Tissue Factor Pathway Inhibitor Protein Research Articles

Efficacy and Safety of the Anti-Tissue Factor Pathway Inhibitor Marstacimab in Participants with Severe Hemophilia without Inhibitors: Results from the Phase 3 Basis Trial

Background:Marstacimab (PF-06741086) is a monoclonal antibody targeted to the tissue factor pathway inhibitor protein to improve hemostasis via the extrinsic pathway of blood coagulation. Previous phase 1/2 studies demonstrated the efficacy and safety of long-term administration of marstacimab up to 450 mg weekly for reducing bleeding episodes in adults with severe hemophilia A (HA) or hemophilia B (HB), with or without inhibitors, compared with on-demand (OD) therapy. We evaluated the efficacy and safety of marstacimab in participants with severe HA or moderately severe to severe HB without inhibitors compared with previous factor replacement therapy. Methods: BASIS (NCT03938792) is an open-label, multicenter, pivotal phase 3 study that enrolled male participants aged ≥12 to ˂75 y with severe HA (factor [F] VIII ˂1%) or moderately severe to severe (FIX ≤2%) HB, with or without inhibitors. Following screening, participants entered a 6-month observational phase (OP) and were categorized by factor replacement treatment: (1) OD or (2) routine prophylaxis (RP). Participants who completed the OP crossed over to 12-month active treatment phase (ATP) and received a single subcutaneous loading dose of 300 mg followed by once weekly 150 mg marstacimab. Primary endpoints were annualized bleeding rate (ABR) for treated bleeds and safety outcomes. Secondary endpoints included incidence of various types of breakthrough bleeds and health-related quality of life (HRQoL) measures. Participants who completed the ATP were eligible to enroll in the long-term extension (LTE) study. Informed consent/ethics committee approvals were obtained. Results for participants without inhibitors are presented. Results: Participants (N=128; 108 adults, 20 adolescents) with HA or HB without inhibitors entered the OP (OD: HA n=29, HB n=8; RP: n=72, HB n=19); of these, 116 entered the ATP. The median age was 30 [range, 13-66] y, most participants were White (50.8%) or Asian (47.7%) and predominately from Europe and India (51.6%). At baseline, 89 participants (69.5%; OD: n=36; RP: n=53) had ≥1 target joint. Mean (range) duration of marstacimab treatment was 12.1 (11.5-13.1) months for OD and 11.6 (0.9-12.8) months for RP. Eighty-eight participants entered the LTE (OD: HA n=22, HB n=7; RP: HA n=45; HB n=13). Data were not available by the cutoff date for 1 RP participant and was not included in the LTE safety analysis set. The mean (range) treatment duration in the LTE was 8.0 (1.2-14.5) months for OD and 6.5 (1.1-16.1) months for RP. In the phase 3 study, the OD group reported 12 (36.4%) adverse events (AEs) during ATP vs 9 (24.3%) in OP whereas the RP group reported 62 (74.7%) AEs in ATP vs 20 (22.0%) in OP. Both groups reported more treatment-related AEs during ATP ( Table 1). ADAs developed in 23/112 participants (20.5% incidence), of which titers were low and resolved in 22 participants by end of study. One RP participant discontinued due to a non-treatment-related SAE, and no deaths or thromboembolic events were recorded in the phase 3 study or the LTE. Mean (95% CI) ABR for treated bleeds was reduced for OD (91.6% [88.1-94.1%]) and RP (35.2% [5.6-55.6%]) participants over the 12-month ATP and marstacimab demonstrated superiority vs OD (P<0.001) and non-inferiority and superiority vs RP (P=0.0376) therapy. Marstacimab was also associated with significant reductions in ABR across all breakthrough bleed categories vs OD, and numerical reductions vs RP (non-inferiority). Overall, mean ABR declined over the first 6 months of ATP, which continued to Month 12 (data not shown). Bleed rates for an additional 16 months of follow-up in the LTE were consistent with those observed during the first 12 months of treatment in the phase 3 study ( Table 2). The ABR reductions observed with marstacimab during ATP were consistent across hemophilia types and age groups for OD and were generally consistent across hemophilia types and age groups for RP, with all point estimates for a difference <2.5 (non-inferiority margin for the ABR of treated bleeds). HRQoL parameters demonstrated non-significant improvements vs OD therapy and non-inferiority vs RP therapy. Conclusion: Compared with previous OD or RP therapy, once weekly subcutaneous marstacimab was safe and effective for reducing bleeding events in participants with severe HA or moderately severe to severe HB without inhibitors beyond 12 months in the phase 3 study and up to an additional 16 months in the LTE study.

Expression of tissue factor pathway inhibitor 2 in the follicles of chicken ovaries and its regulatory mechanism in cultured granulosa cells1

Tissue factor pathway inhibitor 2 (TFPI2) plays a key role in female reproduction. However, its expression and function in chickens are still unclear. In the present study, ovarian tissues from 30 w and 15 w laying chickens were analyzed using RNA-seq to identify the differentially expressed gene TFPI2. The full-length cDNA of TFPI2 was obtained from adult chicken ovaries by rapid-amplification of cDNA ends (RACE), and the putative TFPI2 protein was found to share a highly conserved amino acid sequence with known bird homologs. In addition, TFPI2 was widely expressed in the tissues of adult chicken follicles according to quantitative real-time PCR (qRT–PCR) and Western blotting. Immunohistochemistry suggested that the TFPI2 protein existed in chicken ovary follicles at different developmental states, such as primordial follicles, the ovarian stroma, and the granulosa and theca layers of prehierarchical follicles (6-8 mm) and preovulatory follicles (F1). In vitro, Follicle stimulating hormone or Luteinizing hormone (FSH/LH) stimulated the expression of TFPI2 in chicken granulosa cells. FSH-/LH-induced TFPI2 mRNA expression was mediated by signaling pathways such as the PKA, PKC, PI3K, and mTOR pathways. Functionally, TFPI2 promoted the proliferation and viability of cultured granulosa cells and decreased the secretion of Progesterone (P4) and Estrogen (E2) and the mRNA abundance of key steroidogenic enzymes (STAR, Cyp17a1, Cyp19a1 and 3B-HSD) as well as MMPs (MMP2, 7, 9 and 11). Mechanistically, TFPI2 inhibited the expression of MMP7 via the Wnt signaling pathway. These findings indicate that TFPI2 may play an important role in regulating chicken follicular development and ovulation and suggest the molecular regulation mechanisms.

Tissue Factor Pathway Inhibitors as Potential Targets for Understanding the Pathophysiology of Preeclampsia.

Preeclampsia is a hypertensive disorder of pregnancy that causes maternal and perinatal morbidity and mortality worldwide. Preeclampsia is associated with complex abnormalities of the coagulation and fibrinolytic system. Tissue factor (TF) is involved in the hemostatic system during pregnancy, while the Tissue Factor Pathway Inhibitor (TFPI) is a major physiological inhibitor of the TF-initiated coagulation cascade. The imbalance in hemostatic mechanisms may lead to a hypercoagulable state, but prior research has not comprehensively investigated the roles of TFPI1 and TFPI2 in preeclamptic patients. In this review, we summarize our current understanding of the biological functions of TFPI1 and TFPI2 and discuss future directions in preeclampsia research. A literature search was performed from inception to 30 June 2022 in the PubMed and Google Scholar databases. TFPI1 and TFPI2 are homologues with different protease inhibitory activities in the coagulation and fibrinolysis system. TFPI1 is an essential physiological inhibitor of the TF-initiated extrinsic pathway of coagulation. On the other hand, TFPI2 inhibits plasmin-mediated fibrinolysis and exerts antifibrinolytic activity. It also inhibits plasmin-mediated inactivation of clotting factors and maintains a hypercoagulable state. Furthermore, in contrast to TFPI1, TFPI2 suppresses trophoblast cell proliferation and invasion and promotes cell apoptosis. TFPI1 and TFPI2 may play important roles in regulating the coagulation and fibrinolytic system and trophoblast invasion to establish and maintain successful pregnancies. Concentrations of TF, TFPI1, and TFPI2 in maternal blood and placental tissue are significantly altered in preeclamptic women compared to normal pregnancies. TFPI protein family may affect both the anticoagulant (i.e., TFPI1) and antifibrinolytic/procoagulant (i.e., TFPI2) systems. TFPI1 and TFPI2 may function as new predictive biomarkers for preeclampsia and navigate precision therapy.

Downregulation of microRNA-23a confers protection against myocardial ischemia/reperfusion injury by upregulating tissue factor pathway inhibitor 2 following luteolin pretreatment in rats.

Downregulation of microRNA-23a confers protection against myocardial ischemia/reperfusion injury by upregulating tissue factor pathway inhibitor 2 following luteolin pretreatment in rats.

Serum levels of tissue factor pathway inhibitor: Potential association with Raynaud's phenomenon and telangiectasia in patients with systemic sclerosis.

Vasculopathy is a critical step of systemic sclerosis (SSc) development, bridging between autoimmune inflammation and tissue fibrosis. Impaired coagulation system is a part of SSc vasculopathy, but the role of tissue factor pathway inhibitor (TFPI), a critical regulator of the extrinsic coagulation pathway, remained unknown. Therefore, we evaluated the clinical correlation of serum TFPI levels in SSc patients. Serum TFPI levels were comparable between SSc and control participants, but SSc patients with Raynaud's phenomenon and telangiectasia had significantly lower serum TFPI levels than those without. Importantly, there was a significant positive correlation between serum TFPI levels and protein S activity. These results support the critical role of impaired coagulation system in SSc.

Berberine dose-dependently inhibits the expression of procoagulant and fibrinolytic inhibitory factors in lipopolysaccharide-induced rat type II alveolar epithelial cells

To observe the effects of berberine on procoagulant and fibrinolytic inhibitory factors produced by rat type II alveolar epithelial cell (AEC II) induced by lipopolysaccharide (LPS). AEC II cells (RLE-6TN cells) were cultured in vitro, and the cells in logarithmic growth phase were collected. The cytotoxicity text of berberine was detected by cell counting kit-8 (CCK-8) to determine the drug concentration range according to inhibition concentration of half cells (IC50). The RLE-6TN cells were divided into five groups, the cells in blank control group were cultured in DMEM; the cells in LPS group were stimulated with 5 mg/L LPS; and the cells in berberine pretreatment groups were pretreated with 20, 50 and 80 μmol/L berberine for 1 hour, and then were co-cultured with 5 mg/L LPS. The cells were collected after LPS induced for 24 hours. The protein and mRNA expression levels of tissue factor (TF), tissue factor pathway inhibitor (TFPI) and plasminogen activator inhibitor-1 (PAI-1) in the cells were detected by Western blotting and real-time fluorescence quantification reverse transcription-polymerase chain reaction (RT-qPCR). The levels of activated protein C (APC), precollagen III peptide (PIIIP), thrombin-antithrombin complex (TAT) and antithrombin III (AT III) in the cell supernatant were measured by enzyme linked immunosorbent assay (ELISA). According to the inhibition rate curve, the IC50 of berberine on RLE-6TN cells was 81.16 μmol/L. Therefore, 20, 50 and 80 μmol/L were selected as the intervention concentration of berberine. Compared with the blank control group, the expression and secretion of procoagulant and fibrinolytic inhibitory factors were abnormal in RLE-6TN cells after LPS induced for 24 hours. The protein and mRNA expression levels of TF and PAI-1 in the LPS group were significantly increased, but the protein and mRNA expression levels of TFPI were significantly decreased. Meanwhile, the levels of APC and AT III in the cell supernatant were significantly decreased, while the levels of PIIIP and TAT were significantly increased. After pretreatment with berberine, the abnormal expression and secretion of procoagulant and fibrinolytic inhibitory factors induced by LPS were corrected in a dose-dependent manner, especially in 80 μmol/L. Compared with the LPS group, the protein and mRNA expression levels of TF and PAI-1 in the berberine 80 μmol/L group were significantly decreased [TF protein (TF/GAPDH): 0.45±0.02 vs. 0.55±0.03, TF mRNA (2-ΔΔCt): 0.39±0.08 vs. 1.48±0.11, PAI-1 protein (PAI-1/GAPDH): 0.37±0.02 vs. 0.64±0.04, PAI-1 mRNA (2-ΔΔCt): 1.14±0.29 vs. 4.18±0.44, all P < 0.01] and those of TFPI were significantly increased [TFPI protein (TFPI/GAPDH): 0.53±0.02 vs. 0.45±0.02, TFPI mRNA (2-ΔΔCt): 0.94±0.08 vs. 0.40±0.05, both P < 0.01]. Meanwhile, the levels of APC and AT III in the cell supernatant were significantly increased [APC (μg/L): 1 358.5±26.0 vs. 994.2±23.1, AT III (μg/L): 118.0±7.4 vs. 84.4±2.7, both P < 0.01], while those of PIIIP and TAT were significantly decreased [PIIIP (μg/L): 11.2±0.4 vs. 18.6±0.9, TAT (ng/L): 222.1±2.8 vs. 287.6±7.0, both P < 0.01]. Berberine could inhibit the LPS-induced expressions of procoagulant and fibrinolytic inhibitory factors in rat AEC II cells and promote the expressions of anticoagulant factors in a dose-dependent manner. Berberine may be a new therapeutic target for alveolar hypercoagulability and fibrinolysis inhibition in acute respiratory distress syndrome (ARDS).

Tissue factor pathway inhibitor 2 suppresses the growth of thyroid cancer cells through by induction of apoptosis.

Tissue factor pathway inhibitor 2 (TFPI-2) has been recently identified as a tumor suppressor gene in several human cancers, whereas its role in thyroid cancer has been unclear. The TFPI-2 protein level in thyroid cancer tissues and cell lines (8305C and B-CPAP) were examined using immunohistochemistry and immunoblotting. The TFPI-2 promoter methylation was examined using methylation-specific polymerase chain reaction (MSP). Lentivirus containing TFPI-2 cDNA (Lenti-TFPI-2) was constructed to elevate TFPI-2 expression in 8305C and B-CPAP cells. The effects of Lenti-TFPI-2 on cell proliferation in vitro and in vivo were evaluated by MTT assay and mouse xenograft model. Annexin V/PI double staining assay was performed to detect the effect of Lenti-TFPI-2 on cell apoptosis. TFPI-2 protein level were decreased in cancer tissues and lymph node metastasis, and TFPI-2 protein level is positively associated with survival time. The promoter of TFPI-2 is hypermethylated in cancer tissues. TFPI-2 mRNA and protein levels were abundant in normal human thyroid follicular cell line Nthy-ori 3-1 cells, whereas they were decreased in 8305C and B-CPAP cells. pcDNA-TFPI-2 elevated TFPI-2 mRNA and protein in 8305C and B-CPAP cells. TFPI-2 overexpression suppressed proliferation and induced apoptosis of 8305C and B-CPAP cells. TFPI-2 inactivation may play a role in thyroid cancer tumorigenesis and development. TFPI-2 overexpression suppressed cell proliferation through induction of cell apoptosis, suggesting that TFPI-2 may serve as a novel and effective target for thyroid cancer therapy.

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.

Tissue Factor Pathway Inhibitor Enhances Transendothelial Migration of Chronic Lymphocytic Leukemia Cells through Binding to Glypican-3

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. Cell trafficking and homing of CLL cells play a critical role in organ infiltration and contribute to the clinical course of CLL. Interaction between CLL cells and endothelial cells affects gene expression in CLL cells and further regulates cell trafficking. Endothelial cells are the main source of tissue factor (TF) pathway inhibitor (TFPI), which is the primary inhibitor of TF. However, the role of TFPI in leukemic cell trafficking has not been explored before. CXCL12 and its receptor CXCR4 play an important role in CLL cell trafficking and homing. To assess the effect of TFPI on CXCL12-mediated migration of leukemic cells, we investigated transendothelial migration (TEM) of B cells from CLL patients by using CXCL12 as a chemoattractant. We found that full-length recombinant TFPI (rTFPI) increased CXCL12-mediated migration of CLL cells dose-dependently by using MACS flow cytometry to count the migrated cells. Accordingly, Western blot showed the binding of rTFPI to the CLL cells after 24 h treatment. However, the chemokine receptor CXCR4 expression in CLL cells was not increased or even slightly decreased by rTFPI treatment, suggesting that TFPI enhances leukemic cell migration probably through interacting with other factor(s) than overexpressing CXCR4 in CLL cells. Surface expression and intracellular trafficking of TFPI is regulated by glycosylphosphatidylinositol (GPI)-anchored co-receptor(s). Glypican-3, a member of proteoglycan family, binds to the plasma membrane by a GPI anchor and its heparan sulfate (HS)-type glycosaminoglycan chains are considered to mediate the interaction with other cell membrane proteins. To determine whether TFPI interacts with glypican-3, immunofluorescence staining and co-immunoprecipitation were performed in K562 and HL60 cells. Immunofluorescence analysis revealed colocalization of TFPI and glypican-3 on the surface of K562 and HL60 cells. Co-immunoprecipitation confirmed the binding between TFPI and glypican-3 in K562 cells. Moreover, co-localized TFPI and glypican-3 were found in bone marrow biopsies of leukemia patients. We further employed an antibody against HS chains of glypican-3, which inhibits the activity of glypican-3. Western blot showed that rTFPI treatment failed to increase TFPI protein in CLL cells when glypican-3 inhibitor HS20 was applied. In addition, TEM assay showed that HS20 treatment abolished the effect of TFPI on cell migration, which suggests that inhibition of glypican-3 blocks the binding of TFPI to CLL cells and then abolishes the effect of TFPI on cell migration. It indicates that TFPI affects CLL cell migration through binding to glypican-3. Using a factor Xa generation assay, we found that HS20 suppressed TF activity on the surface of HL60 cells, suggesting that the inhibition of glypican-3 impaired the procoagulant activity of leukemic cells. In addition, Western blot and RT-PCR showed that HS20 downregulated TF mRNA and protein expression in CLL cells. Finally, we explored the role of Wnt/β-catenin signaling, which has been shown to regulate tumor progression and metastasis. We found a dose-dependent increase of activated β-catenin in the CLL cells after rTFPI treatment. Inhibition of Wnt/β-catenin signaling by a potent inhibitor IWP4 strongly impaired the migration of CLL cells induced by rTFPI. In this study, we show that TFPI enhances CLL cell migration through binding to glypican-3 and activating Wnt/β-catenin signaling pathway. Our findings suggest a novel molecular mechanism regulating the transmigration of leukemic cells. It may open a new window for therapeutic targeting in the treatment of organ infiltration of CLL patients. DisclosuresNo relevant conflicts of interest to declare.

Pharmacodynamics, Pharmacokinetics and Safety of Bay 1093884, an Antibody Directed Against Human TFPI, in Patients with Factor VIII or IX Deficiency (With and Without Inhibitors): A Phase 1 Study

Background: BAY 1093884 is a fully human monoclonal antibody directed against the K1 and K2 domains of human tissue factor pathway inhibitor (TFPI). TFPI regulates the initiation of coagulation and is important for regulation of normal hemostatic response. In factor (F)VIII and FIX deficiency (hemophilia A [HA] and B [HB], respectively), lack of adequate amplification (FXa generation) in tandem with intact regulation contributes to reduced clotting. Inhibition of TFPI has the potential to normalize coagulation in hemophilia even in the absence of FVIII or FIX and it is expected to result in normalization of thrombin generation and consequently improvement in clot formation. TFPI is an attractive therapeutic target as it is unaffected by FVIII or FIX inhibitors and can be targeted in both HA and HB. A potential advantage of targeting TFPI is that downstream mechanisms that protect from excessive coagulation (including antithrombin), remain intact.Methods: BAY 1093884 was evaluated in a multicenter, open-label study in patients with severe HA or HB, with or without inhibitors. Safety, pharmacokinetics (PK) and pharmacodynamics (PD) were assessed after single intravenous (IV) (0.3 and 1 mg/kg) and subcutaneous (SC) (1, 3, and 6 mg/kg) doses. A multiple-dose (MD) cohort received BAY 1093884 150 mg SC once weekly for 6 weeks. Total and free TFPI protein levels and TFPI activity were measured. Thrombin generation assay (TGA) parameters were measured using a calibrated automated thrombogram. Clot formation was assessed using rotational thromboelastometry (ROTEM). A mechanistic physiology-based PK/PD (PBPK/PD) model was developed based on single-dose PK data observed in the study, and used to determine dosing in the MD cohort. Data for HA, HB, Japanese and inhibitor patient subgroups were analyzed separately.Results: Thirty-two patients were enrolled; 30 with HA and 2 with HB, of which 4 (3 HA, 1 HB) were included in the MD cohort. At least 4 patients were treated in each administration method/dose group. Clot formation using ROTEM was assessed in 6 patients. Total and free TFPI-related parameters showed dose-dependent effects, with the concentrations being below the lower limit of quantification for longer duration at higher doses. Improvements in TGA parameters were observed following both IV and SC administration; duration of improvement was longer at higher doses. BAY 1093884 led to shortening of the clotting time in all 6 patients tested. Dose-dependent increases in plasma concentration exceeding dose proportionality were seen following IV and SC administration, consistent with expectations of target-mediated drug disposition for BAY 1093884. Moderate accumulation was observed after MD. Limited information on subgroups (patients with inhibitors [n=4], patients with HB [n=2] and Japanese origin [n=1]) suggests that the PK and PD data for these subgroups were consistent with those from non-inhibitor HA patients. The PBPK/PD model adequately described the concentration time course and total TFPI following single-dose IV and SC administration of BAY 1093884; the PK and PD data from the MD cohort were as predicted by the model. The model also supported that the PK/PD of BAY 1093884 are independent of inhibitor status. No serious adverse events, deaths, or adverse events leading to discontinuation were observed. Fibrinogen and platelet counts remained within normal range at most visits and no meaningful changes in levels of antithrombin, protein C, or FV were seen. The mechanisms for anticoagulation and fibrinolysis were intact.Conclusions: Overall, BAY 1093884 gave no safety concerns in the dose range evaluated. Results suggest that BAY 1093884 shifts hemostatic balance towards greater thrombin generation and clot formation as intended. PK/PD characteristics were similar for inhibitor and HB patients as compared with HA patients, potentially supporting use in all of these patient types. The PBPK/PD model developed can be used for dose selection in future studies. Treatment efficacy will be evaluated in a future study. DisclosuresChowdary:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Swedish Orphan Biovitrum AB (Sobi): Honoraria; Baxalta (Shire): Honoraria, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Freeline: Consultancy; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Honoraria. Lissitchkov:Novo Nordisk: Other: Investigator fees as a participant of the clinical trial. Willmann:Bayer: Employment. Schwers:Bayer: Employment. Michaels:Bayer: Employment. Shah:Bayer: Employment.

MiR-616-3p modulates cell proliferation and migration through targeting tissue factor pathway inhibitor 2 in preeclampsia.

Despite improvements in diagnosis and treatment, preeclampsia (PE) continues to pose a significant risk of maternal and foetal morbidity and mortality if not addressed promptly. An increasing number of studies have suggested that tissue factor pathway inhibitor 2 (TFPI2) acts as a suppressor gene, possibly inhibiting multiple serine proteases affecting cell proliferation and migration. It plays an essential role in the occurrence and development of PE, but the pathogenesis remains unclear. In our research, we performed western blotting, immunohistochemistry and qPCR assays to investigate TFPI2 and miR-616-3p expression in preeclamptic placental tissues. Cell assays were performed in HTR-8/SVneo and JEG3 cell lines. Cell proliferation and migration events were investigated by MTT, EdU and transwell assays. In conjunction with bioinformatics analysis, luciferase reporter assays were performed to elucidate the mechanism by which miR-616-3p binds to TFPI2 mRNA. We established that TFPI2 protein levels were significantly upregulated in PE placental tissues. In addition, we found that miR-616-3p binds specifically to the 3'-UTR region of TFPI2 mRNA. Furthermore, miR-616-3p knockdown or TFPI2 overexpression substantially impaired cell growth and migration, whereas miR-616-3p upregulation or TFPI2 knockdown stimulated cell proliferation and migration. This miR-616-3p/TFPI2 axis was also found to affect the epithelial-mesenchymal transition process in PE. Our results demonstrated that TFPI2 plays a vital role in the progression of PE and might provide a prospective therapeutic strategy to mitigate the severity of the disorder.

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.

Hypomethylation of tissue factor pathway inhibitor 2 in human placenta of preeclampsia

ObjectivesTo investigate the expression, DNA methylation status and its regulatory mechanism of tissue factor pathway inhibitor 2 (TFPI-2) in human placenta tissues of preeclampsia (PE). Material and methodsWe studied the mRNA and protein expression and the promoter methylation levels of TFPI-2 in the PE placentas compared with those in the normal pregnant (NP) women. Quantitative real-time polymerase chain reaction, immunohistochemistry, western blot, and Sequenom MassARRAY were used for placenta tissue detection. ResultsThe expressions of TFPI-2 mRNA and protein were significantly elevated in the PE placentas when compared with those in the NP ones (P<0.05). Hypomethylation of the TFPI-2 promoter was detected both in PE patients and NP women, with a significant decrease in PE placentas (P=0.005). The methylation level was significantly decreased at CpG_6 (−168 to −167), CpG_15 (−98 to −97) and CpG_18.19 (−68 to −65) in PE patients than that in normal placentas (P<0.05). However, the expression of DNMT-1 didn't show significant difference between the two groups (P>0.05). ConclusionOver-expression of TFPI-2 and aberrant promoter mythylation status presented in the PE placentas, suggesting that epigenetic mechanism might contribute to the pathogenesis of PE.

Abstract 13974: Loss of Endothelial Primary Cilia Promotes Thrombus Formation in Mice

Introduction: Recent evidence suggest that endothelial cilia may suppress anti-inflammatory signaling and protect against atherosclerosis. Given that atherosclerosis predisposes to thrombosis, we hypothesized that endothelial cilia may also play a fundamental role in thrombogenesis. Hypothesis: We hypothesize that endothelial cilia is important for protection against thrombosis, and that loss of endothelial cilia promotes thrombus formation in mice. Methods: We focused on the intraflagellar transport 88 (IFT88) gene since endothelial deletion of IFT88 has been associated with decreased atherosclerotic lesions in a murine model of atherosclerosis. Key thrombosis-associated molecules were evaluated in IFT88-silenced human umbilical vein endothelial cells (HUVECs). Thrombi formation were monitored in the laser-injured cremasteric arteries of 8 week old male endothelial cell (EC) specific IFT88 knockout (EC-IFT88 -/- ) mice. Results: Compared to scrambled siRNA-treated HUVECs, those exposed to siIFT88 exhibited lower mRNA and protein levels of tissue factor pathway inhibitor (TFPI) and thrombomodulin, two key regulators of blood coagulation (-27% and -20%, respectively; N=3; p &lt; 0.05). We next examined the effect of IFT88 loss on thrombosis in mice. Upon laser-induced injury, we determined, via mean fluorescence intensity, that although the thrombi formed in the cremasteric arteries of EC-IFT88 -/- mice were similar in size to those produced in the wild type (WT) mice (2.2 ± 0.6 and 2.0 ± 0.3 AU, respectively; x10 6 ; N=32 thrombi; p=0.8), the average rate at which thrombi developed was significantly faster in the EC-IFT88 -/- mice relative to that in WT mice (97 ± 11 and 57 ± 3 AU/s, respectively; N=32 thrombi; p&lt;0.001). Conclusions: Loss of endothelial IFT88 results in decreased TFPI mRNA and protein expression in HUVECs. EC-IFT88 -/- mice exhibited earlier thrombi formation in our laser injury model of thrombosis. These results collectively suggest that endothelial cilia intricately regulates thrombus formation in a TFPI-dependent manner and represents a previously unrecognized target to modulate thrombosis susceptibility.

Sulfur mustard inhalation: mechanisms of injury, alteration of coagulation, and fibrinolytic therapy.

Acute lung injury due to sulfur mustard (SM) inhalation causes the formation of airway fibrin casts that obstruct airways at multiple levels, leading to acute respiratory failure and death. These pathophysiological effects are seen in rodent models of acute SM vapor inhalation, as well as in human victims of acute SM inhalation. In rat models, the initial steps in activation of the coagulation system at extravascular sites depend on tissue factor (TF) expression by airway cells, especially in the microparticle fraction, and these effects can be inhibited by TF pathway inhibitor protein. Not only does the procoagulant environment of the acutely injured lung contribute to airway cast formation, but these lesions persist in airways because of the activation of multiple antifibrinolytic pathways, including plasminogen activator inhibitor-1, thrombin-activatable fibrinolysis inhibitor, and α2-antiplasmin. Airway administration of tissue plasminogen activator can overwhelm these effects and save lives by preventing fibrin-dependent airway obstruction, gas-exchange abnormalities, and respiratory failure. In human survivors of SM inhalation, fibrotic processes, including bronchiolitis obliterans and interstitial fibrosis of the lung, are among the most disabling chronic lesions. Antifibrotic therapies may prove useful in preventing either or both of these forms of chronic lung damage.

The role of microRNA‐27a/b and microRNA‐494 in estrogen‐mediated downregulation of tissue factor pathway inhibitor α

Essentials Estrogens are known to influence the expression of microRNAs in breast cancer cells. We looked at microRNAs in estrogenic regulation of tissue factor pathway inhibitor α (TFPIα). Estrogen upregulated microRNA-27a/b and microRNA-494 through the estrogen receptor α. MicroRNA-27a/b and microRNA-494 are partly involved in estrogenic downregulation of TFPIα. Background Tissue factor pathway inhibitor (TFPI) has been linked to breast cancer pathogenesis. We have recently reported TFPI mRNA levels to be downregulated by estrogens in a breast cancer cell line (MCF7) through the estrogen receptor α (ERα). Accumulating evidence also indicates that activation of ERα signaling by estrogens may modulate the expression of target genes indirectly through microRNAs (miRNAs). Objectives To examine if miRNAs are involved in the estrogenic downregulation of TFPIα. Methods Computational analysis of the TFPI 3'-untranslated region (UTR) identified potential binding sites for miR-19a/b, miR-27a/b, miR-494, and miR-24. Transient overexpression or inhibition of the respective miRNAs was achieved by transfection of miRNA mimics or inhibitors. Direct targeting of TFPI 3'-UTR by miR-27a/b and miR-494 was determined by luciferase reporter assay in HEK293T cells. Effects of 17α-ethinylestradiol (EE2) and fulvestrant on relative miR-27a/b, miR-494, and TFPI mRNA levels in MCF7 cells were determined by qRT-PCR and secreted TFPIα protein by ELISA. Transient knockdown of ERα was achieved by siRNA transfection. Results EE2 treatment lead to a significant increase in miR-19a, miR-27a/b, miR-494, and miR-24 mRNA levels in MCF7 cells through ERα. miR-27a/b and miR-494 mimics lead to reduced TFPI mRNA and protein levels. Luciferase assay showed direct targeting of miR-27a/b and miR-494 on TFPI mRNA. Impaired estrogen-mediated downregulation of TFPI mRNA was detected in anti-miR-27a/b and anti-miR-494 transfected cells. Conclusions Our results provide evidence that miR-27a/b and miR-494 regulate TFPIα expression and suggest a possible role of these miRNAs in the estrogen-mediated downregulation of TFPIα.

PO-24 - Determinants of thrombin generation in gynaecological malignancies

Increased thrombin production is associated with malignancy and is a marker for venous thromboembolism (VTE). Our group has shown that thrombin generation is increased in gynaecological malignancies. Although tumour derived Tissue Factor (TF) has been implicated, the precise mechanism by which thrombin production is increased is not fully understood. Our group has shown that gynaecological cancers can alter tumour expression of cogulation proteases. These changes may be implicated in the increased thrombin generation observed. Previous studies in normal control patients by other groups have shown that multiple coagulation factors are implicated in thrombin generation and that the effects depend on assay conditions however free Tissue Factor Pathway Inhibitor (TFPI), Factors V, VIIIc and protein S, were significant determinants of thrombin generation. The aim of this study was to determine the effect of factor V factor, VIIIc, TFPI and free protein S on the thrombin generation in patients with endometrial and ovarian cancer compared with benign controls. Patients with a gynaecological malignancy (n=43) (ovarian or endometrial) were matched with patients with a benign tumour (n=43) gave full and informed consent. Venous blood samples were obtained prior to surgery and chemotherapy. Thrombin generation was was measured using a fluorogenic assay. Lag time, peak thrombin and area under the thrombin generation curve (ETP) was determined and reported for each sample. Free protein S, free TFPI and factor V were determined using ELISA. Factor VIIIc was determined using a hromogenic assay. Factor V and factor VIIIc were significantly increased in the malignant group compared with the benign group (P<0.03; P<0.006). Increased free TFPI levels were also observed in the malignant group but this did not reach significance (P<0.06). There was no difference in free Protein S levels between the groups. Highest levels of peak thrombin generation were observed in the high grade serous and clear cell ovarian cancer patients. Changes were less marked in the endometrial patients. Free TFPI, factor V and factor VIIIc were important determinants of thrombin generation in the malignant group. Coagulation factors V and VIIIc and free TFPI are altered in patients with gynaecological malignancies and contribute to the increased thrombin generation found in these cancer patients. We have previously found increased expression of factor V in tumours from patients with ovarian cancer in addition to increased TF expression. These changes may explain the high rate of venous thromboembolism found in these patients.

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.

EPAS1/HIF-2 alpha-mediated downregulation of tissue factor pathway inhibitor leads to a pro-thrombotic potential in endothelial cells

Neovascularization and hemorrhaging are evident in advanced atherosclerotic plaques due to hypoxic conditions, and mediate the accumulation of metabolic substrates, inflammatory cells, lipids, and other blood born factors inside the plaque. Tissue factor (TF) pathway inhibitor (TFPI) is mainly expressed by endothelial cells and is the endogenous inhibitor of the coagulation activator TF, which together with the downstream product thrombin can drive plaque progression and atherogenesis. We aimed to investigate the effect of hypoxic conditions on endothelial cell expression and activity of TFPI and TF that are important in coagulation initiation.Hypoxia was induced in primary human umbilical vein endothelial cells using chemicals or 1% oxygen tension, and mRNA and protein expressions were measured using qRT-PCR, ELISA, and Western blot analysis. Microscopy of fluorescence-labeled cells was used to visualize cell-associated TFPI. Cell-surface factor Xa (FXa) activity was measured using a two-stage chromogenic substrate method. We found that hypoxia reduced the TFPI mRNA and protein levels and increased the TF mRNA expression in a dose-dependent manner. The effect on TFPI was apparent on all the protein pools of TFPI, i.e., secreted TFPI, cell-surface associated TFPI, and intracellular TFPI, and seemed to be dependent upon hypoxia inducible factor-2α (HIF-2α). An increase in FXa activity was also observed on the endothelial cell surface, reflecting an increase in pro-thrombotic potential of the cells.Our findings indicate that hypoxic conditions may enhance the pro-coagulant activity of endothelial cells, which may promote atherogenesis in addition to clinical events and thus the severity of atherosclerotic disorders.

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.