Mechanism of factor VIIa–dependent coagulation in hemophilia blood

Abstract The ability of factor VIIa to initiate thrombin generation and clot formation in blood from healthy donors, blood from patients with hemophilia A, and in anti–factor IX antibody–induced (“acquired”) hemophilia B blood was investigated. In normal blood, both factor VIIa–tissue factor (TF) complex and factor VIIa alone initiated thrombin generation. The efficiency of factor VIIa was about 0.0001 that of the factor VIIa–TF complex. In congenital hemophilia A blood and “acquired” hemophilia B blood in vitro, addition of 10 to 50 nM factor VIIa (pharmacologic concentrations) corrected the clotting time at all TF concentrations tested (0-100 pM) but had little effect on thrombin generation. Fibrinopeptide release and insoluble clot formation were only marginally influenced by addition of factor VIIa. TF alone had a more pronounced effect on thrombin generation; an increase in TF from 0 to 100 pM increased the maximum thrombin level in “acquired” hemophilia B blood from 120 to 480 nM. Platelet activation was considerably enhanced by addition of factor VIIa to both hemophilia A blood and “acquired” hemophilia B blood. Thus, pharmacologic concentrations of factor VIIa cannot restore normal thrombin generation in hemophilia A and hemophilia B blood in vitro. The efficacy of factor VIIa (10-50 nM) in hemophilia blood is dependent on TF.

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In Hemophilia Α Plasma Treated with Emicizumab, Factor IX Activation By Factor VIIa Drives Thrombin Generation

Blood ◽

10.1182/blood-2020-139712 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 17-17

Author(s):

Dougald Monroe ◽

Mirella Ezban ◽

Maureane Hoffman

Keyword(s):

Factor Viii ◽

Tissue Factor ◽

Plasma Levels ◽

Thrombin Generation ◽

Hemophilia A ◽

Factor Viia ◽

Factor Ix ◽

Factor X ◽

Dose Dependent

Background.Recently a novel bifunctional antibody (emicizumab) that binds both factor IXa (FIXa) and factor X (FX) has been used to treat hemophilia A. Emicizumab has proven remarkably effective as a prophylactic treatment for hemophilia A; however there are patients that still experience bleeding. An approach to safely and effectively treating this bleeding in hemophilia A patients with inhibitors is recombinant factor VIIa (rFVIIa). When given at therapeutic levels, rFVIIa can enhance tissue factor (TF) dependent activation of FX as well as activating FX independently of TF. At therapeutic levels rFVIIa can also activate FIX. The goal of this study was to assess the role of the FIXa activated by rFVIIa when emicizumab is added to hemophilia A plasma. Methods. Thrombin generation assays were done in plasma using 100 µM lipid and 420 µM Z-Gly-Gly-Arg-AMC with or without emicizumab at 55 µg/mL which is the clinical steady state level. The reactions were initiated with low (1 pM) tissue factor (TF). rFVIIa was added at concentrations of 25-100 nM with 25 nM corresponding to the plasma levels achieved by a single clinical dose of 90 µg/mL. To study to the role of factor IX in the absence of factor VIII, it was necessary to create a double deficient plasma (factors VIII and IX deficient). This was done by taking antigen negative hemophilia B plasma and adding a neutralizing antibody to factor VIII (Haematologic Technologies, Essex Junction, VT, USA). Now varying concentrations of factor IX could be reconstituted into the plasma to give hemophilia A plasma. Results. As expected, in the double deficient plasma with low TF there was essentially no thrombin generation. Also as expected from previous studies, addition of rFVIIa to double deficient plasma gave a dose dependent increase in thrombin generation through activation of FX. Interestingly addition of plasma levels of FIX to the rFVIIa did not increase thrombin generation. Starting from double deficient plasma, as expected emicizumab did not increase thrombin generation since no factor IX was present. Also, in double deficient plasma with rFVIIa, emicizumab did not increase thrombin generation. But in double deficient plasma with FIX and rFVIIa, emicizumab significantly increased thrombin generation. The levels of thrombin generation increased in a dose dependent fashion with higher concentrations of rFVIIa giving higher levels of thrombin generation. Conclusion. Since addition of FIX to the double deficient plasma with rFVIIa did not increase thrombin generation, it suggests that rFVIIa activation of FX is the only source of the FXa needed for thrombin generation. So in the absence of factor VIII (or emicizumab) FIX activation does not contribute to thrombin generation. However, in the presence of emicizumab, while rFVIIa can still activate FX, FIXa formed by rFVIIa can complex with emicizumab to provide an additional source of FX activation. Thus rFVIIa activation of FIX explains the synergistic effect in thrombin generation observed when combining rFVIIa with emicizumab. The generation of FIXa at a site of injury is consistent with the safety profile observed in clinical use. Disclosures Monroe: Novo Nordisk:Research Funding.Ezban:Novo Nordisk:Current Employment.Hoffman:Novo Nordisk:Research Funding.

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TFPI Antagonist Aptamer ARC19499 Is a Procoagulant In Vitro and In Vivo.

Blood ◽

10.1182/blood.v116.21.1134.1134 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1134-1134 ◽

Cited By ~ 1

Author(s):

Kathleen E. McGinness ◽

Emily K. Waters ◽

Ryan M. Genga ◽

Karen A. Olson ◽

Jennifer A. Nelson ◽

...

Keyword(s):

Thrombin Generation ◽

Hemophilia A ◽

Specific Interaction ◽

Hemophilia B ◽

Model Systems ◽

Primary Role ◽

Procoagulant Effect ◽

Coagulation Pathway

Abstract Abstract 1134 Hemophilia is a bleeding disorder that results from deficiencies in coagulation factor VIII (FVIII; hemophilia A) or factor IX (FIX; hemophilia B), primarily impacting the intrinsic coagulation pathway. The extrinsic coagulation pathway remains intact in hemophiliacs and is negatively regulated by tissue factor pathway inhibitor (TFPI). The primary role of TFPI is regulation of the FVIIa:tissue factor complex through inhibition of FVIIa and FXa. Inhibition of TFPI may provide an effective treatment for hemophilia by allowing sufficient thrombin generation via the extrinsic coagulation pathway to bypass the defect in clot propagation caused by deficiency of FVIII or FIX. ARC19499 is an aptamer that is a potent, specific inhibitor of TFPI. We have employed a variety of in vitro and in vivo methods to demonstrate that ARC19499 mediates a procoagulant effect in hemophilic plasma and in a model of hemophilia A in non-human primates (NHP). Plasma-based experiments that measure thrombin generation over time demonstrate that ARC19499 mediates a procoagulant response in both hemophilia A and hemophilia B plasma, restoring thrombin generation to near normal levels at 10 – 100 nM aptamer. Experiments in TFPI-depleted plasma and in vitro binding experiments demonstrate that this procoagulant effect is dependent on the specific interaction between ARC19499 and TFPI. ARC19499 restores thrombin generation to a level that is equivalent to or better than 14% FVIII replacement at a concentration of 30 nM in hemophilia A plasma and has an additive effect on thrombin generation when used in combination with FVIII. In a NHP model of hemophilia, cynomolgus monkeys acquire a hemophilia A-like state following administration of an anti-FVIII antibody as measured by ex vivo thromboelastography (TEG). TEG coagulation parameters are corrected following administration of ARC19499. In addition, antibody-mediated FVIII depletion moderately prolongs saphenous-vein bleeding time. ARC19499 corrects bleeding times to normal. These experiments demonstrate that the anti-TFPI aptamer ARC19499 mediates a procoagulant hemostatic effect both in vitro and in vivo in hemophilia model systems that is dependent on specific interaction between the aptamer and TFPI. ARC19499 may provide an effective alternative to replacement factors and bypassing agents for the treatment of hemophilia. Additionally, aptamer therapeutics have the advantage of subcutaneous bioavailability which provides an opportunity for improved treatment regimens in hemophilia. Disclosures: McGinness: Archemix Corporation: Employment. Waters:Archemix Corporation: Employment. Genga:Archemix Corporation: Employment. Olson:Archemix Corporation: Employment. Nelson:Archemix Corporation: Employment. Kurz:Archemix Corporation: Employment. Schaub:Archemix Corporation: Employment.

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

Blood ◽

10.1182/blood.v126.23.4672.4672 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4672-4672 ◽

Cited By ~ 1

Author(s):

Jian-Ming Gu ◽

Chandra Patel ◽

Katalin Kauser

Keyword(s):

Tissue Factor ◽

Healthy Subjects ◽

Tissue Factor Pathway Inhibitor ◽

Hemophilia A ◽

Hemophilia B ◽

Severe Hemophilia ◽

Capture Assay ◽

Healthy Donors ◽

Tissue Factor Pathway

Abstract 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 1. Plasma TFPI Levels in Healthy Subjects and Patients With Severe Hemophilia A and B HealthyHuman Donors(n=30) SevereHem A(n=12) Severe Hem AWith inhibitors(n=9) SevereHem B(n=9) TFPI, ng/mL Mean ± SD 59.5±18.4 62.9±14.6 47.3±4.3 68.1±8.8 Hem=hemophilia; TFPI=tissue factor pathway inhibitor. Disclosures Gu: Bayer HealthCare: Employment. Patel:Bayer HealthCare: Employment. Kauser:Bayer HealthCare LLC: Employment.

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Effects of Recombinant Factor VIIa (NovoSeven®) on Restoring Thrombin Generation in Patients with Hemophilia A and Antibodies to Factor VIII.

Blood ◽

10.1182/blood.v110.11.1161.1161 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1161-1161

Author(s):

Sabine Eichinger ◽

Barbara Lubszcyk ◽

Karl Zwiauer ◽

Andreas Gleiss ◽

Peter Quehenberger ◽

...

Keyword(s):

Factor Viii ◽

Thrombin Generation ◽

Hemophilia A ◽

Factor Viia ◽

Controlled Study ◽

Coagulation Activation ◽

Before And After ◽

Generation Capacity ◽

D Dimer

Abstract Introduction: Development of antibodies (ab) against factor VIII (FVIII) is a serious complication of replacement therapy in patients with hemophilia A. In case of bleeding patients with FVIII ab are treated with agents that induce hemostasis independently of FVIII. Recombinant activated factor VIIa (rVIIa) shows clinical efficacy, but its effects on hemostatic system need still to be fully elucidated. Methods: In an open controlled study, we investigated thrombin generation (peak thrombin) and parameters of coagulation activation [D-Dimer, prothrombin fragment F1+2 (F1+2)] in 5 patients with hemophilia A and FVIII ab, and in 5 healthy age-matched controls before and after intravenous bolus infusion of rVIIa (90 μg/kg bodyweight NovoSeven®, NovoNordisk, Denmark) (in hemophiliacs only). All parameters were measured in plasma before and 0.5, 1, 2, 3, and 4 hours after rVIIa infusion by use of commercially available assays (Technothrombin®TGA, Technoclone, Austria; Asserachrom®D-Di, Diagnostica Stago, France; Enzygnost F1+2, Dade Behring, Germany). Results: At baseline, hemophilia A patients had markedly lower mean (min-max) peak thrombin levels than controls [0.12 (0.0–0.6) nM vs. 186.9 (116.0–254.4) nM]. Mean (min-max) F1+2 levels did not significantly differ between patients and controls [160.7 (89.8–331.3) pmol/l vs. 160.8 (104.4–242.3) pmol/l]. Notably, D-Dimer levels were significantly higher in hemophiliacs than controls [1087.5 (174.8–3882.4) ng/ml vs. 146.3 (87.2–289.8) ng/mL]. FVIIa levels reached a mean (min-max) maximum of 28 (24–32) U/ml after 0.5 hours in all patients. After infusion, a considerable increase in mean (min-max) peak thrombin levels to 40.7 (28.3–51.6) nM was seen. Time to maximum levels was 30 minutes in three patients and 60 minutes in two. For each of the five patients the peak thrombin level was substracted from the level of its matched control at the same time point. The mean of these differences was 168.7 nM (95% CI 82.6–254.8), which translates into 80.2% (95% CI 65.4% – 88.6%) lower peak thrombin levels in haemophiliacs with FVIII ab. F1+2 significantly increased in all patients [mean (min-max) maximum levels 292.5 (175.1–464.3) pmol/l]; time to maximum levels varied from 2 to 4 hours. D-Dimer levels remained almost unchanged in all patients. Conclusion: Patients with hemophilia A and FVIII ab have low in vitro thrombin generation and F1+2 levels. After rVIIa infusion, coagulation activation as measured by F1+2 levels is slightly increased, and thrombin generation capacity is restored by 20% compared to healthy controls. Measurement of peak thrombin could be useful to monitor procoagulant treatment of patients with hemophilia A and FVIII ab.

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Factor VIII Supplementation Improves Recombinant VIIa Initiated Thrombin Generation in Hemophilia A Inhibitor Patient Plasmas

Blood ◽

10.1182/blood.v118.21.28.28 ◽

2011 ◽

Vol 118 (21) ◽

pp. 28-28

Author(s):

Bhavya Doshi ◽

Courtney Cox ◽

Bagirath Gangadharan ◽

Christopher B Doering ◽

Shannon L. Meeks

Keyword(s):

Factor Viii ◽

Thrombin Generation ◽

Hemophilia A ◽

Conflicts Of Interest ◽

Factor Viia ◽

Treatment Options ◽

Lag Time ◽

Time To Peak ◽

Fviii Inhibitor

Abstract Abstract 28 Hemophilia A is an X-linked recessive disorder that is caused by a deficiency or defect of factor VIII (fVIII) coagulant protein. Approximately 20–30% of patients with severe hemophilia A develop antibodies (Abs) against fVIII (inhibitors) following fVIII replacement therapy, which makes bleeding episodes more difficult to control. Patients with inhibitors are treated with fVIII-bypassing agents such as recombinant factor VIIa (rfVIIa) or activated prothrombin-complex concentrate. However for unknown reasons, some patients display poor hemostatic response to bypass therapy and improved treatment options are needed. Thrombin generation assays provide an in vitro methodology for monitoring bypass therapy in hemophilia (Turecek PL et al. Pathophysiol Haemost Thromb 2003; Varadi K et al. Haemophilia 2004). Recently, it was demonstrated by us and others that combination of fVIII and by-passing agents potentiates in vitro thrombin production in hemophilia A inhibitor plasma (Klintman J et al. Br J Haematol 2010). In our study we investigated the potentiation fVIII confers to fVIIa initiated in vitro thrombin generation using a panel of anti-fVIII Abs with known epitopes. We showed that kinetics of inhibition and Ab epitope were the dominant factors influencing ability of fVIII to potentiate in vitro thrombin production. Specifically, monoclonal Abs targeting only 2 of 11 epitopes, 1 of 3 non-overlapping A2 epitopes and 1 of 2 non-overlapping C2 epitopes, inhibited thrombin generation in a manner that could not be recovered by fVIII supplementation. Here, we analyzed in vitro thrombin generation in epitope-mapped plasmas from 10 patients with hemophilia A and long-standing inhibitors after addition of fVIIa alone or in conjunction with fVIII. Methods: FVIII inhibitor plasmas from 10 patients with hemophilia A were obtained as part of an IRB approved study at the Emory Comprehensive Hemophilia Center. FVIII inhibitor titers and inhibitor kinetics were determined using a modified Bethesda assay. Samples were classified as having type II inhibitors if undiluted plasma resulted in incomplete inhibition of residual fVIII activity (Meeks SL et al. Blood 2007). Thrombin generation assays were carried out in the presence of 2.25 μg/ml recombinant fVIIa in the presence or absence of 1 U/ml recombinant full-length fVIII using reagents purchased from DiaPharma (West Chester, OH). The parameters analyzed include endogenous thrombin potential (area under thrombin generation curve), peak thrombin concentration, time to peak thrombin, lag time (time to 1/6th of peak thrombin) and index velocity (Vi-peak thrombin divided by time to peak minus lag time). Domain specific epitope mapping was carried out using direct ELISA and human/porcine domain hybrid fVIII proteins. Results: Domain mapping of the Abs in the plasmas identified 2 plasmas with predominantly anti-A2 Abs, 4 with predominantly anti-C2 Abs, 2 with both anti-A2 and anti-C2 Abs, and 2 with antibodies that were porcine fVIII cross-reactive (see Table). Plasmas with inhibitor titers less than 25 BU/ml were more responsive to fVIII supplementation with 6 of 7 having increased thrombin generation. Plasmas harboring even trace anti-A2 Abs were more resistant to increased thrombin generation with fVIII supplementation than plasmas with anti-C2 Abs alone. Conclusion: This study suggests a more favorable response to fVIII supplementation of rfVIIa may be predicted by the presence of anti-C2 Abs or inhibitory titers less than 25 BU/ml. In conjunction with our previous monoclonal Ab data, further mapping of epitopes within the fVIII A2 and C2 domains may help improve the ability to predict positive responses to fVIII supplementation of by-passing agents.PatientInhibitor Titer (BU/ml)DomainFVIII InhibitorThrombin Generation (fVIII + fVIIa vs. fVIIa)122A2Type IIIncreased242A2Type IIEqual384C2, small A2Type IEqual47C2Type IIncreased58C2Type IIIncreased620C2Type IEqual78C2Type IIncreased842C2, small A2Type IEqual922Porcine cross-reactiveType IIIncreased105.2Porcine cross-reactiveType IIncreased Disclosures: No relevant conflicts of interest to declare.

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An In Vitro Analysis of the Combination of Hemophilia A and Factor VLEIDEN

Blood ◽

10.1182/blood.v90.8.3067 ◽

1997 ◽

Vol 90 (8) ◽

pp. 3067-3072 ◽

Cited By ~ 60

Author(s):

Cornelis van ‘t Veer ◽

Neal J. Golden ◽

Michael Kalafatis ◽

Paolo Simioni ◽

Rogier M. Bertina ◽

...

Keyword(s):

Factor Viii ◽

Tissue Factor ◽

Thrombin Generation ◽

Hemophilia A ◽

Factor Viia ◽

Factor V ◽

Severe Hemophilia ◽

Factor Viii Activity ◽

Thrombin Formation

Abstract The classification of factor VIII deficiency, generally used based on plasma levels of factor VIII, consists of severe (<1% normal factor VIII activity), moderate (1% to 4% factor VIII activity), or mild (5% to 25% factor VIII activity). A recent communication described four individuals bearing identical factor VIII mutations. This resulted in a severe bleeding disorder in two patients who carried a normal factor V gene, whereas the two patients who did not display severe hemophilia were heterozygous for the factor VLEIDEN mutation, which leads to the substitution of Arg506 → Gln mutation in the factor V molecule. Based on the factor VIII level measured using factor VIII–deficient plasma, these two patients were classified as mild/moderate hemophiliacs. We studied the condition of moderate to severe hemophilia A combined with the factor VLEIDEN mutation in vitro in a reconstituted model of the tissue factor pathway to thrombin. In the model, thrombin generation was initiated by relipidated tissue factor and factor VIIa in the presence of the coagulation factors X, IX, II, V, and VIII and the inhibitors tissue factor pathway inhibitor, antithrombin-III, and protein C. At 5 pmol/L initiating factor VIIa⋅tissue factor, a 10-fold higher peak level of thrombin formation (350 nmol/L), was observed in the system in the presence of plasma levels of factor VIII compared with reactions without factor VIII. Significant increase in thrombin formation was observed at factor VIII concentrations less than 42 pmol/L (∼6% of the normal factor VIII plasma concentration). In reactions without factor VIII, in which thrombin generation was downregulated by the addition of protein C and thrombomodulin, an increase of thrombin formation was observed with the factor VLEIDEN mutation. The level of increase in thrombin generation in the hemophilia A situation was found to be dependent on the factor VLEIDEN concentration. When the factor VLEIDEN concentration was varied from 50% to 150% of the normal plasma concentration, the increase in thrombin generation ranged from threefold to sevenfold. The data suggested that the analysis of the factor V genotype should be accompanied by a quantitative analysis of the plasma factor VLEIDEN level to understand the effect of factor VLEIDEN in hemophilia A patients. The presented data support the hypothesis that the factor VLEIDEN mutation can increase thrombin formation in severe hemophilia A.

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Aminoglycoside suppression of nonsense mutations in severe hemophilia

Blood ◽

10.1182/blood-2005-03-1307 ◽

2005 ◽

Vol 106 (9) ◽

pp. 3043-3048 ◽

Cited By ~ 55

Author(s):

Paula D. James ◽

Sanj Raut ◽

Georges E. Rivard ◽

Man-Chiu Poon ◽

Margaret Warner ◽

...

Keyword(s):

Thrombin Generation ◽

Hemophilia A ◽

Aminoglycoside Antibiotic ◽

Bactericidal Effect ◽

Factor Ix ◽

Hemophilia B ◽

Severe Hemophilia ◽

Toxic Agent ◽

Nonsense Mutations ◽

Therapeutic Mechanism

AbstractAminoglycoside antibiotics exhibit their bactericidal effect by interfering with normal ribosomal activity. In this pilot study, we have evaluated the effect of the aminoglycoside antibiotic gentamicin on the factor VIII (FVIII) and IX levels of severe hemophiliacs with known nonsense mutations. Five patients were enrolled and each patient was given 3 consecutive days of gentamicin at a dose of 7 mg/kg intravenously every 24 hours. Two patients (patient no. 1: hemophilia A, Ser1395Stop; and patient no. 5: hemophilia B, Arg333Stop) showed a decrease in their activated partial thromboplastin time (aPTT), an increase in their FVIII (0.016 IU/mL, 1.6%) or FIX (0.02 IU/mL, 2%) levels, and an increase in thrombin generation. The remaining 3 patients (patient no. 2: hemophilia B, Arg252Stop; patient no. 3: hemophilia A, Arg2116Stop; and patient no. 4: hemophilia A, Arg427Stop) showed no response in the aPTTs or factor levels, but one (patient no. 2: hemophilia B, Arg252Stop) showed an increase in the factor IX antigen level (2%-5.5%) that persisted throughout the period of the study and was concordant with an increase in thrombin generation. Gentamicin is unlikely to be an effective treatment for severe hemophilia due to its potential toxicities and the minimal response documented in this report. This study, however, does provide a proof of principle, suggesting that ribosomal interference with a less toxic agent may be a potential therapeutic mechanism for severe hemophilia patients with nonsense mutations.

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A Neutralizing Monoclonal Antibody (PF-06741086) Against Tissue Factor Pathway Inhibitor in Combination with Activated Prothrombin Complex Concentrate Increases Hemostasis in Inhibitor Hemophilia Plasmas without Excessive Thrombin Generation

Blood ◽

10.1182/blood-2018-99-119831 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3779-3779

Author(s):

Swapnil Rakhe ◽

Sheryl Bowley ◽

John E. Murphy ◽

Debra D Pittman

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Tissue Factor Pathway Inhibitor ◽

Prothrombin Complex Concentrate ◽

Hemophilia A ◽

Lag Time ◽

Hemophilia B ◽

Activated Prothrombin Complex Concentrate ◽

Tissue Factor Pathway

Abstract Hemophilia A and B are hereditary bleeding disorders caused by intrinsic coagulation pathway deficiencies of Factor VIII or Factor IX, respectively. Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine protease inhibitor that negatively regulates thrombin generation within the extrinsic pathway of coagulation. PF-06741086 is a fully human monoclonal antibody which binds the Kunitz-2 domain and neutralizes the inhibitory activity of human tissue factor pathway inhibitor 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. Activated prothrombin complex concentrate (aPCC) is used as bypass treatment for the resolution of bleeding in some hemophilia patients with inhibitors. Hemophilia inhibitor patients receiving PF-06741086 have a possibility to also receive treatment with aPCC. The aim of the current study was to assess the potential additive effect of PF-06741086 with aPCC added in vitro to Hemophilia A and B inhibitor plasmas using a thrombin generation assay (TGA). Thrombin generation in the presence of 1 pM tissue factor and 4 µM phospholipid, was measured using the calibrated automated thrombogram (CAT) system in citrated platelet poor hemophilia A inhibitor (88-160 Bethesda Units) donor plasma or hemophilia B inhibitor (FIX immune-depleted and spiked with FIX neutralizing antibody, 14 Bethesda Units) plasma following the addition of PF-06741086 or aPCC (FEIBA) either alone or in combination. All donors had less than 1% coagulation factor activity. Non-hemophilic plasma from healthy donors alone or spiked in vitro with 16 µg/mL of PF-06741086 was also included in the analysis. Non-hemophilic plasma would have the full complement of coagulation factors. Dose-dependent increases in peak thrombin were observed with the addition of aPCC alone or PF-06741086 alone to the hemophilia plasmas. For combination studies, the aPCC concentration of 1 Unit/mL was selected to correspond to plasma levels that could be achieved clinically post-dosing. The concentration of PF-06741086 at 16µg/mL in these studies was chosen to approximate the Cmax concentration following a single 300 mg subcutaneous dose. Both PF-06741086 (16 µg/mL) and aPCC (1 Unit/mL) decreased the lag time in hemophilia plasma, however, there was not an additive decrease in the lag time with the combination of PF-06741086 and aPCC. The addition of PF-06741086 in combination with aPCC to hemophilia plasma resulted in an increase in thrombin generation including a higher peak thrombin concentration compared to the addition of either alone, but was within the range reported in studies for non-hemophilic normal plasma. To summarize, the addition of aPCC (1 Unit/mL) in combination with PF-06741086 (16µg/mL) in vitro resulted in increased thrombin generation in hemophilia A and hemophilia B inhibitor plasmas without inducing excessive coagulation. Disclosures Rakhe: Pfizer: Employment. Bowley:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

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An Antibody to Tissue Factor Pathway Inhibitor (PF-06741086) in Combination with Recombinant Factor VIIa Increases Hemostasis in Hemophilia Plasma without Excessive Thrombin Generation

Blood ◽

10.1182/blood.v128.22.2566.2566 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2566-2566 ◽

Cited By ~ 4

Author(s):

Swapnil Rakhe ◽

Sunita Patel Hett ◽

John E. Murphy ◽

Debra D Pittman

Keyword(s):

Inhibitory Activity ◽

Thrombin Generation ◽

Tissue Factor Pathway Inhibitor ◽

Hemophilia A ◽

Recombinant Factor Viia ◽

Factor Viia ◽

Lag Time ◽

Hemophilia B ◽

Extrinsic Pathway ◽

Tissue Factor Pathway

Abstract Hemophilia is a hereditary bleeding disorder caused by intrinsic coagulation pathway deficiencies of Factor VIII (hemophilia A) or Factor IX (hemophilia B). Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine protease inhibitor that negatively regulates thrombin generation within the extrinsic pathway of coagulation. In hemophilia patients the extrinsic pathway remains intact and thus augmentation of this pathway may circumvent the clotting deficiency in hemophilia. PF-06741086, a monoclonal antibody that binds to and neutralizes the inhibitory activity of TFPI is being developed as a potential treatment for bleeding disorders including hemophilia A and hemophilia B with and without inhibitors. Currently, treatment of inhibitor patients is managed by bypass treatments, such as recombinant Factor VIIa (rFVIIa). The effect of PF-06741086 on thrombin generation in the presence of increasing concentrations of rFVIIa (0.0002 to 20 µg/mL) was studied in severe hemophilia A plasma. A dose-dependent increase in thrombin generation was observed over vehicle control with the addition of rFVIIa to the hemophilia plasma. Addition of a fixed concentration of PF-06741086 (16 µg/mL) in combination with rFVIIa resulted in an increase in thrombin generation including higher peak thrombin and shortening of lag time compared to rFVIIa alone. The TGA profiles with the combination of PF-06741086 and rFVIIa at 0.2, 2, and 20 µg/mL were similar suggesting a saturation of mechanism at these concentrations. The combination of PF-06741086 and rFVIIa restored thrombin generation to normal plasma levels at all rFVIIa concentrations examined. The TFPI inhibitory activity of PF-06741086 on thrombin generation in the presence and absence of rFVIIa was further studied in additional hemophilia A plasmas, including hemophilia A plasmas with inhibitors and hemophilia B plasma. All donors had less than 1% coagulation factor activity. A rFVIIa concentration of 2 µg/mL was selected because it corresponded to plasma levels that could be observed following dosing of FVIIa and because the thrombin generation response in hemophilia plasma was similar with FVIIa added to hemophilia A plasma at 0.2, 2 and 20 µg/mL. The concentration of PF-06741086 was 16 µg/mL in these studies. The effect of PF-06741086 on thrombin generation was also measured in non-hemophilic plasma which would have the full complement of coagulation factors. The addition of PF-06741086 alone or in combination with rFVIIa to hemophilia A and B plasma resulted in an increase in thrombin generation including higher peak thrombin concentration and shortening of lag time compared to addition of rFVIIa alone. In hemophilic plasma samples with inhibitors (3 - 1261 Bethesda Units), PF-06741086 alone also restored thrombin generation. A minimal additive effect in peak thrombin generation was observed with the combination of PF-06741086 (16 µg/mL) and 2 µg/mL rFVIII. The midpoint peak thrombin levels achieved with PF-06741086 alone or in combination with rFVIIa were similar to those observed in non-hemophilic plasma and did not exceed the level observed in non-hemophilic plasma dosed with PF-06741086. To summarize, use of rFVIIa in combination with PF-06741086 results in increased thrombin generation in hemophilia A, hemophilia B and inhibitor plasmas without inducing excessive coagulation. Disclosures Rakhe: Pfizer: Employment. Hett:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

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A Novel Use of Rehydrated, Lyophilized Platelets Increases Recombinant FVIIa Procoagulant Activity in a Model of Hemophilia.

Blood ◽

10.1182/blood.v108.11.1754.1754 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1754-1754

Author(s):

Robert A. Campbell ◽

Thomas H. Fischer ◽

Alisa S. Wolberg

Keyword(s):

Thrombin Generation ◽

Factor Viia ◽

Specific Interaction ◽

Annexin V ◽

Factor Ix ◽

Clot Formation ◽

Generation Rate ◽

Tissue Type ◽

Dependent Manner ◽

Platelet Surface

Abstract The anti-bleeding therapy recombinant factor VIIa (rFVIIa) is used to abrogate bleeding in hemophiliacs with inhibitors, bypassing the need for replacement factors. RFVIIa is hypothesized to work by increasing Xa generation on the platelet’s surface. However, high plasma levels of rFVIIa are required, in part due to the weak binding of rFVIIa to platelets. We hypothesized that the efficacy of the therapy could be improved by administering rFVIIa already bound to platelets. One platelet preparative that may be used in this application is rehydrated, lyophilized (RL) platelets. RL platelets are fixed with paraformaldehyde, which allows them to be frozen and lyophilized while retaining their hemostatic capabilities. Previously, we have shown RL platelets are capable of supporting rFVIIa-mediated thrombin generation and that thrombin generation is increased in a rFVIIa dose-dependent manner (Blood, 106:4057, 2005). In this current study, we have characterized the ability of RL platelets to modulate rFVIIa-mediated thrombin generation and fibrin clot formation in a cell-based complete model of hemophilia. The addition of RL platelets with 50 nM rFVIIa increased the thrombin generation rate in hemophilia 2.8-fold more than 50 nM rFVIIa, alone. Further, the addition of RL platelets with 50 nM rFVIIa normalized clot formation and stability in a fibrinolytic environment, which did not occur in the presence of rFVIIa, alone. In contrast, the addition of RL platelets, alone, to hemophilic conditions had minimal to no effect on thrombin generation rate or the onset of clot formation, suggesting that the effects were due to a specific interaction between rFVIIa and RL platelets. When rFVIIa plus RL platelets were added to platelet-rich plasma from patients with hemophilia A in the presence of tissue-type plasminogen activator, clot formation and stability were improved more than the addition of either agent alone. To examine the mechanism of RL platelets’ augmentation of rFVIIa activity, we titrated the phosphatidylserine (PS) binding protein, annexin V, into reactions with RL platelets in the presence of factors Xa, Va, and prothrombin and measured thrombin generation. The addition of annexin V reduced thrombin generation equally in reactions that contained RL platelets stimulated with or without SFLLRN. Further, thrombin generation was similar on RL platelets simulated with or without SFLLRN in the absence of annexin V. These data suggest that RL platelets already have PS exposed on their surface. We conclude that RL platelets can support rFVIIa-mediated thrombin generation in the absence of factor IX and may enhance rFVIIa activity in part due to PS exposure on the RL platelet surface. We hypothesize that co-administration of RL platelets with rFVIIa may increase the efficacy of rFVIIa, at lower doses of rFVIIa than are currently required to achieve hemostasis.

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