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

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

The activity of gluconeogenesis enzymes.

<p>The activity of PEPCK, F-1, 6-BPase, and G6Pase was tested in un-dialyzed and dialyzed liver homogenates of control and metformin treated RLIP76^+/+ and RLIP76^−/− mice (n = 3) as protocols standardized by us <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024688#pone.0024688-Awasthi1" target="_blank">[1]</a>. *p<0.001, when compared to RLIP76^+/+, and **p<0.005, when compared with metformin treatment in RLIP76^+/+. The enzyme PEPCK, catalyze the conversion of phosphoenolpyruvate to fructose 1,6-biphosphate in a series of steps involving oxidation of NADH to NAD. In this assay, the loss of NADH was determined spectrophotometrically by measuring absorbance at 340 nm, based on the method of Opie and Newsholme <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024688#pone.0024688-Opie1" target="_blank">[28]</a>. To detect F-1, 6-BPase activity, a spectrophotometric coupled enzyme assay was used by a method of Taketa and Pogell <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024688#pone.0024688-Taketa1" target="_blank">[29]</a>. F-1, 6-BPase activity was coupled with phosphoglucose isomerase and NADP dependent glucose 6-phosphate dehydrogenase, and NADPH formation was measured at 340 nm. G6Pase activity was determined spectrophotometrically using the method of Gierow and Jergil <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024688#pone.0024688-Gierow1" target="_blank">[30]</a>. The method is based on a coupled enzyme reaction in which glucose formed is reacted with glucose oxidase and peroxidase and the quinoneimine formed is a colored product and its formation can be followed spectrophotometrically at 510 nm.</p

Differential effect of metformin in RLIP76^{<b>+<b>/</b>+</b>} and RLIP76^−/− mice.

<p><b><u>Panel A</u>:</b> BG was measured prior to and after a single oral dose of metformin (250 mg/kg b.w.) by gavage at various time points (n = 6 mice/group). p<0.001, when compared between RLIP76^+/+ and RLIP76^−/− mice, and metformin treatment in RLIP76^+/+ mice. <b><u>Panel B</u>:</b> Effect of metformin on RLIP76, pAkt, pJNK, PPARγ, and pAMPK expression by Western blot in RLIP76^+/+ and RLIP76^−/− control and metformin treated mouse liver tissue lysates, and developed bands were quantified by scanning densitometry. GAPDH expression was used as loading control. <b><u>Panel C</u>:</b> Inhibition of the transport activity of purified rec-RLIP76 towards ³H-GSHNE by metformin. The experiment was repeated twice and similar results were obtained. WT, wild-type (RLIP76^+/+); KO, RLIP76-knockout (RLIP76^−/−); Met, metformin.</p

Differential effect of rosiglitazone in RLIP76^{<b>+<b>/</b>+</b>}<i>vs.</i> RLIP76^−/− mice.

<p><b><u>Panel A</u>:</b> Effect of RLIP76 depletion by RLIP76 antisense on BG in RLIP76^+/+ mice. p<0.01, when compared to scrambled antisense treatment. <b><u>Panel B</u>:</b> BG was measured prior to and after a single oral dose of rosiglitazone (10 mg/kg b.w.) by gavage at various time points. p<0.001, when compared between RLIP76^+/+ and RLIP76^−/− mice, and rosiglitazone treatment in RLIP76^+/+ mice. <b><u>Panel C</u>:</b> Effect of rosiglitazone on RLIP76 expression (by QRT-PCR), RLIP76 protein content, and PPARγ protein content (by Western blot) in mouse liver tissue lysates. GAPDH expression was used as loading control. <b><u>Panel D</u>:</b> Inhibition of the transport activity of purified recombinant human RLIP76 towards physiological substrate ³H-GSHNE by rosiglitazone. In panels A & B, 5 mice per group were used. These experiments were repeated three times and similar results were obtained.</p