Inhibition Of Tissue Factor Pathway Inhibitor Research Articles

Evolution of Antidrug Antibody Assays During the Development of Anti-Tissue Factor Pathway Inhibitor Monoclonal Antibody Marstacimab.

Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of the extrinsic coagulation pathway. In patients with hemophilia A or B, inhibition of TFPI is an alternative therapeutic approach that augments the extrinsic coagulation pathway. Marstacimab is an investigational fully human monoclonal antibody that binds and neutralizes TFPI and is being evaluated as a prophylactic treatment to prevent or reduce the frequency of bleeding episodes in patients with severe hemophilia A or B, with or without inhibitors (antibodies against coagulation factors). However, the efficacy, safety, and pharmacokinetics of marstacimab may be affected by the induction of antidrug antibody (ADA) responses. Here, we describe the evolution and validation of three quasi-quantitative electrochemiluminescence-based methods to detect marstacimab ADAs, starting from their use in a first-in-human phase 1 study to their use in phase 2 and 3 clinical studies of patients with severe hemophilia. For all three methods, validation criteria evaluated the performance of the assays in screening and confirmatory cut points, precision, selectivity, drug tolerance, target interference, and stability. Additional criteria for validation were dilution linearity (Methods 1 and 2) and low positive control concentration, prozone effect, plate homogeneity, and robustness (Method 3). The three methods met validation criteria and are a potentially valuable tool in detecting the induction of marstacimab ADAs during treatment in patients with hemophilia.

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.

Marstacimab, a tissue factor pathway inhibitor neutralizing antibody, improves coagulation parameters of ex vivo dosed haemophilic blood and plasmas.

Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of the extrinsic pathway that negatively regulates thrombin production during coagulation. Under haemophilic conditions, where the intrinsic coagulation pathway is impaired, inhibition of TFPI may improve clotting. We investigated the ex vivo effects of a human TFPI neutralizing antibody, marstacimab (previously PF-06741086), in coagulation assays including rotational thromboelastometry (ROTEM), thrombin generation assay (TGA) and the dilute prothrombin time (dPT) assay, performed in haemophilic whole blood and plasmas. We compared the effects of marstacimab to the effects of recombinant coagulation factors and investigated the reproducibility of marstacimab in restoring haemostasis by comparing its effect in whole blood collected from the same study participants on differing days. Citrated whole blood and plasmas obtained from haemophilia participants were supplemented ex vivo with vehicle, marstacimab, recombinant FVIII (rFVIII) or recombinant factor IX (rFIX) and analysed in ROTEM, TGA and the dPT assay using low tissue factor concentrations to trigger coagulation. Marstacimab induced pro-coagulant responses in ROTEM parameters including reduction in clotting times and increases in angle. Similarly, participant plasmas supplemented with marstacimab exhibited improvements in TGA parameters, including reduced lag times, increased peak thrombin concentrations and reductions in dPT clotting time. Concentrations of marstacimab tested showed activity comparable to addition of rFVIII or rFIX and were reproducible. These studies show the ex vivo potency of marstacimab in restoring haemostasis in whole blood and plasmas from haemophilia participants and comparability to ex vivo reconstitution with recombination coagulation factors.

The Tissue Factor Pathway Inhibitor Antibody, PF-06741086, Increases Thrombin Generation in Rare Bleeding Disorder and Von Willebrand Factor Deficient Plasmas

Hemophilia A and B are hereditary bleeding disorders that result from deficiencies in the intrinsic coagulation pathway leading to insufficient generation of Factor Xa (FXa) and thrombin to promote stable hemostasis. Coagulation defects are also observed in other inherited rare factor bleeding disorders. The extrinsic pathway of coagulation in these disorders cannot generate sufficient levels of FXa due to the regulation by Tissue Factor Pathway Inhibitor (TFPI). TFPI is a Kunitz-type serine protease inhibitor that negatively regulates thrombin generation by inhibiting the FXa/tissue factor (TF)/Factor VIIa (FVIIa) complex. PF-06741086, a fully human inhibitory monoclonal antibody, binds the Kunitz-2 domain and is currently under development as a potential prophylactic treatment to prevent bleeding episodes in hemophilia A and hemophilia B patients with and without inhibitors. The addition of PF-06741086 in vitro to donor plasma from both healthy normal volunteers and hemophilia patients promoted thrombin generation and restored hemostasis in vivo in murine hemophilia bleeding models. Pharmacological effects of PF-06741086 on thrombin generation were also observed in a healthy volunteer Phase 1 study. Other rare disease coagulopathies also result in the insufficient generation of thrombin. In this study, the potential of PF-06741086 to restore thrombin generation in rare disease plasma was explored.Thrombin generation was measured in citrated platelet poor Factor XI (FXI), Factor V (FV), FVII, von Willebrand Factor (vWF) deficient (Type 1, 2A, 2B and 3) congenital donor plasma following the in vitro addition of PF-06741086 (0, 1, 10 or 100 nM) or a human IgG1 antibody; initiated with 1 pM TF and 4 µM phospholipid. FXI, FV, and FVII donors had less than 1% coagulation factor activity. Non-hemophilic plasma from healthy donors alone was also included in the analysis. In FXI deficient plasmas, a concentration-dependent increase in peak thrombin and a shortening of the lag time was observed with the addition of PF-06741086 normalizing and restoring levels to those observed in the non-hemophilic plasma. A similar response was also observed in all of the vWF deficient plasmas. In one FVII deficient plasma, an increase in peak thrombin was observed at dose of 100 nM PF-06741086, however, the lag time (20 minutes) was significantly extended, relative to healthy volunteer non-hemophilic plasma. As expected, the addition of PF-06741086 to FV deficient plasma did not increase thrombin generation at any concentration.The in vitro addition of the TFPI antibody, PF-06741086, improved thrombin generation in selected coagulation factor deficient plasmas, including vWF deficiency, to the levels observed in normal plasma. This data suggestion that the inhibition of TFPI may promote hemostasis in rare bleeding disorders such as FXI deficiency and vWF deficiencies. DisclosuresRakhe:Pfizer: Employment. Patel-Hett:Pfizer: Employment. Bowley:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

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.

The Chemical Chaperone 4-Phenylbutyrate Increases Secretion and Activity of Missense and Elongated Factor VII Mutants

The F7 gene mutations p.Q160R and p.A354V-p.P464Hfs are associated with very low factor VII (FVII) activity and antigen levels and a bleeding phenotype. We have previously demonstrated reduced secretion of the recombinant (r) variants rFVII-160R and rFVII-354V-464Hfs in vitro, possibly caused by a misfolding mechanism resulting in intracellular retention and increased endoplasmic reticulum (ER) stress. Chemical chaperones are compounds that can enable proteins to recover from a misfolded state and rescue intracellular processing. The aim of the study was to determine whether chemical chaperones could increase the secretion and enhance the biological activity of the above mentioned FVII mutants.Chinese hamster ovary (CHO-K1) cells stably expressing rFVII-160R or rFVII-354V-464Hfs were treated with the chemical chaperones 4-phenylbutyrate (4-PBA), betaine, taurine, taurourosdeoxycholic acid (TUDCA), trimethylamine N-oxide (TMAO) or lumacaftor (VX-809). The intracellular and secreted levels of FVII antigen were measured by ELISA and F7 mRNA levels were assessed by quantitative RT-PCR. Treatment of cells with 4-PBA for 48 hours increased F7 mRNA levels and increased the ratio of secreted/intracellular of both FVII mutants by ~2-2.5-fold, indicating an effect of 4-PBA FVII expression and secretion.4-PBA is known to inhibit the activity of histone deacetylases, thereby inducing hyperacetylation of histones and increasing gene transcription. To measure histone acetylation, we next performed Western blot analysis of acetylated histone H3 (AcH3). We found that the levels of AcH3 were significantly increased after treatment with 4-PBA, indicating that induced transcription of F7 by 4-PBA could be a result of increased global histone acetylation. Since 4-PBA treatment increased the secretion of the FVII mutants, we questioned whether this also would alleviate ER stress. Using an ER stress luciferase assay, we observed that the ER stress levels in 4-PBA treated cells were significantly lower than in non-treated cells.To determine whether 4-PBA treatment could restore the intracellular trafficking of the FVII mutants from the ER to Golgi, confocal immunofluorescence microscopy was utilized The studies revealed that upon treatment with 4-PBA, the p.Q160R mutant had a strong co-localization with the Golgi marker GM130 and with the Golgi stacking protein GRASP55, known to be associated with unconventional protein secretion. A vesicular pattern of staining could be observed in cells expressing the p.A354V-p.P464Hfs. However, these structures did not overlap with endosomal markers or markers of the ER-Golgi transport remaining the secretory route of this FVII mutant elusive.Finally, to assess the activity of FVII mutants, we measured the generation of activated (a) factor X (FXaG) in FVII deficient plasma supplemented with conditioned medium from cells stably expressing rFVII mutants before and after 4-PBA treatment. To increase the assay sensitivity to very low FVII activity levels we also boosted the coagulation initiation phase by inhibiting the tissue factor pathway inhibitor (TFPI) through an anti-TFPI RNA aptamer. We found that treatment of cells with 4-PBA gave rise to a modest, but measurable FXaG activity in conditioned medium for both FVII mutants. Furthermore, inhibition of TFPI proportionally potentiated FXaG activity of both 4-PBA-induced FVII variants.The present study demonstrates that chemical chaperones, such as 4-PBA, can increase intracellular trafficking through the secretory pathway of misfolded FVII mutants. Importantly, the secreted mutant proteins were found to be biologically regulated and active in the coagulation pathway. Thus, 4-PBA could have therapeutic effect in FVII deficiency caused by misfolded FVII mutants. DisclosuresNo relevant conflicts of interest to declare.

Anti-tissue factor pathway inhibitor (TFPI) therapy: a novel approach to the treatment of haemophilia.

Novel approaches to the treatment of haemophilia are needed due to the limitations of the current standard of care, factor replacement therapy. Aspirations include lessening the treatment burden and effectively preventing joint damage. Treating haemophilia by restoring thrombin generation may be an effective approach. A promising target for restoring thrombin generation is tissue factor pathway inhibitor (TFPI), a multivalent Kunitz-type serine protease inhibitor that regulates tissue factor-induced coagulation via factor Xa-dependent feedback inhibition of the tissue factor-factor VIIa complex. Inhibition of TFPI reverts the coagulation process to a more primitive state evolutionarily, whilst regulation by other natural inhibitors is preserved. An aptamer and three monoclonal antibodies directed against TFPI have been investigated in clinical trials. As well as improving thrombin generation in the range associated with mild haemophilia, anti-TFPI therapies have the advantage of subcutaneous administration. However, the therapeutic window needs to be defined along with the potential for complications due to the novel mechanism of action. This review provides an overview of TFPI, its role in normal coagulation, the rationale for TFPI inhibition, and a summary of anti-TFPI therapies, previously or currently in development.

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

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

Elevated plasma TFPI activity causes attenuated TF-dependent thrombin generation in early onset preeclampsia.

Early onset preeclampsia (EOP) is a pregnancy-specific proinflammatory disorder that is characterised by competing thrombotic and bleeding risks. It was the aim of this study to characterise thrombin generation, a major determinant of thrombotic and bleeding risk, in order to better understand the haemostatic balance in patients with EOP. Patients with EOP were recruited at the Rotunda Hospital, Dublin. Twenty-six cases of EOP were recruited over a 21-month period, out of 15,299 deliveries at the Rotunda. Blood samples were collected into sodium citrate plus corn trypsin inhibitor anticoagulated vacutainers, platelet-poor plasma was prepared, and calibrated automated thrombography was used to assess thrombin generation. Results were compared to age and sex-matched non-pregnant controls (n=13) and age- and gestation-matched pregnant controls (n=20). The rate and extent of thrombin generation triggered by low-dose tissue factor (TF) was significantly reduced in patients with EOP compared to pregnant controls, most significantly in cases of severe EOP. EOP patients displayed a trend towards an increased response to endogenous activated protein C and thrombomodulin relative to pregnant controls. Plasma tissue factor pathway inhibitor (TFPI) activity was increased in EOP patients. Inhibition of TFPI abolished the attenuation of thrombin generation stimulated by low-dose TF. In conclusion, patients with EOP are characterised by an attenuated coagulation response characterised by reduced thrombin generation stimulated by low-dose TF and elevated plasma TFPI activity. These changes in coagulation may modulate thrombotic risk and bleeding risk in patients with EOP.

Tissue Factor Pathway Inhibitor Mediates Attenuation of Tissue Factor-Stimulated Thrombin Generation in Early Onset Preeclampsia

IntroductionPregnancy increases the risk of venous thromboembolism (VTE) in women. Interestingly, preeclampsia, an extremely pro-inflammatory disorder specific to pregnancy is associated with a lower than expected increase in thrombotic risk compared to other proinflammatory disorders. The mechanism underling this lower thrombotic risk is unknown. The aim of this study was to investigate the coagulation balance, a major determinant of VTE risk, in early onset preeclampsia (EOP) patients. EOP is the most inflammatory form of preeclampsia, characterised by the development of hypertension and proteinuria prior to 34 weeks gestation.MethodsPlatelet-poor plasma was collected from patients with early onset preeclampsia (EOP n=26), matched pregnant (n=20) and non pregnant controls (n=16). Calibrated automated thrombography, an assay of thrombin generation and TFPI assays were performed. Data are expressed as mean ± standard deviation.ResultsDuring the study period, 15,299 women delivered and 40 patients developed EOP, of whom 26 were successfully recruited with consent. For comparison, 16 non pregnant controls and 20 pregnant controls were also recruited. When artefactual contact activation was inhibited by using corn trypsin inhibitor as anticoagulant, EOP patients were characterised by a decrease in the rate and extent of Tissue Factor (TF) thrombin generation compared to pregnant controls. There was a prolongation of the time to peak thrombin generation (16 ± 4 minutes vs. 13 ± 2 minutes, p < 0.05), a decrease in the velocity index (25 ± 17nM/minute vs. 41 ± 27nM/minute, p < 0.05), and a decrease in peak thrombin generation (150 ± 80 nM IIa vs. 210 ± 70 nM IIa, p < 0.05 ) in EOP compared to pregnant controls. This reduction in the rate and extent of thrombin generation was most amplified in patients with severe EOP (multi-organ involvement) compared with moderate EOP. This lower overall procoagulant state seen was further emphasised by the increase in sensitivity to the anticoagulant activity of exogenously added activated protein C and thrombomodulin observed in EOP. Again, the increased sensitivity to APC and thrombomodulin was most apparent in severe EOP cases. Previous studies have shown that preeclampsia is characterised by a increase in plasma TFPI activity. As such, we investigated whether increases in plasma TFPI activity explained the reduction in TF-dependent thrombin generation. Consistent with previous studies, plasma tissue factor pathway inhibitor (TFPI) levels and plasma TFPI activity significantly increased in EOP, most notably in severe EOP cases. There was a significant inverse correlation between total TFPI levels and peak thrombin generation (r2 = -0.64, p < 0.05) and TFPI activity and peak thrombin generation (r2= -0.52, p < 0.05). The inhibition of TFPI with a polyclonal anti-TFPI antibody abolished the attenuation in thrombin generation seen in severe EOP.ConclusionIn conclusion, TF-dependent thrombin generation is reduced in patients with early onset preeclampsia due to increases in plasma TFPI activity. These findings may partially explain the lower thrombosis risk observed in patients with early onset preeclampsia relative to comparable systemic proinflammatory conditions. These data also have future potential in helping clinicians to manage competing bleeding and thrombotic risks in these very high-risk patients. DisclosuresMaguire:Actelion UK: Research Funding. Ní Áinle:Actelion UK: Research Funding.

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

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

Acute Efficacy of a Novel Anti-Tissue Factor Pathway Inhibitor Antibody BAY 1093884 in Hemophilia A Mouse Severe Tail Bleeding

Tissue factor pathway inhibitor (TFPI) is the major inhibitor of the tissue factor initiated extrinsic coagulation pathway in blood and is intact in patients with hemophilia. The inhibition of TFPI may restore hemostasis in patients with hemophilia. BAY 1093884 is a fully human monoclonal antibody against TFPI developed as a bypass agent for hemophilia patients with or without inhibitors. It restores thrombin burst for stable clot formation in hemophilic conditions in vitro. The goal of these studies was to determine the in vivo acute efficacy of BAY 1093884 in the hemophilia A (HemA) mouse.In the first study, the acute efficacy of BAY 1093884 (3−100 mg/kg) was demonstrated and compared with full-length recombinant factor VIII (rFVIII; 10−100 IU/kg) by a HemA mouse tail clip model, in which blood loss from a severed tail tip was measured over 45 minutes after injury (n=12−27 mice/group). Naive C57/BL6 and HemA mice were used as positive and negative controls, respectively. Whereas isotype control antibody−treated HemA mice had median blood loss of 870 μL, increasing doses of BAY 1093884 to 50 and 100 mg/kg significantly reduced blood loss to a median of 55 and 5 μL. The dose required to reduce blood loss by 50% was 18 mg/kg, approximately equivalent to the efficacy of 20 IU/kg rFVIII.In a second study, we characterized the combined action of BAY 1093884 and activated recombinant factor VII (rFVIIa; n=10−25 mice/group). Low doses of BAY 1093884 (2.5 mg/kg) and rFVIIa (0.5 and 1.0 mg/kg) with minimal efficacies were tested. Untreated HemA mice had median blood loss of 860 μL. As stand-alone treatments, 2.5 mg/kg BAY 1093884, 0.5 mg/kg rFVIIa, and 1 mg/kg rFVIIa provided minimal blood loss protection, with bleeding volume reduced to 675, 830, and 770 μL, respectively. In comparison, the combination of 2.5 mg/kg BAY 1093884 with 0.5 mg/kg rFVIIa or 1.0 mg/kg rFVIIa reduced median blood loss to 215 and 35 μL, respectively. These results showed a combination effect of BAY 1093884 and rFVIIa in this severe acute efficacy model.These studies demonstrate that BAY 1093884 could potently reduce acute blood loss in HemA mice and may offer a new treatment option for hemophilia patients. DisclosuresXu:Bayer HealthCare LLC: Employment. Koellenberger:Bayer Pharma AG: Employment. Laux:Bayer Pharma AG: Employment. Kauser:Bayer HealthCare LLC: Employment. Sim:Bayer HealthCare LLC: Employment.

Correcting thrombin generation ex vivo using different haemostatic agents following cardiac surgery requiring the use of cardiopulmonary bypass.

Recently, lower thrombin generation has been associated with excess bleeding post-cardiopulmonary bypass (CPB). Therefore, treatment to correct thrombin generation is a potentially important aspect of management of bleeding in this group of patients. The objective of the present study was to investigate the effects of fresh frozen plasma (FFP), recombinant factor VIIa (rFVIIa), prothrombin complex concentrate (PCC) and tissue factor pathway inhibitor (TFPI) inhibition on thrombin generation when added ex vivo to the plasma of patients who had undergone cardiac surgery requiring CPB. Patients undergoing elective cardiac surgery were recruited. Blood samples were collected before administration of heparin and 30 min after its reversal. Thrombin generation was measured in the presence and absence of different concentrations of FFP, rFVIIa, PCC and an anti-TFPI antibody. A total of 102 patients were recruited. Thrombin generation following CPB was lower compared with pre-CPB (median endogenous thrombin potential pre-CPB 339 nmol/l per min, post-CPB 155 nmol/l per min, P < 0.0001; median peak thrombin pre-CPB 35 nmol/l, post-CPB 11 nmol/l, P < 0.0001). Coagulation factors and anticoagulants decreased, apart from total TFPI, which increased (55–111 ng/ml, P < 0.0001), and VWF (144–170 IU/dl, P < 0.0001). Thrombin generation was corrected to pre-CPB levels by the equivalent of 15 ml/kg FFP, 45 μg/kg rFVIIa and 25 U/kg of PCC. Inhibition of TFPI resulted in an enhancement of thrombin generation significantly beyond pre-CPB levels. This study shows that FFP, rFVIIa, PCC and inhibition of TFPI correct thrombin generation in the plasma of patients who have undergone surgery requiring CPB. Inhibition of TFPI may be a further potential therapeutic strategy for managing bleeding in this group of patients.

Plasma and Platelet Tissue Factor Pathway Inhibitor (TFPI) in Blood Donors and in People with Hemophilia

Introduction: TFPI is an anticoagulant protein produced primarily by endothelium and megakaryocytes. It circulates in plasma as TFPIα (“free” TFPI; “full-length” TFPI) and as variably truncated forms bound to lipoproteins. TFPIα is also within platelets, and platelet TFPI promotes blood loss in a tail bleeding assay in Factor VIII- deficient mice (Maroney et al., PNAS 2012;109(10):3927-31). Furthermore, evidence suggests that inhibition of TFPI may represent an attractive treatment option for people with hemophilia (Hilden et al., Blood 2012;119(24):5871-8). Plasma and platelet TFPI in people with hemophilia were measured to determine the contribution of these TFPI pools to the severity of this bleeding diathesis, and to aid in the dosing of anti-TFPI compounds. Plasma and platelet TFPI were also measured in a large cohort of blood donors, to determine the demographic factors affecting these pools of TFPI.Methods: High affinity monoclonal antibodies were used to develop ELISAs for plasma and platelet TFPI. Plasma total TFPI and TFPIα, and platelet TFPI, were determined in 427 blood donors (209 male, age 18-87; 218 female, age 19-89) and 58 people with severe hemophilia (47 hemophilia A, 11 hemophilia B). Data obtained from blood donors was stratified by gender, age, and oral contraceptive (OC) use, and reference ranges established (Table 1).Results: Plasma total TFPI correlated with that obtained with a commercially available ELISA (r2 = 0.75) while TFPIα did not (r2 = 0.29). The current TFPIα assay uses a high affinity anti-K3 antibody with almost no dissociation, which may account for this discrepancy. The reference ranges for platelet and plasma TFPI are broad, varying approximately 5-fold. In males, platelet and plasma TFPI were not affected by age. In females, plasma TFPIα increases by ≈1% per year. Females have ≈10% lower plasma total TFPI than males, and ≈10% higher platelet TFPI. OC users have decreased plasma total TFPI and TFPIα compared to non-OC users. Interestingly, platelet TFPI in females did not vary by age or OC use. Preliminary analysis of data from people with hemophilia demonstrated that plasma total TFPI in either hemophilia cohort was not statistically different from male blood donors (Table 2). Plasma TFPIα in people with hemophilia A was approximately 17% lower than male blood donors (Table 2). A similar decrease was observed in people with hemophilia B when compared to male blood donors, though the difference did not reach statistical significance. Platelet TFPI in male blood donors and people with either form of hemophilia was not statistically different.Conclusions: Robust immunoassays for the measurement of plasma and platelet TFPI were developed and validated. Poor correlation between the current TFPIα assay and other established assays suggests that the plasma TFPIα concentration may be underestimated in previous studies. The current study is the first to determine platelet TFPI concentration in a large cohort of normal donors, and in people with hemophilia. Plasma TFPI is affected by gender, age, and oral contraceptive use, whereas platelet TFPI is affected by gender only. People with hemophilia have normal plasma total TFPI and platelet TFPI, and mildly decreased plasma TFPIα. This data will be used to investigate the contribution of plasma and platelet TFPI to the bleeding diathesis in people with hemophilia, and to develop dosing strategies for the administration of anti-TFPI compounds that may be used in their treatment.Abstract 4241. Table 1Reference Ranges for Different TFPI PoolsMales (n = 209)Females 19-50(No OC, n = 95)Females 51+(No OC, n = 74)Mean ± SD (ng/mL)Ref. Range (ng/mL, Mean ± 2 SD)Mean ± SD (ng/mL)Ref. Range (ng/mL, Mean ± 2 SD)Mean ± SD (ng/mL)Ref. Range (ng/mL, Mean ± 2 SD)Plasma Total TFPI61.9 ± 19.323.3-100.551.8 ± 21.58.8-94.862.5 ± 21.818.9-106.1Plasma TFPIα29.4 ± 7.913.6-45.220.4 ± 8.63.2-37.629.6 ± 10.58.6-50.6Platelet TFPIα32.7 ± 11.210.0-55.138.2 ± 12.912.4-64.037.7 ± 11.414.9-60.5Table 2TFPI in Hemophilia PatientsMales (n = 209)Severe Hemophilia A (n = 47)Severe Hemophilia B (n = 11)Mean ± SD (ng/mL)Mean ± SD (ng/mL)p-value (vs Male Blood Donors)Mean ± SD (ng/mL)p-value (vs Male Blood Donors)Plasma Total TFPI61.9 ± 19.364.3 ± 28.0> 0.0579.5 ± 46.8> 0.05Plasma TFPIα29.4 ± 7.924.4 ± 7.3< 0.0125.7 ± 9.2> 0.05Platelet TFPIα32.7 ± 11.237.6 ± 13.9> 0.0539.6 ± 16.5> 0.05 DisclosuresAugustsson:Novo Nordisk: Employment. Andersen:Novo Nordisk: Employment. Hilden:Novo Nordisk: Employment. Mast:Novo Nordisk: Research Funding.

Small Peptides Blocking Inhibition of Factor Xa and Tissue Factor-Factor VIIa by Tissue Factor Pathway Inhibitor (TFPI)

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that inhibits activated factor X (FXa) via a slow-tight binding mechanism and tissue factor-activated FVII (TF-FVIIa) via formation of a quaternary FXa-TFPI-TF-FVIIa complex. Inhibition of TFPI enhances coagulation in hemophilia models. Using a library approach, we selected and subsequently optimized peptides that bind TFPI and block its anticoagulant activity. One peptide (termed compound 3), bound with high affinity to the Kunitz-1 (K1) domain of TFPI (Kd ∼1 nM). We solved the crystal structure of this peptide in complex with the K1 of TFPI at 2.55-Å resolution. The structure of compound 3 can be segmented into a N-terminal anchor; an Ω-shaped loop; an intermediate segment; a tight glycine-loop; and a C-terminal α-helix that is anchored to K1 at its reactive center loop and two-stranded β-sheet. The contact surface has an overall hydrophobic character with some charged hot spots. In a model system, compound 3 blocked FXa inhibition by TFPI (EC50 = 11 nM) and inhibition of TF-FVIIa-catalyzed FX activation by TFPI (EC50 = 2 nM). The peptide prevented transition from the loose to the tight FXa-TFPI complex, but did not affect formation of the loose FXa-TFPI complex. The K1 domain of TFPI binds and inhibits FVIIa and the K2 domain similarly inhibits FXa. Because compound 3 binds to K1, our data show that K1 is not only important for FVIIa inhibition but also for FXa inhibition, i.e. for the transition of the loose to the tight FXa-TFPI complex. This mode of action translates into normalization of coagulation of hemophilia plasmas. Compound 3 thus bears potential to prevent bleeding in hemophilia patients.

Inhibition of tissue factor pathway inhibitor increases the sensitivity of thrombin generation assay to procoagulant microvesicles

Patients with cancer have a seven-fold to 10-fold increased risk of developing venous thromboembolism (VTE). Circulating microvesicles could be a predictive biomarker for VTE in cancer. Thrombin generation assay (TGA) is a useful technique to detect procoagulant activity of microvesicles. However, TGA suffers from a lack of sensitivity due to the presence of tissue factor pathway inhibitor (TFPI) in plasma. The aim of the study was to improve the sensitivity of TGA to tissue factor by limiting the interference of TFPI. Serial dilutions of MDA-MB231 cells were incubated for 45 min at 37°C to generate microvesicles. Samples were then centrifuged and supernatants that contain microvesicles were used for TGA. Normal pooled plasma was incubated with inhibitor of TFPI or was diluted twice to decrease plasma level of TFPI. Lagtime was used as a surrogate marker of TGA to detect procoagulant activity of microvesicles. Inhibition of TFPI decreased twice the cell concentration needed for a significant reduction of lagtime and decreased 2.4-fold the intraassay variability. Plasma dilution had no impact on the TGA sensitivity when TGA was triggered by microvesicles derived from MDA-MB-231. Thrombin generation is a very sensitive method to study the procoagulant activity of tissue factor bearing microvesicles. The sensitivity can be increased by inhibition of TFPI with specific monoclonal antibody against its Kunitz domain I. A two times plasma dilution is an interesting cheaper alternative to study the procoagulant activity of microvesicles by TGA with a good sensitivity, especially when low plasma quantities are available.

Aptamer BAX 499 mediates inhibition of tissue factor pathway inhibitor via interaction with multiple domains of the protein

Tissue factor pathway inhibitor (TFPI) is a multidomain protein that negatively regulates the coagulation cascade. TFPI inhibits the tissue factor (TF)-activated factorVII-activated FX (FXa) complex during TF-mediated coagulation initiation. The aptamer BAX499 binds specifically to TFPI and inhibits its function, mediating a procoagulant effect in both invitro and invivo models of hemophilia. This study sought to identify the regions of TFPI that are critical for BAX499 binding, and to determine how binding mediates aptamer inhibition of TFPI. Invitro biochemical methods were used to evaluate the BAX499 interaction with and inhibition of TFPI. Binding experiments indicated that the full-length TFPI protein is required for tight aptamer binding. Binding-competition experiments implicated the Kunitz1, Kunitz3 and C-terminal domains of TFPI in aptamer binding, a finding that is supported by hydrogen-deuterium exchange experiments, and indicated that aptamer and FXa can bind simultaneously to TFPI. In enzymatic assays, BAX499 inhibited TFPI in a manner that is distinct from domain-specific antibodies, and aptamer inhibitory activity is reduced in the presence of the TFPI cofactor proteinS. These studies demonstrate that BAX499 binds to TFPI via multiple domains of the protein in a manner that is distinct from other TFPI inhibitors, mediating a mechanism of inhibition that does not involve direct competition with FXa. With this unique inhibitory mechanism, BAX499 provides a useful tool for studying TFPI biology in health and disease.

Structure-Activity Relationship of Pro- and Anticoagulant Effects of Fucus Vesiculosus Fucoidan

AbstractAbstract 3353Fucoidans are sulfated polysaccharides extracted from brown algae. Fucoidans have a wide variety of biological activities including pro- and anticoagulant activities, which occur at different concentration ranges. Therefore, fucoidans have also been described as non-anticoagulant sulfated polysaccharides (NASPs, Liu et al. Thromb Haemost 2006; 95:68). Fucoidans have complex structures due to their large molecular weight (Mw), wide Mw distribution, variable degree and pattern of sulfation, diverse monosaccharide composition, branching of the sugar chain and different monomer linkages. This structural complexity challenges identification of the components responsible for fucoidan activities.The aim of the presented study was to fractionate Fucus vesiculosus (F.v.) fucoidan by size and to de- and oversulfate it to obtain compounds of varying Mw or degree of sulfation (DS), respectively. The fucoidans were then to be analyzed for their pro-and anticoagulant activities and structure, and the effect of modified fucoidan on the target protein Tissue Factor Pathway Inhibitor (TFPI) investigated.Two approaches were applied to generate F.v. fucoidan with different structural properties to the original. Firstly, fucoidan was fractionated by size using ultrafiltration. The Mw of the resulting fractions ranged from 8–180 kD. NMR, elemental analysis and HPAEC showed that other structural features, such as sulfate content and monosaccharide composition, were similar to those of the original fucoidan. Thrombin generation (CAT) assays showed an EC50 for procoagulant activity of 0.3–3 μg/mL; aPTT increased by 50% at 4–25 μg/mL. Generally, higher Mw increased procoagulant activity. Below 15 kD, this activity was markedly reduced. The minimum active length of F.v. fucoidan was 70 carbohydrate units.De- and oversulfated F.v. fucoidans were used to investigate the impact of charge on pro- and anticoagulant activities. In the CAT assay, oversulfated fucoidans showed improved procoagulant activity with an EC50 of 0.09–0.12 μg/mL, while desulfated fucoidans demonstrated reduced procoagulant activity compared to the original. A DS of 0.5 was estimated to be the limit for procoagulant activity. Inhibition of TFPI by fucoidan was assessed with a dilute prothrombin time assay (dPT) with added full-length TFPI. TFPI-blocking activity was mainly dependent on the DS and less on the fucoidans’ Mw. Interestingly, oversulfation also stimulated an undesired activation of the contact pathway.The presented study determined the minimal structural requirements for procoagulant activity of fucoidan molecules and identified features causing undesired biological effects. Disclosures:No relevant conflicts of interest to declare.

Comparison of the Use of TFPI Inhibition and Plasma Dilution to Increase the Sensitivity of Thrombin Generation Assay to Measure the Procoagulant Activity of Microvesicles

AbstractAbstract 4392 Introduction:Patients with cancer have a 7- to 10- fold increased risk of developing venous thromboembolism. Circulating microvesicles (MVs) could be a predictive biomarker for venous thromboembolism in cancer. Thrombin generation assay is a useful technique to detect procoagulant activity of MVs. However, thrombin generation assay suffers from a lack of sensitivity due to the presence of Tissue Factor Pathway Inhibitor (TFPI) in plasma. Aims:To improve the sensitivity of thrombin generation assay to tissue factor (TF) by limiting the interference of TFPI. Methods:Serial dilutions of MDA-MB231 cells were incubated for 45 min at 37°C to generate MVs. Samples were then centrifuged and supernatants which contain MVs were used for thrombin generation assay. Normal pooled plasma was incubated with inhibitor of TFPI or was diluted twice to decrease plasma level of TFPI. Lagtime was used as a surrogate marker of thrombin generation assay to detect procoagulant activity of MVs. Results:i) Inhibition of TFPI decreased twice the cell concentration needed for a significant reduction of lagtime and decreased 2.4-fold the intra-assay variability. ii) Plasma dilution had no impact on the thrombin generation assay sensitivity when thrombin generation assay was triggered by MVs derived from MDA-MB-231. Conclusions:Thrombin generation is a very sensitive method to study the procoagulant activity of TF-MVs. The sensitivity can be increased by inhibition of TFPI with specific monoclonal antibody against its Kunitz Domain I. A twice plasma dilution is an interesting alternative to study the procoagulant activity of MVs by thrombin generation assay with a good sensitivity, especially when low plasma quantities are available. Disclosures:No relevant conflicts of interest to declare.

Effect of BAX499 aptamer on tissue factor pathway inhibitor function and thrombin generation in models of hemophilia

IntroductionIn hemophilia, thrombin generation is significantly suppressed due to decreased factor (F)X activation. Clinical studies and experiments with transgenic mice have suggested that the severity of hemophilia is substantially reduced by tissue factor pathway inhibitor (TFPI) deficiency. MethodsWe evaluated the effect of TFPI antagonist aptamer BAX499 (formerly ARC19499) on TFPI function in purified systems and on thrombin generation and clot formation in plasma and blood. ResultsBAX499 effectively neutralized TFPI inhibition of FXa and FXa dependent inhibition of TF/FVIIa by TFPI. BAX499 did not inhibit FXa or TF/FVIIa when used up to 500nM. In the synthetic coagulation proteome with TFPI at its mean physiologic concentration, BAX499 at 1 – 10nM increased thrombin generation triggered with 5 pM relipidated TF in a concentration-dependent manner. In severe hemophilia A or B models using the synthetic coagulation proteome, the addition of BAX499 at 5nM increased thrombin generation to the levels observed in normal control. Thrombin generation measured in induced hemophilia B plasma required ~100nM BAX499 to restore thrombin levels to those seen in untreated plasma. In induced hemophilia B whole blood, BAX499 repaired the clotting time but failed to appreciably impact the propagation phase of thrombin generation. ConclusionThese data suggest that inhibition of TFPI by BAX499 may have potential for hemophilia treatment but requires further study in blood-based hemophilia systems.