Factor VIII Coagulant Activity Research Articles

Shifting Paradigms and Arising Concerns in Severe Hemophilia A Treatment.

The management of hemophilia A has undergone a remarkable revolution, in line with technological advancement. In the recent past, the primary concern associated with Factor VIII (FVIII) concentrates was the risk of infections, which is now almost resolved by advanced blood screening and viral inactivation methods. Improving patients' compliance with prophylaxis has become a key focus, as it can lead to improved health outcomes and reduced health care costs in the long term. Recent bioengineering research is directed toward prolonging the recombinant FVIII (rFVIII) coagulant activity and synthesising higher FVIII yields. As an outcome, B-domain deleted, polyethylene glycolated, single-chain, Fc-fused rFVIII, and rFVIIIFc-von Willebrand Factor-XTEN are available for patients. Moreover, emicizumab, a bispecific antibody, is commercially available, whereas fitusiran and tissue factor pathway inhibitor are in clinical trial stages as alternative strategies for patients with inhibitors. With these advancements, noninfectious complications, such as inhibitor development, allergic reactions, and thrombosis, are emerging concerns requiring careful management. In addition, the recent approval of gene therapy is a major milestone toward a permanent cure for hemophilia A. The vast array of treatment options at our disposal today empowers patients and providers alike, to tailor therapeutic regimens to the unique needs of each individual. Despite significant progress in modern treatment options, these highly effective therapies are markedly more expensive than conventional replacement therapy, limiting their access for patients in developing countries.

Markedly Reduced FVIII Recovery Associated with Anti-FVIII Peg Antibodies after BNT162b2 Sars-Cov-2 Vaccination

Background The covalent link of polyethylene glycol (PEG) chains to recombinant factor VIII (FVIII) molecule is one of the most established approaches to produce novel therapeutic FVIII products with extended half-life (EHL). To date, three PEGyated-FVIII molecules, rurioctocog alfa pegol, turoctocog alfa pegol, and damoctocog alfa pegol are available for treatment of hemophilia A patients. From the pivotal clinical studies data, they showed comparable safety profile and pharmacokinetic with a mean half-live 1.3-1.7 times longer than the standard non-PEGylated FVIII. Although the PEG polymer is considered non immunogenic, antibodies against PEG, called pre-existing anti-PEG antibodies (APAs), have been reported in persons never treated with PEGylated drugs owing to PEG exposure during the activities of daily living. These antibodies can impact the clinical efficacy of PEGylated drug therapies by accelerating the blood clearance and changing pharmacokinetic. Recently, small amounts of PEG are also included in the lipid nanoparticles of the mRNA SARS-Cov-2 vaccines, BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna). Aim To describe a poor plasma FVIII recovery after the first infusion of a PEGylated FVIII product in two male patients with severe hemophilia A without inhibitor at about one month from the second dose of the BNT162b2 SARS-Cov-2 vaccination. Method About two months after the first dose of the SARS-Cov-2 vaccine and one month after the second, before switching from a standard half-life to a PEGylated FVIII product, patients underwent a PK study by infusion of 50 IU/kg of turoctocog alfa pegol (patient 1) or damoctocog alfa pegol (patient 2). FVIII activity was measured by Chromogenix Coamatic FVIII (Werfen) in plasma samples collected before and after the PEGylated FVIII infusion. Anti-PEGylated FVIII antibodies were searched by means of a home-made ELISA in plasma samples collected before administration of SARS-Cov-2 vaccine and at PK study. The assay was performed by using as capturing substrates each of the licensed PEGylated FVIII product (rurioctocog alfa pegol, turoctocog alfa pegol, and damoctocog alfa). Moreover, a non-PEGylated standard FVIII (Advate®) was employed as control substrate. Results FVIII coagulant activity measured after the infusion of 50 IU/kg of the PEGylated FVIII product showed a very low plasma recovery, 5% for patient 1 and 4% for patient 2. Anti-PEGylated FVIII antibodies reacting with each PEGylated FVIII products were found in plasma samples collected at PK baseline and after FVIII infusion but not in a sample obtained before the SARS-Cov-2 vaccination. No reactivity was found in vitro against the non-PEGylated standard FVIII product (figure). Conclusions Our finding of antibodies reacting with the PEGylated part of the FVIII molecule induced or boosted by BNT162b2 SARS-Cov-2 vaccination supports the view that the administration of the mRNA vaccine could impair plasma FVIII recovery after infusion of a PEGylated product. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A high efficient FVIII variant corrects bleeding in hemophilia A mouse model

Hemophilia A is a bleeding disorder caused by quantitative or qualitative deficiencies in coagulation factor VIII (FVIII). Low FVIII expression due to its unstable mRNA and binding with immunoglobulin-binding protein (BiP) compromises gene therapy endeavors in hemophilia A. Site-directed mutagenesis have demonstrated an improvement in the expression of FVIII proteins. In this study, a targeted point mutation of Pro at position 290 to Thr (P290T) enhances the in vitro specific activity of B-domain-deleted factor VIII (BDD-FVIII). Hydrodynamic gene delivery of P290T cDNA into FVIII-deficient (FVIII−/-) mice corrected bleeding symptoms. P290T variant resulted in high plasma FVIII coagulant activity 24 h post-gene delivery. Furthermore, bleeding time and average blood loss was significantly reduced in FVIII−/- mice injected with P290T variant, whereas BDD-FVIII and PBS-injected mice experienced prolonged bleeding and excessive blood loss. Histological analysis of the liver biopsies revealed no apparent signs of liver damage. No signs of potential toxicity were seen in mice following mice bodyweights assessment. Altogether, our results demonstrate that the introduction of P290T mutation increases both in vitro and in vivo FVIII coagulant activity, supporting ongoing efforts to develop more effective replacement therapy for hemophilia A.

X Chromosome inactivation: a modifier of factor VIII and IX plasma levels and bleeding phenotype in Haemophilia carriers.

Haemophilia A and B are X-linked hemorrhagic disorders caused by gene variants in the F8 and F9 genes. Due to recessive inheritance, males are affected, while female carriers are usually asymptomatic with a wide range of factor VIII (FVIII) or IX (FIX) levels. Bleeding tendency in female carriers is extremely variable and may be associated with low clotting factor levels. This could be explained by F8 or F9 genetic variations, numerical or structural X chromosomal anomalies, or epigenetic variations such as irregular X chromosome inactivation (XCI). The aim of the study was to determine whether low FVIII or FIX coagulant activity in haemophilia carriers could be related to XCI and bleeding symptoms. HUMARA assay was performed on 73 symptomatic carriers with low clotting activity ≤50 IU/dL. Bleeding Assessment Tool (BAT) from the International Society on Thrombosis and Haemostasis (ISTH) was used to describe symptoms in the cohort of carriers. In 97% of haemophilia carriers, a specific gene variant in heterozygous state was found, which alone could not justify their low FVIII or FIX levels (≤50 IU/dL). A statistical association between XCI pattern and FVIII and FIX levels was observed. Moreover, female carriers with low coagulant activity (≤20 IU/dL) and high degree of XCI ( ≥ 80:20) had a higher ISTH-BAT score than the carriers with the opposite conditions (>20 IU/dL and <80:20). In our cohort of haemophilia carriers, XCI was significantly skewed, which may contribute to the low expression of clotting factor levels and bleeding symptoms.

Functional polymorphisms in the LDLR and pharmacokinetics of Factor VIII concentrates

BackgroundOptimization of factor VIII (FVIII) infusion in hemophilia A would benefit from identification of FVIII pharmacokinetics (PK) determinants. The low‐density lipoprotein receptor (LDLR) contains an FVIII‐binding site and might influence FVIII clearance. Consistently, LDLR polymorphisms have been associated with FVIII levels. ObjectiveTo investigate the relationships between individual FVIII PK and functional LDLR polymorphisms. Patients/MethodsThirty‐three hemophilia A patients (FVIII coagulant activity [FVIII:C] ≤2 IU/dL) without inhibitors underwent 85 FVIII single‐dose (21.4‐51.8 IU/kg) PKs with different FVIII concentrates. Twenty patients underwent repeated PKs (2‐6). FVIII:C measured up to 72 hours was analyzed by two‐compartment model. Parameters were evaluated in relation to F8 mutations, ABO blood‐group and LDLR genotypes. ResultsF8 mutation types were not associated with PK parameters. ABO and LDLR c.1773C/T polymorphism were associated with Alpha, Alpha HL, CLD2, K1‐2, and K2‐1 parameters, suggesting an influence on the FVIII initial distribution phase. Regression analysis showed an independent association of both ABO and LDLR c.1773C/T with PK parameters (Alpha, β‐coefficient −0.311 vs 0.348; CLD2, β‐coefficient −0.335 vs 0.318), giving rise to an additive effect in subjects stratified by combined phenotypes. Differently, the LDLR c.81C/T was associated with FVIII clearance and volume of distribution at steady state, which could be related to distinct effects of polymorphisms, potentially linked to LDLR intracellular distribution and FVIII binding behavior. ConclusionsWith the limitation of different FVIII concentrates and low number of patients, our data show plausible associations of LDLR polymorphisms with FVIII PK parameters, thus supporting their investigation as candidate functional determinants of FVIII PK.

Laboratory monitoring of replacement therapy for major surgery in von Willebrand disease.

Von Willebrand disease (VWD) is an inherited haemorrhagic disorder caused by a quantitative or qualitative defect of von Willebrand factor (VWF), a multimeric plasma glycoprotein that plays a key role in platelet adhesion to the subendothelium and acts as a carrier of factor VIII (FVIII) in blood. Patients with VWD experience bleeding symptoms that are mainly localized in mucous membranes and soft tissues, and their severity depends on the degree of the primary reduction in VWF and the secondary deficiency of FVIII in plasma. Because VWD patients are also at increased risk of perioperative bleeding, a prophylactic treatment aimed to correct the dual haemostatic defect (i.e. VWF and FVIII) is warranted. This review summarizes knowledge on the current management of patients undergoing major surgery, focusing on the peri-surgical laboratory monitoring of replacement therapy with VWF/FVIII concentrates. We suggest to monitor plasma levels of FVIII coagulant activity in the postoperative period rather than a surrogate maker of platelet-binding VWF activity as the ristocetin cofactor assay and its recent modifications.

Cross-reacting Material-positive Hemophilia A Diagnosed in a Patient with a Spontaneous Thigh Hemorrhage

A 53-year-old man, who had been diagnosed with mild hemophilia A (HA) at 35 years of age, was hospitalized with a thigh hematoma. His bleeding continued despite the administration of recombinant factor VIII (FVIII). The results of an FVIII/von Willebrand factor binding assay were normal. The patient's FVIII coagulant activity (FVIII:C) was low, but his FVIII antigen levels were within the normal limits, suggesting FVIII protein dysfunction. The FVIII:C measurements obtained by one-stage clotting and chromogenic assays were different. An FVIII gene analysis revealed a missense mutation p.Ser308Leu, which is rare in Japan. This case highlights that gene analyses and chromogenic assays are necessary to interpret the discrepancies between FVIII:C and the bleeding phenotype of patients with mild HA.

Thrombin Generation and Bleeding Phenotype during Personalized Prophylaxis with Recombinant Human FVIII in Previously Treated Patients with Severe Hemophilia A

Background and objectivesIn apharmacokinetic (PK)-guided personalized prophylaxis study with human-cl rhFVIII(Nuwiq®), a factor VIII (FVIII) concentrate from a human cell line,58% of the previously treated adult patients (PTPs) with severe hemophilia A received 2 or fewer prophylactic infusions per weekwith a median dosing interval of3.5 days. Seventy-three percent (73%) of patients did not experience any bleeding and 83% had no spontaneous bleeding episodes during the 6-month personalized prophylaxis period. The objective of this analysis was to evaluate the relationship between endogenous thrombin generation (TG) as well as FVIII plasma concentrations with the bleeding phenotype.Study design and methodsThis prospective, open-label, multicenter study included 66 adult PTPs with severe hemophilia A. After the previously given FVIII concentrate was washed out, patients received human-cl rhFVIII(60 IU/kg) for PK evaluation. Individual PK data were analyzed to determine doses and injection intervals that would theoretically result in a trough FVIII plasma level of ≥1%. Individualized prophylaxis lasted 6 months. TG and FVIII:C plasma concentrations were measured before and during the PK assessment, and trough TG and FVIII:C levels were measured 2, 4 and 6 months after the start of personalized prophylaxis. For TG, blood was drawn in trisodium citrate tubes (0.106 M) containing 1.45 µM corn trypsin inhibitor, centrifuged twice to obtain platelet poor plasma, and stored frozen until central analysis. TG was initiated by adding tissue factor (1 pM), and endogenous thrombin potential (ETP) was measured using the calibrated, automated TG assay.FVIII:C was measured using one-stage (automated APTT, Trinity Biotech, Siemens BCX-XP) and chromogenic assays (Coatest SP FVIII Kit, Chromogenix, Siemes BCS-XP).ResultsData on both baseline ETP and bleeding rate during PK-guided individualized prophylaxis were available for 32 patients. Twenty-one patients did not experience any bleeding episode, and 25 patients had no spontaneous bleeding episodes. The mean baseline ETP did not differ between patients with and without any bleeding (373 ± 334 vs 367 ± 168 nmol*min), but was considerably lower in patients who had spontaneous bleeding episodes compared with those without spontaneous bleeding episodes (164 ± 66 vs 426 ± 231 nmol*min). Data on trough ETPs and FVIII:C during personalized prophylaxis were available for 34 patients.Patients with low TG potential tended to experience more frequent spontaneous bleeding episodes during this phase of the protocol (Figure 1), while low FVIII:C levels seemed to be less related to spontaneous bleeding episodes.ConclusionsLow TG capacity seems to be correlated with increased frequency of spontaneous bleeding episodes, irrespective of trough FVIII plasma levels. Further prospective studies should be carried out to evaluate the value of this global coagulation parameter in the personalization prophylactic treatment in patients with hemophilia A in comparison with trough FVIII coagulant activity. [Display omitted] DisclosuresKlamroth:Biogen Idec: Other: honoraria for advisory boards and speaker fees; Baxalta: Other: honoraria for advisory boards and speaker fees ; Bayer: Other: honoraria for advisory boards and speaker fees; CSL Behring: Other: honoraria for advisory boards and speaker fees; NovoNordisk: Other: honoraria for advisory boards and speaker fees; SOBI: Other: honoraria for advisory boards and speaker fees; pfizer: Other: honoraria for advisory boards and speaker fees; Octapharma: Other: honoraria for advisory boards and speaker fees; uniqure: Other: honoraria for advisory boards and speaker fees. Windyga:Biogen: Consultancy, Equity Ownership, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding, Speakers Bureau; Aspen: Consultancy, Equity Ownership, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding, Speakers Bureau; Nordisk: Consultancy, Equity Ownership, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding, Speakers Bureau; Alexion: Other: Speaker’s honorarium; Sanofi: Consultancy, Equity Ownership, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding, Speakers Bureau; Octapharma: Consultancy, Equity Ownership, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding, Speakers Bureau; Baxalta, now part of Shire: Consultancy, Equity Ownership, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Other: Investigator Clinical Studies, Patents & Royalties, Research Funding, Speakers Bureau; CSL Behring: Consultancy, Equity Ownership, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding, Speakers Bureau. Bichler:Octapharma: Employment. Knaub:Octapharma: Employment. Negrier:NovoNordisk: Membership on an entity’s Board of Directors or advisory committees, Other: Travel support, Research Funding; LFB: Membership on an entity’s Board of Directors or advisory committees; Alnylam: Research Funding; Bayer: Membership on an entity’s Board of Directors or advisory committees, Other: Travel support; CSL Behring: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity’s Board of Directors or advisory committees, Other: Travel support, Research Funding; Pfizer: Research Funding; SOBI: Membership on an entity’s Board of Directors or advisory committees; Baxter: Membership on an entity’s Board of Directors or advisory committees.

Badania genetyczne w diagnostyce hemofilii B

Hemophilia A (HA) is one of the most frequently recognized genetically determined bleeding disorders. At its ground is a congenital lack, deficiency or dysfunction of coagulation factor VIII (FVIII), which leads to significant disturbances of hemostatic system. The severity of bleeding diathesis is determined by the degree of FVIII deficiency in plasma. The diagnosis of HA is mainly based on the results of the coagulation plasma test, including measuring the FVIII coagulation activity. Nowadays, it is believed that diagnosis of HA should be extended to genetic testing, to get to know the causative mutation. Determination of the mutation responsible for HA is important for at least two reasons: 1) enables a preliminary assessment of the risk of FVIII inhibitor development and 2) make possible the clear-cut diagnosis of HA carrier status. This paper presents an algorithm forgenetic testing in patients with HA. On top of that characterizes the molecular techniques used indiagnostic process, and discusses the difficulties associated with the interpretation of the results.

Progress of in vitro factor VIII coagulant activity from 0 to 8 hours after reconstitution.

BackgroundContinuous infusion of factor VIII (FVIII) is a more cost-effective method for treating hemophilia A than intermittent bolus injection. However, there is currently no specific data in Korea about the progress of in vitro FVIII coagulant activity (FVIII:C) after reconstitution from its lyophilized form.MethodsThree commercial FVIII concentrate products (two recombinant FVIII and one plasma-derived) were used. In vitro FVIII:C was measured at 0, 2, 4, 6, and 8 hours following reconstitution in both the indoor light-exposed and light-shielded groups.ResultsFor the three drugs, in vitro FVIII:C decreased over the 8 hours following reconstitution (P<0.001). The decline of FVIII:C was linear (P<0.001). In vitro FVIII:C for the indoor light-exposed groups was 95.3±1.9% and 90.6±2.5% after 4 and 8 hours following reconstitution, respectively, compared to baseline activity. In the light-shielded group, FVIII:C was 95.4±1.1% and 90.9±1.7% of the baseline activity after 4 and 8 hours, respectively. There was no statistical difference between FVIII:C in the indoor light-exposed and light-shielded groups (P=0.849).ConclusionIn vitro FVIII:C decreased after reconstitution, but activity was maintained at over 90% of the baseline value during 8 hours. Exposure to indoor light did not accelerate the loss of FVIII:C over the experimental time. This result indicates that CI with FVIII is available in 8-hour intervals, with no indoor light-exposure precautions needed.

Inter-chain disulfide bond improved protein trans-splicing increases plasma coagulation activity in C57BL/6 mice following portal vein FVIII gene delivery by dual vectors

Protein trans-splicing based dual-vector factor VIII (FVIII) gene delivery is adversely affected by less efficiency of protein splicing. We sought to increase the amount of spliced FVIII protein and plasma coagulation activity in dual-vector FVIII transgene in mice by means of strengthening the interaction of inteins, protein splicing elements, thereby facilitating protein trans-splicing. Dual-vector delivery of the FVIII gene in cultured cells showed that replacement of Met226 in the heavy chain and Asp1828 in the light chain with Cys residues could facilitate inter-chain disulfide linking and improve protein trans-splicing, increasing the levels of spliced FVIII protein. In this study, C57BL/6 mice were coadministered dual vectors of intein-fused human FVIII heavy chain and light chain with Cys mutations via portal vein injection into the liver. Forty-eight hours post-injection, plasma was collected and analyzed for FVIII antigen concentration and coagulation activity. These mice showed increased circulating FVIII heavy chain polypeptide (442 ± 151 ng m(L-1) vs. 305 ± 103 ng mL(-1)) and coagulation activity (1.46 ± 0.37 IU m(-1) vs. 0.85 ± 0.23 IU mL(-1)) compared with control mice co-administered dual vectors expressing the heavy and light chains of wild-type FVIII. Moreover, coagulation activity was similar to that of mice receiving a single vector expressing FVIII (1.79 ± 0.59 IU mL(-1)). These findings indicate that improving protein trans-splicing by inter-chain disulfide bonding is a promising approach for increasing the efficacy of dual-vector based FVIII gene transfer.

Surgical Injury Alone Does Not Provoke the Development of Factor VIII Inhibitors in Mouse Models of Hemophilia A

AbstractAbstract 627 Introduction:Inhibitory antibodies (inhibitors) to exogenous factor VIII (FVIII) are the major complication of treatment with recombinant FVIII for patients with hemophilia A. Inflammatory “danger signals”, such as would be produced by surgical injury, have been hypothesized as important acquired determinants of inhibitor risk. Surgery has been associated with an increased inhibitor risk in observational studies of humans. However, it is not possible to experimentally separate surgical injury from intensive FVIII exposure in humans, and this relationship has not previously been explored in animal models of hemophilia A. We investigated the relationship between surgical injury and FVIII inhibitor development in two distinct mouse models of hemophilia A. Methods:The hemophilia A models used were F8 exon 16 knockout (E16KO) mice and F8 exon 17 knockout mice with the humanized major histocompatibility complex II allele HLA-DRB1*1501 (E17KO/hMHC). The model surgical procedure was an open-and-close laparotomy under isofluorane anaesthesia at 6 to 12 weeks of age. Recombinant human FVIII, with or without lipopolysaccharide (LPS), was administered intravenously through the tail vein. Preoperatively, FVIII was given in doses of 2 units (approximately 0.1 mcg/unit) weekly for 4 weeks; postoperatively FVIII was given in doses of 2 to 6 units daily for 3 to 5 days. Blood was obtained via retro-orbital sampling or cardiac puncture, and plasma was separated by centrifugation. Plasma was assayed for IL-1 and IL-6 concentration by ELISA, for anti-FVIII IgG by ELISA, and for inhibition of FVIII coagulant activity (FVIII:C) by Bethesda assay. Single cell suspensions of splenocytes were analyzed by flow cytometry. Data was analyzed using Student t tests, Mann-Whitney U tests, and chi-square tests. All animal experiments were approved by the Animal Care Committee at Queen's University. Results:In E16KO mice, surgery produced greater than 5-fold increases in IL-1 levels (p=0.0005) and 150-fold increases in IL-6 levels (p=0.000023), compared to FVIII controls in the 24 hrs after the surgical injury. Surgery also resulted in significantly increased expression by splenic dendritic cells of CD80 (p=0.0004), but not of CD40 or CD86. All E16KO mice developed high-titre antibodies (by both ELISA and Bethesda assay) and there was no difference in Bethesda titres between surgery and control groups, either for mice FVIII-naïve at time of surgery (p=0.27), or for mice with FVIII exposure prior to surgery (p=0.66). In contrast to E16KO hemophilic mice, FVIII immune responses were seen in only some E17KO/hMHC animals. Proportions of FVIII-naïve E17KO/hMHC mice that did develop detectable antibodies were similar between surgery and control groups (47% vs. 53% for ELISA, p=0.72; 7% vs. 22% for Bethesda, p=0.19); among mice who developed antibodies, ELISA and Bethesda titres were not significantly different between surgery and control groups (p>0.05). E17KO/hMHC mice were exposed to FVIII, and those that were tolerant to FVIII (ie. did not have antibodies detectable by ELISA) then had either surgery with post-operative FVIII exposure, FVIII without surgery, or FVIII and LPS. No FVIII-tolerant E17KO/hMHC developed antibodies detectable by ELISA after surgery or after FVIII exposure alone, but 100% of FVIII-LPS-exposed mice developed antibodies (p=0.001). Conclusions:Although laparotomy produces acute increases in the inflammatory cytokines IL-1 and IL-6 and upregulates expression of the costimulatory molecule CD80 on antigen presenting cells, E16KO mice that underwent laparotomy did not have greater immunologic responses to FVIII than those who did not. FVIII-naïve E17KO/hMHC mice who underwent surgery were no more likely to develop detectable antibodies than those who did not, and surgery did not result in higher-titre immune responses. In E17KO/hMHC mice who were immunologically tolerant to FVIII, surgery did not break this tolerance, although coadministration of LPS and FVIII did; tolerance could be broken in these mice, but was not broken by surgical injury. Given the limitations of existing clinical research in this area, our results indicate that understanding of specific inflammatory stimuli involved in FVIII inhibitor development in humans should be obtained before proceeding with the assumption that surgical injury alone is a significant risk factor for inhibitors in patients with hemophilia A. Disclosures:Moorehead:Baxter Biosciences: Honoraria, Travel funding for educational meetings Other; Bayer: Honoraria, Travel funding for educational meetings, Travel funding for educational meetings Other. Steinitz:Baxter BioScience: Employment. Reipert:Baxter Biosciences: Employment.

Enhanced plasma factor VIII activity in mice via cysteine mutation using dual vectors

Hemophilia A is caused by a genetic mutation in coagulation factor VIII (FVIII) gene and gene therapy is considered to be a promising strategy for its treatment. We recently demonstrated that co-delivery of two vectors expressing M662C mutated heavy and D1828C mutated light chain genes of B-domain-deleted coagulation factor VIII (BDD-FVIII) leads to inter-chain disulfide cross-linking and improved heavy chain secretion in vitro. In this study, co-injection of both M662C and D1828C mutated BDD-FVIII gene expression vectors into mice resulted in increased heavy chain secretion and coagulation activity in plasma in vivo. Approximately (239 ± 56) ng mL(-1) above endogenous levels of transgenic FVIII heavy chain was found in mouse plasma using a chain-specific ELISA. For FVIII coagulation activity, approximately (1.09 ± 0.25) IU mL(-1) above endogenous levels were detected in co-injected transgenic mouse plasma using a chromogenic assay. These data demonstrate that inter-chain disulfide bonds likely increase heavy chain secretion and coagulation activity in the plasma of transgenic mice with an improved efficacy of a dual-vector delivery of BDD-FVIII gene. These findings support our ongoing efforts to develop a gene therapy for hemophilia A treatment using dual-AAV vectors.

A Prospective, Multi-Center, Randomized Clinical Trial to Compare the Pharmacokinetic Properties of Human-Cl rhFVIII, a New Human-Cell Line Derived Recombinant Factor VIII, with Those of a Full-Length Recombinant Factor VIII Expressed in Baby Hamster Kidney Cells,

Abstract 3309▪▪This icon denotes a clinically relevant abstract Introduction:human-cl rhFVIII is the first recombinant factor VIII (FVIII) concentrate expressed in a human cell line (Human Embryonic Kidney (HEK) 293F cells). The manufacturing process includes two steps for virus inactivation and removal (solvent/detergent treatment and a nanofiltration). No animal- or human-derived materials are added to the culture medium or during the purification and final formulation. Objective:The primary objective of this study is to compare the pharmacokinetics (PKs) of human-cl rhFVIII, a B-domain deleted rFVIII product expressed in a human cell line, with a commercially available full-length rFVIII product (Kogenate FS), which is expressed in Baby Hamster Kidney (BHK) cells. Methods:This study is an open, multinational, prospective, multicenter clinical phase-II trial. The PK properties of human-cl rhFVIII and Kogenate FS were determined using a randomized two-period, cross-over design. After a 96 hours or more wash-out phase of any previously administered FVIII product, subjects received a single intravenous dose of 50 IU/kg body weight of one of the products. Blood samples were collected over a 48-h period and FVIII coagulant activity (FVIII:C) was measured by validated chromogenic substrate and one-stage clotting assay in a central laboratory, which also assigned drug potency with the same assays. Following completion of the PK segment of the study, subjects will be followed for at least 50 Exposure Days and at least 6 months of treatment with human-cl rhFVIII to evaluate the efficacy and safety of on-demand treatment. The planned interim analysis stipulates the comparison of the complete PK profile of human-cl rhFVIII and Kogenate FS with the bioequivalence of the AUC as the primary endpoint. The full efficacy and safety profile will be reported when the study is completed. Results:Twenty-two adolescent and adult PTPs with severe hemophilia A (FVIII:C <1%) were enrolled at 10 study centers in the United States and Europe. The key PK parameters (AUC, elimination half-life, in-vivo recovery, clearance) are comparable between human-cl rhFVIII and Kogenate FS both for the one-stage and chromogenic assays. The two products were judged to be bioequivalent as the 90% confidence interval of the ratio of the geometric mean values of the AUC fell within 80–125%. human-cl rhFVIII has been well tolerated and no safety or efficacy issues have been reported so far, in particular no inhibitors or non inhibitory antibodies were detected or reported. Conclusion:The PK data from this study indicate that human-cl rh FVIII is bioequivalent to a licensed rFVIII concentrate measured by the one stage and the chromogenic assays. In this study as well as in the other ongoing clinical studies, which examine the efficacy and safety of human-cl rhFVIII during prophylactic treatment in children and adults with severe hemophilia A, the product has been safe and well tolerated so far. Disclosures:Knaub:Octapharma AG: Employment. Valentino:Baxter Bioscience: Consultancy; GTC Biotherapeutics: Consultancy; Bayer Healthcare: Consultancy; NovoNordisk: Consultancy; Inspiration Bioscience: Consultancy; Biogen: Consultancy. Manco-Johnson:Octapharma AG: Consultancy.

P.Tyr365Cys change in factor VIII: haemophilia A, but not as we know it

Haemophilia A is caused by a reduction in clotting factor VIII (FVIII). FVIII coagulant activity (FVIII:C) can be measured by three methods; the one-stage activated partial thromboplastin time-based clotting assay, the two-stage Xa generation-based clotting assay and the chromogenic Xa generation-based assay. The FVIII:C of most patients with haemophilia A are concordant regardless of the assay method employed. Up to a third of patients show assay discrepancy, usually with the two-stage and chromogenic assays being much lower than the one-stage assay. Very rarely, patients have been reported with lower one-stage compared to two-stage and chromogenic assays, but here we report that the mutation p.Tyr365Cys accounts for most of these patients and, at least in the UK, is not rare. We have identified this mutation in 23 different families. Affected male index individuals had a lower mean one-stage FVIII:C of 27 iu/dl compared to two-stage FVIII:C mean of 77 iu/dl. Affected individuals had minimal or absent bleeding symptoms and when these were present they were usually in patients with another co-inherited bleeding disorder. Affected individuals often had surgery without the need to correct the one-stage FVIII:C.

Development of a new modified Bethesda method for coagulation inhibitors: the Osaka modified Bethesda method

The Nijmegen assay for the factor VIII (F-VIII) inhibitor is recommended by the International Society on Thrombosis and Haemostasis/Scientific and Standardization Committee. However, due to cumbersome and complicated preprocessing, it is presently difficult to introduce this assay into hospital laboratories. We used buffered plasma that was made by addition of 1 volume of 1 mol/l HEPES buffer at pH 7.35 to 9 volumes of plasma to form the test samples. The inhibitor titer was calculated by the remaining rate of F-VIII coagulation activity (F-VIII:C), using the ratio of actual value to the theoretical value. Five hundred microliters of the buffered test plasma and the control (30 mmol/l HEPES buffered saline at pH 7.35) were each mixed with equal volumes (500 μl) of normal pooled plasma in a test tube (11 mm internal diameter and 6.5 ml volume capacity), and incubated at 37°C for 2 h. In our modified Bethesda method, there were no significant changes in pH and F-VIII:C of control and test mixtures after incubation tests for stability. With the modified method, the inhibitor titers (mean, SD) from examining three hemophilia A plasma samples (F-VIII:C, <1-3%) and 40 normal samples (F-VIII:C, 34.5-168.3%) were 0.032, 0.057 and -0.009, 0.057, respectively. By our method, the F-VIII inhibitor titer of type I inhibitor-positive samples was higher than the Nijmegen method, and for type II inhibitor-positive samples, the titer was similar. We believe that our method can be applied to not only the type I inhibitor, but also to assays of type II inhibitor, without cumbersome and complicated preprocessing.

Polymorphisms at LDLR locus may be associated with coronary artery disease through modulation of coagulation factor VIII activity and independently from lipid profile

AbstractHigh levels of coagulation factor VIII (FVIII) have been associated with cardiovascular disease. Low-density lipoprotein receptor (LDLR) has been recently demonstrated to contribute to FVIII clearance from plasma. The aim of this study was to evaluate 3 single nucleotide polymorphisms in SMARCA4-LDLR gene locus (rs1122608, rs2228671, and rs688) and FVIII coagulant activity (FVIII:c) in subjects with (n = 692) or without (n = 291) angiographically confirmed coronary artery disease (CAD). High FVIII:c levels were an independent risk factor for CAD. The rs688 and rs2228671 genotypes were predictors of FVIII:c with T alleles associated with higher FVIII:c levels. The rs2228671T allele was associated also with reduced total and LDL-cholesterol levels. With respect to the risk of CAD, no association was found for rs2228671. Consistently with higher FVIII:c levels, the rs688T allele was associated with CAD, whereas, consistently with a favorable lipid profile, the rs1122608T allele was associated with a decreased CAD prevalence. After adjustment for classic cardiovascular risk factors, including plasma lipids, rs688 remained associated with CAD (OR for T carriers: 1.67 with 95% confidence interval, 1.10-2.54). Haplotype analysis confirmed such results. Our data suggest that polymorphisms at LDLR locus modulate FVIII:c levels and may be associated with CAD risk independently from plasma lipids.

Haemophilia A and von Willebrand’s disease

Deficient or defective coagulation factor VIII (FVIII) and von Willebrand factor (VWF) can cause bleeding through congenital deficiency or acquired inhibitory antibodies. Recent studies on type 1 von Willebrand's disease (VWD), the most common form of the disease, have begun to explain its pathogenesis. Missense mutations of varying penetrance throughout VWF are the predominant mutation type. Other mutation types also contribute while about one-third of patients have no mutation identified. Enhanced clearance and intracellular retention contribute to pathogenic mechanisms. Chromogenic substrate (CS) methods to determine FVIII coagulant activity have several advantages over one-stage methods, which include minimal influence by variable levels of plasma components, notably lupus anticoagulant. Direct proportionality between FVIII activity and FXa generation results in high resolution at all FVIII levels, rendering the CS method suitable for measuring both high and low levels of FVIII activity. FVIII inhibitors in patients with inherited or acquired haemophilia A present several challenges in their detection and accurate quantification. The Nijmegen method, a modification of the Bethesda assay is recommended for inhibitor analysis by the International Society on Thrombosis and Haemostasis. Understanding potential confounding factors including heparin and residual FVIII in test plasma, plus optimal standardization can reduce assay coefficient of variation to 10-20%.These areas are all explored within this article.

Prophylaxis in haemophilia with inhibitors: update from international experience

An antibody response to therapeutically administered factor VIII (FVIII) can occur in up to 30% of patients with haemophilia, resulting in the production of inhibitors that neutralize FVIII coagulant activity. Immune tolerance induction (ITI) therapy can be used to eradicate inhibitors in these patients, allowing them to continue with factor replacement therapy. Patients with inhibitors (prior to initiation of ITI, while on ITI or those who fail ITI) experience more difficulty in treating bleeds than those patients who do not develop inhibitors. Increasingly, prophylaxis with bypassing therapy is being employed in patients who develop inhibitors. Two bypassing agents, a plasma-derived activated prothrombin complex concentrate [aPCC (FEIBA; Baxter AG, Vienna, Austria)] and NovoSeven [recombinant factor VIIa (rFVIIa); NovoNordisk, Denmark] are potentially available for prophylaxis in patients with haemophilia who have developed inhibitors. Results from recent retrospective studies demonstrate the efficacy and safety of both aPCC and rFVIIa in decreasing the frequency of bleeding episodes in patients with haemophilia and inhibitors. In this article, we highlight a number of ongoing studies that aim to identify patients who should be placed on prophylaxis with bypassing agents.

Quantitation of Changes in VWF and FVIII Following Elective Orthopedic Surgery in Normal Individuals.

Abstract 1296Poster Board I-318 BackgroundPatients with inherited bleeding disorders undergoing surgery typically receive therapy to correct the deficient factor to a level of ∼1.0 IU/mL. There is a paucity of well-constructed descriptive data quantifying the changes in coagulation that occur in response to surgical stress in normal individuals, however. Additionally, von Willebrand factor (VWF) and factor VIII (FVIII) are acute phase reactants, and thus, supratherapeutic levels of these proteins that might develop in response to the surgical stress would predispose patients to thrombotic events. Aim: With this in mind, our objective was to quantify the changes in hemostatic variables that occur in response to elective orthopedic surgery in normal individuals. MethodsEligible subjects > 18 years of age undergoing total knee or total hip replacement were recruited. Exclusion criteria included a known bleeding disorder, hormone replacement therapy, use of oral contraceptives or pregnancy. Following informed consent, blood samples were drawn at five time points: 1) baseline - in clinic 1-2 weeks pre-operatively (pre-op), 2) pre-op the morning of surgery – same day admitting center, 3) intra-operatively (intra-op) – 30 minutes after the start of surgery, 4) 30 minutes post-operatively (post-op) - recovery room, and 5) post-op day one (POD 1) – on the ward. Samples were analyzed for complete blood counts (CBC), prothrombin time (PT), activated partial thromboplastin time (aPTT), VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), FVIII coagulant activity (FVIII:C) and whole blood thromboelastography (TEG) without tissue factor activation. Repeated measures analysis of variance was used to evaluate trends over time; paired samples t-tests were done to maximize the sample size for pairwise comparisons. Results37 subjects were recruited (25 females, 12 males) with a mean age of 64 years (range 33 – 87). Means and ranges for all laboratory parameters are displayed in Table 1 in addition to statistical analysis of the change in all variables over the 5 time points. Statistically significant changes were seen in all variables except PTT and TEG angle. In general, VWF and FVIII indices decreased during surgery (presumably due to consumption) and then increased in the post-operative period (presumably due to a stress response). A great deal of variability was seen in post-operative levels of VWF and FVIII between individuals with some showing borderline low levels and some achieving very high levels. ConclusionThese data establish a baseline on which to reconsider current strategies for the peri-operative management of patients with inherited bleeding disorders, and provide relevant information when considering the relative risks of bleeding or thrombosis associated with surgery.Table 1Baseline mean (range)Pre-op mean (range)Intra-op mean (range)Post-op mean (range)POD 1 mean (range)p-value (ANOVA)*WBC X109/L (4.0 – 10.5)6.71 (4.0-10.1)6.84 (4.8-10.3)5.88 (3.3-9.5)9.31 (5.2-20.3)8.56 (5.0-13.8)<0.0001 n=20Hemoglobin g/L (120 – 160)136 (108-160)140 (119-157)120 (99-140)118 (87-146)102 (71-126)<0.0001 n=20Platelet X109/L (150 – 400)265 (160-479)273 (168-499)226 (97-477)241 (158-399)211 (124-327)<0.0001 n=20PT seconds (12.5 – 15.7)13.1 (12.3-13.8)14 (12.9-16.9)14.1 (13.2-15.4)14.5 (13.1-15.9)<0.0001 n=17PTT seconds (23 – 36)29 (25-36)29 (25-36)30 (20-39)30 (26-34)30 (26-36)0.448 n=12VWF:Ag IU/mL (0.50 – 2.00)1.46 (0.5-2.6)1.48 (0.55-2.66)1.22 (0.5-2.21)1.34 (0.54-2.19)1.72 (0.62-4.0)<0.001 n=15VWF:RCo IU/mL (0.50 – 2.00)1.39 (0.57-2.21)1.35 (0.57-2.28)1.12 (0.53-1.79)1.32 (0.49-3.23)1.65 (0.71-3.32)0.004 n=15FVIII:C IU/mL (0.50 – 1.50)1.33 (0.56-2.69)1.31 (0.62-2.91)1.01 (0.43-2.77)1.38 (0.45-4.29)1.54 (0.51-4.50)<0.0001 n=15TEG R minutes (9 – 27)8.9 (8 – 10.2)6.6 (3.7 – 8.2)0.042≠ n=4TEG angle degree (22 – 58)56 (48 – 62)57 (23 – 76)0.743≠ n=4*ANOVAs were performed on subjects with complete data sets with n= shown. Results of paired samples t-tests were generally similar (demonstrating a statistically significant difference between the baseline/intra-op, pre-op/intra-op, and intra-op/POD1 time points).≠paired samples t-tests. DisclosuresNo relevant conflicts of interest to declare.