Recombinant Human Coagulation Factor VIIa Research Articles

COMPARING THE EFFECTIVENESS OF CONCENTRATE OF ALL PROTHROMBIN COMPLEX FACTORS WITH RECOMBINANT HUMAN COAGULATION FACTOR VIIa IN THE TREATMENT OF BLEEDING AFTER CARDIAC SURGERY

Aim: The authors evaluated the effectiveness of treatment with recombinant human coagulation factor VIIa and concentrate of all prothrombin complex factors in patients with massive postoperative bleeding that could not be controlled with traditional therapy. Materials and Methods: In the period from 2020 to 2021, recombinant human coagulation factor VIIa was administered to 18 patients after cardiac surgery (group I), while the concentrate of all prothrombin complex factors was administered to 16 patients postoperatively (group II). During this period, 647 patients were operated on. The patients had normal coagulation screening tests (APTT, INR, TT, fibrinogen level, and PLT level) before surgery. Mean blood loss before and after administration of eptacog alfa and the total prothrombin complex concentrate was assessed. The mean dose of eptacog alfa was 30.95 mcg/kg b.w., and the total prothrombin complex factor concentrate dose was 14.17 mcg/kg b.w. After transfusion with red blood cell concentrate, fresh frozen plasma, and platelet concentrate, in the absence of improvement in the dynamics of postoperative drainage, it was decided to include recombinant human coagulation factor VIIa or a concentrate of all prothrombin complex factors in the treatment. Results: After administration of recombinant human coagulation factor VIIa at a dose of 30.95 mcg/kg b.w., bleeding stopped in 12 patients, but the remaining 6 patients required reoperation due to persistently high drainage. The decision to perform a rethoracotomy was made by a team of cardiothoracic surgeons and anesthesiologists, taking into account the dynamics of drainage (bleeding) and the hemodynamic stability of the patient. After the administration of concentrate of all prothrombin complex factors at a dose of 14.17 U/kg b.w., bleeding stopped in 12 patients. Four patients required reoperation due to persistent bleeding. Conclusions: Treatment with recombinant human coagulation factor VIIa and concentrate of all prothrombin complex factors is effective and safe for cardiac surgery patients.

Perioperative application of recombinant human coagulation factor Ⅶa combined with prothrombin complex in two hemophilia A patients with high titer inhibitor

Perioperative application of recombinant human coagulation factor Ⅶa combined with prothrombin complex in two hemophilia A patients with high titer inhibitor

PRO17 A BUDGET IMPACT ANALYSIS OF ANTI-INHIBITOR COAGULANT COMPLEX USE IN PEDIATRIC HEMOPHILIA A PATIENTS WITH INHIBITORS ACROSS KINGDOM OF SAUDI ARABIA.

Hemophilia is rare genetic disorder affecting blood’s ability to clot due to clotting factor deficiency. Hemophilia A (HA), the most common form of Hemophilia, is due to factor VIII (FVIII) deficiency. HA treatment options include plasma-derived or recombinant-FVIII concentrates. Approximately 30% of HA patients develop inhibitors against FVIII concentrates. Patients with inhibitors are usually managed using bypassing agents, e.g., Anti-Inhibitor Coagulant Complex (ACC) or recombinant human coagulation Factor VIIa. (rFVIIa). In 2016, the Kingdom of Saudi Arabia (KSA) reported 31 pediatric HA inhibitor patients, among whom 26 patients were treated in the ministry of health (MOH) facilities. To assess the budget impact (BI) of switching MOH pediatric HA patients with inhibitors from-rFVIIa to ACC both as prophylaxis and an on-demand therapy. A global BI excel model was adapted from payer’s perspective. Model inputs were retrieved from literature and validated by key experts through face-to-face interviews. The model was adapted for two scenarios: Switching patients from (1) rFVIIa prophylaxis therapy to ACC prophylaxis therapy and (2) rFVIIa on-demand therapy to ACC on-demand therapy. The model considered direct costs such as drug acquisition, drug administration and bleeding episodes management. A sensitivity analysis was performed to assess the robustness of the results. The BI analysis showed that switching patients from rFVIIa prophylaxis to ACC prophylaxis would result in three times (-112 million Saudi Riyal [SAR]) and eight times (-418 million SAR) savings in the annual payers’ budget using rFVIIa standard dose and megadose. Similarly, switching patients from rFVIIa on-demand to ACC on-demand therapy would result in approximately 1.5 times (-15 million SAR) savings in the payers’ budget. Switching pediatric HA patients with inhibitors from rFVIIa to ACC as prophylaxis and on-demand therapies could be cost saving options for KSA payers thereby freeing more resources for other diseases management.

Acquired hemophilia A developing cerebral infarction 36 days after the frequent administration of bypass hemostatic agents.

A 74-years-old male who was a smoker and received treatment for hypertension, dyslipidemia, peripheral arterial disease and idiopathic interstitial pneumonia complained of subcutaneous hemorrhage of the right lower thigh. Marked anemia (hemoglobin 5.5 g/dL) and prolonged activated partial thromboplastin time (≥130 seconds) were noted. The factor VIII activity level was reduced to 1.2%, and the factor VIII inhibitor titer was 285.3 BU/mL, a diagnosis of acquired hemophilia A (AHA) was made. Then, hematomas of 5 intra-muscles were recurred. Hemostasis became difficult despite frequent and high-dose administration of recombinant human coagulation factor VIIa (total: 18 days, 305 mg). Hemostasis was achieved by switching to activated prothrombin complex concentrate (for 3 days, 18,000 units), however, cerebral infarction occurred after 36 days. After the frequent administration of bypass hemostatic agents on elderly AHA patients with several risk factors for ischemic stroke, the risk of subsequent thrombotic events may persist for 1 month.

Efficacy of recombinant human coagulation factor Ⅶ and immunosuppressive agent in patients with acute intestinal graft versus host disease complicated with bleeding after allogeneic hematopoietic stem cell transplantation

Efficacy of recombinant human coagulation factor Ⅶ and immunosuppressive agent in patients with acute intestinal graft versus host disease complicated with bleeding after allogeneic hematopoietic stem cell transplantation

The application of recombinant human coagulation factor VIIa during perioperative period of congenital heart disease in children

Background Refractory hemorrhage is a fatal complication during peri-operative period of congenital heart disease(CHD) in children, which has great influence on the prognosis of patients. Recombinant human coagulation factor Ⅶa(rFⅦa) has been widely used to treat refractory bleeding, but there is no well controlled clinical trial with randomized selected cases or wide accepted guidance to guide clinical application. The application methods and evaluation still vary among different medical centers about the dosage, therapeutic efficacy and adverse effects of rFⅦa in refractory bleeding treatment. Objective This article aims to address the role of rFⅦa played in refractory bleeding treatment to guide clinical applications of rFⅦa. Content This article reviews the index of using rFⅦa, dosage, efficacy and adverse reactions of rFⅦa during the perioperative period of CHD. Trend As more studies are carried out, the use of rFⅦa will be standard and safe. Key words: Recombinant human coagulation factor Ⅶa; Refractory bleeding; Congenital heart disease

Differences in N-glycosylation of recombinant human coagulation factor VII derived from BHK, CHO, and HEK293 cells.

Background & MethodsRecombinant factor VII (rFVII), the precursor molecule for recombinant activated FVII (rFVIIa), is, due to its need for complex post translational modifications, produced in mammalian cells. To evaluate the suitability of a human cell line in order to produce rFVII with post-translational modifications as close as possible to pdFVII, we compared the biochemical properties of rFVII synthesized in human embryonic kidney-derived (HEK)293 cells (HEK293rFVII) with those of rFVII expressed in Chinese hamster ovary (CHO, CHOrFVII) and baby hamster kidney (BHK, BHKrFVII) cells, and also with those of plasma derived FVII (pdFVII), using various analytical methods. rFVII was purified from selected production clones derived from BHK, CHO, and HEK293 cells after stable transfection, and rFVII isolates were analyzed for protein activity, impurities and post-translational modifications.Results & DiscussionThe analytical results showed no apparent gross differences between the various FVII proteins, except in their N-linked glycosylation pattern. Most N-glycans found on rFVII produced in HEK293 cells were not detected on rFVII from CHO and BHK cells, or, somewhat unexpectedly, on pdFVII; all other protein features were similar. HEK293rFVII glycans were mainly characterized by a higher structural variety and a lower degree of terminal sialylation, and a high amount of terminal N-acetyl galactosamines (GalNAc). All HEK293rFVII oligosaccharides contained one or more fucoses (Fuc), as well as hybrid and high mannose (Man) structures.ConclusionsFrom all rFVII isolates investigated, CHOrFVII contained the highest degree of sialylation and no terminal GalNAc, and CHO cells were therefore assumed to be the best option for the production of rFVII.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0205-1) contains supplementary material, which is available to authorized users.

SENSITIZATION TO THE MAMMALIAN OLIGOSACCHARIDE GALACTOSE-ALPHA-1,3- GALACTOSE (ALPHA-GAL): EXPERIENCE IN A FLEMISH CASE SERIES

Background. Recent observations have disclosed that the galactose-α(1,3)-galactose (α-gal) moiety of non-primate glycoproteins can constitute a target for meat allergy.Objective. To describe adults with allergic reactions to mammalian meat, dairy products and gelatin. To investigate whether patients could demonstrate sensitization to activated recombinant human coagulation factor VII ectapog alpha that is produced in baby hamster kidney cells.Methods. Ten adults with mammalian meat, dairy products and gelatin allergies were examined using quantification of specific IgE and/or skin prick test for red meat, milk, milk components, gelatin, cetuximab and eptacog alpha.Results. Most patients demonstrate quite typical clinical histories and serological profiles, with anti-α-gal titers varying from less than 1% to over 25% of total serum IgE. All patients demonstrate negative sIgE for gelatin, except the patient with a genuine gelatin allergy. All patients also demonstrated a negative sIgE to recombinant milk components casein, lactalbumin and lactoglobulin. Specific IgE to eptacog was positive in 5 out of the 9 patients sensitized to α-gal and none of the 10 control individuals.Conclusion. This series confirms the importance of the α-gal carbohydrate moiety as a potential target for allergy to mammalian meat, dairy products and gelatin (oral, topical or parenteral) in a Flemish population of meat allergic adults. It also confirms in vitro tests to mammalian meat generally to be more reliable than mammalian meat skin tests, but that diagnosis can benefit from skin testing with cetuximab. Specific IgE to gelatin is far too insensitive to diagnose α-gal related gelatin allergy. IgE binding studies indicate a potential risk of α-gal-containing human recombinant proteins produced in mammalians.

Rituximab for the treatment of high titre inhibitors in mild haemophilia A.

Rituximab for the treatment of high titre inhibitors in mild haemophilia A.

Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells

Human coagulation factor VII (FVII) plays an important role in the blood coagulation process and exists in micro amounts in human plasma; therefore, any attempt at the large-scale production of FVII in significant quantities is challenging. The purpose of this study was to express and obtain biologically active recombinant FVII (rFVII) from Chinese hamster ovary K1 (CHO-K1) cells. The full-length FVII cDNA was isolated from a HepG2 cell line and then subcloned in pcDNA3.1 to construct an expression vector, pcDNA-FVII. CHO-K1 cells were transfected with 1 µg pcDNA-FVII. The cell line that stably expressed secretory FVII was screened using 900 µg/mL G418. The FVII copy number in CHO-K1 cells was detected by quantitative polymerase chain reaction (qPCR). The rFVII was purified in ligand affinity chromatography medium. The purified protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The biological activity of the purified FVII protein was determined by a prothrombin time assay. Three cell lines that permanently expressed rFVII were screened. The qPCR results demonstrated that each CHO-K1 cell harbored two FVII DNA copies. The SDS-PAGE and Western blot analysis showed that the purified protein was about 50 kDa. The purity of the target protein was 95%. The prothrombin time assay indicated that the FVII-specific activity of rFVII was 2573 ± 75 IU/mg. This method enabled the fast preparation of high-purity rFVII from CHO-K1 cells, and the purified protein had good biological activity.

Use of Recombinant Factor VII in Cardiac Surgery

The off-label use of recombinant human coagulation factor VIIa has been increasing to a number of different treatment areas since its original approval in 1999. Several US patents describe claims for FVIIa utilization in nonhemophilia patients, treatment of bleeding due to trauma, as a means to reverse major bleeding, including intracranial bleeding, induced from fibrinolytic therapy as well as a patent for using FVIIa in the treatment of bleeding for patients with bleeding disorders not caused by hemophilia, but rather bleeding disorders due to thrombocytopenia, platelet disorders, and von Willebrand's disease. Bleeding after cardiac surgery remains a serious complication that can increase both morbidity and mortality. We review the off-label usage of recombinant factor VIIa as a hemostatic agent that may help control bleeding following cardiac surgery.

520 Diagnosis and Treatment Peculiarities in an Infant with Bleeding Disorder

Background and AimsOne of bleeding causes due to vitamin K deficiency is gut flora destruction secondary to antibiotic treatment early in life. Authors emphasize diagnosis and treatment difficulties for an...

Development of a flow cytometric assay for detection of coated platelets in dogs and evaluation of binding of coated platelets to recombinant human coagulation factor VIIa

To develop an antibody-based flow cytometric assay to detect coated platelets in dogs and to characterize the interaction of recombinant human coagulation factor VIIa with activated platelets from dogs with hemophilia A. Platelets from 4 dogs with hemophilia A, 4 dogs with hemophilia B, 4 dogs with von Willebrand disease, and 6 hemostatically normal dogs. Freshly isolated platelets were activated with thrombin, convulxin, or a thrombin-convulxin combination. Resulting platelet phenotypes were resolved on the basis of P-selectin and fibrinogen expression, and binding of recombinant human coagulation factor VIIa to these distinct platelet subpopulations was measured by use of a flow cytometric assay. Coated platelets were identified on the basis of expression of α-granule fibrinogen and were generated in response to stimulation with the thrombin-convulxin combination but not to stimulation with either agonist alone. Approximately 70% of the platelets from dogs with hemophilia A, hemophilia B, and von Willebrand disease and from the control dogs had the coated platelet phenotype. Recombinant human coagulation factor VIIa bound preferentially to coated platelets with a mean ± SD binding equilibrium constant of 2.6 ± 0.5μM. Formation of coated platelets in dogs was similar to that in humans. Recombinant human coagulation factor VIIa bound preferentially to coated platelets from dogs. A similar mechanism of action for recombinant human coagulation factor VIIa may exist in dogs and humans. The potential for use of dogs in the study of bleeding disorders in humans was strengthened.

Characterization of canine coagulation factorVII and its complex formation with tissue factor: canine-human cross-species compatibility.

Canine models have been good predictors of efficacy of hemophilia treatments, including recombinant human coagulation factor (F)VIIa (hFVIIa). However, canine FVIIa and tissue factor (TF) have remained incompletely characterized. To explore canine-human cross-species FVIIa-TF compatibility in order to strengthen the predictive value of canine models in research on FVIIa and TF. Canine FVIIa (cFVIIa) and canine TF((1-217)) [cTF((1-217))] were produced by recombinant techniques, and canine-human cross-species FVIIa-TF interactions were characterized in vitro. Recombinant cFVIIa and soluble cTF((1-217)) were produced and purified to homogeneity. hFVIIa and cFVIIa bound with comparably high affinities to cTF((1-217)) (K(D)=6.0±0.7 nm and K(D)=6.0±0.3 nm, respectively) and to cell surface-expressed cTF (K(D)=8.4±0.4 nm and K(D)=7.2±1.2 nm, for (125) I-labeled hFVIIa and cFVII, respectively). In contrast, cFVIIa bound to human TF (hTF) with decreased affinity, both in solution and on cell surfaces. The decreased binding resulted in reduced activity of cFVIIa in functional assays with hTF((1-209)) . In direct comparison, cFVIIa was more active than hFVIIa, both in the absence and the presence of cognate TF. The present finding that hFVIIa binds to cTF essentially as it does to hTF substantiates the hypothesis that human FVIIa-TF biology can be reliably recapitulated in canine models on administration of hFVIIa to dogs.

High-level expression of functional recombinant human coagulation factor VII in insect cells

Recombinant coagulation factor VII (FVII) is used as a potential therapeutic intervention in hemophilia patients who produce antibodies against the coagulation factors. Mammalian cell lines provide low levels of expression, however, the Spodoptera frugiperda Sf9 cell line and baculovirus expression system are powerful systems for high-level expression of recombinant proteins, but due to the lack of endogenous vitamin K-dependent carboxylase, expression of functional FVII using this system is impossible. In the present study, we report a simple but versatile method to overcome the defect for high-level expression of the functional recombinant coagulation FVII in Sf9 cells. This method involves simultaneous expression of both human gamma-carboxylase (hGC) and human FVII genes in the host. It may be possible to express other vitamin K-dependent coagulation factors using this method in the future.

Expression and purification of recombinant human coagulation factor VII fused to a histidine tag using Gateway technology.

Factor VII (FVII) is a plasma glycoprotein that participates in the coagulation process leading to the generation of fibrin. The aim of this study was to construct, express and purify recombinant FVII fused to a polyhistidine (his) tag using Gateway technology. To construct the entry clone, blunt-end FVII cDNA and subsequent polymerase chain reaction (PCR) product isolated from a HepG2 cell line was TOPO-cloned into a pENTR TOPO vector. To construct the expression clone, a LR recombination reaction was carried out between the entry clone and destination vector, pDEST26. Chinese hamster ovary (CHO) cells were transfected with 1 microg of DNA of PDEST26-FVII using the FuGENE HD transfection reagent. Two cell lines that permanently expressed recombinant FVII were established. The expression of recombinant FVII was confirmed by reverse transcriptase PCR and enzyme-linked immunosorbent assay. Culture medium containing his-tagged FVII was added to the nickel-nitrilotriacetic acid resin column and bound protein was eluted. The purified protein was detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The biological activity of the recombinant FVII was determined by a prothrombin time assay using FVII-depleted plasma. The results showed that human recombinant FVII was successfully cloned and the accuracy of the nucleotide sequence of the gene and its frame in the vector were confirmed by DNA sequencing. Stable clones transfected with the construct expressed FVII mRNA and related protein but no expression was detected in the CHO cells containing an empty vector. A protein of about 52 KDa was detected in SDS-PAGE and was further confirmed by western blot analysis. A three-fold decrease in clotting time was observed using this recombinant FVII. As far as we are aware, this is the first report of expression of recombinant FVII fused with a his-tag through Gateway technology. The next steps, including large scale expression, purification, activation and stabilisation, are underway.

Formulary Management of Recombinant Factor VIIa at an Academic Medical Center

Recombinant human coagulation factor VIIa (rVIIa) is a procoagulant indicated for treatment of bleeding in patients with hemophilia. A large proportion of rVIIa utilization is for off-label administration in nonhemophiliac patients with acute hemorrhage. Concerns of potentially inappropriate use, safety, and cost of rVIIa led to efforts to standardize use of this agent. To comparatively describe the utilization of rVIIa upon implementation of an evidence-based guideline at a university hospital. With advisory direction from a multidisciplinary task force, an evidence-based guideline for use of rVIIa was developed, approved, and fully implemented. Assessment of appropriateness of use and retrospective review were required for all cases. Effects of these actions were evaluated by auditing and comparing rVIIa use in patients treated in two 6-month observation periods before and after guideline implementation. Outcomes assessed were proportions of patients deemed appropriate to receive rVIIa, compliance with dosing recommendations, and acquisition costs. Twenty-two and 29 patients were treated in the periods before and after guideline implementation, respectively. Patient characteristics were similar, except more cardiothoracic surgeries were performed in patients treated before implementation of the guideline. Indications for rVIIa use were judged appropriate in 21 (95.5%) before-cases and in all (100%) after-cases. The dose was compliant in 1 (4.6%) before-case and 27 (93.1%) after-cases (p < 0.001). Mean dosages of rVIIa administered were 81.8 microg/kg and 45.3 microg/kg in before- and after-cases, respectively (p < 0.001). During the respective periods of observation, amounts of rVIIa purchased monthly averaged 42.6 mg and 21.8 mg, a 49% difference. Semiannual expenditures for rVIIa decreased approximately $110,000 following guideline implementation. Patient outcomes were similar. A guideline based on currently available evidence can serve to sustain the clinical appropriateness of rVIIa therapy and substantially decrease costs.

Thromboembolic Adverse Events After Use of Recombinant Human Coagulation Factor VIIa

Thromboembolic Adverse Events After Use of Recombinant Human Coagulation Factor VIIa

Recombinant factor VIIa should be used in massive obstetric haemorrhage

Recombinant activated factor VII, (rFVIIa, NovoSeven®, Novo Nordisk), is licensed in the UK, Europe and US for treatment of haemophilia patients with inhibitors to factors VII and IX, during bleeding episodes occurring spontaneously or caused by trauma or surgery. It has been approved for use in patients with factor VII deficiency and Glanzmann’s thrombasthenia. [1] O’Connell Wood J.J. Wise R.P. et al. Thromboembolic adverse effects after use of recombinant human coagulation factor VIIa. JAMA. 2006; 295: 293-298 Crossref PubMed Scopus (575) Google Scholar It is not licensed for use in any other group of patients.

More rFVIIa-Linked Thromboembolic Events After Off-Label Use

The FDA licensed recombinant human coagulation factor VIIa (rFVIIa) in 1999 for use in patients with hemophilia and inhibitors to factors VII or IX.