Tissue Factor Pathway Inhibitor Gene Research Articles

A Nonsynonymous-Single-Nucleotide Polymorphism (ns-SNP) in TFPI, the Gene Encoding Tissue Factor Pathway Inhibitor (TFPI), is Pleiotropically Associated with Increased Plasma TFPI Levels and a Decreased Risk of Type 2 Diabetes (T2D) in Mexican Americans of South Texas

The ns-SNP, rs7586970 (T>C), which results in an asparagine (Asn [N]) to serine (Ser [S]) substitution at amino acid 221 (221 N>S) in TFPI, has been shown to be associated with coronary heart disease and T2D. We performed an exome scan of plasma TFPI levels in 706 participants in our study of genetic determinants of cardiovascular disease in Mexican Americans of South Texas. Using a linear mixed model approach, while accounting for age, sex, and their interactions, we found a heritability of 34% for plasma TFPI levels (p=4.4E-08). The only variant showing exome-wide significance was the rs7586970 (T>C) variant on chromosome 2 at the TFPI gene (p=5.1E-07; Figure 1A). The quantile-quantile distribution of the observed p-values from all the exome-wide tests against the p-values expected under the null hypothesis of no association shows an overall agreement, indicating that the p-value for the association test for the 221 N>S variant is likely not an artifact (data not shown). The regression coefficient for the polymorphism as a predictor of TFPI levels showed them to be increasing from the individuals homozygous for the major T-allele (T/T: N/N), to the heterozygous individuals (T/C: N/S), to the individuals homozygous for the minor C-allele (C/C: S/S) (Figure 1B). Using a statistical genetic model for the liability to disease conditional on a threshold, which is equivalent to a probit mixed model, we found that the rs7586970 (T>C) polymorphism is significantly associated with a reduction in the risk of T2D (p=0.009) (data not shown). While not shown, the effect of the TFPI variant is to upwardly displace the liability threshold to the right, which results in a reduced prevalence of T2D from 6% to just below 3% in the rare allele homozygotes. While the reduction is a small change, it is statistically significant, nonetheless. Moreover, our observed effect size in terms of T2D risk is consistent with the widely held view that T2D has a multifactorial etiology involving a complex combination of environmental risk factors and genes with small effects sizes. A plausible mechanism for the reduced risk of T2D involves tissue factor (TF), which is the physiologic initiator of coagulation that is normally found outside the vasculature. As such, coagulation is initiated when the blood vessel is disturbed or when vascular and blood cells are induced to secrete and express surface membrane TF, as occurs in inflammation. TF then binds its primary cofactor, FVIIa, which circulates in the blood, TF-FVIIa activates FIX and FX, and the small amount FXa generated converts prothrombin to thrombin. TFPI is the primary inhibitor of TF-induced coagulation by binding and inhibiting TF-FVIIa in a FXa-dependent manner. Coagulation only proceeds when enough FXa is generated to overwhelm the inhibitory potential of TFPI. The rs7586970 ns-SNP conferred substitution of Ser for Asn at amino acid 221, the putative glycosyl-phosphatidylinositol anchor site of TFPI-β, its phospholipid bound form, potentially disrupts the binding of TFPI-β to the surface of endothelial cells resulting in higher levels of free form which has more potent anticoagulant activity. In conclusion, we found that TFPI 221 N>S ns-SNP is pleiotropically associated with plasma TFPI levels and risk of T2D. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genetic variation of the blood coagulation regulator tissue factor pathway inhibitor and venous thromboembolism among middle‐aged and older adults: A population‐based cohort study

BackgroundTissue factor is the main initiator of blood coagulation, and tissue factor pathway inhibitor (TFPI) is the primary inhibitor of the initiation of blood coagulation.The genetic variation of TFPI and the relation to venous thromboembolism (VTE), that is, venous thrombosis and pulmonary embolism, remains to be clarified. This exome sequencing study aimed to determine the molecular epidemiology of the TFPI gene and the relation to VTE in a large population‐based cohort of middle‐aged and older adults. MethodsThe exomes of TFPI were analyzed for variants in 28,794 subjects without previous VTE (born 1923–1950, 60% women), who participated in the Malmö Diet and Cancer Study (1991–1996). Patients were followed until the first event of VTE, death, or 2018. Qualifying variants were defined as loss‐of‐function or nonbenign (PolyPhen‐2) missense variants with minor allele frequency less than 0.1%. ResultsNo common variant was associated with VTE. Nine rare variants (two loss‐of‐function and seven nonbenign missense) were classified as qualifying and included in collapsing analysis. Prevalence of qualifying variants was 0.09%. Five individuals with VTE compared to 17 individuals without VTE carried one qualifying variant. Cox multivariate regression analysis adjusted for age, sex, body mass index, systolic blood pressure, smoking and alcohol consumption, rs6025, rs1799963, and ancestry showed a hazard ratio of 2.9 (95% CI, 1.2–7.1) for rare qualifying variants. ConclusionRare qualifying TFPI variants were associated with VTE, suggesting that rare variants in TFPI contribute to the development of VTE. The qualifying TFPI gene variants were very rare, suggesting a constrained gene.

Platelet FV Deficiency Restores Survival of TFPI Null Mice Independently of Plasma FV

Introduction: Platelets are essential for the hemostatic process, releasing “just in time” prothrombotic molecules, such as factor V (FV) and prothrombin to sites of vascular injury. Paradoxically, platelets also carry potent antithrombotic molecules, including tissue factor pathway inhibitor (TFPI), which may prevent hemostatic platelet plugs from expanding into vascular occluding thrombi. The TFPI gene undergoes alternative splicing to produce several isoforms, including TFPIα and TFPIβ. Platelets are the primary location of TFPIα in the murine circulation. TFPIα has a basic 9 amino acid sequence in its C-terminal region nearly identical to a basic sequence in the FV B domain. The TFPIα basic region tightly binds to an acidic region of the FV B domain. This binding takes place only when the FV basic region is absent, as occurs following proteolysis by factor Xa (FXa), in some forms of FV naturally present in platelet α-granules, or in genetically altered forms of FV, such as FV-short (associated with East Texas bleeding disorder). This interaction provides an essential exosite for TFPIα inhibition of the prothrombinase complex (FXa-FVa) assembled with forms of FVa retaining the B domain acidic region. Since prothrombinase assembles on the surface of activated platelets, we hypothesized that inhibition of prothrombinase via the platelet TFPIα-platelet FV interaction is a physiologically important biochemical mechanism for the inhibiting early stages of coagulation. We probed this hypothesis using TFPI K1 deleted (TFPI-/-), FV deleted (F5-/-) and FV transgenic (F5Tg) mice to uncover whether specifically reducing or removing platelet FV would rescue TFPI-/- mice from embryonic lethality.Methods/Results: We performed crossbreeding experiments using F5-/- mice carrying liver- or platelet-specific transgenes. F5LiverTg produces 50% plasma FV with no FV in platelets, whereas F5PltTg produces 3% platelet FV with no FV in plasma. F5-/- F5LiverTgand F5-/- F5PltTgmice were bred into TFPI+/- mice. Offspring from this cross were bred to determine the effects of the liver or platelet transgene on the survival of mice lacking TFPI.F5-/- is embryonic lethal in mice. F5-/- mice are rescued by transgenes expressing either liver-specific FV or platelet specific FV. When we bred F5+/- TFPI+/- x F5+/- F5LiverTg TFPI+/-, neither F5-/- TFPI-/- nor F5+/- TFPI-/- mice without the liver transgene were viable. Liver transgene expression did not rescue F5+/- TFPI-/- mice, however, it produced grossly normal pups lacking TFPI (F5-/- F5LiverTg TFPI-/-) that lived to adulthood with no overt phenotypes. We further characterized this phenotype by crossbreeding F5+/- TFPI+/- x F5-/- F5LiverTg TFPI-/-. In this cross, F5-/- F5LiverTg TFPI-/- pups survived with ~80% penetrance (45 of 234 total mice, p<5.0 x10-12). Next, we bred F5+/- TFPI+/- x F5+/- F5PltTgTFPI+/-. Platelet transgene expression also produced grossly normal pups lacking TFPI (F5-/- F5PltTg TFPI-/-) that lived to adulthood with no overt phenotypes. Further crossbreeding, as described for the liver transgene, revealed a similar ~80% survival of F5-/- F5PltTg TFPI-/- pups (56 of 291 total mice, p<9.0 x10-18). Both male and female platelet and liver specific F5-/- F5Tg TFPI-/- were fertile, and mothers supported pups until weaning.Conclusions: Neither total loss of FV nor concerted 50% reduction in plasma and platelet FV rescued TFPI-/- embryonic lethality, indicating that TFPI anticoagulant activity is not simply counterbalanced by FV procoagulant activity (or vice versa) during embryogenesis. Remarkably, either the total lack of platelet FV with 50% normal plasma FV, or the total lack of plasma FV with 3% normal platelet FV, resulted in the nearly complete rescue of F5-/- TFPI-/- mice with pups surviving to adulthood without grossly observable pathology. These observations indicate that severe reduction of platelet FV, independent of the plasma FV concentration, produces this extraordinary rescue of TFPI-/- embryonic lethality. Collectively, these findings expose the critical role for platelet TFPIα in regulating procoagulant activity of prothrombinase assembled with platelet FV. This platelet FV-platelet TFPIα interaction highlights the important biological activity of pro and anticoagulant proteins located within platelets in regulating hemostatic balance during murine development and into adulthood. DisclosuresMast:Novo Nordisk: Research Funding.

A Re-Evaluation of Murine TFPI and Comparison to TFPI in Man: Murine Plasma TFPI Is Derived from TFPIγ

Tissue factor pathway inhibitor (TFPI) inhibits FXa and produces FXa-dependent feedback inhibition of FVIIa/TF activity. TFPI is expressed as isoforms α and β in man, and α, β and γ in the mouse. TFPIα consists of an acidic N-terminal peptide followed by three Kunitz-type protease inhibitory domains (K1K2K3) and a basic C-terminal peptide (CTP). TFPIβ lacks the K3 and CTP of TFPIα and instead contains a sequence that directs the attachment of a GPI membrane anchor to TFPIβ. Murine TFPIγ encodes a two-Kunitz domain TFPI with a short, unique C-terminal region.TFPI in murine plasma circulates at ~20 times the level in man and is not detectably affected by heparin treatment: ~31.5 + 3.7 nM before and 29.7 + 4.1 nM (n=10) 15 min after 2 units of heparin (IP). The major form of murine plasma TFPI migrates on SDS-PAGE as a ~41,000 MW protein but elutes from size-exclusion column chromatography with at an apparent MW of ~300,000. It binds to Cab-O-Sil and floats at 1.25 g/mL KBr consistent with an association with lipoproteins like the TFPI in human plasma. Mass spectroscopy (MS) analysis identifies this major form as a two-Kunitz domain form lacking detectible C-terminal isoform-specific sequences. In contrast, MS analysis of total TFPI isolated from murine plasma, detects both TFPIα-specific and TFPIγ-specific peptides, but no TFPIβ-specific peptides. This is the first identification of TFPI γ protein. MS analysis of human lipoprotein-associated TFPI was also found to be a two-Kunitz domain form lacking isoform-specific sequences. Analysis of total TFPI from human plasma detected TFPIα-specific, but no TFPIβ- and no TFPIγ-specific sequences.Sequences homologous with the mouse γ exon are present within the TFPI gene of many species. mRNA amplified by RT-PCR from a variety of human tissues contained the TFPIγ homologous sequence, but in each case it was part of TFPIβ 3‘-untranslated message. The 3‘ splice acceptor sequence for the TFPI γ exon in mouse, rat, Chinese hamster and squirrel is CAG, which is commonly used for splicing (71%), and rat ESTs encoding TFPIγ have been reported. The corresponding sequence in man and other primates, GAG, is rarely utilized (<1%) for splicing.TFPI α exon-deleted, β exon-deleted, β/γ exons-deleted and γ exon-disrupted mouse strains were generated using CRISPR/Cas9 technology. The KO mice, born at expected ratios, appear healthy. Using quantitative RT-PCR, validated with isoform-deleted mice, relative amounts of TFPI α, β, and γ mRNA in WT murine lung are 30%, 20%, and 50%, respectively. Plasma TFPI levels in TFPIα(-/-) and TFPIβ(-/-) mice are similar to WT mice (~100%). They are ~50% in TFPIγ(+/-) and TFPIβ/γ(+/-) mice and <2% in TFPIγ(-/-) and TFPIβ/γ(-/-) mice. Thus, the major form of murine plasma TFPI is derived form TFPI γ .Four samples of platelets isolated from 5-10 mice, revealed the blood level of TFPI in murine platelets (at 7.5 x 108/mL) was 74.6 + 16.0 pM. Present at WT levels in TFPIβ/γ KO mice, platelet TFPI is absent in TFPIα KO mice, confirming that in mice, like humans, platelet TFPI is TFPIα. Studies of mice with combined TFPIβ/γ(-/-) gene deletion (expressing α only) show a plasma level of TFPIα of 66.8 + 12.9 pM that increases 7.3 + 2.5-fold after heparin treatment. Our baseline TFPIα measurement represents a total value and the distribution of TFPIα derived “free” and lipoprotein-associated forms remains to be determined. The heparin-releasable TFPIα is likely endothelial-derived but this remains to be determined. Wild type mice presumably also release TFPIαwith heparin treatment but, in our experience, this response is masked due to the high circulating level of TFPIγ.Our results for TFPI in the m ouse differ from, and add to, previous reports in important respects: 1. The majority of TFPI circulating at high levels in mouse plasma is derived from TFPIγ, not α or β. 2. Sequences homologous to the mouse TFPIγ exon are present in many species, but the 3' acceptor splice site sequence in man and other primates is unfavorable for splicing. 3. Relative levels of TFPI α, β, and γ mRNA in mouse lung are 30%, 20%, and 50%, respectively. 4. Like in man, TFPIα circulates at a low level in mouse plasma, which increases several-fold following heparin treatment. 5. A two-Kunitz domain form of TFPI, lacking isoform-specific C-terminal sequences, is bound to plasma lipoproteins in both mouse and man. 6. Studies in TFPIγ deleted mice (expressing only α and β) should provide results most relevant to humans. DisclosuresNo relevant conflicts of interest to declare.

Inhibitory Effect of Eukaryotic Expression Vector Bearing TFPI-2 Gene on SHI-1 Cell Growth

To construct a eukaryotic expression vector of human tissue factor pathway inhibitor-2 (TFPI-2) and to investigate the effect of TFPI-2 gene on the growth of acute monocytic leukemia cell line (SHI-1). The cDNA of TFPI-2 was obtained by genetic chemical synthesis, the TFPI-2 gene and the linear vector fragment were ligated and inserted into the multiple cloning site of PEGFP-N1 vector, and the eukaryotic expression vector PEGFP-N1-TFPI-2 was transfected SHI-1 cells, then the obtained SHI-1 cells was observed by fluorescence microscopy; MTT assay was used to detect the effect of TFPI-2 gene on the relative growth rate of SHI-1 cells at the different time-point; RT-PCR was used to detect TFPI-2 mRNA expression levels in the cells of each group before and after TFPI-2 transfection; TFPI-2 protein expression was detected by Western blot. The cells which successfully transfected with PEGFP-N1-TFPI-2 vector were named as SHI-1-TFPI-2 (experimental group), and the cells transfected with the empty vector pEGFP-N1 and the untransfected cells were named as SHI-1-V and SHI-1-P and used as the control group. The human TFPI-2 gene eukaryotic expression vector PEGFP-N1-TFPI-2 was successfully constructed, then the transfected into SHI-1 cells, observed by fluorescence microscopy 24 hours later, as a result, the PEGFP-N1-TFPI-2 was successfully transferred into SHI-1 cells, and the number of fluorescent cells increased after 48 h and 72 h. RT-PCR showed that the gray scale ratio of TFPI-2 gene to β- actin in the experimental group was higher than that in the control group. The gray scale ratio was 0.51±0.04 in SHI-1-V group, 0.52±0.03 in SHI-1-P group, 0.87±0.08 in SHI-1-TFPI-2 group, and the difference between SHI-1-TFPI-2 and SHI-1-V, SHI-1-P group was statistically significant (P＜0.05). The expression of TFPI-2 gene in PEGFP-N1-TFPI-2 can inhibit the growth of SHI-1 cells, which provides a research direction for gene therapy of leukemia in the future.

The Tissue Factor Pathway Inhibitor Gamma Knockout Mouse Is a Better Model Than the Wild Type Mouse for Studying Tissue Factor-Related Human Diseases

BACKGROUND. Tissue factor (TF) is critical for hemostasis. However, TF also plays a pathogenic role in a multitude of diseases, including: thrombosis, coagulation-associated inflammation, multi-organ failure associated with infections, tumor growth, angiogenesis and metastasis. Tissue Factor Pathway Inhibitor (TFPI), the regulator of TF actions, produces FXa-dependent feedback inhibition of the FVIIa/TF catalytic complex, directly inhibits FXa, and suppresses cellular signaling by FVIIa/TF/FXa complexes. In man, TFPI is expressed as isoforms TFPIα and TFPIβ. The pathophysiologic impacts of the individual α and β isoforms have yet to be elucidated. Mouse models have been used extensively to provide insight into human physiology and disease, including abnormalities concerning hemostasis and TF-associated diseases. Like man, mice express TFPI α and β, but unlike man, mice express a third transcript encoding TFPIγ. In fact, we recently reported that the vast majority (98%) of TFPI in mouse plasma is derived from TFPIγ. We also reported that anti-TFPI antibodies enhance in vitro thrombin generation in hemophilia mouse plasma that contains only the TFPIγ isoform, which demonstrated that TFPIγ possesses anticoagulant activity. RESULTS. We report herein that, while complete TFPI gene deletion is 100% embryonic lethal, TFPIγ only mice [TFPIαβ(-/-)] are viable and reproduce. Offspring genotypes from parents with intact TFPIγ(+/+) exons and TFPIαβ(+/-) were 33 TFPIαβ(+/+), 57 TFPIαβ(+/-) and 34 TFPIαβ(-/-) at weaning. Also, TFPIγ only mice produced similar numbers of pups per litter as wild type mice. The viability of TFPIγ only mice proves endogenous mouse TFPIγ possesses substantial functional activity. TFPIα is considered to be a much more potent anticoagulant than TFPIγ. Our preliminary thrombin generation assay data suggests that the sum total anticoagulant activity of the endogenous low level (0.07 nM) of TFPIα is similar to the sum total anticoagulant activity of the endogenous high level (3.5 nM) of TFPIγ. The presence of active TFPIγ in mice, not found in man, confounds attempts to translate results from wild type mouse studies to the human condition. The extents to which TFPIγ influences the various wild type mouse models of human diseases are unknown. The presence of mouse TFPIγ also precludes our ability to properly distinguish the roles of TFPI α and β isoforms independently. Deletion of the TFPIγ exon eliminates this confounder. Fortunately, in TFPIαβ mice the levels, properties and location of TFPIα and TFPIβ in mice reflect those in man. Importantly, we can now report that our CRISPR-Cas9-generated TFPIγ KO strains - TFPIαβ mice, TFPIα only mice, and TFPIβ only mice are viable and reproduce. To illustrate the potential influence of TFPIγ in a mouse model, we used a tail vein-rebleeding assay to assess bleeding in TFPI isoform-specific mice in a FVIII KO background. Reported as mean number of clots per 15 minutes, results were: 4.8 for hemophilia controls; 25.4 for controls with FVIII replacement; 4.2 for TFPIα KOs; and 13.5 for TFPIβγ KOs. (The S.E.M. of each group was &amp;lt;1.0 ). We find that deletion of TFPIα (including in platelets!) does not affect bleeding (P&amp;gt;0.5 vs controls), whereas deletion of TFPIβ and γ improves hemostasis ( P&amp;lt;0.001 vs controls). In hemophilia mice, suppressing TFPIα was without effect. We don't expect this observation to translate to man. Rather, suppressing TFPIα in man is anticipated to enhance coagulation in hemophilia patients. (Note, multiple trials show that TFPI antibodies that inhibit all TFPI forms enhance coagulation). Moreover, in the absence of the confounding influence of TFPIγ, we anticipate suppression of TFPIα will improve coagulation and reduce bleeding in TFPIγ deleted hemophilia mice. Further studies in our TFPIγ deleted strains are necessary to determine if this prediction is correct and could influence future anti-TFPI approaches for man. CONCLUSIONS. 1.) Endogenous mouse TFPIγ possesses significant functional activity. 2.) TFPIγ confounds the extrapolation of results in wild type mouse models to human diseases. 3.) The TFPIγ KO mouse possesses a TFPIαβ profile that is similar to man. 4.) For essentially all studies of human TF-associated diseases in mouse models, it would be prudent to delete TFPIγ. Disclosures No relevant conflicts of interest to declare.

Transcription factor FOXP3: A repressor of the TFPI gene?

Single nucleotide polymorphisms (SNPs) may play an important role in the risk of certain diseases. We have previously shown that the -287T/C SNP of the tissue factor pathway inhibitor (TFPI) gene promoter region exerts differential impact on TFPI mRNA expression; the C allele being associated with higher TFPI expression, which in turn is associated with reduced risk of thrombosis. In the present study, we aimed to reveal the underlying molecular mechanisms using human embryonic kidney 293 (HEK293) and Michigan Cancer Foundation-7 (MCF7) cells that both express TFPI. Transfecting the cells with luciferase reporter gene constructs containing the TFPI promoter with either the T or the C allele of -287T/C resulted in increased luciferase activity with the C allele relative to the T allele. Three potential candidate transcription factors for binding to the two -287 alleles were predicted using the ALGGEN PROMO software, and results from electrophoretic mobility shift assays indicated that forkhead box protein 3 (FOXP3), initially identified as a functional marker of T regulator cells, bound more specifically to the T allele compared with the C allele. By chromatin immunoprecipitation assays analysis it was confirmed that FOXP3 was able to bind to the DNA region that contains the SNP. Knockdown or overexpression of FOXP3 resulted in increased or decreased TFPI levels, respectively, in both cell types. In conclusion, this study indicates that FOXP3 most likely is involved in the increased levels of TFPI observed with the -287C allele and also that FOXP3 might be a repressor for TFPI expression.

Interaction between tissue factor pathway inhibitor-2 gene polymorphisms and environmental factors associated with coronary atherosclerosis in a Chinese Han

To investigate the association of single nucleotide polymorphisms (SNPs) within tissue factor pathway inhibitor-2 (TFPI-2) gene polymorphisms and additional gene-environment interaction with coronary atherosclerosis risk. Generalized multifactor dimensionality reduction (GMDR) was used to screen the best interaction combination among 4 SNPs, smoking and alcohol drinking. Logistic regression was performed to investigate association between 4 SNPs within TFPI-2 gene and coronary atherosclerosis risk. Coronary atherosclerosis risk was significantly higher in carriers with the A allele of rs34489123 within TFPI-2 gene than those with GG genotype (GA+AA versus GG), adjusted OR (95% CI) = 1.70 (1.20-2.31), and was also higher in carriers with the G allele of rs4264 within TFPI-2 gene than those with AA genotype (AG+GG versus AA), adjusted OR (95% CI) = 1.62 (1.21-2.11). GMDR model shown the best models for gene-environment interaction were rs34489123 and smoking after adjusting the covariates, which scored 10 out of 10 for cross-validation consistency and 0.0010 for the sign test. Heavy LD was found for SNPs rs34489123 and rs59805398 (D' value was more than 0.8). Compared to control individuals, the AG haplotypes appeared to be significantly associated with increased coronary atherosclerosis risk, OR (95% CI) = 1.73 (1.22-2.32). We found that the A allele of rs34489123 and the G allele of rs4264 within TFPI-2 gene, interaction between rs34489123 and smoking and AG haplotypes were all associated with increased coronary atherosclerosis risk.

Lysine‑specific demethylase 2 contributes to the proliferation of small cell lung cancer by regulating the expression of TFPI‑2.

The present study aimed to investigate the effect of lysine-specific demethylase 2 (LSD2) in small cell lung cancer (SCLC) and explore its underlying regulatory mechanism. Cell growth was tested by MTT assay and mRNA and protein expression was determined by quantitative polymerase chain reaction (q-PCR) and western blot analysis, respectively. Chromatin immunoprecipitation (ChIP) was used to investigate the degree of H3K4me2 enrichment in the promoter region of tissue factor pathway inhibitor-2 (TFPI-2). SCLC tissues and cell lines presented significantly higher expression of LSD2 and DNA methyltransferase 3B (DNMT3B) and lower expression of TFPI-2 compared with the controls. In H1417 cells LSD2 overexpression increased the mRNA and protein expression of DNMT3B, while inhibiting the mRNA and protein expression of TFPI-2. Following transfection with short interfering (si) RNA-DNMT3B, the expression of TFPI-2 increased in H1417 cells. The results of ChIP demonstrated that compared with the controls, H3K4me1 enrichment in the TFPI-2 promoter region was to a lower degree in the H1417 cells with LSD2 overexpression and a higher degree in the H1417 cells with LSD2 silencing. MTT assays revealed that LSD2 overexpression significantly promoted the growth of H69, DMS-114 and H1417 cells, which was contradictory to the effect on LSD2 silencing. Compared with the LSD2 overexpression cells, SCLC cells with simultaneous overexpression of LSD2 and TFPI-2 demonstrated a decreased proliferation. These results suggest that LSD2 achieves a promoting effect on SCLC by indirectly regulating TFPI-2 expression through the mediation of DNMT3B expression or through the regulation of the demethylation of H3K4me1 in the promoter region of the TFPI-2 gene.

Abstract 410: Statin Use May Alleviate Coagulation- and Inflammation-associated Gene Expression in Circulating Classical Monocytes in Women With Chronic HIV-1 Infection

During virally-suppressed chronic human immunodeficiency virus (HIV) infection, persistent inflammation contributes to the development of cardiovascular disease, a major comorbidity in people living with HIV. Monocytes play a key role in atherosclerotic plaque development, inflammation, and stability, but their contribution to the CVD under viral suppression in people living with HIV remains unknown. Here, we investigated the transcriptomes of classical (CD14 ++ CD16 - ) blood monocytes from 92 women with and without chronic HIV infection and subclinical cardiovascular disease (sCVD), defined as the presence of focal carotid artery plaque, from the Women's Interagency HIV Study (WIHS). Differential gene expression, based on four two-way comparisons among participant groups (HIV-sCVD-, HIV+sCVD-, HIV-sCVD+, and HIV+sCVD+, 23 subjects each), identified large pro-inflammatory gene signatures for both sCVD and virally-suppressed HIV. These findings were further corroborated by Ingenuity Pathway Analysis. We found that classical monocytes persistently express common CVD-related markers of inflammation, including IL6, IL1β, and IL12B; overlapping with many transcripts identified in sCVD+ participants. In comorbid disease (HIV+sCVD+), those reporting statin use showed dramatically reduced pro-coagulant tissue factor (F3) and tissue factor pathway inhibitor (TFPI and TFPI2) gene expression to a level comparable with healthy participants. Cytokine expression profiles associated with the tissue factor pathway were also modified in participants on statins, suggesting that statins may benefit women with chronic HIV infection by limiting pro-coagulation and inflammation pathways in classical monocytes.

Significant genetic association of a functional TFPI variant with circulating fibrinogen levels and coronary artery disease.

The tissue factor pathway inhibitor (TFPI) gene encodes a protease inhibitor with a critical role in regulation of blood coagulation. Some genomic variants in TFPI were previously associated with plasma TFPI levels, however, it remains to be further determined whether TFPI variants are associated with other coagulation factors. In this study, we carried out a large population-based study with 2313 study subjects for blood coagulation data, including fibrinogen levels, prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT). We identified significant association of TFPI variant rs10931292 (a functional promoter variant with reduced transactivation) with increased plasma fibrinogen levels (P=0.017 under a recessive model), but not with PT, APTT or TT (P>0.05). Using a large case-control association study population with 4479 CAD patients and 3628 controls, we identified significant association between rs10931292 and CAD under a recessive model (OR 1.23, P=0.005). For the first time, we show that a TFPI variant is significantly associated with fibrinogen levels and risk of CAD. Our finding contributes significantly to the elucidation of the genetic basis and biological pathways responsible for fibrinogen levels and development of CAD.

Epigallocatechin gallate inhibits the growth and promotes the apoptosis of bladder cancer cells.

Epigallocatechin gallate (EGCG) has been revealed to inhibit the proliferation and induce the apoptosis of several types of tumor, in addition to inhibiting DNA methyltransferase activity, leading to CpG demethylation. Tissue factor pathway inhibitor 2 (TFPI-2) expression is downregulated in bladder cancer. The present study revealed that this downregulation was partly due to hypermethylation of the TFPI-2 gene promoter, which was decreased by EGCG treatment. In addition, the present study demonstrated that EGCG could inhibit the viability and invasion, and induce the apoptosis, of bladder cancer T24 cells. Furthermore, western blot analysis and reverse transcription-quantitative polymerase chain reaction analyses demonstrated that EGCG could upregulate the expression of TFPI-2. These results suggest that EGCG inhibits the growth and induces the apoptosis of bladder cancer cells through restoring TFPI-2 expression. Thus, EGCG is a potential therapeutic candidate for the treatment of bladder cancer.

A novel hypoxia response element regulates oxygen-related repression of tissue factor pathway inhibitor in the breast cancer cell line MCF-7

BackgroundHypoxia is one of the most pervasive physiological stresses in solid tumors. We have previously demonstrated that tissue factor (TF) pathway inhibitor (TFPI) expression was transcriptionally repressed by the activation of hypoxia inducible factor (HIF)-1α under hypoxic conditions. However, the role of HIF-2α, also known as endothelial PAS domain-containing protein 1 (EPAS1), on TFPI expression remains unclear. AimTo explore the role of HIF-2α/EPAS1 in the regulation of TFPI expression under hypoxia in breast cancer cells. Methods and resultsQuantitative RT-PCR showed that total TFPI mRNA and protein levels were decreased by the overexpression of HIF-2α/EPAS1 in MCF7 cells. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay indicated a HIF-2α/EPAS1 responsive region located in the TFPI promoter region at −170 to +21 relative to the transcriptional start site. Subsequent mutagenesis demonstrated a functional hypoxia response element (HRE) 5′-AAACAGGA-3′ for HIF-2α/EPAS1 within the TFPI promoter located at −45 to −38. In breast cancer patients, a positive correlation between HIF-2α/EPAS1 and total TFPI mRNA expression was observed by using gene expression analysis. ConclusionsThis study provides evidence that HIF-2α/EPAS1 is involved in the regulation of TFPI gene expression in breast cancer cells, suggesting that the activation of coagulation and the increased risk of thrombosis observed in breast cancer patients may correlate with local hypoxic regulation of coagulation factors and their inhibitors.

Effect of hypoxia on tissue factor pathway inhibitor expression in breast cancer

ESSENTIALS: A hypoxic microenvironment is a common feature of tumors that may influence activation of coagulation. MCF-7 and SK-BR-3 breast cancer cells and breast cancer tissue samples were used. The results showed transcriptional repression of tissue factor pathway inhibitor expression in hypoxia. Hypoxia-inducible factor 1α may be a target for the therapy of cancer-related coagulation and thrombosis. Activation of coagulation is a common finding in patients with cancer, and is associated with an increased risk of venous thrombosis. As a hypoxic microenvironment is a common feature of solid tumors, we investigated the role of hypoxia in the regulation of tissue factor (TF) pathway inhibitor (TFPI) expression in breast cancer. To explore the transcriptional regulation of TFPI by hypoxia-inducible factor (HIF)-1α in breast cancer cells and their correlation in breast cancer tissues. MCF-7 and SK-BR-3 breast cancer cells were cultured in 1% oxygen or treated with cobalt chloride (CoCl2 ) to mimic hypoxia. Time-dependent and dose-dependent downregulation of TFPI mRNA (quantitative RT-PCR) and of free TFPI protein (ELISA) were observed in hypoxia. Western blotting showed parallel increases in the levels of HIF-1α protein and TF. HIF-1α inhibitor abolished or attenuated the hypoxia-induced downregulation of TFPI. Luciferase reporter assay showed that both hypoxia and HIF-1α overexpression caused strong repression of TFPI promoter activity. Subsequent chromatin immunoprecipitation and mutagenesis analysis demonstrated a functional hypoxia response element within the TFPI promoter, located at -1065 to -1060 relative to the transcriptional start point. In breast cancer tissue samples, gene expression analyses showed a positive correlation between the mRNA expression of TFPI and that of HIF-1α. This study demonstrates that HIF-1α is involved in the transcriptional regulation of the TFPI gene, and suggests that a hypoxic microenvironment inside a breast tumor may induce a procoagulant state in breast cancer patients.

Methylation of tissue factor pathway inhibitor 2 as a prognostic biomarker for hepatocellular carcinoma after hepatectomy.

Methylation of tissue factor pathway inhibitor 2 (TFPI2) gene has been detected in hepatocellular carcinoma (HCC). However, the clinicopathologcial significance and prognostic value of TFPI2 methylation in HCC remains largely unknown. This study aimed to investigate the prognostic value of TFPI2 methylation in HCC after hepatectomy. Methylation status of TFPI2 gene was examined in 178 surgical specimens of HCC and 20 normal liver samples using methylation-specific polymerase chain reaction. Methylation of TFPI2 gene was detected in 44.9% (80 of 178) of primary HCC samples, 10.7% (19 of 178) of the corresponding non-tumorous liver samples, and 5.0% (1/20) of the normal liver samples. The mRNA concentrations of TFPI2 in primary HCC tissues were significantly lower than those in corresponding non-tumorous liver tissues and those in normal liver tissues. TFPI2 methylation was significantly associated with higher TNM stage. Patients with TFPI2 methylation demonstrated a significantly poorer prognosis than those without TFPI2 methylation for both overall survival and disease-free survival (P < 0.001, respectively). Multivariate analyses confirmed that TFPI2 methylation was an independent prognostic factor for both overall survival (P = 0.002) and disease-free survival (P = 0.000) in HCC after hepatectomy. Moreover, TFPI2 methylation was found to be the only independent predictor for early tumor recurrence of HCC after resection based on multivariate analysis (P = 0.002). Methylation of TFPI2 predicts high risk of advanced tumor stage, early tumor recurrence, and poor prognosis, and it could be a potential prognostic biomarker in patients with HCC after hepatectomy.

Effects of glycosaminoglycan from Mactra veneriformis on the protein C system and expression of relevant factors in human umbilical vein endothelial cells.

The aim of this study was to examine whether glycosaminoglycan (GAG) from Mactra veneriformis had an impact on the protein C (PC) system in rats and expression of relevant factors in human umbilical vein endothelial cells. Isotonic saline, heparin sodium (4 mg/kg), and GAG (1, 4, and 16 mg/kg) were administered to Wistar rats through caudal vein at the total volume of 0.5 ml for each injection, and the anticoagulant blood was prepared. The activities of PC and protein S, and the concentrations of activated protein C inhibitor (APCI) and thrombomodulin were detected according to the kit instructions. The expression levels of tissue factor, tissue factor pathway inhibitor and thrombomodulin genes in human umbilical vein endothelial cells were detected by the reverse transcription polymerase chain reaction method. GAG could significantly reduce the activity of PC (P < 0.05, P < 0.01) and slightly increase the concentration of APCI, and high concentration of GAG could significantly decrease the activity of protein S (P < 0.05) and significantly increase the concentration of thrombomodulin (P < 0.05). GAG could significantly downregulate the expression level of tissue factor gene (P < 0.05, P < 0.01) and significantly upregulate the expression levels of tissue factor pathway inhibitor and thrombomodulin genes (P < 0.05, P < 0.01). GAG showed the inhibitory effect on PC system in vivo. GAG might play an important role in the resistance to extrinsic coagulation pathway.

Lethality of Mouse Tissue Factor Pathway Inhibitor Gene Disruption Is Rescued By Transgenic Human Tissue Factor Pathway Inhibitor Knock in

Background: Tissue factor pathway inhibitor (TFPI) is a key regulator of the extrinsic coagulation pathway. It inhibits FXa generation by forming a quaternary complex containing factor VIIa (FVIIa), tissue factor (TF), factor Xa (FXa), and TFPI. Two TFPI isoforms, TFPI alpha (TFPI a) and TFPI beta (TFPI b), have been identified, which differ in their C-terminal part due to alternative mRNA splicing events. TFPI a consists of three Kunitz domains (KD), while TFPI b contains two KDs and a C terminal GPI anchor linking the protein to endothelial cell surface. Deletion of the first Kunitz domain of TFPI, which is present in TFPI a and TFPI b in mice is known to be incompatible with viability due to intrauterine lethality (Huang et al., 1997).Aim: To generate transgenic humanized TFPI mice in which mouse (m)-TFPI is entirely replaced by human (hu)-TFPI, in order to facilitate analysis of specific anti hu-TFPI antagonists without interference from m-TFPI.Methods: Integration of the targeting vector, consisting of the m TFPI signal sequence, followed by the human TFPI cDNA and subsequent breeding analysis, was followed by genomic PCR. A sophisticated breeding strategy was used to entirely delete m-TFPI exon 4, which encodes KD1, in humanized transgenic mice. Expression of hu-/m-TFPI a and b mRNAs was analyzed by reverse transcription, cloning, and sequencing of the obtained DNA fragments. Protein levels of hu- and m-TFPI in plasma of transgenic and wild-type (wt) mice were analyzed using species specific ELISAs. Immunoprecipitation experiments in plasma and various mouse tissues are being performed to obtain more information on the presence and distribution of the hu-TFPI protein in transgenic mice.Results: Homozygous humanized TFPI mice were viable and exhibited no obvious abnormalities. Animals showed normal litter size with equal numbers of female and male pups. Genomic PCRs revealed proper integration of the targeting vector into the mouse chromosome and the homozygous status with the expected deletion of m-TFPI exon 4. Expression analyses of humanized TFPI mice on mRNA level demonstrated the absence of full length m-TFPI a and the presence of the humanized TFPI mRNA. Alternative spliced m-TFPI b messages lacking exons three and four were identified, likely leading to a nonfunctional protein. Full length hu-TFPI a mRNA was detected in various tissues in the humanized TFPI mice. The TFPI protein level in plasma from humanized mice was below the detection limit of the ELISA and at least ~300 fold below that for wt mice.Conclusion: Low levels of hu-TFPI may compensate the function of m-TFPI in vivo and circumvent embryonic lethality. Furthermore, we established a new mouse model which allows the regulation of physiologic and pathologic pathways to be assessed at TFPI plasma concentrations below the limit of detection. DisclosuresHoellriegl:Baxalta Innovations GmbH: Employment. Scheiflinger:Baxalta Innovations GmbH: Employment.

Generation and Initial Characterization of Three Murine Models with Altered Platelet TFPI Expression

Introduction: Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein that inhibits tissue factor and prothrombinase during early stages of the procoagulant response. It is produced in alternatively spliced isoforms. In humans, TFPIα is within platelets and released upon activation and also is present in plasma, while TFPIβ is bound to the endothelial surface. Mice differ from humans in that they do not have TFPIα in plasma, making them an optimal model system for study of the function of platelet TFPIα. Studies of murine hematopoietic cell TFPI using fetal liver transplantation models have found that platelet TFPI dampens platelet accumulation in a growing thrombus following vascular injury and alters bleeding severity in mice with hemophilia. However, results of these studies are potentially confounded by altered TFPI expression on other hematopoietic cells, such as monocytes. To better understand the function of platelet TFPI, murine models of total TFPIα deletion (TFPIα KO), of platelet-specific TFPIα deletion (pTFPIα KO) and platelet-specific TFPIα over expression (pTFPIα high) were produced and characterized.Methods: A TFPIα deleted mouse was generated by inserting a stop codon within exon 10 of the TFPI gene effectively blocking translation of the highly conserved basic C-terminal region of TFPIα, but not altering TFPIβ. The PF4-Cre transgene was bred into the floxed-TFPI(K1) mouse to produce mice specifically lacking platelet TFPIα. A transgenic mouse over expressing platelet TFPIα under control of the GP1b promoter was produced. Mice were characterized for platelet and plasma TFPI antigen by ELISA, for activity by TF-FVIIa inhibition and whole blood platelet aggregation, and for bleeding by tail clip.Results: All 3 strains were viable and reproduced. Platelet TFPI antigen was decreased approximately 25-fold in the TFPIα KO and pTFPIα KO mice and elevated approximately 5-fold in the pTFPIα high mice with corresponding changes in TF-FVIIa inhibitory activity. Plasma TFPI antigen was no different between pTFPIα KO (20.6±4.4 nM) and wild type (21.5±6.2 nM) littermates. Plasma thrombin-antithrombin complex levels were not elevated in any strain. In tail clip bleeding time assays, there was no difference between pTFPIα KO (0.23±0.16 mg/ml Hb) and wild type (0.23±0.37 mg/ml Hb), but there was a trend towards increased blood loss in pTFPIα high mice (1.25±1.8 mg/ml Hb). The pTFPIα high mice also had altered whole blood platelet aggregation that was evident primarily in a prolonged time for initiation of aggregation with 6.50uM ADP (81±12 sec vs. 6.1±5.6 sec for control) or 0.8ug/ml collagen (35±17 sec vs. 16±21 sec for control). Finally, the pTFPIα high transgene was bred into TFPI heterozygous mice which were then bred to determine if over expression of platelet TFPIα would rescue the TFPI null embryonic lethal phenotype. However, no live TFPI null pups were observed following genotyping of over 50 pups from these matings.Conclusions: Three new mouse models of altered TFPI expression have been made providing valuable tools to study the role of platelet TFPI in bleeding and clotting disorders. Plasma TFPI is unchanged in mice lacking platelet TFPI demonstrating that platelet TFPI does not contribute to plasma TFPI in mice. Platelet TFPIα antigen and activity correlate in all three models. Deletion of the basic C-terminal region in TFPIα in the TFPIα KO mice produces a TFPIα hypomorphic mouse, with only trace amounts of truncated TFPIα present in platelets. This lack of TFPIα does not produce a generalized procoagulant state. The 5-fold excess TFPIα antigen in the pTFPIα high mice substantially delays the onset of platelet aggregation in whole blood assays initiated with collagen or ADP. Results from tail clip bleeding assays suggest that over expression of platelet TFPIα may prolong bleeding in adult mice, but it is not sufficient to rescue the embryonic lethality of TFPI null mice. DisclosuresMast:Novo Nordisk: Honoraria, Research Funding.

Tissue Factor Pathway Inhibitor-Coated Stents Inhibit Restenosis in a Rabbit Carotid Artery Model.

Our aim was to study the efficacy and safety of tissue factor pathway inhibitor (TFPI)-coated stents in inhibiting restenosis in a rabbit carotid artery model. Subculture was conducted in aorta smooth muscle cell, which was taken from male Wistar rat, and the 3-5-generation cells were taken for plasmid transfection and cytotoxicity experiment. TFPI microspheres were made of a TFPI plasmid which was enwrapped by poly-l-glutamic acid (PLGA). TFPI-coated stents (n = 7) and bare metal stents (n = 6) were implanted into prepared carotid artery stenosis model of New Zealand white rabbits. The transfection efficiency of TFPI gene and its influence on animal tissue, restenosis inhibition, and biochemical indicator were observed. Tissue factor pathway inhibitor microspheres can transfect successfully into cells, and present no cytotoxicity. Autopsy results showed no pathological changes in liver and spleen of rabbits after implanting TFPI-coated stents. TFPI gene could transfect and express successfully in vessel wall cells, and thrombus was found in some lumens of bare metal stents group after 7 day, while no such thrombus was observed in coated stents group. Degree of hyperplasia of coronary endarterectomy in bare metal stents group was evidently higher than those in coated stents group. Obvious stent restenosis was discovered only in one case in bare metal stents group (diameter stenosis ≥50%). However, no case in coated stents group showed with stent restenosis. Tissue factor pathway inhibitor-coated stents could successfully transfect TFPI gene into vessel wall cells, thereby inhibiting restenosis without obvious side effect in the rabbit carotid artery model.

Tissue factor pathway inhibitor gene transfer prevents vascular smooth muscle cell proliferation by interfering with the MCP-3/CCR2 pathway

Increased vascular smooth muscle cell (VSMC) proliferation substantially contributes to the pathogenesis of atherosclerosis and intimal hyperplasia after vascular injury. The importance of inflammation in VSMC proliferation is now being recognized. Preventing the inflammatory response is one therapeutic strategy that can be used to inhibit atherosclerosis in the clinic. The present study, using RNA interference and gene transfer techniques, was conducted to investigate the effect of monocyte chemotactic protein-3 (MCP-3) on VSMC proliferation that is a result of TNF-α stimulation, and whether overexpression of the tissue factor pathway inhibitor (TFPI) gene could prevent VSMC proliferation by blocking the MCP-3/CC chemokine receptor 2 (CCR2) pathway. Mouse VSMCs were infected in vitro with recombinant adenoviruses containing either mouse MCP-3-shRNA (Ad-MCP-3-shRNA), the TFPI gene (Ad-TFPI), or the negative control, which was shRNA encoding the sequence for EGFP (Ad-EGFP) or DMEM only. The cells were then stimulated with TNF-α for different time periods on the third day after gene transfer. The data show that VSMC proliferation in the Ad-MCP-3-shRNA and Ad-TFPI groups was markedly decreased using BrdU ELISA and MTT assays; MCP-3-shRNA and TFPI inhibited the expression of MCP-3 and CCR2 after long-term stimulation and inhibited the phosphorylation of ERK1/2 and AKT after short-term stimulation, as shown by ELISA and western blot analysis. This study provides convincing evidence that clarifies the effect of the proinflammatory factor MCP-3 in promoting VSMC proliferation. Our data also show, for the first time, that TFPI has an anti-proliferative role in TNF-α stimulated-VSMCs at least partly by interfering with the MCP-3/CCR2 pathway and then via suppression of the ERK1/2 and PI3K/AKT signaling pathways. We conclude that TFPI gene transfer may be a safe and effective therapeutic tool for treating atherosclerosis and intimal hyperplasia.