ALBERT

Description: Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like proenzyme that, once activated by thrombin, the thrombin-thrombomodulin complex, or plasmin, attenuates fibrinolysis. Aberrant regulation of the TAFI pathway alters the balance between coagulation and fibrinolysis and may underlie severe haemostatic disorders such as thrombosis and hemophilia. Indeed, high plasma TAFI levels have been associated with several cardiovascular diseases. It is important to note that there is a large inter-individual variability in plasma TAFI levels within the population, and this variation is primarily due to non-genetic factors. Therefore, variation in TAFI gene expression is a risk factor for thrombotic disorders and may be an important means by which TAFI responds to environmental and physiological stimuli. Novel associations between plasma TAFI levels and sex hormones have triggered interest in determining the role of TAFI as a mediator of the cardioprotective effects of estrogens and progestagens, or as a mediator of the increased thrombotic risk that accompanies use of oral contraceptives or hormone replacement therapy. Plasma TAFI concentrations rise with age in women but not in men, and are elevated in post-menopausal women compared to pre-menopausal women. In addition, plasma TAFI levels have been shown to be decreased by selective estrogen receptor modulators such as HMR 3339 and raloxifene, estradiol plus trimegestone, transdermal estradiol, and oral estradiol plus gestodene. On the other hand, some studies have reported minimal to no change in plasma TAFI levels occurring with the use of oral contraceptive, Raloxifene, or Tamoxifen. Paradoxically, it has been shown that both plasma TAFI levels and clot lysis time rise during pregnancy and then promptly return to basal levels after delivery. These studies illustrate the controversies surrounding the role of sex steroids in modulating plasma TAFI levels. In the present study, we have attempted to directly measure the effect of sex steroids on hepatic TAFI gene expression, and to uncover the molecular mechanisms underlying these regulatory events. HepG2 (human hepatocellular carcinoma) cells were cultured in the presence or absence of progesterone and b-estradiol and TAFI mRNA abundance was measured using real-time RT-PCR. We found that both of these hormones significantly decrease endogenous TAFI mRNA abundance in a dose-dependant manner. To assess the ability of these hormones to influence transcription of the gene encoding TAFI, we treated HepG2 cells that had been transiently transfected with luciferase reporter plasmids containing the 5′-flanking region of the TAFI gene. Interestingly, the change in promoter activity closely paralleled changes in mRNA abundance, suggesting that the effect of the hormones is mediated at the level of transcription. Furthermore, changes in TAFI mRNA abundance following treatments with estrogen were not associated with a decrease in TAFI mRNA stability when compared to the untreated control. TAFI protein levels were also decreased in a dose-dependent manner as assessed by western blot analysis. Inspection of the sequence of the TAFI 5′-flanking region does not show any consensus estrogen responsive elements, although we cannot exclude a role for more complex transcriptional system such as an estrogen response unit. The effect of estrogen could also be performed indirectly through the modulation of other transcription factors such SP-1 or members of the basal transcriptional machinery. We also investigated whether progesterone decreases TAFI gene expression via the binding of the progesterone receptor to the established glucocorticoid responsive element (GRE) within the TAFI promoter. Our results showed that progesterone generates the same decrease in promoter activity even when the GRE site was mutated, indicating that progesterone may act through a different site. In conclusion, our studies are beginning to reveal the molecular basis for the apparent relationship between female sex steroids and plasma concentrations of TAFI: specifically, a direct downregulatory effect on transcription of the gene encoding TAFI.

Description: Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase zymogen defining a pathway that functions as a molecular link between coagulation and fibrinolysis. Activation by thrombin, the thrombin-thrombomodulin complex, or plasmin, the resultant enzyme (TAFIa) affects the balance between these two cascades by attenuating positive feedback in the fibrinolytic cascade, thereby inhibiting fibrin clot lysis. Plasma TAFI antigen levels vary significantly between individuals, which has implicated TAFI as a risk factor for thrombotic diseases. TAFIa can also inactivate pro-inflammatory peptides such as the anaphylatoxins and bradykinin, suggesting a role for the TAFI pathway as a link between coagulation and inflammation. TAFI expression in cultured hepatic cells is decreased by interleukins −1 and −6, and plasma TAFI levels in human are decreased in experimental endotoxemia. Although the liver is the main source of plasma TAFI, TAFI has also been identified in platelets, and TAFI mRNA has been detected in the Dami (megakaryoblastic) cell line (but not the MEG-01 cell line). TAFI mRNA has also been detected in adipocytes of patients with type 2 diabetes; however, TAFI mRNA expression in human umbilical vein endothelial cells is still a point of controversy. It has been hypothesized that platelet TAFI arises from TAFI gene expression in megakaryocytes (MK). Using RT-PCR and real-time RT-PCR, we not only confirmed the presence of TAFI mRNA in Dami cells, but also found that TAFI mRNA abundance was increased throughout Dami cell differentiation along the megakaryocytes/platelet lineage (up to 8 fold increase after 48 hours) stimulated by phorbol myristate acetate (PMA) treatment. The quantitative real-time RT-PCR experiments revealed that TAFI mRNA is present in differentiated Dami cells at a level that is only one-hundredth of that observed in HepG2 (hepatoma) cells. Using transfection experiments with luciferase reporter plasmids containing progressive deletions of the human TAFI 5′-flanking region, we identified the sequence between −438 and −257 (relative to the initiator methionine codon) to be responsible for the enhanced TAFI gene transcription as Dami cells differentiate into more mature MK-like cells. Moreover, using western blot analysis, we detected TAFI protein expression in the medium of differentiated Dami cells, but not untreated Dami cells. Together, these data provide further evidence supporting the idea that platelet TAFI is generated from TAFI gene expression in megakaryocytes rather than by uptake from the plasma. To study TAFI gene regulation in monocytes and macrophages, RT-PCR and realtime RT-PCR were used to detected and quantify, respectively, TAFI mRNA expression in both THP-1 and THP-1 cells that have been differentiated into macrophage-like cells (THP-1ma) by PMA treatment. TAFI mRNA abundance was similar in THP-1 cells as what was observed in differentiated Dami cells. In addition, we found a progressive decrease in TAFI mRNA abundance throughout the THP-1 differentiation with an 85% decrease after 24 hours of PMA treatment. Transfection experiments using luciferase reporter plasmids representing progressive deletions of the human TAFI 5′-flanking region identified sequences between −151 and −121 as harboring key promoter elements for the differentiation-associated decrease in TAFI gene expression as THP-1 differentiate into macrophage-like cells. However, no TAFI protein was detected in either THP-1 or THP-1ma conditioned medium using western blot analyses. Nonetheless, extra-hepatic expression of TAFI, such as platelet, monocytes and macrophages, suggests novel roles for TAFI pathway beyond regulation of fibrin clot breakdown.

Description: Thrombin-activable fibrinolysis inhibitor (TAFI) is a plasma zymogen that acts as a molecular link between coagulation and fibrinolysis. Numerous single nucleotide polymorphisms (SNPs) have been identified in CPB2, the gene encoding TAFI, and are located in the 5′-flanking region, in the coding sequences, and in the 3′-untranslated region (UTR) of the CPB2 mRNA transcript. Associations between CPB2 SNPs and variation in plasma TAFI antigen concentrations have been described, but the identity of SNPs that are causally linked to this variation is not known. In the current study, we investigated the effect of the SNPs in the 5′-flanking region on CPB2 promoter activity and SNPs in the 3′-UTR on CPB2 mRNA stability. Whereas the 5′-flanking region SNPs (with 2 exceptions) did not have a significant effect on promoter activity, either alone or in haplotypic combinations seen in the human population, all of the 3′-UTR SNPs substantially affected mRNA stability. We speculate that these SNPs, in part, contribute to variation in plasma TAFI concentrations via modulation of CPB2 gene expression through an effect on mRNA stability.

Description: Lilliput was discovered in 2005 as the southernmost known hydrothermal field along the Mid-Atlantic Ridge. It is exceptional in that it lacks high-temperature venting probably because of a thickened crust. The absence of thermophilic and hyperthermophilic prokaryotes in emissions supports the argument against the presence of a hot subsurface at Lilliput, as is typically suggested for diffuse emissions from areas of high-temperature venting. The high phylogenetic diversity and novelty of bacteria observed could be because of the low-temperature influence, the distinct location of the hydrothermal field or the Bathymodiolus assemblages covering the sites of discharge. The low-temperature fluids at the Lilliput are characterized by lowered pH and slightly elevated hydrogen (16 nM) and methane (∼2.6 μM) contents compared with ambient seawater. No typical hydrogen and methane oxidizing prokaryotes were detected. The higher diversity of reverse tricarboxylic acid genes and the form II RubisCO genes of the Calvin Benson-Bassham (CBB) cycle compared with the form I RubisCO genes of the CBB cycle suggests that the chemoautotrophic community is better adapted to low oxygen concentrations. Thiomicrospira spp. and Epsilonproteobacteria dominated the autotrophic community. Sulfide is the most abundant inorganic energy source (0.5 mM). Diverse bacteria were associated with sulfur cycling, including Gamma-, Delta- and Epsilonproteobacteria, with the latter being the most abundant bacteria according to fluorescence in situ hybridization. With members of various Candidate Divisions constituting for 25% of clone library sequences we suggest that their role in vent ecosystems might be more important than previously assumed and propose potential mechanisms they might be involved in at the Lilliput hydrothermal field.