Induced by Oleic Acid in Mice.

BACKGROUND/AIMS: This study was conducted to investigate the relationship between differentially expressed proteins (DEPs) and the pathogenesis of oleic acid (OA)-induced acute lung injury (ALI) in mice. METHODS: Eight-week-old male C57BL/6 mice were injected with OA through the tail vein and sacrificed 6 hours after OA administration to identify protein expression levels in lung tissue using isobaric tags for relative and absolute quantification (iTRAQ) technology. Then, DEPs such as antithrombin III (AT III), 12-lipoxygenase (12-LO), dedicator of cytokinesis 2 (DOCK2), polycystin-2 and plasminogen were identified by western blotting. Subsequently, we focused on investigating the effect of AT III on endothelial integrity using siRNA interference technology. The levels of IL-6, IL-1ß, TNF-α and TGF-ß expression were detected using an enzyme-linked immunosorbent assay (ELISA). Alterations in the tight junction component ZO-1 and the phosphorylation of myosin light chain (pMLC) were determined by western blotting. The stress fiber F-actin were also detected by immunofluorescence staining. In addition, endothelial permeability was determined via a transwell permeability assay. RESULTS: A total of 5152 proteins were found to be expressed in lung tissues from the OA-treated and saline-treated mice. Among these proteins, 849 were differentially expressed between the two groups, including 545 upregulated and 304 downregulated proteins. After AT III knockdown, the levels of inflammatory factors and endothelial permeability were elevated, the expression of ZO-1 was decreased, and the expression of F-actin and pMLC was increased. All these results illustrated that AT III knockdown exaggerated the disruption of endothelial integrity mediated by OA. CONCLUSION: These findings using iTRAQ technology demonstrate, for the first time, differences in the lung tissue expression levels of proteins between OA-treated mice and saline-treated mice. This study reveals that 12-LO, DOCK2 and especially AT III may be candidate biomarkers for OA-induced acute lung injury.

Effect of Sulfation and Molecular Weight on Anticoagulant Activity of Dextran.

Sulfation (to 2.8) of dextrans with molecular weight of 150 and 20 kDa was followed by the appearance of anticoagulant activity that increased with decreasing their molecular weight and did not depend on antithrombin, plasma inhibitor of serine proteases of the blood coagulation system. Antithrombin activity of dextran sulfate with a molecular weight of 20 kDa reached 12.6-15.3 U/mg. Dextran sulfates with molecular weights of 20 and 150 kDa did not potentiate ADP-induced human platelet aggregation.

Dietary trans fats enhance doxorubicin-induced cardiotoxicity in mice.

This study investigated the combined effects of trans fat diet (TFD) and doxorubicin upon cardiac oxidative, inflammatory, and coagulatory stress. TFD increased trans fatty acid deposit in heart (P < 0.05), and decreased protein C and antithrombin-III activities in circulation (P < 0.05). TFD plus doxorubicin treatment elevated activities of plasminogen activator inhibitor-1, lactate dehydrogenase, and creatine phosphokinase (P < 0.05). This combination also raised xanthine oxidase activity, and enhanced cardiac levels of reactive oxygen species, interleukin (IL)-6, IL-10, tumor necrosis factor-alpha, and monocyte chemoattractant protein-1 than TFD or doxorubicin treatment alone (P < 0.05). TFD alone increased cardiac nuclear factor kappa B (NF-κB) activity (P < 0.05), but failed to affect expression of NF-κB and mitogen-activated protein kinase (MAPK) (P > 0.05). Doxorubicin treatment alone augmented cardiac activity, mRNA expression, and protein production of NF-κB and MAPK (P < 0.05). TFD plus doxorubicin treatment further upregulated cardiac expression of NF-κB p65, p-p38, and p-ERK1/2 (P < 0.05). These findings suggest that TFD exacerbates doxorubicin-induced cardiotoxicity.

Potentiation of anti-angiogenic activity of heparin by blocking the ATIII-interacting pentasaccharide unit and increasing net anionic charge.

Heparin, a potent anticoagulant used for the prevention of venous thromboembolism, has been recognized as a tumor angiogenesis inhibitor. Its limitation in clinical application for cancer therapy, however, arises from its strong anticoagulant activity, which causes associated adverse effects. In this study, we show the structural correlation of LHT7, a previously developed heparin-based angiogenesis inhibitor, with its influence on VEGF blockade and its decreased anticoagulant activity. LHT7 was characterized as having average seven molecules of sodium taurocholates conjugated to one molecule of low-molecular-weight heparin (LMWH). This study showed that the conjugation of sodium taurocholates selectively blocked interaction with antithrombin III (ATIII) while enhancing the binding with VEGF. This resulted in LHT7 to have negligible anticoagulant activity but potent anti-angiogenic activity. Following up on this finding, we showed that the bidirectional effect of sodium taurocholate conjugation was due to its unique structure, that is, the sterane core hindering the ATIII-binding pentasaccharide unit of LMWH with its bulky and rigid structural characteristics while the terminal sulfate group interacts with VEGF to produce stronger binding. In addition, we showed that LHT7 was localized in the tumor, especially on the endothelial cells. One explanation for this might be that LHT7 was delivered to the tumor via platelets.

Direct inhibitors of coagulation proteins - the end of the heparin and low-molecular-weight heparin era for anticoagulant therapy?

Heparins, either unfractionated or low-molecular-weight (UFH and LMWHs), and vitamin K antagonists (VKAs) are currently the anticoagulants of choice for the prevention of post-operative venous thromboembolism (VTE) and for the treatment of acute venous and arterial thromboembolism. While VKAs are widely used in the US, LMWHs are the standard of care in the EU. Although efficacious, these agents are associated with a number of drawbacks, such as the risk of heparin-induced thrombocytopenia, the need for frequent coagulation monitoring in the case of UFH and VKAs, and the parenteral mode of administration in the case of heparins, which can lead to problems associated with patient compliance. There is a need for new anticoagulants that overcome these limitations. Direct, small-molecule inhibitors of coagulation proteins targeting a single enzyme in the coagulation cascade - particularly thrombin or Factor Xa - have been developed in recent years. Two agents, the direct thrombin inhibitor dabigatran and the direct Factor Xa inhibitor rivaroxaban, have recently been approved in the EU and several other countries for the prevention of VTE after total hip or knee replacement surgery. Here we will review data that suggest that the antithrombin-independent mechanism of action of these agents, particularly that of direct Factor Xa inhibitors, leads to increased efficacy with similar safety profiles compared with the antithrombin-dependent heparins. Although the end of the heparins era is not to be expected, the new anticoagulants presented in this review potentially represent the future of anticoagulation.

Inhibition of antithrombin by hyaluronic acid may be involved in the pathogenesis of rheumatoid arthritis.

Thrombin is a key factor in the stimulation of fibrin deposition, angiogenesis, proinflammatory processes, and proliferation of fibroblast-like cells. Abnormalities in these processes are primary features of rheumatoid arthritis (RA) in synovial tissues. Tissue destruction in joints causes the accumulation of large quantities of free hyaluronic acid (HA) in RA synovial fluid. The present study was conducted to investigate the effects of HA and several other glycosaminoglycans on antithrombin, a plasma inhibitor of thrombin. Various glycosaminoglycans, including HA, chondroitin sulfate, keratan sulfate, heparin, and heparan, were incubated with human antithrombin III in vitro. The residual activity of antithrombin was determined using a thrombin-specific chromogenic assay. HA concentrations ranging from 250 to 1000 mug/ml significantly blocked the ability of antithrombin to inhibit thrombin in the presence of Ca2+ or Fe3+, and chondroitin A, B and C also reduced this ability under the same conditions but to a lesser extent. Our study suggests that the high concentration of free HA in RA synovium may block antithrombin locally, thereby deregulating thrombin activity to drive the pathogenic process of RA under physiological conditions. The study also helps to explain why RA occurs and develops in joint tissue, because the inflamed RA synovium is uniquely rich in free HA along with extracellular matrix degeneration. Our findings are consistent with those of others regarding increased coagulation activity in RA synovium.

Fondaparinux: an update on new study results.

Fondaparinux (Arixtra) is the first selective factor Xa inhibitor approved for use in thromboprophylaxis after orthopaedic surgery. New recently completed trials have also demonstrated the potential of fondaparinux in the prevention of venous thromboembolism (VTE) in other surgical and medical settings and in the treatment of established VTE. In the randomized double-blind PEGASUS study in high-risk abdominal surgery patients, fondaparinux reduced the incidence of VTE from 6.1% with dalteparin to 4.6% (odds ratio reduction = 25.8%, P = 0.14), without increasing the bleeding risk. In the randomized double-blind ARTEMIS trial in acutely ill medical patients, fondaparinux reduced the incidence of VTE from 10.5% with placebo to 5.6% (odds ratio reduction = 49.5%, P = 0.029), without increasing the bleeding risk; there was no pulmonary embolism in the fondaparinux group compared with five, all fatal, in the placebo group (P = 0.029). In the two MATISSE trials, both the efficacy and safety of once daily fondaparinux were at least as good as enoxaparin in the treatment of deep-vein thrombosis (MATISSE-DVT) and unfractionated heparin in the treatment of pulmonary embolism (MATISSE-PE). In patients with coronary artery disease, promising results were obtained in phase II trials and large phase III trials are ongoing. In conclusion, fondaparinux may further improve and simplify the prevention and treatment of thrombosis in a large range of medical and surgical settings.

[The dependence of lipid peroxidation and blood hemostasis from antioxidant activity of different tissues]. / Zalezhnist' reaktsii perekysnoho okysnennia lipidiv i hemostazu vid antyoksydantnoï aktyvnosti riznykh tkanyn.

The experiments with rabbits show that animal maintenance on the antioxidant-free diet leads to the peroxidative lipid oxidation (PLO), hemostasis activation and to the superoxidedismutase (SOD) level reducing in a blood and tissues (of cerebrum, heart, kidney, stomach). The greatest SOD activity decreasing is observed in the cerebrum and heart tissues. Probably, these organs make the most considerable contribution in PLO and hemostasis course in the antioxidative reducing conditions.

The inactivation of antithrombin III by serum elastase in patients with surgical infections.

The relationship between serum elastase and antithrombin III was determined in septic surgical patients as a possible mechanism for intravascular thrombosis and hypercoagulability during sepsis. Eighteen patients with surgical infections and elevated white blood cell counts had their blood assayed daily for white blood cell count, serum elastase, and antithrombin III, until the patient's white blood cell count returned to normal. Antithrombin III was significantly lower (0.87%) when elastase was above the normal range (greater than 14.2 micrograms/ml). Elastase was significantly higher (30.6 micrograms/ml), when antithrombin III was less than normal. These data indicate that elevated serum elastase is associated with a significant reduction in circulating antithrombin III. Stimuli that increase serum elastase, i.e. surgery, trauma, or sepsis may promote intravascular thrombosis by the inhibition of antithrombin III at the blood-endothelial cell interface.

Heparin binding domain of human antithrombin III inferred from the sequential reduction of its three disulfide linkages. An efficient method for structural analysis of partially reduced proteins.

Human antithrombin III (AT-III) was partially reduced under mild conditions in the absence or presence of low molecular weight heparin. Quantitation of reduced disulfide bonds was facilitated by the application of a water-soluble color reagent, 4-N,N-dimethylaminoazobenzene-4'-iodoacetamido-2'-sulfonic acid (S-DABIA). The study shows that the three disulfide linkages of AT-III can be sequentially reduced, with Cys8-Cys128 being the most sensitive, followed by Cys21-Cys95, while Cys247-Cys430 is the most resistant to the mild reduction conditions. The rate of reduction of Cys8-Cys128 and Cys21-Cys95 was significantly decreased in the presence of heparin. The reduction of Cys8-Cys128 was also found to correlate quantitatively with the loss of heparin-accelerated antithrombin activity, heparin binding affinity, and heparin-induced fluorescence enhancement. These results suggest that Cys8-Cys128 is required for the integrity of the heparin binding domain of AT-III and support previous findings that lysyl residues surrounding Cys128 (Lys107, Lys114, Lys125, and Lys136) constitute an important part of the heparin binding site in AT-III.

A fragment of antithrombin that binds both heparin and thrombin.

In order to identify the regions of antithrombin that interact with heparin and thrombin, it was degraded with CNBr and the activities of the isolated products were investigated. These fragments did not exhibit direct thrombin-neutralizing activity; however, one unique fragment was found to bind to heparin-Sepharose and also to interfere with the inhibition of thrombin by intact antithrombin. This fragment was identified as the one consisting of three disulphide-linked polypeptide chains containing residues 1-17, 104-251 and 424-432. At a concentration of 46 nM, this product decreased the heparin-enhanced thrombin-inhibitory activity of antithrombin by half, and completely abolished this inhibition when above 300 nM. In the absence of heparin, the action of antithrombin was not completely nullified by the fragment, even when present at relatively high concentrations. At a given fragment concentration, the extent of inhibition was independent of antithrombin concentration over the range tested. It was found that the fragment decreased the second-order rate constant for the antithrombin-thrombin reaction. Reduction and alkylation of the fragment showed that the above properties reside primarily in the peptide with residues 104-251. It is concluded that this peptide possesses portions of the antithrombin molecule that bind to heparin as well as to a site on thrombin.

Inhibition of thrombin-neutralizing activity of antithrombin III by steroid hormones.

Estrogens in high doses have been shown to inhibit, in vitro, the thrombin-neutralizing action of antithrombin III (AT III). In this study we investigate the effect of estrogens on AT III in greater detail. To increase the sensitivity of measurement of AT III activity in the absence of heparin, we have developed an assay system utilizing human platelets, AT III and thrombin. The two proteins derived from human plasma were prepared in high purity. Platelet aggregation was induced by approximately 0.02 NIH U of thrombin. AT III was added in amounts that suppressed 95% of the aggregation-inducing effect of thrombin. Estrogens blocked the thrombin-neutralizing effect of AT III in dose-dependent manner. This effect was shown to be specific for AT III. Neither aggregability of platelets nor aggregating effect of thrombin were affected by the steroid hormone. Evidence for binding of estrogen to AT III was obtained from changes in intrinsic fluorescence of AT III. ACtivity of AT III was also reduced in increasing order of effectiveness by cholesterol, cortisone, testosterone and progesterone. Our studies suggest a direct effect of estrogens and other steroids on AT III, altering its specific neutralization of thrombin.