TFPI is a colonic crypt receptor for TcdB from hypervirulent clade 2 C. difficile.

The emergence of hypervirulent clade 2 Clostridioides difficile is associated with severe symptoms and accounts for >20% of global infections. TcdB is a dominant virulence factor of C. difficile, and clade 2 strains exclusively express two TcdB variants (TcdB2 and TcdB4) that use unknown receptors distinct from the classic TcdB. Here, we performed CRISPR/Cas9 screens for TcdB4 and identified tissue factor pathway inhibitor (TFPI) as its receptor. Using cryo-EM, we determined a complex structure of the full-length TcdB4 with TFPI, defining a common receptor-binding region for TcdB. Residue variations within this region divide major TcdB variants into 2 classes: one recognizes Frizzled (FZD), and the other recognizes TFPI. TFPI is highly expressed in the intestinal glands, and recombinant TFPI protects the colonic epithelium from TcdB2/4. These findings establish TFPI as a colonic crypt receptor for TcdB from clade 2 C. difficile and reveal new mechanisms for CDI pathogenesis.

Tissue factor pathway inhibitor-2 (TFPI-2)-an underappreciated partaker in cancer and metastasis.

The coagulation system is known to play an important role in cancer development and metastasis, but the precise mechanisms by which it does so remain incompletely understood. With this in mind, we provide an updated overview of the effects of TFPI-2, a protease inhibitor, on cancer development and metastasis. TFPI-2 interacts with the thrombin cascade and also employs other mechanisms to suppress cancer growth and dissemination, which include extracellular matrix stabilization, promotion of caspase-mediated cell apoptosis, inhibition of angiogenesis and transduction of intracellular signals. Down-regulation of TFPI-2 expression is well documented in numerous types of neoplasms, mainly via promoter methylation. However, the exact role of TFPI-2 in cancer progression and possible approaches to up-regulate TFPI-2 expression warrant further studies. Strategies to reactivate TFPI-2 may represent a promising direction for future anticancer studies and therapy development.

Relationship between plasma tissue Factor Pathway Inhibitor (TFPI) levels, thrombin generation and clinical risk of bleeding in patients with severe haemophilia A or B.

INTRODUCTION: Bleeding severity in severe haemophilic patients, with low thrombin generation (TG) capacity, can vary widely between patients, possibly reflecting differences in tissue factor pathway inhibitor (TFPI) level. AIM: To compare free TFPI (fTFPI) levels in patients with severe haemophilia A (sHA) and severe haemophilia B (sHB) and to investigate in these patients as a whole the relationships between bleeding and TG potential, between TG potential and fTFPI level and between fTFPI level and bleeding tendency. METHODS: Data on bleeding episodes retrospectively recorded during follow-up visits over 5-10 years were collected and used to calculate the annualised joint bleeding rate (AJBR). fTFPI levels and basal TG parameters were determined in platelet-poor plasma (PPP) and platelet-rich plasma (PRP) using calibrated automated tomography (CAT). RESULTS: Mean fTFPI levels did not differ significantly between sHA (n = 34) and sHB (n = 19) patients. Mean values of endogenous thrombin potential (ETP) and thrombin peak (peak) in PPP and PRP were two-fold higher when fTFPI levels < 9.4 versus > 14.3 ng/mL. In patients treated on demand, ETP and peak in PRP were doubled when AJBR was ≤ 4.9 $ \le 4.9$ , AJBR being halved in patients with a low fTFPI level (9.4 ng/mL). In patients on factor prophylaxis, no association was found between TG parameters and either fTFPI level or AJBR. CONCLUSION: In patients treated on demand, bleeding tendency was influenced by fTFPI levels, which in turn affected basal TG potential. In patients on prophylaxis, bleeding tendency is probably determined primarily by the intensity of this treatment.

Antithrombin supplementation attenuates heparin resistance in plasma spiked with Gla-domainless factor Xa S195A in vitro.

BACKGROUND: Andexanet alfa is a Gla-domainless mutant (S195A) factor Xa (GDXa) approved for acute reversal of oral factor Xa inhibitors. Cardiac surgery patients exposed to andexanet before cardiopulmonary bypass often exhibit severe heparin resistance. There is a paucity of data on the effectiveness and optimal dosage of antithrombin use in this setting. The objective of this study was to evaluate the in vitro effect of increased heparin with antithrombin levels on attenuating heparin resistance induced by GDXa. METHODS: Heparinised normal pooled plasma and cardiopulmonary bypass plasma were spiked with GDXa 4 µM. Tissue factor-activated thrombin generation was used to assess heparin reversal effects of GDXa and restoration of anticoagulation with additional heparin with and without antithrombin. Serum thrombin-antithrombin complex, antithrombin activity, and tissue factor pathway inhibitor were also measured in tissue factor-activated, recalcified cardiopulmonary bypass plasma spiked with GDXa. RESULTS: In normal pooled plasma, GDXa-induced heparin reversal was mitigated by maintaining a high heparin concentration (12 U ml-1) and supplementing antithrombin (1.5-4.5 µM) based on peak and velocity of thrombin generation. Heparin reversal by GDXa was also demonstrated in cardiopulmonary bypass plasma, but supplementing both heparin (8 U ml-1) and antithrombin (3 µM) attenuated GDXa-induced changes in peak and velocity of thrombin generation by 72.5% and 72.2%, respectively. High heparin and antithrombin levels attenuated thrombin-antithrombin complex formation in tissue factor-activated, GDXa-spiked cardiopulmonary bypass plasma by 85.7%, but tissue factor pathway inhibitor remained depleted compared with control cardiopulmonary bypass plasma. CONCLUSIONS: Simultaneous supplementation of heparin and antithrombin mitigate GDXa-induced heparin resistance by compensating for the loss of tissue factor pathway inhibitor.

Mutational analysis of pig tissue factor pathway inhibitor α to increase anti-coagulation activity in pig-to-human xenotransplantation.

Blood coagulation mediated by pig tissue factor (TF), which is expressed in pig tissues, causes an instant blood-mediated inflammatory reaction during pig-to-human xenotransplantation. Previously, we generated a soluble pig tissue factor pathway inhibitor α fusion immunoglobulin (TFPI-Ig) which inhibits pig TF activity more efficiently than human TFPI-Ig in human plasma. In this study, we generated several pig TFPI-Ig mutants and tested the efficacy of these mutants in preventing pig-to-human xenogeneic blood coagulation. Structurally important amino acid residues of pig TFPI-Ig were changed into different residues by site-directed mutagenesis. Subsequently, a retroviral vector encoding each cDNA of several pig TFPI-Ig mutants was cloned and transduced into CHO-K1 cells. After establishing stable cell lines expressing each of the pig TFPI-Ig mutants, soluble proteins were produced and purified for evaluating their inhibitory effects on pig TF-mediated blood coagulation in human plasma. The replacement of K36 and K257 with R36 and H257, respectively, in pig TFPI-Ig more efficiently blocked pig TF activity in human plasma when compared with the wild-type pig TFPI-Ig. These results may provide additional information to understand the structure of pig TFPIα, and an improved pig TFPI-Ig variant that more efficiently blocks pig TF-mediated blood coagulation during pig-to-human xenotransplantation.

Regulation of factor V by the anticoagulant protease activated protein C: Influence of the B-domain and TFPIα.

Activated protein C (APC) is an important anticoagulant protein that regulates thrombin generation through inactivation of factor V (FV) and activated factor V (FVa). The rate of APC inactivation of FV is slower compared to FVa, although proteolysis occurs at the same sites (Arg306, Arg506, and Arg679). The molecular basis for FV resistance to APC is unknown. Further, there is no information about how FV-short, a physiologically relevant isoform of FV with a shortened B-domain, is regulated by APC. Here, we identify the molecular determinants which differentially regulate APC recognition of FV versus FVa and uncover how FV-short can be protected from this anticoagulant pathway. Using recombinant FV derivatives and B-domain fragments, we show that the conserved basic region (BR; 963-1008) within the central portion of the B-domain plays a major role in limiting APC cleavage at Arg506. Derivatives of FV lacking the BR, including FV-short, were subject to rapid cleavage at Arg506 and were inactivated like FVa. The addition of a FV-BR fragment reversed this effect and delayed APC inactivation. We also found that anticoagulant glycoprotein TFPIα, which has a C-terminal BR homologous to FV-BR, protects FV-short from APC inactivation by delaying cleavage at Arg506. We conclude that the FV-BR plays a major role in protecting FV from APC inactivation. Using a similar mechanistic strategy, TFPIα also shields FV-short from APC. These findings clarify the resistance of FV to APC, advance our understanding of FV/FVa regulation, and establish a mechanistic framework for manipulating this reaction to alter coagulation.

Longitudinal observations of TFPI levels in paediatric Haemophilia A patients.

INTRODUCTION: Inhibition of tissue factor pathway inhibitor (TFPI) is a potential new mode of action to achieve haemostasis in haemophilia A and B patients. AIM: Knowledge about potential developmental changes of TFPI levels during childhood are a prerequisite to translate adult doses of TFPI inhibitors to doses in paediatric patients. METHODS: In this study we present longitudinal data for total TFPI concentrations (TFPI-T) and TFPI activity (TFPI-A) from 48 paediatric Haemophilia A patients in the age range from 3 to 18 years (2-12 observations per patient). RESULTS: TFPI-T and TFPI-A tend to decrease over age during childhood. Lowest values were observed between 12 and <18 years. On average, TFPI-T and TFPI-A were lower in adolescent haemophilia patients than in adult haemophilia patients. CONCLUSION: In summary, the presented information on TFPI levels in children adds to the current knowledge of developmental haemostasis and it can be helpful in evaluating how children respond to haemophilia treatment including the new class of anti-TFPI compounds.

Targeted Therapy of Non-Small Cell Lung Cancer and Liver Cancer: Functional Nanocarriers for the Delivery of Cisplatin and Tissue Factor Pathway Inhibitor-2.

INTRODUCTION: The aim of the study was to construct folic acid-modified PEGylated paramagnetic nanoparticles (MNPs) co-carrying tissue factor pathway inhibitor-2 (TFPI-2) and cisplatin (CDDP), and to study the molecular-targeting and inhibitory effects of the nanocomposite on non-small cell lung cancer (NSCLC) and liver cancer. METHODS: Nanocomposites were prepared using amino-modified iron oxide nanoparticles as carriers, co-loading CDDP and PEGylated FA/TFPI-2. Transmission electron microscopy, UV absorption spectrum, and dynamic light scattering were employed to characterize the morphology, structure, particle size, and zeta potential of the nanocomposite. The phenylenediamine method was used to detect the loading of CDDP, and the CCK-8 assay was used to detect the toxic effect of the nanocomposite on HUVECs, A549, and NCI-H460 cells. In tumor-bearing mice models, the antitumor effects of the nanocomposites were assessed using TUNEL staining (at the molecular level), reverse transcriptase quantitative polymerase chain reaction (at the gene level), hematoxylin and eosin staining (at the cellular level), and the appearance of the mice models. RESULTS: The synthesized FA-MNP/CDDP/TFPI-2 nanocomposite was uniformly dispersed and spherical in shape (approximate diameter: 10 nm). The zeta potential of particles was -9.44 mV, and the average particle size was 25 nm. The loading amount of CDDP was 70.24 µg/mL (23.33%). The nanocomposite was nontoxic to HUVECs, while it showed a favorable inhibitory effect on A549 and NCI-H460 cells. In vivo experiments in mice demonstrated satisfactory imaging properties and therapeutic effects of nanocomposite against liver cancer. DISCUSSION: FA-MNP/CDDP/TFPI-2 may provide insights for the development of new chemotherapeutic drugs.

Tissue factor pathway inhibitor 2: Current understanding, challenges, and future perspectives.

AIM: Tissue factor pathway inhibitor 2 (TFPI2) is a structural homolog of tissue factor pathway inhibitor 1 (TFPI1). Since TFPI2 is a placenta-derived protein, dynamic changes in TFPI2 levels may be related to pregnancy-related diseases. Furthermore, TFPI2 has been reported to be a novel serum biomarker for detecting ovarian cancer, especially clear cell carcinoma (CCC). This review aims to summarize the current knowledge on the biological function of TFPI2, highlight the major challenges that remain to be addressed, and discuss future research directions. METHODS: Papers published up to March 31, 2023 in the PubMed and Google Scholar databases were included in this review. We also provide novel complementary information to what is known about the action of TFPI2. RESULTS: Since TFPI2 concentrations in the blood of pregnant women, preeclampsia patients, and cancer patients vary greatly, its pathophysiological functions have attracted attention. Downregulation of TFPI2, a tumor-suppressor gene, by hypermethylation may contribute to the progression of several cancers. On the other hand, TFPI2 overexpressed in CCC is a risk factor for the development of thrombosis, possibly through inhibition of plasmin activity. However, agreement on the biological function of TFPI2 is still lacking and there are many scientific questions to be addressed. In particular, the lack of international standardization for the quantification of TFPI2 concentrations makes it difficult for researchers and clinicians to evaluate, pool, and compare data from different studies across countries. DISCUSSION: This review summarizes current understandings and challenges in TFPI2 research and discusses future perspectives.

The role of tissue factor pathway inhibitor 2 in the coagulation and fibrinolysis system.

AIM: Tissue factor (TF), the primary initiator of the extrinsic coagulation pathway, contributes to the generation of a hypercoagulable and prothrombotic state in cancer patients. TF pathway inhibitor (TFPI) is a major inhibitor of TF-mediated coagulation pathway. The two proteins, TFPI1 and TFPI2, are encoded by separate genes. Indeed, various cancer patients with venous thromboembolism (VTE) had significantly lower TFPI1 levels than those without VTE. In contrast, serum TFPI2 level was found to increase in ovarian cancer patients with VTE. It remains unclear why TFPI2, unlike TFPI1, is elevated in ovarian cancer patients with VTE. The aim of this review is to explore the pathophysiological role of TFPI2 on the coagulation and fibrinolysis system. METHODS: A literature search was performed from inception to April 30, 2022 in the PubMed and Google Scholar databases. RESULTS: TFPI1 and TFPI2 are homologs with different protease inhibitory activities in the coagulation and fibrinolysis system. TFPI1 inhibits TF/factor VIIa (FVIIa) catalyzed factor X (FX) activation. On the other hand, TFPI2 is unlikely to affect TF-initiated thrombin generation, but it has strong inhibitory activity against plasmin. Plasmin is involved in fibrin degradation, clot lysis, and inactivation of several coagulation factors (such as FV, FVIII, FIX, and FX). TFPI2 may increase the risk of VTE by inhibiting plasmin-dependent fibrinolysis. CONCLUSION: TFPI1 and TFPI2 may have different key functions in regulating the coagulation and fibrinolytic systems.

Studies on activation and regulation of the coagulation cascade in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Increased activation of the coagulation cascade and diminished fibrinolysis combine to promote fibrin deposition and polyp formation in chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP). More information is needed concerning mechanisms of coagulation in CRSwNP. OBJECTIVE: We investigated the mechanisms as well as the initiation and regulation of coagulation cascade activation in CRS. METHODS: Samples were collected from 135 subjects with CRSwNP, 80 subjects with chronic CRS without nasal polyps (NP), and 65 control subjects. The levels of activated factor X (FXa), prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex, tissue factor (TF), and TF pathway inhibitor (TFPI) were monitored in CRS by real-time PCR, ELISA, immunohistochemistry, or immunofluorescence. Heteromeric complexes of TF with activated factor VII (FVII) and TF with activated FVII and FXa were assessed by coimmunoprecipitation and Western blotting. RESULTS: Increased levels of FXa, F1+2, and thrombin-antithrombin complex were detected in NP tissue compared to uncinate tissue from CRS and control subjects. Although free TF protein levels were not increased in NP, immunoprecipitation of TF in NP tissue revealed increased complexes of TF with FVII. Local expression of FVII was detected in sinonasal mucosa, and the ratio of TFPI to FXa was lower in NP tissue. CONCLUSION: The coagulation cascade is associated with NP compared to control and uncinate tissue from CRS patients, and TF and FVII are produced locally in sinonasal mucosa in patients. TF and FVII can activate the extrinsic coagulation pathway, suggesting that this pathway may activate fibrin deposition in CRSwNP. Reduced formation of the complex of FXa and TFPI in NP may reduce natural suppression of the extrinsic coagulation pathway in CRSwNP.

Long Non-Coding RNAs in Venous Thromboembolism: Where Do We Stand?

Venous thromboembolism (VTE), a common condition in Western countries, is a cardiovascular disorder that arises due to haemostatic irregularities, which lead to thrombus generation inside veins. Even with successful treatment, the resulting disease spectrum of complications considerably affects the patient's quality of life, potentially leading to death. Cumulative data indicate that long non-coding RNAs (lncRNAs) may have a role in VTE pathogenesis. However, the clinical usefulness of these RNAs as biomarkers and potential therapeutic targets for VTE management is yet unclear. Thus, this article reviewed the emerging evidence on lncRNAs associated with VTE and with the activity of the coagulation system, which has a central role in disease pathogenesis. Until now, ten lncRNAs have been implicated in VTE pathogenesis, among which MALAT1 is the one with more evidence. Meanwhile, five lncRNAs have been reported to affect the expression of TFPI2, an important anticoagulant protein, but none with a described role in VTE development. More investigation in this field is needed as lncRNAs may help dissect VTE pathways, aiding in disease prediction, prevention and treatment.

Regulation of factor V and factor V-short by TFPIα: Relationship between B-domain proteolysis and binding.

Coagulation factor V (FV) plays an anticoagulant role but serves as a procoagulant cofactor in the prothrombinase complex once activated to FVa. At the heart of these opposing effects is the proteolytic removal of its central B-domain, including conserved functional landmarks (basic region, BR; 963-1008 and acidic region 2, AR2; 1493-1537) that enforce the inactive FV procofactor state. Tissue factor pathway inhibitor α (TFPIα) has been associated with FV as well as FV-short, a physiologically relevant isoform with a shortened B-domain missing the BR. However, it is unclear which forms of FV are physiologic ligands for TFPIα. Here, we characterize the binding and regulation of FV and FV-short by TFPIα via its positively charged C-terminus (TFPIα-BR) and examine how bond cleavage in the B-domain influences these interactions. We show that FV-short is constitutively active and functions in prothrombinase like FVa. Unlike FVa, FV-short binds with high affinity (Kd ∼1 nM) to TFPIα-BR, which blocks procoagulant function unless FV-short is cleaved at Arg1545, removing AR2. Importantly, we do not observe FV binding (µM detection limit) to TFPIα. However, cleavage at Arg709 and Arg1018 displaces the FV BR, exposing AR2 and allowing TFPIα to bind via its BR. We conclude that for full-length FV, the detachment of FV BR from AR2 is necessary and sufficient for TFPIα binding and regulation. Our findings pinpoint key forms of FV, including FV-short, that act as physiologic ligands for TFPIα and establish a mechanistic framework for assessing the functional connection between these proteins.

Befovacimab, an anti-tissue factor pathway inhibitor antibody: Early termination of the multiple-dose, dose-escalating Phase 2 study due to thrombosis.

INTRODUCTION: Befovacimab (formerly BAY 1093884) is a fully human monoclonal antibody able to bind to tissue factor pathway inhibitor (TFPI) and developed as a non-replacement therapy for individuals with haemophilia A/B, with or without inhibitors. AIM: To assess the safety of multiple escalating doses of befovacimab in individuals with severe haemophilia A/B with or without inhibitors. METHODS: In this non-randomised, open-label Phase 2 study (NCT03597022), adult males with <1% factor VIII or <2% factor IX and ≥4 bleeds in the previous six months were enrolled in three dose cohorts (100/225/400 mg). Participants received befovacimab subcutaneously once weekly. The primary endpoint was safety; secondary endpoints included annualised bleeding rate (ABR) and pharmacokinetics/pharmacodynamics (PK/PD) of befovacimab. RESULTS: A total of 24 participants (n = 8 in each dose cohort) were treated for 2-47 weeks. Patients treated with 100 mg and 225 mg doses of befovacimab demonstrated improved bleeding control compared with pre-study bleeding rates, with a dose-dependent effect. Dosing was suspended and the study prematurely terminated following three drug-related thrombotic serious adverse events (SAEs): two at the 225 mg dose and one at the 400 mg dose. These occurred in the absence of bleeding episodes or concomitant use of replacement/bypass therapies. No laboratory abnormalities were observed, and PK/PD data did not show correlation between SAE occurrence and levels of circulating befovacimab or free TFPI. CONCLUSION: Despite favourable initial results from preclinical and clinical studies, a positive safety profile of befovacimab was not confirmed. The lack of SAE-related laboratory abnormalities or differentiating PK/PD characteristics in participants experiencing SAEs raises concerns about the predictability of thrombosis following befovacimab treatment and emphasises the need for further investigation into the therapeutic window of anti-TFPI treatment.

NETosis and SARS-COV-2 infection related thrombosis: a narrative review.

BACKGROUND: Coronavirus disease 2019 (COVID-19) infection is related to immune hyperactivity, the release of inflammatory cytokines, and immunothrombosis. Among the underlying mechanisms in COVID-19 thrombosis, neutrophil extracellular traps (NETs) formation, NETosis, may have a significant role. COVID-19 thrombi obtained from extracorporeal membrane oxygenation contained an accumulation of neutrophils and in a higher amount of NETs when compared with non-COVID-19 thrombi specimens. MAIN BODY: During sepsis and inflammatory status, NETs released from neutrophils and histones and nucleosomes extruded into the extracellular space and take part in the host innate immunity defense, inflammation, and thrombosis. Excessive NETosis is related to clinical progression and respiratory failure in infections and sepsis. NETosis act as a scaffold for thrombus formation, and new associative data support the relation between deregulated immune responses with thrombus formation and organ failure. NETosis is reported in COVID-19 patients. In COVID-19 infection, overproduction of tissue factor (TF) by neutrophils has a role in immunothrombosis. Additionally, NETs can trap TF pathway inhibitor (TFPI) as the only endogenous protein that effectively inhibits the activity of the significant proteases- complexes, TF-FVIIa and prothrombinase. CONCLUSION: Because of NETosis can induce intrinsic and extrinsic coagulation cascade activation through the production of TF, activation of FXII, and inhibition of TFPI and fibrinolysis and induce immunothrombosis, targeting NETosis may diminish thrombus formation related to NETs in COVID-19 patients.

Tissue Factor Pathway Inhibitor 2: A Novel Biomarker for Predicting Asymptomatic Venous Thromboembolism in Patients with Epithelial Ovarian Cancer.

OBJECTIVES: Patients with asymptomatic venous thromboembolism (VTE) are associated with an increased risk of pulmonary thromboembolism events. However, due to low specificity and high false-positive rates, D-dimer testing cannot be used alone to diagnose VTE. Tissue factor pathway inhibitor 2 (TFPI2), a new serodiagnostic marker for ovarian cancer, plays a role in blood coagulation system regulation. We hypothesized that combining D-dimer and TFPI2 would improve its utility in diagnosing VTE. This study aimed to look into the clinical utility of serum D-dimer and TFPI2 levels in detecting asymptomatic VTE in patients with epithelial ovarian cancer (EOC). DESIGN: From January 2008 to December 2015, researchers at Nara Medical University Hospital's Department of Gynecology conducted a single-center retrospective study. The receiver operating characteristic (ROC) curve analysis was used to determine the diagnostic value of preoperative D-dimer, TFPI2, and D-dimer combined with TFPI2 in distinguishing VTE patients from those who did not have VTE. PARTICIPANTS: This study included 122 patients with EOC who met the inclusion and exclusion criteria out of 223 admitted to the hospital with EOC. The patients were divided into two groups: VTE (n = 25) and non-VTE (n = 97). RESULTS: There were significant differences in D-dimer, TFPI2, and CA125 levels and residual tumor between the VTE and non-VTE groups. The D-dimer level was found to be significantly related to age, body mass index, VTE, massive ascites, residual tumor, histology, and Federation of Gynecology and Obstetrics stage, whereas the TFPI2 level was only related to VTE. Multivariate analysis revealed that D-dimer (the optimal cutoff value, 3.5 µg/mL) and TFPI2 (the optimal cutoff value, 400 pg/mL) are independent risk factors for preoperative VTE. ROC analysis revealed that the area under the curve was 0.8266 for D-dimer, 0.7963 for TFPI2, and 0.8495 for the combination of D-dimer and TFPI2. When compared to the D-dimer test alone, the combination of D-dimer and TFPI2 had higher specificity (77.3-96.9%) and positive predictive value (48.8-81.2%) for the diagnosis of VTE. LIMITATIONS: This is a single-center retrospective study. CONCLUSION: The combination of D-dimer and TFPI2 may be useful to safely exclude VTE and select patients at high risk of VTE.

Tissue factor pathway inhibitor 2: A potential diagnostic marker for discriminating benign from malignant ovarian tumors.

OBJECTIVES: Carbohydrate antigen 125 (CA125), CA19-9, carcinoembryonic antigen (CEA), human epididymis protein 4 (HE4), and the Risk of Ovarian Malignancy Algorithm (ROMA) are widely used as tumor markers and algorithms for the diagnosis of ovarian cancer (OC). Tissue factor pathway inhibitor 2 (TFPI2) has been developed as a potential serodiagnostic marker for OC in Japan. The aim of this study is to evaluate the diagnostic accuracy of the six markers alone and in combination to find the best marker for discriminating between benign and malignant ovarian tumors. METHODS: Frozen serum samples collected from 484 patients were divided into three groups based on histopathological results: OC (n = 119), borderline ovarian tumors (BR) (n = 48), and benign ovarian tumors (BN) (n = 317). Diagnostic accuracy was calculated with an area under a receiver operating characteristic (AUC) curve. RESULTS: TFPI2 achieved the highest discrimination between the OC + BR group versus the BN group (AUC 0.8076). ROMA values best discriminated patients with OC from those with BN (AUC, 0.8966), which was equivalent to TFPI2 (AUC, 0.8937). For discriminating the OC group from the BR + BN group, the highest AUC value was achieved by ROMA values (AUC, 0.8884), and TFPI2 also showed comparable diagnostic accuracy (AUC, 0.8845). Combining TFPI2 with ROMA had the highest AUC (0.8420-0.9357). CONCLUSION: TFPI2 may be a clinically useful single marker comparable to conventional ROMA values for discriminating between benign and malignant ovarian tumors.

Expression of tissue factor and tissue factor pathway inhibitors during ovulation in rats: a relevance to the ovarian hyperstimulation syndrome.

BACKGROUND: Blood coagulation has been associated with ovulation and female infertility. In this study, the expression of the tissue factor system was examined during ovulation in immature rats; the correlation between tissue factor and ovarian hyperstimulation syndrome (OHSS) was evaluated both in rats and human follicular fluids. METHODS: Ovaries were obtained at various times after human chorionic gonadotropin (hCG) injection to investigate the expression of tissue factor system. Expression levels of ovarian tissue factor, tissue factor pathway inhibitor (Tfpi)-1 and Tfpi-2 genes and proteins were determined by real-time quantitative polymerase chain reaction (qPCR), and Western blot and immunofluorescence analyses, respectively. Expression levels of tissue factor system were also investigated in ovaries of OHSS-induced rats and in follicular fluid of infertile women. RESULTS: The expression of tissue factor in the preovulatory follicles was stimulated by hCG, reaching a maximum at 6 h. Tissue factor was expressed in the oocytes and the preovulatory follicles. Tfpi-2 mRNA levels were mainly increased by hCG in the granulosa cells whereas the mRNA levels of Tfpi-1 were decreased by hCG. Human CG-stimulated tissue factor expression was inhibited by the progesterone receptor antagonist. The increase in Tfpi-2 expression by hCG was decreased by the proliferator-activated receptor γ (PPARγ) antagonist. Decreased expression of the tissue factor was detected in OHSS-induced rats. Interestingly, the tissue factor concentrations in the follicular fluids of women undergoing in vitro fertilization were correlated with pregnancy but not with OHSS. CONCLUSIONS: Collectively, the results indicate that tissue factor and Tfpi-2 expression is stimulated during the ovulatory process in rats; moreover, a correlation exists between the levels of tissue factor and OHSS in rats but not in humans.

An in vitro pharmacodynamic spiking study of befovacimab, a tissue factor pathway inhibitor monoclonal antibody, in blood samples from patients with severe FVIII deficiency.

INTRODUCTION: Tissue factor pathway inhibitor (TFPI) is an endogenous protein that inhibits the extrinsic (tissue factor) pathway and negatively regulates thrombin production during coagulation. Inhibiting TFPI may become a useful target for haemophilia drug development to allow greater thrombin generation without use of the intrinsic (contact) pathway. AIMS: The in vitro effects of befovacimab, a humanized TFPI neutralizing antibody, were studied in whole blood and plasma samples from patients with severe FVIII deficiency. METHODS: Blood and plasma obtained from participants was supplemented in vitro with befovacimab (0.5, 1, 5, 10 and 100 nM) or recombinant factor VIII (rFVIII) 5-, 10- and 40% and analysed using rotational thromboelastometry (ROTEM), thrombin generation assay (TGA) and the dilute prothrombin time (dPT) assay. The in vitro coagulation effects of befovacimab were compared to samples supplemented with rFVIII. RESULTS: Befovacimab induced consistent pro-coagulant responses in ROTEM parameters including reduction in clotting times and increases in α-angle; induced reductions in dPT clotting time; and improvements in TGA parameters (reduced lag time and increased thrombin generation parameters). There was a modest concentration-dependent response generally from 0.5- to 10 nM, after which, the pharmacodynamic effect plateaued through the 100 nM concentration. Befovacimab concentrations of 5 to 10 nM showed pro-coagulant activity comparable to blood samples supplemented with rFVIII 10-40%. CONCLUSIONS: Befovacimab has modest dose-response effects from 0.5 to 10 nM with minimal improvement with higher concentrations. In vitro befovacimab blood concentrations of 5 to 10 nM had pro-coagulant effects similar to blood supplemented with rFVIII 10- to 40%.

Novel treatments for hemophilia through rebalancing of the coagulation cascade.

Hemophilia A and B are inherited hemorrhagic disorders that result from alterations in the coagulation cascade. Aside from spontaneous bleeding, the main complication of hemophilia is hemarthrosis. Progress over the last three decades, specifically prophylaxis using recombinant factor, has prevented hemarthrosis and lengthened patient life expectancies. However, many treatments require frequent dosing up to three times a week, and alloantibodies (inhibitors) against replacement factor continues to be an issue. These problems call for novel treatments for patients with hemophilia. Although there has been progress in extended half-life factors and mimetics of factor VIII, an alternative treatment methodology is to rebalance the activities of pro- and anticoagulant factors through inhibition of the natural anticoagulants: antithrombin, tissue factor pathway inhibitor, protein C, and protein S. This review will explore the efficacy of targeting these inhibitory pathways from preclinical development through clinical trials, and delve into concerns of thrombotic risk.