Recombinant Factor VIIa Does not Induce Hypercoagulability In Vitro

1999 ◽  
Vol 81 (02) ◽  
pp. 245-249 ◽  
Author(s):  
Gerhard Cvirn ◽  
Wolfgang Muntean ◽  
Siegfried Gallistl
Keyword(s):  
Factor Viii ◽  
Thrombin Generation ◽  
Recombinant Factor Viia ◽  
Factor Viia ◽  
Factor Ix ◽  
Control Factor ◽  
Prothrombin Complex Concentrates ◽  
Thrombotic Complications ◽  
Activated Prothrombin Complex Concentrates

SummaryRecombinant factor VIIa (rVIIa) has been reported to be clinically effective and safe in haemophilic patients with inhibitor antibodies. Compared to activated prothrombin complex concentrates the risk of thrombotic complications seems to be very low after rVIIa administration. Determination of free thrombin generation has been shown to identify hypercoagulability. Therefore, free thrombin and prothrombinase activity (Xa generation) were assessed after extrinsic activation of rVIIa supplemented factor VIII and factor IX deficient plasma. Free thrombin generation was also determined after supplementation of (activated) prothrombin complex concentrates. Addition of 150 U rVIIa/ml shortened the clotting times markedly in control, factor VIII, and factor IX deficient plasma. In contrast, free thrombin and Xa generation were not different in the absence or presence of 150 U rVIIa/ml. Addition of (activated) prothrombin complex concentrates resulted in a marked increase of free thrombin generation in all investigated plasmas. Although in vitro studies cannot reflect specific clinical circumstances our results support the notion that rVIIa does not induce a hypercoagulable state as sporadically observed after administration of (activated) prothrombin complex concentrates.

scholarly journals Management of the Bleeding Patient Receiving New Oral Anticoagulants: A Role for Prothrombin Complex Concentrates

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Lisa M. Baumann Kreuziger ◽  
Joseph C. Keenan ◽  
Colleen T. Morton ◽  
David J. Dries
Keyword(s):  
Recombinant Factor Viia ◽  
Factor Viia ◽  
Oral Anticoagulants ◽  
New Oral Anticoagulants ◽  
Coagulation Cascade ◽  
Clotting Factors ◽  
Prothrombin Complex Concentrates ◽  
Clotting System ◽  
Clinical Trial Evidence

Ease of dosing and simplicity of monitoring make new oral anticoagulants an attractive therapy in a growing range of clinical conditions. However, newer oral anticoagulants interact with the coagulation cascade in different ways than traditional warfarin therapy. Replacement of clotting factors will not reverse the effects of dabigatran, rivaroxaban, or apixaban. Currently, antidotes for these drugs are not widely available. Fortunately, withholding the anticoagulant and dialysis are freqnently effective treatments, particularly with rivaroxaban and dabigatran. Emergent bleeding, however, requires utilization of Prothrombin Complex Concentrates (PCCs). PCCs, in addition to recombinant factor VIIa, are used to activate the clotting system to reverse the effects of the new oral anticoagulants. In cases of refractory or emergent bleeding, the recommended factor concentrate in our protocols differs between the new oral anticoagulants. In patients taking dabigatran, we administer an activated PCC (aPCC) [FELBA] due to reported benefit in human in vitro studies. Based on human clinical trial evidence, the 4-factor PCC (Kcentra) is suggested for patients with refractory rivaroxaban- or apixaban-associated hemorrhage. If bleeding continues, recombinant factor VIIa may be employed. With all of these new procoagulant agents, the risk of thrombosis associated with administration of factor concentrates must be weighed against the relative risk of hemorrhage.

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

Blood ◽  
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.

Feiba And Autoplex: A Comparison Of These Two Activated Prothrombin Complex Concentrates

1981 ◽  
Author(s):  
E Lechler ◽  
B Eggeling ◽  
D Meyer-Börnecke ◽  
H Stoy
Keyword(s):  
Hemophilia A ◽  
Gel Filtration ◽  
Activated Partial Thromboplastin Time ◽  
Factor Ix ◽  
Prothrombin Complex Concentrates ◽  
Minor Degree ◽  
Activated Prothrombin Complex Concentrates ◽  
A Minor

These activated concentrates are used for the treatment of patients with factor VIII inhibitors . Both shorten the activated and non-activated partial thromboplastin time of inhibitor plasma and hemophilia A plasma in vitro. They do not or only to a minor degree improve the prothrombin consumption of hemophilia A plasma in vitro. In gel filtration of AUTOPLEX the activity which shortens the PTT of hemophilia A plasma eluted in a volume higher than that of the nonactivated factors of the prothrombin complex and contains activated factor IX. The activity of FEIBA elutes at a lower filtration volume in a rather broad peak together with the factors of the the non-activated prothrombin complex. BaSO4- adsorbed plasma and purified antithrombin (Behring) abolish the activity of AUTOPLEX more readily than of FEIBA. Both concentrates have only a low amidolytic effect (S 2222) and are not inhibited with SBTI and PMSF. In the crossed two-dimensional immunelectrophoresis with heparin in the agarose of the first dimension and anti-antithrombin (Behring) in the agarose of the second dimension (method of Sas), a mixture of AUTOPLEX and antithrombin results into a two peak precipitation of antithrombin, whereas with FEIBA a broadened intermediate peak develops. In vivo both concentrates do not improve the prothrombin consumption and AUTOPLEX shortens the PTT for at least 90 minutes. In summary, these two concentrates differ considerably.

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

Blood ◽  
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.

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

Blood ◽  
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.

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

Blood ◽  
1997 ◽  
Vol 90 (8) ◽  
pp. 3067-3072 ◽  
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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