Plasma Tissue Factor Pathway Inhibitor Levels 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

Thrombin generation abnormalities in commonly encountered platelet function disorders.

IntroductionStudies of thrombin generation (TG) with platelet‐rich plasma (PRP) and platelet‐poor plasma (PPP) have provided insights on bleeding disorders. We studied TG for a cohort with commonly encountered platelet function disorders (PFD).MethodsParticipants included 40 controls and 31 with PFD due to: nonsyndromic dense granule (DG) deficiency (PFD‐DGD, n = 9), RUNX1 haploinsufficiency (n = 6) and aggregation defects from other, uncharacterized causes (n = 16). TG was tested with PRP and PPP samples. As DG store ADP and polyphosphate that enhance platelet‐dependent TG, PFD‐DGD PRP TG was tested for correction with ADP, polyphosphate and combined additives. Tissue factor pathway inhibitor (TFPI), platelet factor V (FV), and platelet TFPI and ANO6 transcript levels were also evaluated. Findings were tested for associations with TG endpoints and bleeding.ResultsPFD samples had impaired PRP TG, but also impaired PPP TG, with strong associations between their PRP and PPP TG endpoints (P ≤ .005). PFD‐DGD PRP TG endpoints showed associations to PPP TG endpoints but not to DG counts, and were improved, but not fully corrected, by adding polyphosphate and agonists. PFD participants had increased plasma TFPI and reduced platelet TFPI (P ≤ .02) but normal levels of platelet FV, and platelet TFPI and ANO6 transcripts levels. PFD plasma TFPI levels showed significant association to several PPP TG endpoints (P ≤ .04). Several PFD PRP TG endpoints showed significant associations to bleeding symptoms, including wound healing problems and prolonged bleeding from minor cuts (P ≤ .04).ConclusionTG is impaired in commonly encountered PFD, with their PRP TG findings showing interesting associations to symptoms.

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.

Biomarkers of Endothelial Function in Sepsis-Associated Disseminated Intravascular Coagulation

Introduction: Sepsis-associated disseminated intravascular coagulation (DIC) is a complex clinical syndrome with high mortality. The pathogenesis of DIC involves significant interactions between multiple pathophysiologic processes including coagulation, inflammation, infection response, platelets and endothelial dysfunction. The role of the endothelium in DIC is multifaceted and may contribute significantly to its severity and outcome. In sepsis, increased inflammation can lead to activation of the endothelium and the development of a procoagulant phenotype, characterized in part by elevated levels of cell surface tissue factor (TF). The endothelial response to inflammation is also characterized by the production of other factors that may be descriptive of the status of the endothelium, including endocan. The endothelium is also largely responsible for the production of endogenous anticoagulants, such as Tissue Factor Pathway Inhibitor (TFPI) and Protein C. The derangement of the endogenous anticoagulant system is critical to the pathogenesis of DIC and may also be reflective of the state of the endothelium. Investigation of factors that may prove damaging to the endothelium, including extracellular nuclear material such as nucleosomes and the protein High Mobility Group Box 1 (HMGB-1) may also provide insight into endothelial status and associated patient prognosis. The purpose of this study was to quantify measures of endothelial function, including markers of activation (TF and endocan), endogenous anticoagulants (TFPI and Protein C), and damage-associated factors (nucleosomes and HMGB-1) in the plasma of patients with sepsis and DIC and to determine the relationship of these factors with severity of illness and outcome.Materials and Methods: Citrated plasma samples were prospectively collected from 103 patients with sepsis and suspected DIC at ICU admission (day 0) and again serially on ICU days 4 and 8 under an IRB approved protocol. DIC score was determined using the ISTH scoring algorithm (e.g. platelet count, PT/INR, fibrinogen, and D-Dimer). Plasma from healthy individuals was purchased from a commercial laboratory (George King Biomedical, Overland, KS). Plasma levels of endocan, TF, TFPI, nucleosomes, and HMGB-1 were evaluated using commercially available ELISA kits. Protein C levels were measured using a functional clot-based assay.Results: In the DIC patients, endocan, TF, TFPI, nucleosomes, and HMGB-1 were significantly elevated compared to normal on ICU days 0, 4, and 8. Protein C was significantly reduced compared to normal at all time points.Patients were stratified into groups of sepsis and DIC based on the ISTH DIC score, and levels of markers were compared between groups. Markers reflective of overall endothelial activation (endocan and TF) showed no variation based on DIC score. Protein C showed significant variation based on DIC score, with a marked drop in Protein C levels with increasing severity of illness; however, TFPI showed no variation. Increasing levels of factors associated with endothelial damage (nucleosomes and HMGB-1) were observed with increasing severity of illness. Results are shown in Table 1.Differences in biomarker levels between survivors and non-survivors were also evaluated. Endocan (p = 0.0252) and HMGB-1 (p = 0.0306) showed significant increases in non-survivors compared to survivors while Protein C showed a significant reduction (p = 0.0093) in non-survivors compared to survivors. TF, TFPI, and nucleosomes showed no variation based on survival.Conclusion: Overall, levels of markers of endothelial activation, endogenous anticoagulants, and factors contributing to endothelial damage were elevated in the plasma of patients with sepsis-associated DIC compared to the healthy population. In particular, factors associated with endothelial damage, namely the toxic nuclear factors nucleosomes and HMGB-1, showed elevated levels with increasing severity of coagulopathy, suggesting that the endothelial damage caused by these factors may contribute to the development of coagulation dysfunction. Levels of endocan, Protein C, and HMGB-1 also showed significant association with mortality, underscoring the relevance of all aspects of endothelial physiology to patient outcome in DIC. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tissue factor pathway inhibitor upregulates CXCR7 expression and enhances CXCL12-mediated migration in chronic lymphocytic leukemia

The infiltration of chronic lymphocytic leukemia (CLL) cells into lymphoid organs correlates with disease severity. CXCL12 is a key chemotactic factor for the trafficking of CLL. Tissue factor pathway inhibitor (TFPI) is a serine protease inhibitor and plays a role in CXCL12-mediated hematopoietic stem cell homing. We aim to explore the role of TFPI in CXCL12-mediated migration of CLL cells. In this study, plasma TFPI concentrations were measured by ELISA. CLL cells were isolated from patients and used for trans-endothelial migration (TEM) assays. Quantitative RT-PCR and Western blotting were used to detect the expression of CXCR7, CXCR4 and β-catenin. Immunofluorescence and co-immunoprecipitation was used to detect the binding of TFPI and glypican-3 (GPC3). We found that plasma TFPI levels in CLL patients were higher than in healthy controls, particularly in the patients with advanced disease. TFPI enhanced CXCL12-mediated TEM of CLL cells by increasing the expression of the CXCL12 receptor CXCR7, but not of the CXCL12 receptor CXCR4. The effect of TFPI on TEM was abolished by the CXCR7 inhibitor, CCX771, while the CXCR4 inhibitor AMD3100 strongly increased TEM. TFPI co-localized with GPC3 on the cell surface. An antibody to GPC3, HS20, decreased CXCR7 expression and abolished the effect of TFPI on TEM. TFPI activated β-catenin and the Wnt/β-catenin inhibitor IWP4 repressed the effect of TFPI on CXCR7 expression and TEM. We conclude that TFPI may contribute to organ infiltration in CLL patients.

Changes of Tissue Factors and Tissue Factor Pathway Inhibitors in the Acute Phase of Cardiogenic Stroke in Patients With Atrial Fibrillation

Abstract Background To investigate changes of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in the acute phase of cardiogenic stroke in patients with atrial fibrillation (AF). Methods A total of 152 patients with nonvalvular persistent AF, 103 cardiogenic stroke patients with nonvalvular persistent AF, and 126 cases with noncardiogenic stroke were enrolled. Non-AF patients (n = 138) without stroke served as controls. The clinical data, biochemical indicators, and plasma levels of von Willebrand factor (vWF), TF, and TFPI were determined. Results Compared with controls, the plasma levels of vWF and TF were significantly higher, while TFPI and TFPI/TF ratio were lower in noncardiogenic stroke patients (all P &amp;lt; 0.05). Compared with cardiogenic stroke patients, noncardiogenic stroke subjects showed significantly higher plasma vWF and TF levels while TFPI levels and TFPI/TF ratio were significantly lower (all P &amp;lt; 0.05). Compared with AF patients, plasma levels of vWF, TF, and TFPI were higher while TFPI/TF ratios were decreased in the cardiogenic ischemic stroke subjects (all P &amp;lt; 0.05). Logistic regression analysis showed that a lower TFPI/TF ratio was an independent predictor of cardiogenic stroke in patients with nonvalvular AF (odds ratio = 3.78, 95% confidence interval: 1.86–7.31). Conclusions During the acute stage of stroke, TFPI/TF ratio is decreased in both cardiogenic and noncardiogenic stroke patients while plasma TFPI levels show divergent changes.

87 Evaluating Endothelial Dysfunction in Thermally-injured Patients with syndecan-1, Tissue Factor Pathway Inhibitor, and Thrombomodulin as Predictors of Mortality

Abstract Introduction Endotheliopathy in burn patients is largely uncharacterized. Syndecan-1 (SDC-1), thrombomodulin (TM), and tissue factor pathway inhibitor (TFPI) are components of the vascular endothelial glycocalyx. Proteolytic cleavage of these moieties may yield biomarkers for endothelial damage. The aim of this study is to evaluate endotheliopathy after burn injury by monitoring plasma levels of these biomarkers over time to investigate potential relationship to mortality. Methods Burn injured patients presenting to a regional burn center from 2012 to 2017 were prospectively enrolled. Blood samples were collected at 0, 2, 4, 8, 12, 24, 36, 48, 60, and 72 hours from admission. Plasma SDC-1, TM, and TFPI levels were quantified by ELISA. Demographic data and injury characteristics were obtained from the medical chart. Patients with concomitant inhalation injury, trauma, or &amp;lt; 10% total body surface area (TBSA) burns were excluded. Statistical analysis was performed using mixed-effect models with Sidak’s correction for multiple comparisons. Significance was set at p =0.05. Data are presented as mean ± standard deviation. Results A cohort of 22 patients was identified with an average age of 45±14 years, TBSA of 30±15%, with 6 patients who died from their injuries. The deceased group was older (59±13 vs. 40±10 years, p = 0.01), and there was no significant difference in burn size. Mean SDC-1 levels were higher in the deceased group at all time points (p=0.0004) and this difference was significant at hour 12 (106±11 vs. 41±31 ng/mL, p = 0.0002), hour 24 (160±39 vs. 35±20 ng/mL, p = 0.04) and hour 72 (100±3 vs. 35±38 ng/mL, p = 0.01). Mean soluble TM levels were higher in the deceased group after hour 12 (p = 0.04) and there was a trend towards higher TFPI levels after hour 12 in the deceased group. Conclusions Biomarkers are elevated in patients following burn injury who die, when inhalation injury and trauma are excluded. Given equivalent TBSA, older patients appear more sensitive to thermally induced glycocalyx degradation. SDC-1 shows the greatest promise as a prognostic indicator as levels tend to be higher among deceased patients on admission and are significantly higher as early as hour 12. Applicability of Research to Practice Reliable assessment of the patient’s endothelial damage may hold predictive value for clinicians and could assist in clinical decision making. Further research must investigate endotheliopathy in burn patients.

The effects of citrate dialysate in hemodialysis on polymorphonuclear elastase interaction with tissue factor and its inhibitor.

This study aimed to investigate whether hemodialysis (HD) affects tissue factor (TF), tissue factor pathway inhibitor (TFPI), and polymorphonuclear elastase (PMNE) in endstage renal disease (ESRD) patients when eliminating the effects of heparin. Also, to explore the interaction of TF, TFPI, and PMNE throughout a single HD session. We enrolled 57 ESRD patients who had undergone hemodialysis for >3 months as an experimental group. Plasma levels of TF, TFPI and PMNE were measured by ELISA in 24 ERSD patients on intermittent HD using low-molecular-weight heparin (LMWH) as anticoagulation (LMWH group) and 33 ESRD patients using citrate as anticoagulation (citrate group) at the start and at 1, 2 and 5 h of the HD session. Meanwhile,28 ESRD patients not on dialysis were enrolled as a control group and fasting venous blood samples were taken in the morning. Compared with the control group, the plasma TFPI levels of the LMWH group and the citrate group were significantly higher (P=0.000, P=0.002, respectively) under baseline conditions as well as the plasma PMNE levels (P=0.001, P=0.02, respectively), whereas TF showed no difference (P=0.186). During HD with citrate, plasma TFPI decreased slightly (P=0.012) and PMNE increased significantly (P=0.008) at 1 h. The plasma TFPI levels of the citrate group correlate with PMNE at 2 and 5 h (P=0.001, P=0.008, respectively). ESRD patients on HD have significantly higher TFPI and PMNE levels compared to patients not on HD under baseline conditions, while TF levels were similar between the three groups. TFPI and PMNE are differently regulated, but the plasma levels correlated during HD in the citrate group. It might be possible that PMNE plays a role in anticoagulative activity through TFPI.

Levels of tissue factor pathway inhibitor in patients with inflammatory bowel disease.

INTRODUCTION Endothelial dysfunction has been reported to be involved in the pathogenesis of inflammatory bowel disease (IBD) and concomitant thromboembolic complications. Inflammation stimulates the expression of tissue factor and tissue factor pathway inhibitor (TFPI) by endothelial cells. OBJECTIVES This study assessed the relationship between TFPI levels and disease activity in patients with IBD. PATIENTS AND METHODS A total of 50 consecutive adult patients with ulcerative colitis (UC), 50 patients with Crohn disease (CD), and 50 healthy controls were enrolled to the study. Plasma levels of total TFPI, free TFPI, and von Willebrand factor were measured. Associations among these levels, disease activity, and inflammatory marker levels were assessed. RESULTS Total TFPI levels were higher in patients with IBD (median, 68.5 [IQR, 60.2-80.1] ng/ml) than in controls (median, 61.1 ng/ml [IQR, 54.3-74.2]; P = 0.01). Free TFPI levels were higher in patients with active UC (median, 12.8 ng/ml [IQR, 11.1-15.4]), inactive UC (median, 9.9 ng/ml [IQR, 7.3-11.5]), active CD (median, 11.7 [IQR, 9.7-14.4] ng/ml), and inactive CD (median, 9.7 ng/ml [IQR, 8.6-11.6]) than in controls (median, 5.5 ng/ml [IQR, 4.3-7.2]; P <0.001). In the CD and UC groups, free TFPI levels correlated with the levels of inflammatory markers and disease activity. The von Willebrand factor level was higher in patients with UC (median, 143.4 IU/dl [IQR, 115.5-170.4]) and those with CD (median, 151.8 IU/dl [IQR, 112.8-189.4]) than in controls (85.1 IU/dl [IQR, 77.1-101.5]; P <0.001 for both comparisons). CONCLUSIONS The anticoagulant TFPI pathway is activated during remissions and flares in patients with IBD. The free TFPI level correlates with biochemical markers of inflammation and disease activity.

Clinical Significance of Detecting Tissue Factor and Tissue Factor Pathway Inhibitor in Thalassemia Patients

To investigate the changes of E-selectin, thrombin-antithrombin complex（TAT）, prothrombin fragment 1+2（F1+2）, tissue factor（TF）and tissue factor pathway inhibitor（TFPI）before and one year after splenectomy in thalassemia patients. A total of 30 thalassemia patients undergoing electric laparoscopic splenectomy and 30 normal controls（NC） were enrolled in the study．Plasma levels of E-selectin, TAT, F1+2, TF and TFPI were detected by enzyme-linked immuno sorbent assay（ELISA）. One year after splenectomy，the plasma concentrations of E-selectin, TAT, F1+2, TF, TFPI were significantly higher than those in both preoperative and NC groups．Levels of E-selectin, TAT, F1+2 before splenectomy were significantly higher than those in NC groups. In addition, there was a positive correlation between plasma TF and TFPI level before and after splenectomy, and the levels of TF and TFPI positively correlated with TAT and F1+2, respectively. After splenectomy, the platelet count increases, the activity of endothelial cells is injured, the procoagulant factor increases, the blood is in procoagulant state, the TF/TFPI shows an importent role in the thrombosis of thalassemia patieints after splenectomy and may be used to evaluate the prothrombotic state of this diasease.

Inhibition of Tissue Factor Pathway Inhibitor (TFPI) as a Treatment for Haemophilia: Rationale with Focus on Concizumab.

Replacement therapy with missing factor (F) VIII or IX in haemophilia patients for bleed management and preventative treatment or prophylaxis is standard of care. Restoration of thrombin generation through novel mechanisms has become the focus of innovation to overcome limitations imposed by protein replacement therapy. Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type serine protease inhibitor that regulates tissue factor (TF)-induced coagulation through a FXa-dependent feedback inhibition of the TF.FVIIa complex in plasma and on endothelial surfaces. Concizumab is a monoclonal, humanised antibody, specific for the second Kunitz domain of TFPI that binds and inhibits FXa, abolishing the inhibitory effect of TFPI. Concizumab restored thrombin generation in FVIII and FIX deficient plasmas and decreased blood loss in a rabbit haemophilia model. Phase 1 single and multiple dose escalation studies in haemophilia patients demonstrated a dose dependent decrease in TFPI levels and a pro-coagulant effect with increasing d-dimers and prothrombin fragment 1 + 2. A dose dependent increase in peak thrombin and endogenous thrombin potential was observed with values in the normal range when plasma TFPI levels were nearly undetectable. A few haemophilia patients in the highest dose cohorts with complete inhibition of plasma TFPI showed a decreased fibrinogen concentration with normal levels of anti-thrombin and platelets and no evidence of thrombosis. Pharmacokinetic parameters were influenced by binding to the target (TFPI), demonstrating target mediated drug disposition. A trend towards decreasing bleeding tendency was observed and this preventative effect is being studied in Phase 2 studies with additional data gathered to improve our understanding of the therapeutic window and potential for thrombosis.

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.

Leveraging cell type specific regulatory regions to detect SNPs associated with tissue factor pathway inhibitor plasma levels.

Tissue factor pathway inhibitor (TFPI) regulates the formation of intravascular blood clots, which manifest clinically as ischemic heart disease, ischemic stroke, and venous thromboembolism (VTE). TFPI plasma levels are heritable, but the genetics underlying TFPI plasma level variability are poorly understood. Herein we report the first genome-wide association scan (GWAS) of TFPI plasma levels, conducted in 251 individuals from five extended French-Canadian Families ascertained on VTE. To improve discovery, we also applied a hypothesis-driven (HD) GWAS approach that prioritized single nucleotide polymorphisms (SNPs) in (1) hemostasis pathway genes, and (2) vascular endothelial cell (EC) regulatory regions, which are among the highest expressers of TFPI. Our GWAS identified 131 SNPs with suggestive evidence of association (P-value < 5 × 10-8 ), but no SNPs reached the genome-wide threshold for statistical significance. Hemostasis pathway genes were not enriched for TFPI plasma level associated SNPs (global hypothesis test P-value = 0.147), but EC regulatory regions contained more TFPI plasma level associated SNPs than expected by chance (global hypothesis test P-value = 0.046). We therefore stratified our genome-wide SNPs, prioritizing those in EC regulatory regions via stratified false discovery rate (sFDR) control, and reranked the SNPs by q-value. The minimum q-value was 0.27, and the top-ranked SNPs did not show association evidence in the MARTHA replication sample of 1,033 unrelated VTE cases. Although this study did not result in new loci for TFPI, our work lays out a strategy to utilize epigenomic data in prioritization schemes for future GWAS studies.

A Pilot Study of Tissue Factor-Tissue Factor Pathway Inhibitor Axis and Other Selected Coagulation Parameters in Broiler Chickens Administered in Ovo with Selected Prebiotics*.

The tissue factor (TF) - tissue factor pathway inhibitor (TFPI) axis plays a major role in hemostasis. Disorders of the coagulation system are commonly diagnosed with the help of screening tests such as prothrombin time (PT), activated partial thromboplastin time (aPTT), and plasma fibrinogen concentration (PFC). However, the effect of prebiotics on the hemostasis system has not been characterized in poultry yet. This study was designed to determine the effect of in ovo administration ofprebiotics on blood coagulation parameters of broiler chickens depending on their age. The study was conducted with 180 broiler chick embryos, the air cells of which were injected on day 12 of incubation with prebiotics (experimental groups: Bi2tos, DiNovoo and RFO) or physiological saline solution (control group). At 1, 21 and 42 days of rearing, blood was sampled from 15 broiler chickens from each group. An enzyme immunoassay was performed to determine plasma TF and TFPI levels, and PT, aPTT and PFC were determined in the chicken blood. We demonstrated that: 1) total TF levels increased with age in the experimental groups, 2) prebiotics had no significant effect on TF levels between the groups at a particular age, 3) total TFPI levels differed between both the type of in ovo injected substance and the broiler chicken age, 4) in the control group, PT and aPTT were found to increase with age whilst fibrinogen concentration decreased. The main conclusion from this pilot study is that total TF and TFPI levels change with age, however no clear patterns regarding TFPI were detected yet. The levels of PT, aPTT and PFC varied with the prebiotics administered in ovo as well as with the age of broiler chickens.

Single nucleotide polymorphisms in an intergenic chromosome 2q region associated with tissue factor pathway inhibitor plasma levels and venous thromboembolism

Essentials Tissue factor pathway inhibitor (TFPI) regulates the blood coagulation cascade. We replicated previously reported linkage of TFPI plasma levels to the chromosome 2q region. The putative causal locus, rs62187992, was associated with TFPI plasma levels and thrombosis. rs62187992 was marginally associated with TFPI expression in human aortic endothelial cells. Click to hear Ann Gil's presentation on new insights into thrombin activatable fibrinolysis inhibitor SUMMARY: Background Tissue factor pathway inhibitor (TFPI) regulates fibrin clot formation, and low TFPI plasma levels increase the risk of arterial thromboembolism and venous thromboembolism (VTE). TFPI plasma levels are also heritable, and a previous linkage scan implicated the chromosome 2q region, but no specific genes. Objectives To replicate the finding of the linkage region in an independent sample, and to identify the causal locus. Methods We first performed a linkage analysis of microsatellite markers and TFPI plasma levels in 251 individuals from the F5L Family Study, and replicated the finding of the linkage peak on chromosome 2q (LOD = 3.06). We next defined a follow-up region that included 112 603 single nucleotide polymorphisms (SNPs) under the linkage peak, and meta-analyzed associations between these SNPs and TFPI plasma levels across the F5L Family Study and the Marseille Thrombosis Association (MARTHA) Study, a study of 1033 unrelated VTE patients. SNPs with false discovery rate q-values of < 0.10 were tested for association with TFPI plasma levels in 892 patients with coronary artery disease in the AtheroGene Study. Results and Conclusions One SNP, rs62187992, was associated with TFPI plasma levels in all three samples (β = + 0.14 and P = 4.23 × 10-6 combined; β = + 0.16 and P = 0.02 in the F5L Family Study; β = + 0.13 and P = 6.3 × 10-4 in the MARTHA Study; β = + 0.17 and P = 0.03 in the AtheroGene Study), and contributed to the linkage peak in the F5L Family Study. rs62187992 was also associated with clinical VTE (odds ratio 0.90, P = 0.03) in the INVENT Consortium of > 7000 cases and their controls, and was marginally associated with TFPI expression (β = + 0.19, P = 0.08) in human aortic endothelial cells, a primary site of TFPI synthesis. The biological mechanisms underlying these associations remain to be elucidated.

Tissue factor-dependent pathway in severe preeclampsia revisited: a Brazilian cohort study.

Previously we investigated the tissue factor (TF)-dependent coagulation pathway and key haemostatic cofactors in white women with preeclampsia (P-EC) and suggested that plasma factor VII (FVII) levels can differentiate women with P-EC from healthy nonpregnant women or normal pregnant women, at the same trimester, with high sensitivity, specificity, positive and negative predictive values. Here we re-examine the TF-dependent pathway in a large cohort of Brazilian women. A total of 240 women were studied. These included healthy nonpregnant women (n = 79), normotensive pregnant women (n = 80) and women with severe P-EC (n = 81). Commercially available enzyme-linked immunosorbent assays were used to measure plasma FVII, activated factor VII (FVIIa), TF and tissue factor pathway inhibitor (TFPI). All study participants were matched for age. Pregnant women (with/without P-EC) were matched for gestational age and parity. Plasma levels of FVII, FVIIa and TFPI were significantly increased in women with severe P-EC compared with healthy nonpregnant women (P < 0.01) or normotensive pregnant women (P < 0.01). FVIIa was also higher in normotensive pregnant women compared with nonpregnant women (P < 0.01). However, no such significant trends were observed for plasma TF levels (P = 0.074). In conclusion, circulating FVII, FVIIa and TFPI were significantly elevated in women with severe P-EC in the absence of comparable changes in plasma TF levels. The present work is in agreement with our previous report on FVII levels in white women with P-EC. Thus, this lends further support to the notion that plasma FVII levels are potentially valuable diagnostic marker for P-EC, irrespective of ethnicity.

Plasma Tissue Factor Pathway Inhibitor (TFPI) Levels in Healthy Subjects and Patients with Hemophilia A and B

BAY 1093884 is a fully human monoclonal antibody against tissue factor pathway inhibitor (TFPI) developed as a potential bypass agent for patients with hemophilia with or without inhibitors. It restores insufficient thrombin burst, leading to stable clot formation in hemophilic conditions in vitro, and effectively stops bleeding in vivo. TFPI is a potent inhibitor of factor Xa (FXa) and the factor VIIa tissue factor complex in the extrinsic pathway. The majority of TFPI is associated with vascular endothelial cells. The mean plasma TFPI concentration in healthy individuals is ~70 ng/mL (1.6 nM) and about 80% of the circulating TFPI is bound to lipoproteins [Dahm, et al. Blood. 2003;101(11):4387-4392; Broze,et al. Front Biosci. 2012;17:262-280]. Some reports indicate that patients with hemophilia B have lower free TFPI levels than patients with hemophilia A, irrespective of phenotypic severity (Tardy-Poncet, et al. Haemophilia 2011;17:312-313). The objective of this study is to determine the plasma TFPI concentration in healthy donors and patients with hemophilia by a newly developed functional TFPI capture assay and to evaluate this assay with inhibition of TFPI by anti-TFPI neutralizing antibody (BAY 1093884) in vitro.A quantitative enzyme-linked immunosorbent assay using FXa as capture agent was developed and validated to measure TFPI levels in human plasma. The assay shows very good precision, accuracy, and reproducibility and should capture all coagulation-relevant forms of TFPI from plasma. Plasma TFPI was determined in 30 healthy donors (15 males and 15 females) and 30 patients with severe hemophilia (hemophilia A [n=12], hemophilia A with inhibitors [n=9], hemophilia B [n=9]).The plasma TFPI levels (mean ± SD) in healthy individuals, patients with severe hemophilia A without and with inhibitors, and severe hemophilia B were 59.5±18.4 ng/mL, 62.9±14.6 ng/mL, 47.3±4.3 ng/mL, and 68.1±8.8 ng/mL, respectively (Table 1). No statistical differences were found based on sex or race (Hispanic, African American, white) in the healthy population and between patients with hemophilia with and without inhibitors. TFPI levels were also not affected by addition of corn trypsin inhibitor (CTI) in citrate plasma. Furthermore, the concentration that inhibits 50% of TFPI levels (IC50) of anti-TFPI antibody (BAY 1093884) was determined to be 4.76 nM in normal human plasma using this assay.In conclusion,plasma TFPI does not appear to be affected by sex or race in healthy subjects, or the deficiency of factor VIII or IX in patients with hemophilia. The functional TFPI capture assay could potentially be used as a pharmacodynamic marker for monitoring plasma TFPI levels after the administration of anti-TFPI antibody and guide dosing strategies.Table 1Plasma TFPI Levels in Healthy Subjects and Patients With Severe Hemophilia A and BHealthyHuman Donors(n=30)SevereHem A(n=12)Severe Hem AWith inhibitors(n=9)SevereHem B(n=9)TFPI, ng/mL Mean ± SD59.5±18.462.9±14.647.3±4.368.1±8.8Hem=hemophilia; TFPI=tissue factor pathway inhibitor. DisclosuresGu:Bayer HealthCare: Employment. Patel:Bayer HealthCare: Employment. Kauser:Bayer HealthCare LLC: Employment.

Endothelial cell-anchored tissue factor pathway inhibitor regulates tumor metastasis to the lung in mice.

Tissue factor pathway inhibitor (TFPI) is a physiological inhibitor of the tissue factor (TF)-initiated coagulation pathway. Both circulating and tumor cell-associated TFPI significantly reduce tumor cell-induced coagulation activation and lung metastasis. However, the significance of endothelial cell-anchored TFPI in cancer biology remains largely unexplored. We generated mice with full-length disruption of TFPI (including TFPIα and TFPIβ isoforms) in endothelial cells, using a Cre-LoxP system and gene inactivation (GI) strategy. Experimental pulmonary tumor metastasis models were used with TFPI-deficient mice to evaluate the role of endothelial cell-anchored TFPI in cancer progression. Finally, lung microvascular permeability and microenvironment were investigated. TFPI-deficient mice were viable and fertile, and showed decreased plasma TFPI levels and lung TFPI levels as compared with their control littermates. TFPI deficiency in endothelial cells promoted pulmonary tumor metastasis with an increased vascular permeability and altered lung microenvironment. Our observations suggest that endothelial cell-anchored TFPI controls lung tumor metastasis, and does so largely through the inhibition of local TF-induced thrombin generation and the regulation of the lung microenvironment in mice.

Tumor expression, plasma levels and genetic polymorphisms of the coagulation inhibitor TFPI are associated with clinicopathological parameters and survival in breast cancer, in contrast to the coagulation initiator TF.

IntroductionHypercoagulability in malignancy increases the risk of thrombosis, but is also involved in cancer progression. Experimental studies suggest that tissue factor (TF) and tissue factor pathway inhibitor (TFPI) are involved in cancer biology as a tumor- promoter and suppressor, respectively, but the clinical significance is less clear. Here, we aimed to investigate the clinical relevance of TF and TFPI genetic and phenotypic diversity in breast cancer.MethodsThe relationship between tumor messenger RNA (mRNA) expression and plasma levels of TF and TFPI (α and β), tagging single nucleotide polymorphisms (tagSNPs) in F3 (TF) (n = 6) and TFPI (n = 18), and clinicopathological characteristics and molecular tumor subtypes were explored in 152 treatment naive breast cancer patients. The effect of tumor expressed TF and TFPIα and TFPIβ on survival was investigated in a merged breast cancer dataset of 1881 patients.ResultsProgesterone receptor negative patients had higher mRNA expression of total TFPI (α + β) (P = 0.021) and TFPIβ (P = 0.014) in tumors. TF mRNA expression was decreased in grade 3 tumors (P = 0.003). In plasma, total TFPI levels were decreased in patients with larger tumors (P = 0.013). SNP haplotypes of TFPI, but not TF, were associated with specific clinicopathological characteristics like tumor size (odds ratio (OR) 3.14, P = 0.004), triple negativity (OR 2.4, P = 0.004), lymph node spread (OR 3.34, P = 0.006), and basal-like (OR 2.3, P = 0.011) and luminal B (OR 3.5, P = 0.005) molecular tumor subtypes. Increased expression levels of TFPIα and TFPIβ in breast tumors were associated with better outcome in all tumor subtypes combined (P = 0.007 and P = 0.005) and in multiple subgroups, including lymph node positive subjects (P = 0.006 and P = 0.034).ConclusionsThis study indicates that genetic and phenotypic variation of both TFPIα and TFPIβ, more than TF, are markers of cancer progression. Together with the previously demonstrated tumor suppressor effects of TFPI, the beneficial effect of tumor expressed TFPI on survival, renders TFPI as a potential anticancer agent, and the clinical significance of TFPI in cancer deserves further investigation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0548-5) contains supplementary material, which is available to authorized users.

Genetic variation in the tissue factor gene is associated with clinical outcome in severe sepsis patients.

IntroductionActivation of inflammation and coagulation was closely related and mutually interdependent in sepsis. Tissue factor (TF) and its endogenous inhibitor, tissue factor pathway inhibitor (TFPI) was the main regulators of the initiation of coagulation process. Altered plasma levels of TF and TFPI have been related to worse outcome in sepsis. The objective of this study was to investigate whether single nucleotide polymorphisms (SNPs) in the TF and TFPI genes were associated with risk and outcome for patients with severe sepsis.MethodsSeventeen SNPs in TF and TFPI were genotyped in samples of sepsis (n =577) and severe sepsis patients (n =476), and tested for association in this case–control collection. We then investigated correlation between the associated SNPs and the mRNA expression, and protein level of the corresponding gene. The mRNA levels of TF were determined using real-time quantitative reverse transcription-polymerase chain reaction and the soluble plasma levels of TF were measured using enzyme linked immunosorbent assay (ELISA) method.ResultsAssociation analysis revealed that three TF SNPs in perfect linkage disequilibrium, rs1361600, rs3917615 and rs958587, were significantly associated with outcome of severe sepsis. G allele frequency of rs1361600 in survivor patients was significantly higher than that in nonsurvivor severe sepsis patients (P =4.91 × 10-5, odds ratio (OR) =0.48, 95% confidence interval (CI) 0.33 to 0.69). The association remained significant after adjustment for covariates in multiple logistic regression analysis and for multiple comparisons. Lipopolysaccharide-induced TF-mRNA expression levels in peripheral blood mononuclear cells from subjects carrying rs1361600 AG and GG genotypes, were significantly lower than those subjects carrying AA genotype (P =0.0012). Moreover, severe sepsis patients of GG and GA genotypes showed lower serum levels of TF than patients with AA genotype (Padj =0.02). The plasma levels of TF were also associated with outcome of severe sepsis patients (Padj =0.01). However, genotype and allele analyses did not show any significant difference between sepsis and severe sepsis patients.ConclusionsOur findings indicate that common genetic variation in TF was significantly associated with outcome of severe sepsis in Chinese Han population.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-014-0631-9) contains supplementary material, which is available to authorized users.