Tissue Factor Pathway Inhibitor Research Articles

Abstract WP212: Association Of Thrombin Generation With Leukocyte Inflammatory Profile In Patients With Acute Ischemic Stroke

Thrombosis is central to the pathogenesis of acute ischemic stroke, with higher thrombin generation being associated with increased stroke risk. The immune system may contribute to thrombin generation in stroke and thus may offer novel strategies for stroke prevention. This study addresses the research question regarding the relationship of thrombin generation to leukocyte gene expression in patients with acute ischemic stroke. We isolated RNA from whole blood and examined the relationship to thrombin generation capacity in patients with acute ischemic stroke. Due to its effects on thrombin generation, patients on anticoagulants were excluded from the study. The relationship of gene expression with peak thrombin was evaluated by analysis of covariance across peak thrombin quartiles adjusted for sex and age. In 97 patients with acute ischemic stroke, peak thrombin was variable, ranging from 252.0 to 752.4 nM. Increased thrombin generation was associated with differences in thromboinflammatory leukocyte gene expression, including a decrease in ADAM metallopeptidase with thrombospondin type 1 motif 13 and an increase in nuclear factor B (NF-kB)-activating protein, protein disulfde isomerase family A member 5, and tissue factor pathway inhibitor 2. Pathways associated with peak thrombin included interleukin 6 signaling, thrombin signaling, and NF-kB signaling. A linear discriminant analysis model summarizing the immune activation associated with peak thrombin in a first cohort of stroke could distinguish patients with low peak thrombin from high peak thrombin in a second cohort of 112 patients with acute ischemic stroke.The identified genes and pathways support a role of the immune system contributing to thrombus formation in patients with stroke. These may have relevance to antithrombotic strategies for stroke prevention.

Abstract B006: Does TFPI1 drive development of multiple drug resistant cancer and can the anaphase promoting complex prevent it

Abstract Chemotherapy is often rendered ineffective by the development of multiple drug resistance (MDR). Given time MDR occurs almost universally and once developed applies in a non-specific manner. The lack of MDR specificity means many drugs, including those a patient has no pervious exposure to, are affected which limits treatment options. The goal of this research is to show nuclear localized tissue factor pathway inhibitor 1 (TFPI1) plays a driving role in development of MDR, and that the Anaphase Promoting Complex (APC) is capable of inhibiting MDR by targeting TFPI1 for degradation. A coagulation regulator, TFPI1 normally inhibits the tissue factor pathway to prevent blood clot formation. However, TFPI1 is highly elevated in many types of MDR cancer where we believe it mediates resistance development. Previous work in our lab has shown plasmid overexpression of TFPI1 induces MDR in MCF7 breast cancer cells, in MDR cells TFPI1 does not localize to the cell surface but instead to the nucleus, and a putative TFPI1 nuclear localization sequence (NLS) was identified. To confirm the putative NLS is required for TFPI1’s localization to nucleus, and subsequent MDR development, NLS mutant or wild type TFPI1-GFP were overexpressed in MCF7 cells. Localization of TFPI1-GFP was observed using widefield and confocal microscopy. Wild type TFPI1-GFP localized to the nucleus while the NLS mutant TFPI1 did not. These results help to confirm the putative NLS and its importance to TFPI1s role in MDR development. The APC is a nuclear E3 ubiquitin ligase and highly conserved regulator of cell cycle progression. Defects in APC function are associated with reduced longevity and cancer. Other work in our lab focuses on the APC’s role in longevity using a yeast model. However, we believe the APC also plays a role in TFPI1’s promotion of MDR. Our observations suggest APC dysfunction exists within MDR cancer cells where APC substrates accumulate, as does TFPI1. We hypothesize that nuclear TFPI1 is an APC substrate, and that APC activation will result in TFPI1 degradation slowing MDR development. Our lab has isolated several small peptides we believe bind and activate the APC, and have used these peptides in conjugation with known APC activators such as Mad2 Inhibitor-1 (M2I-1) to show that APC activation is capable of reducing TFPI1 abundance in MCF7 cells treated with the chemotherapeutic Doxorubicin (Dox). These results suggest TFPI1 is an APC substrate. To summarize, we believe TFPI1 drives development of MDR cancer, for which TFPI1’s accumulation within the nucleus is important. We have shown TFPI1’s NLS is required for localization of the protein to the nucleus, and activation of the APC reduces nuclear accumulation of TFPI1. Moving forward our goal is to demonstrate the effect TFPI1 NLS knockout and APC activation have on the development of MDR in MCF7 cells undergoing drug selection with Dox. By exploring TFPI1’s role in MDR we may elucidate means to slow resistance development and extend chemotherapy effectiveness. Citation Format: Mathew Lubachowski, Troy Harkness. Does TFPI1 drive development of multiple drug resistant cancer and can the anaphase promoting complex prevent it [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B006.

Abstract B043: Tissue Factor Pathway Inhibitor (TFPI), a novel invasion and therapy target in glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM) is a deadly brain cancer with a 4-10% five-year survival rate. Even with the advent of temozolomide and tumor treating fields, at best GBM patients can hope for a median survival of approximately 25 months. The major cause of poor survivorship is the recurrence of inoperable therapy resistant secondary tumors. With the poor survivorship, there is an urgent need for novel therapy regimens. We propose a novel therapy and metastatic target for GBM treatment. Tissue Factor Pathway Inhibitor (TFPI) is a serine proteinase inhibitor. Classically, TFPI inhibits FXa and/or TF-VIIa to reduce blood coagulation. Recently, TFPI expression have been correlated with solid tumors such as breast, pancreatic, colorectal and brain tumors. However, it is unknown how TFPI contributes to cancer disease. We observed that TFPI increased expression correlated with GBM tumor samples. Additionally, GBM patients with high expression of TFPI had a poorer survivorship than those with low expression of TFPI. Using live cell imaging, we found that TFPI is required for cell motility, an initial and required step in cancer cell invasion. Additionally, we found that loss of TFPI increased 53bp1 foci formation. Increased 53bp1 foci formation is an indication of defunct DNA double stranded break repair. We found that loss of TFPI sensitizes cells to ionizing radiation another indication of a dysfunctional DNA double stranded break repair program. We found that loss of TFPI sensitizes cells to Parp-1 inhibition, a hallmark of a defunct homologous recombination repair program. Homologous recombination and non-homologous end-joining are the two major repair pathways for DNA double stranded breaks. Based on these preliminary data, we hypothesize that TFPI plays role in motility/invasion and DNA repair, which leads to therapy resistance and metastasis in GBM. TFPI is an attractive radio-sensitizer for GBM treatment since radiation is a first-line therapy modality. Additionally, Parp-1 inhibition is currently in clinical trials for GBM treatments. Lastly, there is an FDA approved inhibitor for TFPI, ANDEXXA and several TFPI antibodies in clinical trials. Our future endeavors are to elucidate the mechanisms by which TFPI regulates DNA repair and cancer cell invasion. These findings will lay down the foundation for using TFPI inhibitors as a concurrent treatment plan to standard of care. Citation Format: Tuyen T. Dang, Julio C. Morales. Tissue Factor Pathway Inhibitor (TFPI), a novel invasion and therapy target in glioblastoma multiforme [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B043.

Haemostasis and Inflammatory Parameters as Potential Diagnostic Biomarkers for VTE in Trauma-Immobilized Patients.

Venous thromboembolism (VTE), which encompasses deep venous thrombosis (DVT) and pulmonary embolism (PE), is a major public health concern due to its high incidences of morbidity and mortality. Patients who have experienced trauma with prolonged immobilization are at an increased risk of developing VTE. Plasma D-dimer levels have been known to be elevated in trauma patients, and they were closely correlated with the number of fractures. In other words, plasma D-dimer levels cannot be used as the only indicator of VTE in trauma cases. Given the limitations, further study is needed to explore other potential biomarkers for diagnosing VTE. To date, various established and novel VTE biomarkers have been studied in terms of their potential for predicting VTE, diagnostic performance, and improving clinical therapy for VTE. Therefore, this review aims to provide information regarding classic and essential haemostasis (including prothrombin time (PT), activated partial thromboplastin time (aPTT), D-dimer, fibrinogen, thrombin generation, protein C, protein S, antithrombin, tissue factor pathway inhibitor, and platelet count) and inflammatory biomarkers (C-reactive protein, erythrocyte sedimentation rate, and soluble P-selectin) as potential diagnostic biomarkers that can predict the risk of VTE development among trauma patients with prolonged immobilization. Thus, further advancement in risk stratification using these biomarkers would allow for a better diagnosis of patients with VTE, especially in areas with limited resources.

Tissue factor pathway inhibitor is a potential modifier of bleeding risk in factor XI deficiency

BackgroundFactor (F) XI deficiency is associated with increased bleeding risk in some individuals. Neither FXI levels nor clinical clotting assays predict the bleeding risk. Compared with controls, FXI-deficient bleeders have reduced clot formation, decreased fibrin network density, and increased susceptibility to fibrinolysis. Tissue factor pathway inhibitor (TFPI) was recently implicated as a modifying factor in individuals with bleeding of unknown cause. ObjectivesTo determine the potential of TFPI in modifying the bleeding risk in FXI-deficient individuals. MethodsThe effects of TFPI on thrombin generation and clot formation, structure, and fibrinolysis in FXI-deficient plasma were measured in vitro in the absence or presence of inhibitory anti-TFPI antibody or exogenous recombinant TFPIα. Total plasma TFPI concentration was measured in 2 independent cohorts of controls and FXI-deficient individuals classified as bleeders or nonbleeders (cohort 1: 10 controls and 16 FXI-deficient individuals; cohort 2: 48 controls and 57 FXI-deficient individuals) and correlated with ex vivo plasma clot formation and fibrinolysis parameters associated with bleeding risk. ResultsIn an in vitro FXI deficiency model, inhibition of TFPI enhanced thrombin generation and clot formation, increased the network density, and decreased fibrinolysis, whereas an increase in TFPI had the opposite effects. Compared with controls, plasma from FXI-deficient bleeders had higher TFPI concentration. Total plasma TFPI concentrations correlated with parameters from ex vivo clotting and fibrinolysis assays that differentiate FXI-deficient bleeders and nonbleeders. ConclusionCoagulation and fibrinolysis parameters that differentiate FXI-deficient nonbleeders and bleeders were altered by plasma TFPIα. Total plasma TFPI was increased in FXI-deficient bleeders. TFPI may modify the bleeding risk in FXI-deficient individuals.

The influence of lower-leg injury and knee arthroscopy on natural anticoagulants and fibrinolysis

BackgroundPatients with lower-leg injuries and those undergoing knee arthroscopy are at increased risk of developing venous thromboembolism. The mechanism is unknown, including the influence of lower-leg injury and knee arthroscopy on natural anticoagulant factors and fibrinolysis. ObjectivesTo study the effect of lower-leg injury and knee arthroscopy on plasma levels of anticoagulant and fibrinolytic factors. MethodsWe applied the following 2 designs to investigate this effect: a cross-sectional study for lower-leg trauma and a before-and-after study for knee arthroscopy. Plasma samples of POT-CAST– and POT-KAST–randomized clinical trial participants (collected shortly after lower-leg trauma or before or after arthroscopy) were analyzed for clot lysis time and levels of antithrombin, tissue factor pathway inhibitor, protein C, free protein S, plasminogen, tissue plasminogen activator, plasminogen activator inhibitor 1, antiplasmin, thrombin activatable fibrinolysis inhibitor, plasmin-antiplasmin, and D-dimer. For the effect of lower-leg injury, samples of 289 patients were compared with preoperative samples of 293 arthroscopy patients, acting as controls using linear regression and adjusting for age, sex, body mass index, comorbidities, and diurnal variation. For the effect of knee arthroscopy, mean changes were calculated for 277 patients using linear mixed models adjusted for diurnal variation. Parameters other than CLT and D-dimer were measured in smaller subsets. ResultsIn lower-leg injury patients, most parameters were stable, whereas D-dimer increased. After arthroscopy, most parameters decreased (especially clot lysis time, D-dimer, plasminogen, and anticoagulant factors), whereas tissue plasminogen activator and thrombin activatable fibrinolysis inhibitor slightly increased. ConclusionIn contrast to lower-leg injury, knee arthroscopy was associated with decreased natural anticoagulant factor levels. Neither lower-leg injury nor knee arthroscopy affected in vivo fibrinolysis.

Uric Acid Induces a Proatherothrombotic Phenotype in Human Endothelial Cells by Imbalancing the Tissue Factor/Tissue Factor Pathway Inhibitor Pathway.

Several evidence show that elevated plasma levels of uric acid (UA) are associated with the increased risk of developing atherothrombotic cardiovascular events. Hyperuricemia is a risk factor for endothelial dysfunction (ED). ED is involved in the pathophysiology of atherothrombosis since dysfunctional cells lose their physiological, antithrombotic properties. We have investigated whether UA might promote ED by modulating the tissue factor (TF)/TF pathway inhibitor (TFPI) balance by finally changing the antithrombotic characteristics of endothelial cells. Human umbilical vein endothelial cells were incubated with increasing doses of UA (up to 9 mg/dL). TF gene and protein expressions were evaluated by real-time polymerase chain reaction (PCR) and Western blot. Surface expression and procoagulant activity were assessed by FACS (fluorescence activated cell sorting) analysis and coagulation assay. The mRNA and protein levels of TFPI were measured by real-time PCR and Western blot. The roles of inflammasome and nuclear factor-κB (NF-κB) as possible mechanism(s) of action of the UA on TF/TFPI balance were also investigated. UA significantly increased TF gene and protein levels, surface expression, and procoagulant activity. In parallel, TFPI levels were significantly reduced. The NF-κB pathways appeared to be involved in modulating these phenomena. Additionally, inflammasome might also play a role. The present in vitro study shows that one of the mechanisms by which high levels of UA contribute to ED might be the imbalance between TF/TFPI levels in endothelial cells, shifting them to a nonphysiological, prothrombotic phenotype. These UA effects might hypothetically explain, at least in part, the relationship observed between elevated plasma levels of UA and cardiovascular events.

Markers of Hereditary Thrombophilia with Unclear Significance.

Thrombophilia leads to an increased risk of venous thromboembolism. Widely accepted risk factors for thrombophilia comprise deficiencies of protein C, protein S, and antithrombin, as well as the factor V "Leiden" mutation, the prothrombin G20210A mutation, dysfibrinogenemia, and, albeit less conclusive, increased levels of factor VIII. Besides these established markers of thrombophilia, risk factors of unclear significance have been described in the literature. These inherited risk factors include deficiencies or loss-of-activity of the activity of ADAMTS13, heparin cofactor II, plasminogen, tissue factor pathway inhibitor (TFPI), thrombomodulin, protein Z (PZ), as well as PZ-dependent protease inhibitor. On the other hand, thrombophilia has been linked to the gain-of-activity, or elevated levels, of α2-antiplasmin, angiotensin-converting enzyme, coagulation factors IX (FIX) and XI (FXI), fibrinogen, homocysteine, lipoprotein(a), plasminogen activator inhibitor-1 (PAI-1), and thrombin-activatable fibrinolysis inhibitor (TAFI). With respect to the molecular interactions that may influence the thrombotic risk, more complex mechanisms have been described for endothelial protein C receptor (EPCR) and factor XIII (FXIII) Val34Leu. With focus on the risk for venous thrombosis, the present review aims to give an overview on the current knowledge on the significance of the aforementioned markers for thrombophilia screening. According to the current knowledge, there appears to be weak evidence for a potential impact of EPCR, FIX, FXI, FXIII Val34Leu, fibrinogen, homocysteine, PAI-1, PZ, TAFI, and TFPI on the thrombotic risk.

Beyond uterine atony: characterizing postpartum hemorrhage coagulopathy

Postpartum hemorrhage is a leading cause of morbidity and mortality worldwide, yet the associated early coagulopathy is not well defined. We hypothesized that women who develop postpartum hemorrhage have a distinct derangement of thrombin generation and coagulation factors compared with postpartum women without postpartum hemorrhage. This prospective study of pregnant patients with postpartum hemorrhage was completed at a single urban hospital. Blood was drawn on postpartum hemorrhage diagnosis and 2 and 4 hours later. Assays of patients with postpartum hemorrhage included thrombelastography, whole blood thrombin generation, coagulation factor activity, tissue factor levels and activity, and tissue factor pathway inhibitor levels, which were compared with that of patients without postpartum hemorrhage. A total of 81 patients were included in this study. Of those patients, 66 had postpartum hemorrhage, and 15 served as controls. Compared with patients without PPH, patients with postpartum hemorrhage had lower fibrinogen levels (469.0 mg/dL vs 411.0 mg/dL; P=.02), increased tissue plasminogen activator resistance (fibrinolysis 30 minutes after maximal clot strength: 8.7% vs 4.2%; P=.02), decreased peak thrombin concentration (150.2 nM vs 40.7 nM; P=.01), and decreased maximal rate of thrombin generation (60.1 nM/minute vs 2.8 nM/minute; P=.02). Furthermore, compared with patients without postpartum hemorrhage, patients with postpartum hemorrhage had decreased tissue factor levels (444.3 pg/mL vs 267.1 pg/mL; P=.02) and increased tissue factor pathway inhibitor levels (0.6 U/mL vs 0.8 U/mL; P=.04), with decreased tissue factor pathway inhibitor ratios (624 vs 299; P=.01). PPH is not only an issue of uterine tone and mechanical bleeding but also a distinct coagulopathy that is characterized by decreased fibrinogen level, clot breakdown resistance, and markedly low thrombin generation. This pathology seemed to be driven by low tissue factor and high tissue factor pathway inhibitor levels.

SARS-CoV-2 vaccines are not associated with hypercoagulability in apparently healthy people

BackgroundSARS-CoV-2 adenoviral vector DNA vaccines have been linked to the rare but serious thrombotic postvaccine complication vaccine-induced immune thrombotic thrombocytopenia. This has raised concerns regarding the possibility of increased thrombotic risk after any SARS-CoV-2 vaccines. ObjectivesTo investigate whether SARS-CoV-2 vaccines cause coagulation activation leading to a hypercoagulable state. MethodsThis observational study included 567 health care personnel; 521 were recruited after the first dose of adenoviral vector ChAdOx1-S (Vaxzevria, AstraZeneca) vaccine and 46 were recruited prospectively before vaccination with a messenger RNA (mRNA) vaccine, either Spikevax (Moderna, n = 38) or Comirnaty (Pfizer-BioNTech, n = 8). In the mRNA group, samples were acquired before and 1 to 2 weeks after vaccination. In addition to the prevaccination samples, 56 unvaccinated blood donors were recruited as controls (total n = 102). Thrombin generation, D-dimer levels, and free tissue factor pathway inhibitor (TFPI) levels were analyzed. ResultsNo participant experienced thrombosis, vaccine-induced immune thrombotic thrombocytopenia, or thrombocytopenia (platelet count <100 × 109/L) 1 week to 1 month postvaccination. There was no increase in thrombin generation, D-dimer level, or TFPI level in the ChAdOx1-S vaccine group compared with controls or after the mRNA vaccines compared with baseline values. Eleven of 513 (2.1%) participants vaccinated with ChAdOx1-S had anti-PF4/polyanion antibodies without a concomitant increase in thrombin generation. ConclusionIn this study, SARS-CoV-2 vaccines were not associated with thrombosis, thrombocytopenia, increased thrombin generation, D-dimer levels, or TFPI levels compared with baseline or unvaccinated controls. These findings argue against the subclinical activation of coagulation post-COVID-19 vaccination.

A vasculogenic mimicry prognostic signature associated with immune signature in human gastric cancer.

Gastric cancer (GC) is one of the most lethal malignant tumors worldwide with poor outcomes. Vascular mimicry (VM) is an alternative blood supply to tumors that is independent of endothelial cells or angiogenesis. Previous studies have shown that VM was associated with poor prognosis in patients with GC, but the underlying mechanisms and the relationship between VM and immune infiltration of GC have not been well studied. In this study, expression profiles from VM-related genes were retrieved from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Cox regression was performed to identify key VM-related genes for survival. Subsequently, a novel risk score model in GC named VM index and a nomogram was constructed. In addition, the expression of one key VM-related gene (serpin family F member 1, SERPINF1) was validated in 33 GC tissues and 23 paracancer tissues using immunohistochemistry staining. Univariate and multivariate Cox regression suggested that SERPINF1 and tissue factor pathway inhibitor 2 (TFPI2) were independent risk factors for the prognosis of patients with GC. The AUC (> 0.7) indicated the satisfactory discriminative ability of the nomogram. SsGESA and ESTIMATE showed that higher expression of SERPINF1 and TFPI2 is associated with immune infiltration of GC. Immunohistochemistry staining confirmed that the expression of SERPINF1 protein was significantly higher in GC tissues than that in paracancer tissues. A VM index and a nomogram were constructed and showed satisfactory predictive performance. In addition, VM was confirmed to be widely involved in immune infiltration, suggesting that VM could be a promising target in guiding immunotherapy. Taken together, we identified SERPINF1 and TFPI2 as immunologic and prognostic biomarkers related to VM in GC.

Inhibition of platelet-surface-bound proteins during coagulation under flow I: TFPI

Blood coagulation is a self-repair process regulated by activated platelet surfaces, clotting factors, and inhibitors. Tissue factor pathway inhibitor (TFPI) is one such inhibitor, well known for its inhibitory action on the active enzyme complex comprising tissue factor (TF) and activated clotting factor VII. This complex forms when TF embedded in the blood vessel wall is exposed by injury and initiates coagulation. A different role for TFPI, independent of TF:VIIa, has recently been discovered whereby TFPI binds a partially cleaved form of clotting factor V (FV-h) and impedes thrombin generation on activated platelet surfaces. We hypothesized that this TF-independent inhibitory mechanism on platelet surfaces would be a more effective platform for TFPI than the TF-dependent one. We examined the effects of this mechanism on thrombin generation by including the relevant biochemical reactions into our previously validated mathematical model. Additionally, we included the ability of TFPI to bind directly to and inhibit platelet-bound FXa. The new model was sensitive to TFPI levels and, under some conditions, TFPI could completely shut down thrombin generation. This sensitivity was due entirely to the surface-mediated inhibitory reactions. The addition of the new TFPI reactions increased the threshold level of TF needed to elicit a strong thrombin response under flow, but the concentration of thrombin achieved, if there was a response, was unchanged. Interestingly, we found that direct binding of TFPI to platelet-bound FXa had a greater anticoagulant effect than did TFPI binding to FV-h alone, but that the greatest effects occurred if both reactions were at play. The model includes activated platelets’ release of FV species, and we explored the impact of varying the FV/FV-h composition of the releasate. We found that reducing the zymogen FV fraction of this pool, and thus increasing the fraction that is FV-h, led to acceleration of thrombin generation.

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

Tissue Factor Pathway Inhibitor Gamma Deletion Corrects the Bleeding Phenotype in Hemophilia Mice

Tissue factor (TF) is a transmembrane receptor that initiates coagulation and, via protease-activated receptors, enhances inflammation. Tissue factor pathway inhibitor (TFPI) is the endogenous regulator of TF activities. Anti-TFPI antibodies show promise as a hemophilia treatment but must be tittered to avoid potential thrombogenic side effects. From a single gene, through alternative splicing, humans express TFPI transcripts that encode for α and β isoforms; while mice express transcripts that encode for α and β, and also a third isoform γ, which is the most abundant. It is hypothesized that TFPIα is the dominant isoform anti-coagulant in man, whereas cell-anchored TFPIβ is the dominant anti-inflammatory. The main goal of our study is to further understand the impact of the different TFPI isoforms in hemophilic mice. To assess the functions of the α, β, and γ mouse-specific TFPI isoforms, using CRISPR/Cas9 technology, we generated TFPI α-, β-, γ-, α/β-, α/γ-, and β/γ- deleted mouse strains. Each knock-out strain is viable, and the offspring were born with expected Mendelian ratios. To evaluate the hemostatic function of the individual isoforms, all TFPI mice strains were bred on a hemophilia (FVIII-null) background and evaluated with a tail-vein rebleeding assay (TVRB). In this TVRB, a lateral tail vein is transected to cause bleeding; time to clot formation is recorded, then the clot is immediately dislodged, and the process is repeated over 15 minutes. In this assay, C57/Bl6 mice clot on average 26 +1.3 (mean+SD) times, whereas F8 null mice clot 5 + 2.6 times on average. Surprisingly, in the F8 null background, deletion of TFPIγ (14 + 6.5), alone or in combination as α/γ-deleted or β/γ-deleted, significantly reduced bleeding (p < 0.001), whereas TFPIα deletion had no impact on hemophilia bleeding (5 + 3). We next performed ex-vivo thrombin generation assays (TGA). The blood was collected via the inferior vena cava into corn trypsin inhibitor plus citrate syringes. Platelet-poor plasma was extracted and added into 96-well plates in addition to TF, phospholipids, calcium, and substrate. Endogenous thrombin potential was calculated from the area under the curve for generated thrombin for each TFPI mice strain on a hemophilia background. In agreement with the in vivo model, TFPIγ-deletion on the F8 null background significantly improved endogenous thrombin potential (P=0.037), while additional deletion of TFPIα (α/γ-deletion) did not provide any additional procoagulant effect. In conclusion, both in vivo and ex vivo hemostasis mouse models showed that, rather than three Kunitz TFPIα or cell-anchored TFPIβ, soluble two-Kunitz domain TFPIγ is the dominant mouse plasma isoform with respect to anti-coagulant function. Studies using mouse models that investigate the role of TFPI in hemostasis, thrombosis, and inflammation must carefully consider and address the impact of mouse TFPIγ on outcomes before extrapolating findings to human TFPI in health and disease.

Mathematical Modeling to Identify Clotting Factor Combinations That Modify Thrombin Generation in Hemophilia

Background: In previous work, we used our mathematical model to identify modifiers of thrombin generation in hemophilia A (Link, JTH, 18, 2020). We performed thousands of simulations that varied clotting factors and inhibitors between 50 and 150% of their baseline plasma levels. From those simulations, we identified the combinations of factors and inhibitors that led to enhanced thrombin generation. We found that factor V (FV), at low normal levels (50%-75%), was a major contributor of the thrombin enhancement. The model elucidated an underlying mechanism to be substrate competition between factors V and VIII for activated factor X (FXa), which occurred early in the thrombin generation process. That study was focused on hemophilia A. Aims: To identify factor combinations that enhance or attenuate thrombin generation in mild, moderate, and severe hemophilia A, B, and C. Methods: Plasma levels for factors II, V, VII, VIII, IX, X, XI, tissue factor pathway inhibitor (TFPI), and antithrombin (AT) were fixed at one of three values: 50%, 100%, or 150% of normal. We simulated all possible combinations of these three factor levels for normal blood, and mild, moderate, and severe hemophilia A, B, and C type blood, characterized by deficiencies of 10, 5, and 1% of normal plasma levels. For each combination of factor levels, tissue factor (TF) was varied, and combinations of factor levels were identified as thrombin enhancing (or attenuating) when they resulted in at least a 10-fold increase (or decrease) in detectable thrombin levels. We used an updated version of our mathematical model that includes interactions between TFPI and partially cleaved FV. Results: For simulations of severe hemophilia A and B, combinations of low normal levels (50%) of FV and TFPI and high normal levels (150%) of FII and FX lead to an increase in total thrombin. Interestingly, the opposite combinations (high normal FV and TFPI, low normal FII and FX) led to a decrease in total thrombin for simulations of mild hemophilia A and B. In some of the severe deficiency cases, high normal FVIII levels in hemophilia B and low normal FIX levels in hemophilia A were found to enhance thrombin generation. In normal blood and hemophilia C, combinations of low normal levels (50%) of FV and TFPI and high normal levels (150%) of FVIII and FIX led to an increase in total thrombin, and again the opposite levels attenuated thrombin generation. In some cases, low normal levels of AT also enhanced thrombin generation. Conclusion(s): Our mathematical model identified new and testable combinations of parameters that enhance thrombin generation in severe hemophilia and attenuated thrombin generation in mild hemophilia.

An Antibody Targeting Human FV Promotes Thrombin Generation and Reduces Bleeding in a Hemophilic Mouse Model

Hemophilia is a bleeding disorder that results from a deficiency of factor VIII (Hemophilia A; HA) or factor IX (Hemophilia B; HB). Protein replacement therapy ameliorates bleeding but can result in the development of neutralizing antibodies. Innovative nonfactor therapies are in development for hemophilia which target anticoagulant proteins such as tissue factor pathway inhibitor (TFPI), antithrombin, and activated protein C (APC). While these approaches show promise, they influence multiple coagulation factors and pathways which could raise safety concerns. Considering its essential role in coagulation in enhancing thrombin generation (TG), we hypothesized that altering the functional properties of FV/FVa could be an attractive approach for HA and HB treatment. Activated FV (FVa) assembles with FXa on activated cellular surfaces and enhances the kinetics of TG by several orders of magnitude. Here we assess whether monoclonal antibodies targeting all forms of FV are effective in promoting TG in hemophilia using both in vitro and in vivo models in hemophilic mice. Using an initial TG assay screen, we identified a procoagulant antibody (GB5) that binds with high affinity (Kd <0.5 nM) to human (h)FV, FVa, and FV-short and investigated its mechanism of action. Using proteolytic fragments of FV and western blotting experiments, we found that GB5 likely binds in the middle of the light chain region. To evaluate the functional effects of GB5 in vitro, we first employed a plasma-based TG assay using HA or HB plasma triggered with low (0.2 pM) tissue factor. The presence of saturating GB5 increased TG about 3-fold in HA plasma. Similar results were observed with HB plasma. Interestingly, when TG was essentially eliminated in HA and HB plasma with addition of exogenous APC, GB5 restored TG. These results suggest that the procoagulant effect of GB5 neutralizes the anticoagulant effect of APC and enhanced TG in hemophilic plasma To begin to assess the mechanism, the effect of GB5 on FV activation in the TG assay was evaluated. TG assay samples were removed over time, quenched, and analyzed by immunoblotting using labeled FV specific antibodies. In the presence of GB5, ~95% of the starting full-length FV was converted to FVa within 3 min compared to 5 min in the absence of GB5. These results show that FV is activated faster in the presence of GB5 in the TG assay. To extend these data, we next assessed the influence of GB5 in the TG assays using FV-deficient plasma supplemented with either FV or FVa and TFPI⍺. The data show that GB5 had essentially no effect on the TG assay when FV deficient plasma was reconstituted with FVa, but enhanced TG at least 3-fold when supplemented with FV. Consistent with this, the kinetics of prothrombin activation with FXa-FVa-membranes in the presence or absence of GB5 were the same. These data suggest that GB5 impacts reactions that influence FV, with no direct effect on FVa procoagulant function. To evaluate whether GB5 has a procoagulant effect in vivo, a tail vein transection (TVT) model was employed in HA mice. In this model, one lateral tail vein is transected, and blood is collected over 40 min with clots at the vessel wall disrupted every 10 min as needed. Since GB5 interacts with human and not mouse FV, plasma derived human FV was infused into HA mice. We found that blood loss in the TVT model using HA mice infused with human FV alone (5 μg/mouse) or GB5 alone (20 μg/mouse) was no different than mice infused with PBS (n = 3-5 mice/group except GB5 alone where n = 1: ~600 μl blood loss). In contrast to these data, when human FV and GB5 were co-administered, blood loss was reduced ~3-fold (~200 μl) which was statistically significant relative to the controls but did not reach the level of wild-type mice (blood loss ~50 μl). Overall, the results show that GB5 is a procoagulant monoclonal antibody that binds with high affinity to human FV and influences reactions that lead to its activation and inactivation. We hypothesize that the net effect of GB5 is the enhanced production of FVa which has a major impact on TG. In plasma-based systems with and without the protein C system and an in vivo hemophilic model, GB5 is procoagulant and can reduce bleeding. Our work shows that targeting the common coagulation pathway at the level of FV has benefit in the context of hemophilia and may be a viable and safe therapeutic approach.

Treatment Burden and Patient Preference in Patients with Hemophilia A or B with Inhibitors on Concizumab Prophylaxis: Results from the Phase 3 explorer7 Study

Introduction: Concizumab is a humanized, recombinant monoclonal antibody that acts to inhibit tissue factor pathway inhibitor to enhance coagulation. Patients with hemophilia A or B with inhibitors (HAwI/HBwI) received concizumab as a once-daily subcutaneous prophylactic treatment in the phase 3 explorer7 study (NCT04083781). In the explorer7 primary analysis, concizumab prophylaxis was shown to reduce the annualized bleeding rate for patients with either HAwI or HBwI when compared with no prophylaxis. Concizumab was also considered to be safe and well tolerated for HAwI/HBwI, with the latter patient population having the greatest unmet medical need. Subcutaneous administration of concizumab using a pen device may contribute to a lower treatment burden when compared with intravenous factor replacement therapy. Here, we present data on patient-reported treatment burden and patient preference from patients with HAwI/HBwI treated with concizumab in explorer7. Methods: A total of 133 patients aged ≥12 years were either randomized 2:1 to concizumab prophylaxis (arm 2; n=33) or no prophylaxis (arm 1; n=19) or assigned to non-randomized concizumab prophylaxis treatment (arms 3 or 4; n=81) based on the treatment they were receiving before entering the study. Patients were prompted to complete the Hemophilia Treatment Experience Measure (Hemo-TEM; Brod et al. abstract presented at ISTH 2022 [PB0654]) questionnaire at baseline (Week 0) and Week 24. In addition, patients were asked to complete the hemophilia patient preference questionnaire (H-PPQ) at Week 24. Results from both questionnaires were reported as exploratory endpoints in the study. Due to technical reasons, a number of patient-reported outcomes records could not be collected for statistical analysis. Analysis of covariance was applied to estimate mean change in Hemo-TEM domain and total scores from baseline to Week 24 in arms 1 and 2, with treatment, type of haemophilia and bleeding frequency prior to screening as factors, and the baseline value as a covariate. Descriptive statistics were used to describe the results of the H-PPQ questionnaire. Results: The Hemo-TEM total score improved in patients on concizumab prophylaxis (n=19) from baseline to Week 24 (mean estimate -17.0 points [95% CI: -23.7; -10.3]), while patients on no prophylaxis (n=6) did not experience an improvement (mean estimate 3.0 points [95% CI: -9.4; 15.3]). The estimated treatment difference in Hemo-TEM total score between the two arms was statistically significant (-19.9 points [95% CI: -34.3; -5.6]; P=0.009; Figure 1). Improvements were observed across all Hemo-TEM domains for patients on concizumab prophylaxis, with significant improvements in the domains "injection difficulties", "interference", and "emotional impact" (Figure 1). A total of 99 patients received the H-PPQ questionnaire; 77 (78%) indicated that they preferred concizumab over their previous treatment, while only one patient (1%) preferred their previous treatment (16 patients did not respond, and 5 patients had no preference). Out of the 83 patients who responded, 93% preferred concizumab over their previous treatment. The three main reasons for their treatment preference were "fewer bleeds" (75%), "require less time" (43%), and "less painful to inject" (33%). Conclusion: A lower treatment burden was observed for daily concizumab prophylaxis as patients generally reported better Hemo-TEM domain and total scores. For the Hemo-TEM total score, the estimated treatment difference between concizumab and no-prophylaxis arms was statistically significant. Most patients who responded to the H-PPQ questionnaire preferred concizumab prophylaxis over their previous treatment. Intravenous injection of replacement factor products poses a major burden to hemophilia patients. The observed improvements in treatment burden with subcutaneous administration of concizumab may have the potential to also improve treatment adherence and thereby achieve better clinical outcomes for patients with hemophilia. The combination of effective prophylaxis and reduced bleeding (especially in HBwI) together with lower treatment burden of subcutaneous delivery resulted in a clear patient preference in favor of concizumab. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants

Toxin B (TcdB) is a major exotoxin responsible for diseases associated with Clostridioides difficile infection. Its sequence variations among clinical isolates may contribute to the difficulty in developing effective therapeutics. Here, we investigate receptor-binding specificity of major TcdB subtypes (TcdB1 to TcdB12). We find that representative members of subtypes 2, 4, 7, 10, 11, and 12 do not recognize the established host receptor, frizzled proteins (FZDs). Using a genome-wide CRISPR-Cas9-mediated screen, we identify tissue factor pathway inhibitor (TFPI) as a host receptor for TcdB4. TFPI is recognized by a region in TcdB4 that is homologous to the FZD-binding site in TcdB1. Analysis of 206 TcdB variant sequences reveals a set of six residues within this receptor-binding site that defines a TFPI binding-associated haplotype (designated B4/B7) that is present in all TcdB4 members, a subset of TcdB7, and one member of TcdB2. Intragenic micro-recombination (IR) events have occurred around this receptor-binding region in TcdB7 and TcdB2 members, resulting in either TFPI- or FZD-binding capabilities. Introduction of B4/B7-haplotype residues into TcdB1 enables dual recognition of TFPI and FZDs. Finally, TcdB10 also recognizes TFPI, although it does not belong to the B4/B7 haplotype, and shows species selectivity: it recognizes TFPI of chicken and to a lesser degree mouse, but not human, dog, or cattle versions. These findings identify TFPI as a TcdB receptor and reveal IR-driven changes on receptor-specificity among TcdB variants.

Gene variants in pro-coagulant and anti-coagulant genes could be prognostic genetic markers of COVID-19 susceptibility

BackgroundPresent study aimed to identify DNA polymorphisms (variants) which can modulate the risk of COVID-19 infection progression to severe condition. TaqMan based SNP genotyping assay was performed for 11 single nucleotide polymorphisms (SNPs) in pro-coagulant and anti-coagulant genes. MethodologyA total of 33 COVID-19 patients, including dead, severe and moderately infected individuals were compared to 35 healthy controls. Both alleles in the SNP were labelled with two different fluorescent dyes (FAM and VIC) during assay formulation. DNA of study subjects were mixed with SNP assay and TaqMan master mix on 96 well PCR plate according to manufacturer’s protocol and RT-PCR was performed. Allelic discrimination assay gave clear results for presence of specific allele in each sample. Three SNPs were located in the pro-coagulant genes, another three involved in blood clot dissolution while rest five were in the genes encoding natural anti-coagulants. COVID-19 infected patients were further sub-divided into three groups, deceased (n = 16), severe (n = 10) and moderately infected (n = 7). ResultsSNP genotyping showed significant differences between COVID-19 patients and controls in two SNPs, rs6133 in Selectin-P (SELP) and rs5361 in Selectin-E (SELE) gene. Also, rs2020921 and rs8176592, in clot dissolution genes, tissue Plasminogen activator (tPA) and tissue factor pathway inhibitor (TFPI) respectively showed significant genotypic and allelic difference in patients of COVID-19 compared to healthy controls. Further three SNPs rs2227589, rs757583846, and rs121918476 in natural anti-coagulant genes anti-thrombin III (ATIII), protein C (PROC), and protein S (PROS) respectively showed statistically significant difference between the study groups. ConclusionOur findings indicate that gene variants, those involved in coagulation and anti-coagulation may play a major role in determining individual susceptibility to COVID-19.

RTFPI Protects Cardiomyocytes from Hypoxia/Reoxygenation Injury through Inhibiting Autophagy and the Class III PI3K/Beclin-1 Pathway.

Autophagy plays various roles at different stages of ischemia reperfusion (I/R) injury in cardiomyocytes. It has been reported that tissue factor pathway inhibitor (TFPI) has a protective effect on I/R injury. This study aimed to determine the roles of TFPI in autophagy during the I/R injury process in cardiomyocytes and the possible mechanisms. An isolated hypoxia/reoxygenation (H/R) pattern of cardiomyocytes was established by the MIC101 system. The cell viability and oxidative stress of cardiomyocytes were detected by an MTT assay and ROS assay, respectively. The autophagy level was measured by Ad-mCherry-GFP-LC3B and MDC. We detected the expression levels of autophagy-related proteins by western blotting. After 2 h of hypoxia and 12 h of reoxygenation, the cardiomyocyte viability in the H/R group was significantly lower than that in the control group (p < 0.05) than in the H/R group. According to intracellular ROS production, the fluorescence intensity in the H/R group was enhanced compared with that in the negative control group, and it was weaker in the H/R + rTFPI group compared with the H/R group. The level of autophagy and the expression levels of autophagy-related proteins (LC3-II/LC3-I, Beclin-1 and PI3K) were markedly increased in the H/R group compared to the control group (p < 0.05) whereas the levels were markedly decreased in the H/R + rTFPI group compared to the H/R group (p < 0.05). TFPI could relieve cardiomyocyte injury by inhibiting the Class III PI3K/Beclin-1 pathway and oxidative stress; thus, TFPI decreased autophagy and protected cardiomyocytes induced by H/R injury. In conclusion, TFPI may be a new direction for the prevention of myocardial I/R injury.