Inactivation of Factor VIIa by Antithrombin In Vitro, Ex Vivo and In Vivo: Role of Tissue Factor and Endothelial Cell Protein C Receptor

Recent studies have suggested that antithrombin (AT) could act as a significant physiologic regulator of FVIIa. However, in vitro studies showed that AT could inhibit FVIIa effectively only when it was bound to tissue factor (TF). Circulating blood is known to contain only traces of TF, at best. FVIIa also binds endothelial cell protein C receptor (EPCR), but the role of EPCR on FVIIa inactivation by AT is unknown. The present study was designed to investigate the role of TF and EPCR in inactivation of FVIIa by AT in vivo. Low human TF mice (low TF, ∼1% expression of the mouse TF level) and high human TF mice (HTF, ∼100% of the mouse TF level) were injected with human rFVIIa (120 µg kg−1 body weight) via the tail vein. At varying time intervals following rFVIIa administration, blood was collected to measure FVIIa-AT complex and rFVIIa antigen levels in the plasma. Despite the large difference in TF expression in the mice, HTF mice generated only 40–50% more of FVIIa-AT complex as compared to low TF mice. Increasing the concentration of TF in vivo in HTF mice by LPS injection increased the levels of FVIIa-AT complexes by about 25%. No significant differences were found in FVIIa-AT levels among wild-type, EPCR-deficient, and EPCR-overexpressing mice. The levels of FVIIa-AT complex formed in vitro and ex vivo were much lower than that was found in vivo. In summary, our results suggest that traces of TF that may be present in circulating blood or extravascular TF that is transiently exposed during normal vessel damage contributes to inactivation of FVIIa by AT in circulation. However, TF’s role in AT inactivation of FVIIa appears to be minor and other factor(s) present in plasma, on blood cells or vascular endothelium may play a predominant role in this process

Role of Tissue Factor in Mycobacterium tuberculosis-Induced Inflammation and Disease Pathogenesis

Tuberculosis (TB) is a chronic lung infectious disease characterized by severe inflammation and lung granulomatous lesion formation. Clinical manifestations of TB include hypercoagulable states and thrombotic complications. We previously showed that Mycobacterium tuberculosis (M.tb) infection induces tissue factor (TF) expression in macrophages in vitro. TF plays a key role in coagulation and inflammation. In the present study, we investigated the role of TF in M.tb-induced inflammatory responses, mycobacterial growth in the lung and dissemination to other organs. Wild-type C57BL/6 and transgenic mice expressing human TF, either very low levels (low TF) or near to the level of wild-type (HTF), in place of murine TF were infected with M.tb via aerosol exposure. Levels of TF expression, proinflammatory cytokines and thrombin-antithrombin complexes were measured post M.tb infection and mycobacterial burden in the tissue homogenates were evaluated. Our results showed that M.tb infection did not increase the overall TF expression in lungs. However, macrophages in the granulomatous lung lesions in all M.tb-infected mice, including low TF mice, showed increased levels of TF expression. Conspicuous fibrin deposition in the granuloma was detected in wild-type and HTF mice but not in low TF mice. M.tb infection significantly increased expression levels of cytokines IFN-γ, TNF-α, IL-6 and IL-1ß in lung tissues. However, no significant differences were found in proinflammatory cytokines among the three experimental groups. Mycobacterial burden in lungs and dissemination into spleen and liver were essentially similar in all three genotypes. Our data indicate, in contrast to that observed in acute bacterial infections, that TF-mediated coagulation and/or signaling does not appear to contribute to the host-defense in experimental tuberculosis

RLIP76, a Glutathione-Conjugate Transporter, Plays a Major Role in the Pathogenesis of Metabolic Syndrome

PURPOSE: Characteristic hypoglycemia, hypotriglyceridemia, hypocholesterolemia, lower body mass, and fat as well as pronounced insulin-sensitivity of RLIP76⁻/⁻ mice suggested to us the possibility that elevation of RLIP76 in response to stress could itself elicit metabolic syndrome (MSy). Indeed, if it were required for MSy, drugs used to treat MSy should have no effect on RLIP76⁻/⁻ mice. RESEARCH DESIGN AND METHODS: Blood glucose (BG) and lipid measurements were performed in RLIP76⁺/⁺ and RLIP76⁻/⁻ mice, using Ascensia Elite Glucometer® for glucose and ID Labs kits for cholesterol and triglycerides assays. The ultimate effectors of gluconeogenesis are the three enzymes: PEPCK, F-1,6-BPase, and G6Pase, and their expression is regulated by PPARγ and AMPK. The activity of these enzymes was tested by protocols standardized by us. Expressions of RLIP76, PPARα, PPARγ, HMGCR, pJNK, pAkt, and AMPK were performed by Western-blot and tissue staining. RESULTS: The concomitant activation of AMPK and PPARγ by inhibiting transport activity of RLIP76, despite inhibited activity of key glucocorticoid-regulated hepatic gluconeogenic enzymes like PEPCK, G6Pase and F-1,6-BP in RLIP76⁻/⁻ mice, is a salient finding of our studies. The decrease in RLIP76 protein expression by rosiglitazone and metformin is associated with an up-regulation of PPARγ and AMPK. CONCLUSIONS/SIGNIFICANCE: All four drugs, rosiglitazone, metformin, gemfibrozil and atorvastatin failed to affect glucose and lipid metabolism in RLIP76⁻/⁻ mice. Studies confirmed a model in which RLIP76 plays a central role in the pathogenesis of MSy and RLIP76 loss causes profound and global alterations of MSy signaling functions. RLIP76 is a novel target for single-molecule therapeutics for metabolic syndrome

Abstract 526: 4-hydroxy-2-nonenal (HNE), a Lipid Peroxidation Product, Exerts Both Pro- and Anti-thrombotic Effects on Vascular Cells

Background: Oxidative stress and generation of lipid peroxidation (LPO) products are detrimental in the pathogenesis of atherosclerosis and associated acute thrombotic events. However, recent studies suggest that moderate oxidative stress and low levels of LPO products can induce adaptive immune responses and exert beneficial effects. Tissue factor (TF) is a critical initiator of coagulation and aberrant TF expression on vascular cells under inflammation triggers intravascular thrombosis. HNE, a highly reactive LPO product and TF have been shown to be associated with atherosclerosis. Recently, we demonstrated that HNE decrypts procoagulant activity of pre-existing TF on activated monocytes and endothelial cells and generates TF+ microparticles. Here, we investigated the effect of HNE on induction of TF and cell adhesion molecules in monocytes and endothelial cells that were not perturbed earlier. Methods and results: THP-1 monocytic cells and endothelial cells (HUVEC) were stimulated with LPS and TNF-α/IL1-β, respectively, in the presence of a control vehicle or varying concentrations of HNE that are pathophysiologically relevant. TF induction was measured at mRNA (by qRT-PCR), protein (by immunoblotting) and activity levels (in factor X activation assay). Pre-treating cells with HNE inhibited TNF-α/IL1-β- or LPS-stimulated TF procoagulant activity in a dose-dependent manner. THP-1 and HUVEC varied in their sensitivities to HNE (THP-1&gt; HUVEC). HNE-mediated inhibition of TF activity correlated with lower TF mRNA and protein levels. Our results demonstrate that HNE prevents TNF-α/IL1-β- and LPS-induced IKKβ degradation and thereby inhibits NFκβ activation. In addition to inhibiting TF expression, HNE significantly reduced monocyte adhesion to endothelial cells through downregulating TNF-α/IL1-β-induced expression of endothelial adhesion molecules VCAM-1 and ICAM-1. Conclusion: HNE may play a dual role in regulating TF activity in atherosclerosis. HNE could act as a prothrombotic mediator by increasing coagulant activity of pre-existing TF through decryption process. HNE can also elicit anti-thrombotic and anti-inflammatory effect by inhibiting TF and adhesion molecules in response to stimulus by impairing the NF-ĸB pathway. </jats:p

Abstract 4334: Targeting p53 null neuroblastomas through RLIP76

Abstract The search for p53-independent mechanism of cancer cell killing is highly relevant to pediatric neuroblastomas, where successful therapy is limited by its transformation into p53 mutant and a highly drug-resistant neoplasm. Our studies on the drug-resistant p53 mutant as compared with drug-resistant p53 wild-type neuroblastoma revealed a novel mechanism for resistance to apoptosis: a direct role of p53 in regulating the cellular concentration of pro-apoptotic alkenals by functioning as a specific and saturable allosteric inhibitor of the alkenal-glutathione-conjugate transporter, RLIP76. The RLIP76-p53 complex was demonstrated both by using immuno-precipitation analyses of purified proteins as well as by immuno-fluorescence analysis. Drug transport studies revealed that p53 inhibited both basal and PKCα stimulated transport of glutathione-conjugates of 4HNE (GS-HNE) and cisplatin. Drug resistance was significantly greater for p53 mutant as compared with p53 wild-type neuroblastoma cell lines, but both were susceptible to depletion of RLIP76 by antisense alone. In addition, inhibition of RLIP76 significantly enhanced the cytotoxicity of cisplatin. Taken together, these studies provide powerful evidence for a novel mechanism for drug and apoptosis resistance in p53 mutant neuroblastoma, based on a model of regulation of p53 induced apoptosis by RLIP76, where p53 is a saturable and specific allosteric inhibitor of RLIP76, and p53 loss results in over-expression of RLIP76; thus, in the absence of p53, the drug and glutathione-conjugate transport activities of RLIP76 are enhanced. Most importantly, our findings strongly indicate RLIP76 as a novel target for therapy of drug-resistant and p53 mutant neuroblastoma. (Supported in part by NIH grant CA 77495 (SA), CA 155350, Cancer Research Foundation of North Texas, and Institute for Cancer Research &amp; the Joe &amp; Jessie Crump Fund for Medical Education (SSS)) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4334. doi:10.1158/1538-7445.AM2011-4334</jats:p

Inactivation of factor VIIa by antithrombin in vitro, ex vivo and in vivo: role of tissue factor and endothelial cell protein C receptor.

Recent studies have suggested that antithrombin (AT) could act as a significant physiologic regulator of FVIIa. However, in vitro studies showed that AT could inhibit FVIIa effectively only when it was bound to tissue factor (TF). Circulating blood is known to contain only traces of TF, at best. FVIIa also binds endothelial cell protein C receptor (EPCR), but the role of EPCR on FVIIa inactivation by AT is unknown. The present study was designed to investigate the role of TF and EPCR in inactivation of FVIIa by AT in vivo. Low human TF mice (low TF, ∼ 1% expression of the mouse TF level) and high human TF mice (HTF, ∼ 100% of the mouse TF level) were injected with human rFVIIa (120 µg kg(-1) body weight) via the tail vein. At varying time intervals following rFVIIa administration, blood was collected to measure FVIIa-AT complex and rFVIIa antigen levels in the plasma. Despite the large difference in TF expression in the mice, HTF mice generated only 40-50% more of FVIIa-AT complex as compared to low TF mice. Increasing the concentration of TF in vivo in HTF mice by LPS injection increased the levels of FVIIa-AT complexes by about 25%. No significant differences were found in FVIIa-AT levels among wild-type, EPCR-deficient, and EPCR-overexpressing mice. The levels of FVIIa-AT complex formed in vitro and ex vivo were much lower than that was found in vivo. In summary, our results suggest that traces of TF that may be present in circulating blood or extravascular TF that is transiently exposed during normal vessel damage contributes to inactivation of FVIIa by AT in circulation. However, TF's role in AT inactivation of FVIIa appears to be minor and other factor(s) present in plasma, on blood cells or vascular endothelium may play a predominant role in this process