Evaluation of intravenous direct thrombin inhibitor monitoring tests: Correlation with plasma concentrations and clinical outcomes in hospitalized patients.

The parenterally administered direct thrombin inhibitors (DTIs) argatroban and bivalirudin are effective anticoagulants for acute heparin-induced thrombocytopenia (HIT) treatment. The activated partial thromboplastin time (aPTT) has classically been used as the monitoring test to assess degree of anticoagulation, however concerns exist with using aPTT to monitor DTI therapy. In this observational study plasma samples from DTI treated patients were analyzed by aPTT, dilute thrombin time (dTT) and ecarin chromogenic assay (ECA) to delineate results into concordant and discordant groups. Discordant samples were further analyzed via liquid chromatography with tandem mass spectrometry (LC MS/MS). In total 101 patients with 198 samples were evaluated. Discordance between tests were frequent (59% of DTI treated patients). Bivalirudin aPTT vs dTT discordance was observed in 45% (57/126) of samples. Amongst bivalirudin samples with test discordance dTT results were more likely to be concordant with LC MS/MS than the aPTT (77% vs 9%, p < 0.0001). Argatroban aPTT vs dTT discordance was observed in 43% (31/72) and aPTT vs ECA discordance was observed in 40% (29/72) of samples. Amongst argatroban samples with test discordance both the dTT and ECA tests were more likely to have concordant results with LC MS/MS than the aPTT (88% vs 9%, p < 0.0001 for both dTT and ECA tests). There were no differences between discordant and concordant patient groups in a composite outcome of bleeding/thrombosis rate (23% vs 27%, p = 0.689). Further investigation is warranted to elucidate the effect of suitable monitoring assays on patient outcomes in the setting of DTI therapy.

Activation of peroxisome proliferator-activated receptor α (PPARα) suppresses hypoxia-inducible factor-1α (HIF-1α) signaling in cancer cells.

Activation of peroxisome proliferator-activated receptor α (PPARα) has been demonstrated to inhibit tumor growth and angiogenesis, yet the mechanisms behind these actions remain to be characterized. In this study, we examined the effects of PPARα activation on the hypoxia-inducible factor-1α (HIF-1α) signaling pathway in human breast (MCF-7) and ovarian (A2780) cancer cells under hypoxia. Incubation of cancer cells under 1% oxygen for 16 h significantly induced HIF-1α expression and activity as assayed by Western blotting and reporter gene analysis. Treatment of the cells with PPARα agonists, but not a PPARγ agonist, prior to hypoxia diminished hypoxia-induced HIF-1α expression and activity, and addition of a PPARα antagonist attenuated the suppression of HIF-1α signaling. Activation of PPARα attenuated hypoxia-induced HA-tagged HIF-1α protein expression without affecting the HA-tagged HIF-1α mutant protein level, indicating that PPARα activation promotes HIF-1α degradation in these cells. This was further confirmed using proteasome inhibitors, which reversed PPARα-mediated suppression of HIF-1α expression under hypoxia. Using the co-immunoprecipitation technique, we found that activation of PPARα enhances the binding of HIF-1α to von Hippel-Lindau tumor suppressor (pVHL), a protein known to mediate HIF-1α degradation through the ubiquitin-proteasome pathway. Following PPARα-mediated suppression of HIF-1α signaling, VEGF secretion from the cancer cells was significantly reduced, and tube formation by endothelial cells was dramatically impaired. Taken together, these findings demonstrate for the first time that activation of PPARα suppresses hypoxia-induced HIF-1α signaling in cancer cells, providing novel insight into the anticancer properties of PPARα agonists.

MicroRNA-19 (miR-19) regulates tissue factor expression in breast cancer cells.

Tissue factor has been recognized as a regulator of tumor angiogenesis and metastasis. The tissue factor gene is selectively expressed in highly invasive breast cancer cells, and the mechanisms regulating tissue factor expression in these cells remain unclear. This study demonstrates that microRNA-19 (miR-19) regulates tissue factor expression in breast cancer cells, providing a molecular basis for the selective expression of the tissue factor gene. Tissue factor protein was barely detectable in MCF-7, T47D, and ZR-75-1 cells (less invasive breast lines) but was expressed at a significantly higher level in MDA-MB-231 and BT-20 cells (invasive breast lines) as assayed by Western blot. The tissue factor gene promoter was activated, and forced expression of tissue factor cDNA was achieved in MCF-7 cells, implying that the 3'-UTR of the tissue factor transcript is responsible for the suppression of tissue factor expression. Bioinformatics analysis predicted microRNA-binding sites for miR-19, miR-20, and miR-106b in the 3'-UTR of the tissue factor transcript. Reporter gene assay using the TF-3'-UTR luciferase reporter construct confirmed that the 3'-UTR negatively regulates gene expression in MCF-7 cells, an effect reversed by deletion of the miR-19-binding site. Application of the miR-19 inhibitor induces endogenous tissue factor expression in MCF-7 cells, and overexpression of miR-19 down-regulates tissue factor expression in MDA-MB-231 cells. RT-PCR analysis using cDNA made from Ago2-immunoprecipitated RNA samples confirmed that Ago2 binds preferentially to tissue factor 3'-UTR in MCF-7 cells, as compared with MDA-MB-231 cells, consistent with the observation that miR-19 levels are higher in MCF-7 cells.

Trolox enhances curcumin's cytotoxicity through induction of oxidative stress.

Curcumin, a natural polyphenol in the spice turmeric, has been found to exhibit anticancer activity. Although curcumin is generally considered an antioxidant, it is also able to elicit apoptosis through the generation of ROS, thereby functioning as a pro-oxidant in cancer cells. The present study investigated the effects of antioxidant pretreatment on curcumin-induced cytotoxicity in the human cancer cell lines A2780, MCF-7, and MDA-MB-231. Cytotoxicity was enhanced by trolox, vitamin C or vitamin E; trolox, a water soluble vitamin E derivative, was the most potent. The combination of curcumin (10 µM) and trolox (10-50 µM) induced apoptosis of cancer cells as evidenced by PARP cleavage and caspase-3 activation. Furthermore, expression of the pro-apoptotic protein Bad was up-regulated and expression of the anti-apoptotic proteins Bcl-2 and Bcl-xl was down-regulated in cells that had been treated with trolox plus curcumin. ROS generation was detected in curcumin-treated cells and was significantly enhanced when cells were treated with trolox plus curcumin. Exogenous catalase or SOD1 did not alter cytotoxicity, while over-expression of either catalase or SOD1 did, pointing to the importance of intracellular hydrogen peroxide generation in cell killing. In conclusion, we demonstrated for the first time that antioxidants such as trolox can potentiate cancer cell killing by curcumin, a finding which may help in the development of novel drug combination therapies.

Phosphatidylserine is an overlooked mediator of COVID-19 thromboinflammation.

A ubiquitous component of cell membrane, phosphatidylserine (PS), is likely to play a major, but as yet unrecognized, role in the thromboinflammation of COVID-19 and other critical illnesses. PS is present in all plasma membranes but is "hidden" on the inner surface by the action of an ATP-requiring enzyme. Failure of PS to be sequestered on the inner surface of cell membranes, release of PS-containing microparticles from cells, or shedding of enveloped viruses allows it to interact with extracellular proteins, including those of the coagulation and complement systems. Detection and quantification of circulating PS is not standardized, and current methodologies have either focused on circulating cellular elements or subcellular plasma components, but not both. PS may also promote thromboinflammation without circulating if expressed on the surface of endothelial cells, a condition that might only be documented if novel imaging techniques are developed. Research into the role of PS in inflammation and coagulation, called here a "procoagulant phospholipidopathy" may provide novel insights and therapeutic approaches for patients with a variety of illnesses.

Clioquinol targets zinc to lysosomes in human cancer cells.

We have previously demonstrated that clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) acts as a zinc ionophore and induces apoptosis of human cancer cells; however, the mechanisms of clioquinol/zinc-induced apoptotic cell death remain to be elucidated further. Using fluorescence-labelled probes, the present study has examined intracellular zinc distribution after clioquinol treatment in human cancer cells in order to identify cellular targets for zinc ionophores. DU 145, a human prostate cancer line, was chosen as a model system for the present study, and results were confirmed in other human cancer cell lines. Although treatment of cancer cells with 50 microM ZnCl2 for 3 days had no effect on cell viability, addition of clioquinol dramatically enhanced the cytotoxicity, confirming our previous observations. The ionophore activity of clioquinol was confirmed using fluorescence microscopy. Intracellular free zinc was found to be concentrated in lysosomes, indicating that lysosomes are the primary target of zinc ionophores. Furthermore, lysosomal integrity was disrupted after addition of clioquinol and zinc to the cells, as shown by redistribution of both Acridine Orange and cathepsin D. Clioquinol plus zinc resulted in a cleavage of Bid (BH3-interacting domain death agonist), a hallmark of lysosome-mediated apoptotic cell death. Thus the present study demonstrates for the first time that clioquinol generates free zinc in lysosomes, leading to their disruption and apoptotic cell death.

Metal ionophores - an emerging class of anticancer drugs.

Compounds that bind metals such as copper and zinc have many biological activities, including the ability to induce apoptosis in cancer cells. Although some of these compounds have been considered to act as chelators of metals, decreasing their bioavailability, others increase intracellular metal concentrations. We review recent work regarding the recognition of the biological effects of metal ionophores with different structures, particularly with regard to their actions upon cancer cells focusing on dithiocarbamates, pyrithione, and the 8-hydroxyquinoline derivative, clioquinol. We provide a biologically based classification of metal-binding compounds that allows an experimental distinction between chelators and ionophores that can be readily used by biologists, which may lead to further study and classification of metal-binding drugs. Metal ionophores may kill cancer cells by a number of mechanisms, including lysosomal disruption and proteasome inhibition, and likely others. Because some of these compounds have been safely administered to animals and humans, they have the potential to become clinically useful anticancer agents.

Copper deficiency after gastric surgery: a reason for caution.

BACKGROUND: : Acquired copper deficiency in adults leads to hematological and neurological manifestations that mimic vitamin B12 deficiency. A significant number of patients with copper deficiency syndrome have a history of gastric surgery, often remote. We sought to determine whether copper deficiency is present in a population of individuals with longstanding partial gastric resection. METHODS: : Serum copper, ceruloplasmin, and zinc levels were determined in 20 patients with a history of partial gastric resection and 50 controls, randomly selected from the Oklahoma City Veterans Affairs Medical Center electronic database. RESULTS: : Hypocupremia and symptoms of copper deficiency were detected in patients with partial gastric resection in contrast to controls (3/20 versus 0/50, P = 0.02). Serum copper and ceruloplasmin levels were significantly lower in individuals with partial gastric resection than in controls (P = 0.04 and P = 0.001, respectively). The mean interval between gastric surgery and testing was 20.7 years. CONCLUSIONS: : Our results indicate that a significant number of individuals with longstanding history of partial gastric resection have undiagnosed hypocupremia. Screening for copper deficiency after gastric surgery may prevent the development of hematological and neurological complications in these patients.

Phospholipid hydroperoxide glutathione peroxidase plays a role in protecting cancer cells from docosahexaenoic acid-induced cytotoxicity.

Docosahexaenoic acid (DHA; 22:6, n-3) is known to exert cytotoxic effects against various types of tumors via lipid peroxidation. Whereas several enzymes influence the response of cells to oxidative stress, only one enzyme, phospholipid hydroperoxide glutathione peroxidase (GPx-4), directly reduces lipid hydroperoxides in mammalian cells. The present study was designed to examine the involvement of GPx-4 in determining the effects of DHA addition to various human cancer cell lines. Although baseline levels of GPx-4 did not correlate with the relative sensitivity of human cancer cell lines to DHA, DHA reduced the level of protein expression of GPx-4 by at least 50% in all six lines. Knockdown of GPx-4 by small interfering RNA technique in a human ovarian cancer cell line significantly enhanced the cytotoxic effect of DHA in a time- and concentration-dependent manner. This cytotoxic effect of DHA was reversed by pretreatment with vitamin E, suggesting that the enhanced toxicity of GPx-4 knockdown is due to changes in the ability of the cells to handle oxidative stress. Neither baseline superoxide dismutase-1 nor catalase expression correlated with the relative sensitivity of the cells to DHA treatment. These results illustrate that susceptibility to the oxidative stress imposed by DHA, and possibly other therapeutic agents, is due to complex interactions among multiple antioxidant systems. The modulation of GPx-4 levels by DHA administration is of potential importance and may influence the cellular response to other oxidant stresses.

Thrombin Generation Biomarkers Decline With Parenteral Anticoagulation-An Overlooked Means of Anticoagulation Monitoring?

Anticoagulation therapy is administered to patients to prevent or stop thrombin generation in vivo. Although plasma tests of in vivo thrombin generation have been available for more than 2 decades, they are not routinely used in clinical trials or practice to monitor anticoagulation therapy. We observed a fall in one such marker, the D-dimer antigen, in patients receiving anticoagulation therapy. We therefore conducted a systematic review of the medical literature to document the change in serum biomarkers of thrombin generation following the initiation of anticoagulation therapy. Using a defined search strategy, we screened PubMed and Embase citations and identified full-length articles published in English. Eighteen articles containing serial changes in 1 of 3 markers of thrombin generation (D-dimer antigen, thrombin-antithrombin complexes, and prothrombin fragment 1+2 antigen levels) in the 14 days following the initiation of anticoagulation were identified. Even though the assays used varied considerably, each of the 3 markers of thrombin generation declined in the initial period of anticoagulation therapy, with changes evident as early as 1 day after beginning therapy. These observations provide a rationale for further exploration of these markers as measures of the adequacy of anticoagulation using classic as well as novel anticoagulants. Particular patient groups that would benefit from additional means of monitoring anticoagulation therapy are discussed.

Anticancer activity of the antibiotic clioquinol.

Clioquinol, a metal chelator, has been used for many years as an antimicrobial agent and more recently as a potential treatment for Alzheimer's disease. Because it binds copper and zinc, metals essential for the activity of the enzyme superoxide dismutase-1 (SOD1), a potential target for anticancer drug development, we investigated its effects on human cancer cells. Treatment with clioquinol reduced the viability of eight different human cancer cell lines in a concentration-dependent manner, with IC(50) values in the low micromolar range. Biochemical analysis revealed that clioquinol induced cancer cell death through apoptotic pathways that require caspase activity. Although clioquinol induced modest inhibition of SOD1 activity in treated cells, comparable inhibition by a known SOD1 inhibitor, diethyldithiocarbamate, did not result in cytotoxicity. The addition of copper, iron, or zinc did not rescue cells from cliquinol-induced cytotoxicity but enhanced its killing, arguing against metal chelation as its major mechanism of action. To test if clioquinol might act as an ionophore, a fluorescent probe was used to monitor intracellular zinc concentrations. The addition of clioquinol resulted in elevated levels of intracellular zinc, indicating that clioquinol acts as a zinc ionophore. In an in vivo xenografts mouse model, clioquinol inhibited tumor growth of xenografts over a 6-week period, without inducing visible toxicity. Our results show that clioquinol has anticancer effects both in vitro and in vivo. Transition metal ionophores may be a subclass of metal chelators with anticancer activity deserving of further development.

Clioquinol and docosahexaenoic acid act synergistically to kill tumor cells.

Clioquinol, an 8-hydroxyquinoline derivative (5-chloro-7-iodo-8-hydroxyquinoline) with antimicrobial properties, has recently been found to have cytotoxic activity towards human cancer cell lines at concentrations achieved by oral administration. This study was initiated to determine whether clioquinol could potentiate the antitumor effects of two drugs, doxorubicin and docosahexaenoic acid (DHA), believed to act in part via the generation of reactant oxidant species. At low micromolar concentrations, clioquinol had little effect upon cell viability and did not potentiate doxorubicin's cytotoxicity. Clioquinol significantly enhanced DHA's cytotoxic effects, an interaction that was shown to be synergistic by isobolographic analysis. Clioquinol exhibited a synergistic interaction with DHA in reducing nuclear factor-kappaB activity and inducing apoptosis, and the combination reduced the level of several molecules that promote cell survival, including Akt, p65, and Bcl-2. Interestingly, clioquinol neither induced lipid peroxidation itself nor increased peroxidation brought about by the addition of DHA. However, when cells were pretreated with antioxidant vitamin E, the synergism of clioquinol and DHA was blocked, indicating the essential role of lipid peroxidation for their action. These findings reveal a novel antitumor drug combination that synergistically targets major cell survival signaling pathways.

Evaluation of a Reflex Testing Algorithm for Suspected Heparin-Induced Thrombocytopenia.

OBJECTIVES: We implemented a policy of reflex serotonin-release assay (SRA) testing for all patients with a positive heparin-induced thrombocytopenia (HIT) immunoassay. METHODS: We identified all patients who had SRA testing sent as a consequence of a positive HIT immunoassay test. We reviewed charts of patients to calculate the 4Ts clinical score, determined the effect of testing on clinical management, and documented the change in utilization of direct thrombin inhibitors (DTIs). RESULTS: The likelihood of a positive SRA varied with the optical density (OD) of the immunoassay. The performance of the immunoglobulin G (IgG)-specific and polytypic enzyme-linked immunosorbent assay was not statistically different. Both OD and 4Ts score correlated with the likelihood of a positive SRA but demonstrated poor specificity. Discontinuation of DTIs in patients with negative SRAs resulted in decreased drug utilization. CONCLUSIONS: The IgG-specific HIT immunoassay OD correlates with the likelihood of a positive SRA but does not achieve high specificity. The reflex testing algorithm allows for definitive classification of patients, and the cost of such a reflex testing program may be offset by decreased utilization of DTIs.

Evaluation of a Heparin-Calibrated Antifactor Xa Assay for Measuring the Anticoagulant Effect of Oral Direct Xa Inhibitors.

The introduction of oral direct anti-Xa anticoagulants apixaban and rivaroxaban has significantly impacted the treatment and prevention of thromboembolic disease. Clinical scenarios exist in which a quantitative assessment for degree of anticoagulation due to these agents would aid management. The purpose of this work was to evaluate the chromogenic antifactor Xa assay calibrated with heparin standards at our institution for assessment of intensity of anticoagulation with rivaroxaban or apixaban in addition to its current use for unfractionated heparin or low-molecular-weight heparin. We also aimed to propose expected steady state peak and trough antifactor Xa activities for these agents based upon dosing regimens approved for nonvalvular atrial fibrillation. Antifactor Xa activity correlated very strongly with apixaban and rivaroxaban concentration in both spiked samples and treated patient plasma samples (r (2) = .99, P < .001). This correlation was observed over a broad range (20-500 ng/mL) of drug concentrations, as sample dilution with pooled normal plasma significantly extended the range of quantitative assessment. Based on drug concentrations previously published in pharmacokinetic studies, the expected steady state peak and trough antifactor Xa activity ranges for apixaban are 1.80 to 2.20 IU/mL and 0.70 to 1.10 IU/mL, respectively. For rivaroxaban, these ranges are 3.80 to 6.20 IU/mL and 0.60 to 1.00 IU/mL, respectively. In conclusion, our findings demonstrate that heparin-calibrated antifactor Xa activity correlates strongly with apixaban and rivaroxaban concentration. The dilution of samples allowed for this correlation to be extended over the majority of on-therapy drug concentrations.

Differential sensitivity of cancer cells to docosahexaenoic acid-induced cytotoxicity: the potential importance of down-regulation of superoxide dismutase 1 expression.

Docosahexaenoic acid (DHA, 22:6 n-3), a polyunsaturated fatty acid found in fish oil, exerts cytotoxic effects on cancer cells. Although DHA was toxic toward five human cancer cell lines (MCF-7, MDA-MB-231, SiHa, Raji, and DHL-4), the lines were not uniformly sensitive. DHL-4, a bcl-2 overexpressing lymphoid line, was the most sensitive (IC50, 5.2 micromol/L) and the cervical cancer cell line, SiHa, was the most resistant (IC50, >300 micromol/L). Lipid peroxidation has been cited by others as an important component of DHA toxicity, and we confirmed that vitamin E prevents the cytotoxic effects of DHA. Lipid peroxidation was greater following DHA treatment of the sensitive DHL-4 cells than in the resistant SiHa cells, as assessed by thiobarbituric acid reactive substance generation. DHL-4 cells treated with DHA for 20 hours showed a 3.5-fold increase in thiobarbituric acid reactive substances, whereas SiHa cells showed no increase. Reverse transcription-PCR analysis detected a down-regulation of the expression of the major antioxidant enzyme, superoxide dismutase (SOD) 1, in DHL-4 cells but not in SiHa cells after DHA treatment. Knockdown of SOD1 expression in SiHa cells with small interfering RNA significantly enhanced lipid peroxidation and cytotoxicity on exposure to DHA. These results show that DHL-4 cells are highly sensitive to the cytotoxic effect of DHA and that regulation of SOD1 expression may play an important role in determining the sensitivity of different tumor cells to the cytotoxic effects of DHA.

Zinc Protoporphyrin Suppresses ß-Catenin Protein Expression in Human Cancer Cells: The Potential Involvement of Lysosome-Mediated Degradation.

Zinc protoporphyrin (ZnPP) has been found to have anticancer activity both in vitro and in vivo. We have recently demonstrated that ZnPP diminishes ß-catenin protein expression in cancer cells. The present study examined the cellular mechanisms that mediate ZnPP's suppression of ß-catenin expression. We demonstrate that ZnPP induces a rapid degradation of the ß-catenin protein in cancer cells, which is accompanied by a significant inhibition of proteasome activity, suggesting that proteasome degradation does not directly account for the suppression. The possibility that ZnPP induces ß-catenin exportation was rejected by the observation that there was no detectable ß-catenin protein in the conditioned medium after ZnPP treatment of cancer cells. Further experimentation demonstrated that ZnPP induces lysosome membrane permeabilization, which was reversed by pretreatment with a protein transportation inhibitor cocktail containing Brefeldin A (BFA) and Monensin. More significantly, pretreatment of cancer cells with BFA and Monensin attenuated the ZnPP-induced suppression of ß-catenin expression in a concentration- and time-dependent manner, indicating that the lysosome protein degradation pathway is likely involved in the ZnPP-induced suppression of ß-catenin expression. Whether there is cross-talk between the ubiquitin-proteasome system and the lysosome pathway that may account for ZnPP-induced ß-catenin protein degradation is currently unknown. These findings provide a novel mechanism of ZnPP's anticancer action and reveal a potential new strategy for targeting the ß-catenin Wnt signaling pathway for cancer therapy.

Correlates of thrombin generation in patients with advanced prostate cancer.

Thrombin generation is increased in men with advanced prostate cancer. Thrombin has the ability to interact with, and affect the biology of, a variety of cell types including prostate cancer cell lines. We therefore looked for correlations between thrombin generation and other markers of disease activity in spot urine samples obtained from men with advanced prostate cancer. Excretion of part of the prothrombin activation peptide F(1+2) (called here iF2), interleukin-6 (IL-6), the bone turnover marker deoxypyridinoline (DpD), and vascular endothelial growth factor (VEGF) were quantitated in spot urine samples collected from 37 men with hormone-refractory prostate cancer. Following log transformation of the data, significant correlations were found by univariate analysis between the excretion of a marker of thrombin generation (iF2) and IL-6, DpD and VEGF, as well as between IL-6 and DpD or VEGF excretion. No correlation was found between any marker and serum PSA level. After multivariate analysis, a significant correlation remained between thrombin generation and IL-6 excretion. Analysis of a second urine specimen obtained from 19 of the subjects 1 to 7 months after the first also revealed a significant correlation between thrombin generation and IL-6, DpD, and VEGF excretion. These data provide evidence of a correlation between thrombin generation/coagulation system activation and IL-6 generation in patients with cancer. They provide a rationale for studying the effects of inhibitors of thrombin generation upon the biology of prostate cancer.

Comparison of the aPTT with alternative tests for monitoring direct thrombin inhibitors in patient samples.

OBJECTIVES: The activated partial thromboplastin time (aPTT) test has been used for years to monitor parenteral direct thrombin inhibitors (DTIs) and unfractionated heparin. Because the aPTT correlates poorly with unfractionated heparin levels, we hypothesized that the aPTT may not be the best test for monitoring parenteral DTIs. METHODS: Using 235 excess plasma specimens from 82 adult patients receiving treatment with DTIs (argatroban, bivalirudin, or dabigatran), we compared the aPTT with the ecarin chromogenic assay (ECA), the dilute thrombin time (dTT) test, and the prothrombinase-induced clotting time (PiCT) test. RESULTS: The aPTT correlated poorly with each of the other tests in both bivalirudin- and argatroban-containing samples (r(2) = 0.04-0.23). The ECA and dTT exhibited the best correlations (r(2) = 0.66-0.93). Intermediate correlations were seen when the results of the PiCT were plotted against the dTT or ECA (r(2) = 0.46-0.58). Nineteen specimens obtained from six patients receiving dabigatran showed a good correlation between the dTT and the ECA (r(2) = 0.92). CONCLUSIONS: The aPTT does not correlate well with other tests that might be used to monitor parental DTI administration. Further studies are needed to evaluate the clinical usefulness of alternative tests and their correlation with clinical outcomes.

Characterization of docosahexaenoic acid (DHA)-induced heme oxygenase-1 (HO-1) expression in human cancer cells: the importance of enhanced BTB and CNC homology 1 (Bach1) degradation.

The effect of docosahexaenoic acid (DHA) on heme oxygenase-1 (HO-1) expression in cancer cells has never been characterized. This study examines DHA-induced HO-1 expression in human cancer cell model systems. DHA enhanced HO-1 gene expression in a time- and concentration-dependent manner, with maximal induction at 21 h of treatment. This induction of HO-1 expression was confirmed in vivo using a xenograft nude mouse model fed a fish-oil-enriched diet. The increase in HO-1 gene transcription induced by DHA was significantly attenuated by the antioxidant N-acetyl cysteine, suggesting the involvement of oxidative stress. This was supported by direct measurement of lipid peroxide levels after DHA treatment. Using a human HO-1 gene promoter reporter construct, we identified two antioxidant response elements (AREs) that mediate the DHA-induced increase in HO-1 gene transcription. Knockdown of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression compromised the DHA-induced increase in HO-1 gene transcription, indicating the importance of the Nrf2 pathway in this event. However, the nuclear protein levels of Nrf2 remained unchanged upon DHA treatment. Further studies demonstrated that DHA reduces nuclear Bach1 protein expression by promoting its degradation and attenuates Bach1 binding to the AREs in the HO-1 gene promoter. In contrast, DHA enhanced Nrf2 binding to the AREs without affecting nuclear Nrf2 expression levels, indicating a new cellular mechanism that mediates DHA's induction of HO-1 gene transcription. To our knowledge, this is the first characterization of DHA-induced HO-1 expression in human malignant cells.