New Perspectives in Hemophilia Treatment

Hematology ◽  
2005 ◽  
Vol 2005 (1) ◽  
pp. 429-435 ◽  
Author(s):  
Craig M. Kessler
Keyword(s):  
Factor Viia ◽  
Medical Condition ◽  
High Titer ◽  
Parvovirus B19 ◽  
Pathogen Transmission ◽  
Factor Ix ◽  
Bleeding Complications ◽  
Severe Bleeding ◽  
Pathogen Inactivation ◽  
Viral Pathogen

Abstract A variety of factor concentrates are currently available for replacement therapy for patients with hemophilia. These differ by several parameters, including source (pooled from pooled blood vs recombinant), purity, pathogen inactivation, and by the presence or absence of extraneous proteins such as albumin. The choice of replacement product reflects both safety issues of pathogen transmission or inhibitor development, and personal preferences of the patient and the physician. In general, currently available products are viral pathogen-free, although there is debate about the risk of transmission of parvovirus B19 and prion pathogens. Because of this very small risk, recombinant factor is the treatment of choice in previously untreated patients. In addition, a subset of concentrates contain factor that is activated during manufacture, yielding activated products that can be used in the treatment of patients with inhibitors. Such activated products, especially recombinant factor VIIa (rFVIIa), have also acquired several off-label indications in the management of bleeding in non-hemophiliac patients. The management of hemophilia patients with inhibitors is an ongoing challenge. Immune tolerance induction using a desensitization technique is successful in up to 90% of patients with alloantibodies against factor VIII, with greatest success seen in patients with low titer inhibitors who are treated soon after detection of an alloantibody and in whom treatment includes administration of immunosuppression along with repeated infusions of high titer concentrates. Such therapy is less successful in patients with factor IX alloantibodies. Non-hemophiliac patients with acquired inhibitors represent a unique patient population that requires special management. These patients have a mortality rate that approaches 25% because of the association of acquired inhibitors with severe bleeding complications, occurrence in a largely elderly population, and the frequent presence of an underlying, often serious, primary medical condition. Treatment consists of immunosuppression with steroids, chemotherapy, or intravenous immunoglobulin. Recent studies using rituximab for selective B-cell depletion in these patients have been very promising, although prospective controlled studies have not yet been performed. Finally, although hemophilia A and B appear to be ideal diseases to target with gene therapy approaches, the promise of this therapy remains to be realized.

An Analysis of Cleavage of the Factor IX Activation Sites by Factor XIa

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3088-3088 ◽  
Author(s):  
David Gailani ◽  
Stephen B. Smith ◽  
Sayeh Agah ◽  
S. Paul Bajaj
Keyword(s):  
Active Site ◽  
Factor Viia ◽  
Exchange Chromatography ◽  
Factor Ix ◽  
Severe Bleeding ◽  
Western Blots ◽  
Factor Xia ◽  
Gla Domain ◽  
Activation Peptide ◽  
Time Courses

Abstract During blood coagulation, the plasma zymogen factor IX (fIX) is converted to the active protease factor Ixaβ (fIXaβ). The severe bleeding disorder associated with deficiency of fIX (hemophilia B) attests to the importance of this protein in hemostasis. Conversion of fIX to fIXaβ requires two proteolytic cleavages after Arg145 and Arg180, releasing an activation peptide. This process is mediated by the proteases factor VIIa (fVIIa) and factor XIa (fXIa). FVIIa in complex with tissue factor initially cleaves fIX after Arg145 forming an intermediate, factor IXα (fIXα), which is then cleaved after Arg180 to form fIXaβ. Western blots of activation time courses demonstrate fIXα accumulation during this process, indicating cleavage at Arg180 is rate limiting. In contrast, little intermediate accumulation occurs during fIX activation by fXIa. Previously, we showed that fXIa also cleaves fIX initially after Arg145, generating fIXα (Smith et al., J. Biol. Chem.283;6696:2008). To account for the lack of intermediate accumulation, then, the subsequent cleavage after Arg180 must occur at least as rapidly as the initial cleavage. We examined the relative rates of conversion of fIX, fIXα, and the alternative intermediate factor IXaα (fIXaα - cleaved after Arg180) to fIXaβ by fXIa. FIXα or fIXaα were prepared from tritium-labeled fIX by incubation with fXIa-Pro192 (discussed below) or Russell’s Viper Venom protease, respectively, and purified by anion exchange chromatography. Conversion to fIXaβ was determined by measuring release of the tritiated activation peptide. FXIa converted fIX to fIXaβ with a kcat of 29.4 ± 0.4/min, a value reflecting cleavage at both activation sites. Kcat for conversion of fIXα and fIXaα to fIXaβ were 29.9 ± 0.5 and 30.0 ± 1.0/min, respectively. The rate of conversion of fIX to fIXα, estimated by measuring tritiated activation products separated by SDS-PAGE, was 30.0 ± 0.4/min. Recently, we showed that amino acid substitutions in fXIa for the conserved active site residue Gly193 (chymotrypsin numbering) decreased kcat for fIX activation 7–1000 fold, with residues with long branched side-chains having the greatest effect (Schmidt et al. Biochemistry47;1326:2008). Gly193 substitutions had a modestly larger detrimental effect (1.2–1.5 fold) on cleavage of fIX after Arg180 compared to Arg145 that was associated with varying degrees of fIXα accumulation. Similar effects were noted with substitutions for the adjacent residue Lys192. FXIa with Pro192 cleaved fIX after Arg180 >10-fold more slowly than after Arg145, generating fIXα with little subsequent conversion to fIXaβ. Cumulatively, these data support the premise that the rates for the two sequential reactions required for normal fIX activation by fXIa are comparable. Therefore, perturbations causing a greater effect on cleavage after Arg180 compared to Arg145, even if relatively small, result in fIXα accumulation. Initial recognition of fIX by fXIa involves substrate binding exosites distinct from the enzyme active site. At least one exosite appears to be located in the fXIa third apple (A3) domain, and may interact with an epitope on the fIX Ca2+-binding Gla-domain. The rate of fIX activation to fIXaβ by fXIa was significantly reduced in the presence of an antibody to the fXIa A3 domain or by mutations in the A3 domain. Similarly, rates of activation were decreased in the absence of Ca2+, in the presence of an antibody to the fIX Gla-domain, or when fIX with a decarboxylated Gla-domain was the substrate. In all cases, significant fIXα accumulation was noted in time courses, indicating that interfering with this particular substrate-exosite interaction has a significantly greater effect on cleavage after Arg180 than after Arg145. These findings raise the possibility that the exosite on fXIa A3 plays a larger role in conversion of fIXα to fIXaβ than in initial fIX conversion to fIXα, and are consistent with the possibility, recently proposed by Sinha et al. (Biochemistry46;9830:2007), that a second fIX binding exosite is present on fXIa.

Bypassing Agents in Children with Inhibitors: How Much Do We Use and How Much Is Too Much?

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1217-1217
Author(s):  
Deana Gordon ◽  
Michael Wang ◽  
Katherine Ruegg ◽  
Elizabeth Villalobos-Menuey ◽  
Marilyn J. Manco-Johnson
Keyword(s):  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Factor Viia ◽  
High Titer ◽  
Tolerance Induction ◽  
Treatment Center ◽  
Severe Bleeding ◽  
Bleeding Events ◽  
Dose Frequency ◽  
Bypassing Agents

Abstract Abstract 1217 Background: Children with hemophilia A and high-titer inhibitors suffer frequent severe bleeding events and often respond to bypassing agents more poorly than adults resulting in increased dosing and cost. Aims: This study was conducted to determine the amount and variability of bypass agent therapy currently given to children with inhibitors at a large combined adult and pediatric hemophilia treatment center in comparison to treatment of adult inhibitor patients. Methods: All subjects were enrolled onto a single institution prospective inceptional cohort study of bleeding disorders with appropriate IRB-approved consent and assent. All children who developed high-titer inhibitors were treated with immune tolerance induction, and children treated with bypassing agents primarily represent difficult inhibitors. Data regarding treatment agents, dose and duration were extracted for 24 bleeding events in 6 children in comparison to comparable data on 26 bleeding events in 5 adults with high-titer inhibitors. Treatment decisions were made clinically by academic hematologists in consultation with hemophilia nurses, patients and parents based on perceived severity of bleeding events and therapeutic response. Treatment was given as recombinant factor VIIa (NovoSeven, N, n=28), prothrombin complex concentrates (FEIBA, F, n=10) or alternating doses of both (A, n=12). Median results for individual children and aggregated adult data are shown on Table 1. Children received seven times the treatment doses and duration compared with adults. In addition, children received both N and F for half of the bleeding events, while this was used for only one adult event. Treatment duration in children was not affected by initial dose or dose frequency of N (p > 0.05 for both). Bleeding events were severe and difficult to manage; significant long-term morbidity in these 6 children include ankle arthropathy in 3, elbow contracture following compartment syndrome in 2 and quadrucep pseudotumor in 1. Discussion: Children with hemophilia A and inhibitors experience bleeding episodes that are poorly responsive to both N and F, resulting in higher dosing, longer duration and enormous costs. Children require better inhibitor therapies. Clinical trials of new bypassing agents designed specifically for children are urgently needed. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Factor IX p.A37V mutation causes severe bleeding in a patient with phenprocoumon therapy

2021 ◽  
Vol 26 (1) ◽  
Author(s):  
Nils Mülling ◽  
Vivian Rosery ◽  
H. Christian Reinhardt ◽  
Maher Hanoun
Keyword(s):  
Clinical Course ◽  
Vitamin K Antagonists ◽  
Oral Anticoagulants ◽  
Factor Ix ◽  
Bleeding Complications ◽  
Severe Bleeding ◽  
Nucleotide Position ◽  
Missense Mutations ◽  
Case Presentation ◽  
Sequencing Part

Abstract Background Bleeding is the most common complication of oral anticoagulants, due to inadequate dosing. Case presentation This report describes the clinical course of a patient who developed severe bleeding under therapy with phenprocoumon, despite an INR in the lower therapeutic range. Strikingly, aPTT was prolonged, while factor IX activity was significantly reduced. Acquired hemophilia was excluded, due to missing detection of inhibitors. Finally, sequencing part of the factor IX gene including nucleotide position c.110 revealed a hemizygous factor IX mutation c.110C > T p (Ala37Val). Conclusions In rare cases, missense mutations in factor IX propeptide are associated with severe bleeding complications. The substitution of alanin at position 37 to either valin or threonin (Ala37Val or Ala37Thr) leads to hypersensitivity to vitamin k antagonists.

Anticoagulation in addition to dual antiplatelet therapy has no impact on long-term follow-up after endovascular treatment of (sub)acute lower limb ischemia

VASA ◽  
2019 ◽  
Vol 48 (4) ◽  
pp. 321-329
Author(s):  
Mariya Kronlage ◽  
Erwin Blessing ◽  
Oliver J. Müller ◽  
Britta Heilmeier ◽  
Hugo A. Katus ◽  
...  
Keyword(s):  
Endovascular Treatment ◽  
Antiplatelet Therapy ◽  
Dual Antiplatelet Therapy ◽  
Limb Ischemia ◽  
Bleeding Complications ◽  
Severe Bleeding ◽  
Short Term ◽  
Long Term Follow Up

Summary. Background: To assess the impact of short- vs. long-term anticoagulation in addition to standard dual antiplatelet therapy (DAPT) upon endovascular treatment of (sub)acute thrombembolic occlusions of the lower extremity. Patient and methods: Retrospective analysis was conducted on 202 patients with a thrombembolic occlusion of lower extremities, followed by crirical limb ischemia that received endovascular treatment including thrombolysis, mechanical thrombectomy, or a combination of both between 2006 and 2015 at a single center. Following antithrombotic regimes were compared: 1) dual antiplatelet therapy, DAPT for 4 weeks (aspirin 100 mg/d and clopidogrel 75 mg/d) upon intervention, followed by a lifelong single antiplatelet therapy; 2) DAPT plus short term anticoagulation for 4 weeks, followed by a lifelong single antiplatelet therapy; 3) DAPT plus long term anticoagulation for > 4 weeks, followed by a lifelong anticoagulation. Results: Endovascular treatment was associated with high immediate revascularization (> 98 %), as well as overall and amputation-free survival rates (> 85 %), independent from the chosen anticoagulation regime in a two-year follow up, p > 0.05. Anticoagulation in addition to standard antiplatelet therapy had no significant effect on patency or freedom from target lesion revascularization (TLR) 24 months upon index procedure for both thrombotic and embolic occlusions. Severe bleeding complications occurred more often in the long-term anticoagulation group (9.3 % vs. 5.6 % (short-term group) and 6.5 % (DAPT group), p > 0.05). Conclusions: Our observational study demonstrates that the choice of an antithrombotic regime had no impact on the long-term follow-up after endovascular treatment of acute thrombembolic limb ischemia whereas prolonged anticoagulation was associated with a nominal increase in severe bleeding complications.

scholarly journals A Graft-Based Chemotherapy Method for Screening Effective Molecules and Rescuing Huanglongbing-Affected Citrus Plants

2012 ◽  
Vol 102 (6) ◽  
pp. 567-574 ◽  
Author(s):  
Muqing Zhang ◽  
Charles A. Powell ◽  
Ying Guo ◽  
Melissa S. Doud ◽  
Yongping Duan
Keyword(s):  
Survival Rate ◽  
Transmission Rate ◽  
High Titer ◽  
Critical Factor ◽  
Transmission Efficiency ◽  
Pathogen Transmission ◽  
Citrus Industry ◽  
Chemical Treatments ◽  
Bacterial Titer ◽  
Transmission Studies

Huanglongbing (HLB) is the most devastating disease of citrus. The global citrus industry is in urgent need of effective chemical treatments for HLB control because of its rapid spreading worldwide. Due to the fastidious nature of the pathogens, and the poor permissibility of citrus leaf surfaces, effective screening of chemicals for the HLB control can be challenging. In this study, we developed a graft-based chemotherapy method to rapidly screen potential HLB-controlling chemical compounds. In addition, we improved transmission efficiency by using the best HLB-affected scion–rootstock combination, and demonstrated the HLB bacterial titer was the critical factor in transmission. The HLB-affected lemon scions had a high titer of HLB bacterium, survival rate (83.3%), and pathogen transmission rate (59.9%). Trifoliate, a widely used commercial rootstock, had the highest survival rate (>70.0%) compared with grapefruit (52.6%) and sour orange (50.4%). Using this method, we confirmed a mixture of penicillin and streptomycin was the most effective compounds in eliminating the HLB bacterium from the HLB-affected scions, and in successfully rescuing severely HLB-affected citrus germplasms. These findings are useful not only for chemical treatments but also for graft-based transmission studies in HLB and other Liberibacter diseases.

scholarly journals Inhibition of tissue factor/factor VIIa activity in plasma requires factor X and an additional plasma component

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 204-212
Author(s):  
NL Sanders ◽  
SP Bajaj ◽  
A Zivelin ◽  
SI Rapaport
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Additional Material ◽  
Plasma Reaction ◽  
Progress Curve ◽  
Peptide Release ◽  
Release Data

A study was carried out to explore requirements for the inhibition of tissue factor-factor VIIa enzymatic activity in plasma. Reaction mixtures contained plasma, 3H-factor IX or 3H-factor X, tissue factor (vol/vol 2.4% to 24%), and calcium. Tissue factor-factor VIIa activity was evaluated from progress curves of activation of factor IX or factor X, plotted from tritiated activation peptide release data. With normal plasma, progress curves exhibited initial limited activation followed by a plateau indicative of loss of tissue factor-factor VIIa activity. With hereditary factor X-deficient plasma treated with factor X antibodies, progress curves revealed full factor IX activation. Adding only 0.4 micrograms/mL factor X (final concentration) could restore inhibition. Inhibition was not observed in purified systems containing 6% to 24% tissue factor, factor VII, 0.5 micrograms/mL, factor IX, 13 micrograms/mL, and factor X up to 0.8 micrograms/mL, but could be induced by adding barium-absorbed plasma to the reaction mixture. Thus, both factor X and an additional material in plasma were required for inhibition. The amount of factor X needed appeared related to the concentration of tissue factor; adding more tissue factor at the plateau of a progress curve induced further activation. These results also indicate that inhibited reaction mixtures contained active free factor VII(a). Preliminary data suggest that inhibition may stem from loss of activity of the tissue factor component of the tissue factor- factor VII(a) complex.

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.

scholarly journals Inhibition of tissue factor/factor VIIa activity in plasma requires factor X and an additional plasma component

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 204-212 ◽  
Author(s):  
NL Sanders ◽  
SP Bajaj ◽  
A Zivelin ◽  
SI Rapaport
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Additional Material ◽  
Plasma Reaction ◽  
Progress Curve ◽  
Peptide Release ◽  
Release Data

Abstract A study was carried out to explore requirements for the inhibition of tissue factor-factor VIIa enzymatic activity in plasma. Reaction mixtures contained plasma, 3H-factor IX or 3H-factor X, tissue factor (vol/vol 2.4% to 24%), and calcium. Tissue factor-factor VIIa activity was evaluated from progress curves of activation of factor IX or factor X, plotted from tritiated activation peptide release data. With normal plasma, progress curves exhibited initial limited activation followed by a plateau indicative of loss of tissue factor-factor VIIa activity. With hereditary factor X-deficient plasma treated with factor X antibodies, progress curves revealed full factor IX activation. Adding only 0.4 micrograms/mL factor X (final concentration) could restore inhibition. Inhibition was not observed in purified systems containing 6% to 24% tissue factor, factor VII, 0.5 micrograms/mL, factor IX, 13 micrograms/mL, and factor X up to 0.8 micrograms/mL, but could be induced by adding barium-absorbed plasma to the reaction mixture. Thus, both factor X and an additional material in plasma were required for inhibition. The amount of factor X needed appeared related to the concentration of tissue factor; adding more tissue factor at the plateau of a progress curve induced further activation. These results also indicate that inhibited reaction mixtures contained active free factor VII(a). Preliminary data suggest that inhibition may stem from loss of activity of the tissue factor component of the tissue factor- factor VII(a) complex.

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