scholarly journals Reducing Antithrombin in Plasma to Levels Observed in Fitusiran-Treated Patients Does Not Interfere with Coagulation Assays

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 10-10
Author(s):  
Shaobin Wang ◽  
Sravya Kattula ◽  
Ayman Ismail ◽  
Nina Leksa ◽  
Arjan van Der Flier ◽  
...  
Keyword(s):  
Hemophilia A ◽  
Clotting Factor ◽  
Publicly Traded ◽  
Coagulation Assays ◽  
One Stage ◽  
Endogenous Expression ◽  
Relevant Effect ◽  
Fviii Activity ◽  
Potential Impact ◽  
Plasma Activity

Introduction: Fitusiran is being evaluated in phase 3 clinical studies as a once monthly subcutaneously administered RNA interference therapy designed to decrease endogenous expression of the anticoagulant antithrombin (AT). Reducing the anticoagulant potential of AT in the plasma of Hemophilia A and B patients, with or without inhibitors, restores the hemostatic balance and improves thrombin generation potential. Fitusiran and other non-Factor replacement molecules, such as emicizumab, use novel mechanisms to restore hemostasis. Consequently, some standard coagulation assays typically used to measure factor levels, such as the chromogenic, one-stage, and APTT assays have been shown to be affected by emicizumab. In order to assess the potential impact of fitusiran treatment on our ability to measure clotting factor activity in individuals with hemophilia A (HemA), we investigated the effect of decreased AT levels on standard coagulation laboratory assays. The following four assays were evaluated: one stage (OSA) and chromogenic (CSA) factor VIII (FVIII) activity assays, activated partial thromboplastin time (aPTT), and the prothrombin clotting time assay (PT). These assays are relevant to determine the FVIII plasma levels in patients dosed with FVIII replacement factors while treating potential breakthrough bleeds or undergoing surgery. Method and Results: Pooled human HemA plasma was used directly or after AT depletion using an affinity-based method to remove 95% AT activity, resulting in HemA plasma with 5% AT activity. Additional batches of HemA/AT levels were created by AT reconstitution to 10 and 20% AT plasma activity. Subsequently, the 4 HemA plasma pools (one with normal -100 %- AT and three with reduced levels of AT-5, 10, 20%) were prepared with FVIII spiked to 0, 5, 10, 20, 30, 50, and 100% . FVIII plasma activity was measured by OSA and CSA against Siemens normal standard plasma. Compared to FVIII plasma activity for all FVIII spikes in normal HemA plasma containing 100% AT, respective FVIII spikes in HemA/AT depleted plasma samples (5, 10, and 20% AT) did not show therapeutic significant effects of AT levels on measured FVIII activity by OSA and chromogenic FVIII activity assay. Additionally, we evaluated the effect of the various AT levels and FVIII spikes on clotting assays, aPTT and PT, among the HemA plasma with varying (5-100%) AT levels. AT level did not affect the aPTT for respective FVIII spikes in HemA and Hem/AT depleted plasma. Furthermore, no therapeutic relevant effect of ATIII and FVIII level was observed in PT values between HemA and Hem/AT depleted plasma. Conclusions: Our results demonstrate that a reduction of AT levels in plasma does not affect standard coagulation assays, suggesting these assays can be utilized to assess hemostasis and factor levels in individuals treated with fitusiran. The ability to monitor FVIII levels in patients treated with non-replacement therapies is important as these patients require factor to treat breakthrough bleeds and during some surgeries. Disclosures Wang: sanofi: Current Employment, Current equity holder in publicly-traded company. Kattula:Sanofi: Current Employment, Current equity holder in publicly-traded company. Ismail:Sanofi: Current Employment, Current equity holder in publicly-traded company. Leksa:Sanofi: Current Employment, Current equity holder in publicly-traded company. van Der Flier:sanofi: Current Employment, Current equity holder in publicly-traded company. Salas:sanofi: Current Employment, Current equity holder in publicly-traded company.

scholarly journals Factor VIII Level Comparison in Patients with Severe Hemophilia a on Emicizumab with Inhibitors with One Stage, Bovine and Human Chromogenic Assays and the Factor VIII Equivalency of Emicizumab Using In Vivo Global Hemostasis Assays

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4234-4234
Author(s):  
Hande Kizilocak ◽  
Elizabeth Marquez-Casas ◽  
Joshua Brown ◽  
Jemily Malvar ◽  
Guy Young
Keyword(s):  
Factor Viii ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Activated Partial Thromboplastin Time ◽  
Clotting Factor ◽  
Normal Range ◽  
One Stage ◽  
Chromogenic Assay ◽  
Coagulation Defect ◽  
Fviii Activity

Abstract Introduction Emicizumab is a recombinant, humanized bispecific monoclonal antibody that mimics the function of factor VIII (FVIII) which results in a significant reduction in the annualized bleeding rate in patients with hemophilia A (HA), however, the degree with which emicizumab corrects the coagulation defect remains unclear. The objective of this study was to compare the current available laboratory methods in clinical practice; one-stage clotting factor assays (OSCA), bovine and human chromogenic FVIII activity (bovCHR and humCHR, respectively) and FVIII Equivalency of Emicizumab by Thrombin Generation (F8EmT). Aims The aim of this study is to address the differences of FVIII activity with different techniques in patients with severe HA with inhibitors on emicizumab. Materials and Methods Factor VIII levels are determined with an activated partial thromboplastin time (aPTT), OSCA using SynthASil on the ACL TOP 500 (Instrumentation Laboratory, Bedford, MA). Factor VIII activity is also determined photometrically via the Chromogenix Coatest® SP4 FVIII chromogenic assay kit (bovCHR, Diapharma Group, West Chester, OH) and the Biophen FVIII:C chromogenic assay kit (humCHR, Aniara Diagnostica, West Chester, OH). For F8EmT, linear regression was utilized to model the FVIII levels as a function of the endogenous thrombin potential (ETP) and peak thrombin values for patients with mild/moderate hemophilia. Then, we used the ETP and peak thrombin results of the severe HA patients on emicizumab with the calibration curve to calculate their F8EmT. Association between patient weight and their F8EmT were also examined and evaluated by linear regression. Results Data is presented for eight patients with severe HA with inhibitors on emicizumab in the non-bleeding state (Table-1). All patients' FVIII levels measured with OSCA are in or above the normal range (94.0-289.1). Bovine chromogenic FVIII activity is in the severe hemophilia range for five out of eight patients, for the rest it is in the moderate hemophilia range. Human chromogenic FVIII activity ranged between 12.5-49.8%. Factor VIII Equivalency of Emicizumab by Thrombin Generation is either in the mild hemophilia or normal range in all participants of the study. Conclusion One-stage clotting factor assays demonstrated falsely high results as expected since it is activated partial thromboplastin time based. Bovine chromogenic FVIII activity results were consistent with the severe HA range of the patients though a few had results slightly above that level. Previous literature has stated that the humCHR in patients on emicizumab results in FVIII levels of ∼30% when emicizumab is at its therapeutic concentration (∼50 mcg/ml). This study also demonstrated similar results with 5/8 patients having levels 30-50%. F8EMT levels were mostly consistent with the humCHR. In conclusion, understanding the degree to which emicizumab corrects the coagulation defect of is an important goal as it has clinical implications.Certainly, additional studies with higher participant numbers are needed to confirm these findings. Figure 1 Figure 1. Disclosures Young: Apcintex, BioMarin, Genentech/Roche, Grifols, Novo Nordisk, Pfizer, Rani, Sanofi Genzyme, Spark, Takeda, and UniQure: Consultancy; Genentech/Roche, Grifols, and Takeda: Research Funding.

scholarly journals Laboratory monitoring of hemophilia A treatments: new challenges

2020 ◽  
Vol 4 (9) ◽  
pp. 2111-2118 ◽  
Author(s):  
Peter J. Lenting
Keyword(s):  
Tissue Factor Pathway Inhibitor ◽  
Hemophilia A ◽  
Laboratory Monitoring ◽  
Coagulation Assays ◽  
Fviii Activity ◽  
Different Types ◽  
Tissue Factor Pathway ◽  
Level Of Difficulty ◽  
New Challenges ◽  
Proper Interpretation

Abstract Monitoring factor VIII (FVIII) activity has traditionally been complicated by discrepancies between assays for the various sorts of FVIII molecules. The advent of novel nonfactor therapies (emicizumab, fitusiran, and anti-tissue factor pathway inhibitor antibodies) in hemophilia A poses a new level of difficulty on the laboratory monitoring of these patients. To use the correct assays and for a proper interpretation of their results, it is pertinent to understand the mode of action of these nonfactor agents. Furthermore, the biochemical consequences for the different types of activity assays (whether it be specific FVIII activity assays or global coagulation assays) should be taken into account as well. In this review, these aspects will be discussed. In addition, the use of various animal models to estimate FVIII-equivalence of the nonfactor therapies will be presented.

N1922S Mutation In the Factor VIII A3 Domain Produces a Rate-Limiting, Domain-Specific Folding Defect Leading to Hyposecretion of a Functional Protein

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2209-2209
Author(s):  
Ryan J. Summers ◽  
Shannon L. Meeks ◽  
John F. Healey ◽  
Harrison C. Brown ◽  
Ernest T Parker ◽  
...  
Keyword(s):  
Factor Viii ◽  
Intracellular Trafficking ◽  
Hemophilia A ◽  
Activity Level ◽  
Reactive Material ◽  
Domain Specific ◽  
One Stage ◽  
Fviii Activity ◽  
Coagulation Assay ◽  
Specific Activities

Abstract Abstract 2209 Factor VIII (fVIII) contains a domain sequence designated A1-A2-B-ap-A3-C1-C2. Mutation of Asn1922 to Ser (N1922S) in the A3 domain results in moderate to severe hemophilia A. However, it is unclear whether this mutation leads to secretion of cross-reactive material positive dysfunctional protein or decreased secretion of fVIII protein. We investigated the fVIII activity and antigen levels in a N1922S patient and found an activity level of 1.7% and an antigen of <4 ng/ml suggesting a secretion defect. To investigate this further, we constructed a B-domain deleted human fVIII cDNA encoding this mutation, designated N1922S fVIII, and compared its heterologous expression to non-mutated “wild-type” fVIII (wt-fVIII) in a baby hamster kidney-derived cell line. Levels of fVIII expression in cell culture media measured by antigen-capture ELISA were 0.011 and 0.73 mg/ml for N1922S fVIII and wt-fVIII, respectively. The corresponding media levels of fVIII activity measured by one-stage coagulation assay were 0.03 and 3.5 U/ml for N1922S fVIII and wt-fVIII, respectively. These values correspond to specific activities of 2800 and 4800 U/mg for N1922S fVIII and wt-fVIII, respectively. Consistent with this, both N1922S fVIII and wt-fVIII were over twenty-fold activatable by thrombin in the one-stage coagulation assay. These comparable coagulant activities of N1922S fVIII and wt-fVIII indicate that the N1922S mutation produces a kinetic block in the synthesis of a functionally normal fVIII protein. In contrast to media levels of fVIII, in-cell Western analysis revealed that intracellular levels of N1922S fVIII were similar to wt-fVIII. However, specific activities of N1922S fVIII and wt-fVIII in cell lysates were 290 and 6800 U/mg, respectively, indicating the presence of large amounts of a non-functional N1922S fVIII folding intermediate. Immunofluorescence microscopy demonstrated co-localization of wt-fVIII with both endoplasmic reticulum (ER)- and Golgi-resident proteins. In contrast, N1922S fVIII co-localized only with ER-resident proteins, indicating a kinetic block in intracellular trafficking between the ER and the Golgi. To investigate further whether the defect in N1922S fVIII trafficking was related to protein misfolding, we compared lysate-to-media antigenic ratios of N1922S fVIII and wt-fVIII using a panel of non-overlapping monoclonal antibodies (MAbs) consisting of one anti-A1, one anti-A2, three anti-A3, one anti-C1 and two anti-C2 MAbs. Lysate-to-media antigenic ratios for the anti-A1, anti-A2 and anti-C2 MAbs were similar between N1922S fVIII and wt-fVIII. In contrast, lysate-to-media ratios of the three anti-A3 MAbs and the anti-C1 MAb were markedly decreased for N1922S fVIII compared to wt-fVIII. This result indicates that the A1, A2 and C2 domains in N1922S fVIII fold independently into antigenically intact tertiary structures, but that folding is stalled in mutant A3 domain and its contiguous C1 domain. Because Asn1922 is buried in the interface of the two cupredoxin-like A3 subdomains in the two available X-ray structures of fVIII (Shen BW et al. Blood 2008;111:1240-1247; Ngo JC, et al. Structure 2008;16:597-606), the kinetic defect associated with this mutation may be due to slow association of intact A3 subdomains. This domain-specific defect in protein folding and intracellular trafficking is a novel mechanism for secretion defects leading to hemophilia A. Disclosures: No relevant conflicts of interest to declare.

scholarly journals One-Stage FVIII Activity Is Inversely Correlated with Heavy Menstrual Bleeding in Hemophilia a Carriers

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3542-3542
Author(s):  
Robert F. Sidonio ◽  
Shannon L. Meeks ◽  
Hilary Baker Whitworth
Keyword(s):  
Thrombin Generation ◽  
Hemophilia A ◽  
Menstrual Bleeding ◽  
Bleeding Tendency ◽  
One Stage ◽  
Chromogenic Assay ◽  
Fviii Activity ◽  
Coagulation Assay ◽  
Bleeding Score ◽  
Risk For Bleeding

Abstract Introduction: The majority of carriers of hemophilia A have historically been considered to have normal hemostasis (FVIII:C >50%) and thus not have an increased bleeding tendency. However, our research group has demonstrated that some hemophilia A carriers can experience increased bleeding compared to normal women despite this normal FVIII activity. In our recently published study in adult hemophilia A carriers there was no correlation between FVIII:C, as measured by a one-stage coagulation assay, and bleeding phenotype, as measured by the MCMDM-1 VWD bleeding score. In this follow up study we sought to determine which of these hemophilia A carriers with normal FVIII:C are at risk for bleeding. The goal of this study is to determine the relationship between FVIII assays (one-stage and chromogenic) and thrombin generation with both menstrual bleeding and an overall bleeding tendency. Methods: We recruited mothers of children with hemophilia A in the Emory University pediatric bleeding disorders clinic. We included only adult (>18 year of age) obligate hemophilia A carriers that did not have a concomitant bleeding disorder or chronic disease that would increase their bleeding tendency. We gathered basic demographic information and evaluated the overall bleeding tendency using the ISTH Bleeding Assessment Tool (ISTH BAT), a semi-quantitative assessment of bleeding scored from 0 to 56. We considered a score of 6 or higher consistent with pathologic bleeding. In addition, we inventoried menstrual bleeding with the Pictorial Bleeding Assessment Chart (PBAC), a visual representation of menstrual blood loss. We considered a PBAC greater than 100 consistent with heavy menstrual bleeding. Plasma samples were collected from each subject at the time of the survey in sodium citrate tubes without corn trypsin inhibitor. Samples were double spun at 2,500 rpm for 15 minutes and stored at -80°C.Laboratory evaluation for each subject included FVIII:C, measured by one-stage coagulation assay (aPTT reagent with micronized silica) and chromogenic assay (COATEST SP4 FVIII kit, Chromogenix), as well as thrombin generation (Calibrated Automated Thrombogram, PPP-Reagent Low, FluCa Kit) to evaluate endogenous thrombin potential (ETP, nM*min), peak thrombin (nM) and lag time (min). We performed linear regression using GraphPad Prism; p<0.05 was considered significant. Results: Over a three-month period, we approached 32 adult obligate hemophilia A carriers; 23 agreed to participate in the survey, 16 consented to blood draw. One carrier was excluded in extreme outlier analysis and due to an autoimmune disorder, leaving 15 evaluable subjects. Our cohort is relatively young with a median age of 41 years (range 23-51), predominantly Caucasian (53%, 8/15) and the majority carry a severe mutation (10/15 severe, 2/15 moderate, 3/15 mild). Reproductive bleeding (post-partum hemorrhage and/or menstrual bleeding) was commonly reported (93%, 14/15). The median ISTH BAT bleeding score for subjects was 2 (range 1-8). The median PBAC score was 148 (range 10-608). The carriers had a relatively normal FVIII:C by one-stage assay with a median of 0.78 U/mL (range 0.30 - 1.06) and by chromogenic assay with a median of 1.15 U/mL (range 0.66 - 1.9). FVIII:C by one-stage assay was inversely correlated to PBAC (r2 =0.4; p=0.01, figure 1A). Conversely, FVIII:C by chromogenic assay did not correlate with PBAC, however trended toward significance (r2 = 0.19; p=0.10, figure 1A). Additionally, there was no correlation found between FVIII:C (one-stage or chromogenic assay) and ISTH BAT bleeding score (figure 1B). There was also no correlation found between bleeding score or PBAC and the parameters of the thrombin generation assay or between severity of the closest male relative's hemophilia and any of the assays performed. Conclusions: In our cohort of adult obligate hemophilia A carriers, as the FVIII:C (one-stage assay) decreased, the menstrual bleeding tendency increased as measured by the PBAC. We were unable to find a correlation between other measures of hemostasis, including the chromogenic assay and thrombin generation, and commonly used bleeding assessment tools. Further larger studies are warranted to help determine which hemophilia A carriers would be at risk for bleeding. Disclosures No relevant conflicts of interest to declare.

A novel mechanism of Enhancing the Haemostatic Effect in the Combination with Recombinant Factor VIII and Activated Prothrombin Complex Concentrate(APCC) in Hemophilia A Patients with Inhibitor

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1178-1178
Author(s):  
Koji Yada ◽  
Keiji Nogami ◽  
Kenichi Ogiwara ◽  
Midori Shima
Keyword(s):  
Combination Therapy ◽  
Research Funding ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Specific Inhibitor ◽  
Activity Level ◽  
The Other ◽  
One Stage ◽  
Fviii Activity

Abstract Abstract 1178 We have reported that factor (F)VIIa/tissue factor (TF) rapidly activated FVIII by proteolysis of the heavy chain (HCh), and that appeared to serve physiologically as an activator for up-regulation of FVIII activity in very early-timed coagulation phase (J Thromb Haemost. 2010;8:2494). Furthermore, the activation phase could be observed even in the presence of anti-FVIII inhibitors, independently of their types of kinetic and epitope of inhibitors, whilst the inactivation was moderated by anti-C2 with type 1 behavior (Thromb Haemost. 2011;105:989). More recently, the other group has reported that the combination of FVIII and bypassing agent, APCC, also potentiated the thrombin generation in hemophilia A plasmas with inhibitors (Klintman et al. Br J Haematol. 2010;151:381), but the mechanisms have remained unknown. In this study, we investigated the hemostatic effect of the combination therapy with APCC and FVIII. We first examined FVIII activation catalyzed by APCC in one-stage clotting assay. The activity level of FVIII (10 nM) elevated rapidly by ∼3-fold within 1 min after addition of APCC (0.05 U/mL)/TF (0.5 nM), and subsequently decreased to the initial level within 10 min. However, the addition of APCC without TF little affected FVIII activity within 10 min, but after then gradually elevated its activity. The presence of E-76, FVIIa-specific inhibitor, significantly moderated the reaction triggered by APCC/TF, but hirudin, FIIa-specific inhibitor, little affected this activation. We further evaluated the velocity of APCC-induced thrombin generation in the presence or absence of FVIII. The increase rate of thrombin production triggered by APCC/TF in the presence of FVIII was greater than that by APCC/TF in the absence of FVIII by ∼1.6-fold, whilst the increase was little observed in the absence of TF. To clarify its enhancing effects, we performed APCC-catalyzed FVIII cleavage in SDS-PAGE and Western blot. APCC contains FVII (mainly active form) and FII, FIX and FX (mainly non-active forms). In general, FVIIa/TF, FIIa, and FXa rapidly proteolyze the HCh at Arg372 (and Arg740), and FVIIa/TF and FXa proteolyze at Arg336. FVIII proteolysis by FVIIa/TF is dependent on the presence of PL, but not FIIa. Interestingly, APCC/TF proteolyzed the HCh at Arg372 and Arg740, followed by at Arg336 in the presence of PL, whilst did not proteolyze in its absence. The presence of TF accelerated the proteolysis by ∼6.6-fold compared to the absence of TF. However, the addition of E-76 significantly delayed these cleavages. These findings supported that APCC as well as rFVIIa possessed a potential to activation/inactivation of FVIII in early-timed coagulation phase, and that FVIIa in APCC appeared to play a major role in APCC-catalyzed FVIII activation/inactivation. Furthermore, to confirm this enhancing effect in the presence of anti-FVIII inhibitors, we prepared the anti-FVIII inhibitor IgGs (3 of anti-A2, 4 of anti-C2 with type 1, and 2 of anti-C2 with type 2). The presence of anti-FVIII inhibitors did not significantly affect the APCC-catalyzed FVIII activation (by ∼3-fold), independently of their epitopes, in one-stage clotting assay. Of surprise, anti-C2 with type 1 significantly moderated APCC-catalyzed FVIIIa inactivation, and the peak level of FVIIIa retained over 30 min. In contrast, the other inhibitors little affected this inactivation, similar to FVIIa/TF-catalyzed FVIII reaction. In conclusion, we demonstrated the putative mechanism of enhancing hemostatic effects in the combination therapy using FVIII and APCC. In addition, only a small amount of APCC relative to the standard dosage (1–2 U/mL) for clinical use could activate FVIII even in the presence of anti-FVIII inhibitors, and this combination therapy would provide new therapeutic strategy in congenital hemophilia A with inhibitor and/or acquired hemophilia A. Disclosures: Yada: Baxter Hemophilia Scientific Research and Education Fund 2011: Research Funding. Nogami:Bayer Award 2009: Research Funding.

scholarly journals Population Pharmacokinetic Modeling and Simulation of Recombinant Single-Chain Factor VIII (r VIII-SingleChain) in Patients with Hemophilia a

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2814-2814
Author(s):  
Ying Zhang ◽  
Tharin Limsakun ◽  
Debra M. Bensen-Kennedy ◽  
Alex Veldman ◽  
Zhenling Yao
Keyword(s):  
Body Weight ◽  
Factor Viii ◽  
Half Life ◽  
Hemophilia A ◽  
Volume Of Distribution ◽  
Population Pharmacokinetic ◽  
Fviii Activity ◽  
Dosing Regimens ◽  
Trough Levels ◽  
Plasma Activity

Abstract Introduction: Hemophilia A is a rare but serious X-linked recessive bleeding disorder that affects males and is characterized by a deficiency in the plasma protein known as coagulation Factor VIII. rVIII-SingleChain is a proprietary, lyophilized formulation of clotting factor VIII (FVIII) produced by recombinant technology. As part of the clinical development of rVIII-SingleChain, a population pharmacokinetic (PK) analysis was undertaken, utilizing data from Study CSL627_1001 in subjects with hemophilia A, with the objectives of (a) characterizing the PK of rVIII-SingleChain at a population level, (b) assessing the ability of various patient characteristics (e.g., von Willebrand factor, VWF) to describe variability in the PK parameters, and (c) enable population-based simulations of rVIII-SingleChain dosing regimens that may provide improved prophylaxis coverage compared with octocog alfa (Advate®). Methods: Twenty-seven male subjects (aged 19-60 years) enrolled in Study CSL627_1001 (Part 1) received a single 50 IU/kg IV infusion of Advate®, followed by a single 50 IU/kg IV infusion of rVIII-SingleChain, with a minimum 4-day washout period. Plasma PK samples (for the determination of FVIII activity) were collected over 72 hours for both Advate® and rVIII-SingleChain (at pre-specified time points) and were measured by a validated chromogenic assay. Population PK models were developed separately for rVIII-SingleChain and Advate®, using the NONMEM 7 with FOCEI method. Various covariates, including VWF, body weight, and effect of age on clearance (CL) and volume of distribution were tested. Bootstrap and visual predictive check (VPC) were used for model evaluation. Simulations of different dosing regimens were performed to evaluate the FVIII activity plasma exposure profiles that may provide improved prophylaxis coverage. Results: A two-compartmental model with first-order elimination was developed to describe FVIII plasma activity data for both rVIII-SingleChain and Advate®. VWF was found to be a significant covariate influence on FVIII plasma activity CL, whilst body weight influenced both CL and volume of distribution in the central compartment. Population parameter estimates indicated a lower CL (2.02 vs 2.49 dL/h) and longer half-life (13.1 vs 9.3 h) for rVIII-SingleChain compared with Advate®. The results of bootstrap and VPC implied that the model was stable, and the parameters were estimated with good precision. PK simulations indicated that rVIII-SingleChain, at the same doses and frequencies, resulted in higher FVIII plasma activities throughout the dosing period, as reflected in higher area-under-the-curve (AUC). The dosing regimens for the simulations were designed based on the dosing recommendations of the Advate® label and rVIII-SingleChain phase III study. The results showed that rVIII-SingleChain provided a higher percentage of subjects with trough levels of at least 1% FVIII plasma activity, compared with Advate® at the same dosing regimen. Every 3 days dosing at 40-50 IU/kg rVIII-SingleChain was predicted to achieve similar prophylaxis protection compared with Advate® every 2 days (i.e., about 90% of subjects with trough levels of at least 1% FVIII plasma activity). In addition, 73-90% of subjects were predicted to achieve trough levels of at least 1% FVIII plasma activity with twice weekly dosing (4- and 3-day schedule) at 50 IU/kg rVIII-SingleChain, compared with 65-80% of subjects dosed with Advate® using the same regimen. Conclusion: The population model shows that rVIII-SingleChain has a longer half-life, lower CL and higher AUC compared with Advate®. Simulations demonstrated that rVIII-SingleChain resulted in higher trough concentrations when compared with Advate®, indicating the possibility of greater prophylaxis coverage. Disclosures Zhang: CSL Behring: Employment. Limsakun:CSL Behring: Employment. Bensen-Kennedy:CSL Behring: Employment. Veldman:CSL Behring GmbH: Employment. Yao:CSL Behring: Employment.

scholarly journals Ultrasound Combined with Microbubbles Mediated Factor VIII Plasmid Gene Therapy in Hemophilia a Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2039-2039
Author(s):  
Shuxian Song ◽  
James Harrang ◽  
Bryn Smith ◽  
Carol H. Miao
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Factor Viii ◽  
Hemophilia A ◽  
Liver Perfusion ◽  
Transfer Efficiency ◽  
Clotting Factor ◽  
Gene Transfer Efficiency ◽  
Fviii Activity

Abstract Hemophilia A is a genetic bleeding disorder resulted from a deficiency of blood clotting factor VIII. In order to develop the efficient approach to gene therapy for hemophilia A, we previously explored reporter gene transfer mediated by ultrasound (US) combined with microbubbles (MBs). It was demonstrated that US/MB can significantly enhance gene transfer efficiency and serve as an efficient non-viral physical delivery strategy. In this study, we further delivered a therapeutic FVIII plasmid into the livers of hemophilia A (HA) mice. In consideration of FVIII synthesis from multiple tissues/cell lines, we first explored the distribution of gene expression using a pGL4.13 [luc2/SV40] luciferase plasmid driven by a ubiquitous promoter. One day following gene transfer, hepatocytes and endothelia cells were isolated from treated lobes by liver perfusion and centrifuge method. Evaluation of luciferase levels in two cell populations indicated that luciferase predominantly expressed in hepatocytes (5.35´104 RLU/107 cells vs. 1.46´103 RLU/107 cells in endothelia cells). Furthermore, gene transfer of pGFP (driven by a ubiquitous CMV promoter) mediated by US/MB also showed fluorescence distribution mostly in hepatocytes. These results indicate that hepatocyte is the predominant site of gene expression following US/MB mediated gene transfer into the liver. Based on these results, a hepatocyte-specific human FVIII plasmid (pBS-HCRHP-hFVIII/N6A) was used for US/MB mediated gene transfer in HA mice. In the short-term experiment, FVIII activity levels of treated HA mice ranged from 4-40% of normal FVIII activity. To follow FVIII expression for longer term, HA mice were pretreated with IL-2/IL-2 mAb (JES6-1) complexes on day −5, −4, and −3 to prevent immune response. In addition, the mice were infused with normal mouse plasma and human FVIII protein prior to gene transfer to maintain hemostasis. Subsequently, FVIII plasmids and 5 Vol% NUVOX MBs were injected into the mouse liver under simultaneous US exposure (1.1MHz transducer H158A driven by a pulse generator and high-power radio frequency amplifier capable of generating up to 1000W). Blood and liver samples were collected at serial time points after treatment to determine FVIII activity in plasma and liver damage. Following gene transfer, 10-30% of FVIII activity was achieved on day 4 and persisted in the average level of 20% by day 28. In a separate long-term follow-up experiment (n=3), 2 of 3 mice still maintained 10-30% activity after 120 days. Both transaminase levels (alanine aminotransferase and aspartate aminotransferase) and histological examination showed that the procedure of plasmid/MBs portal-vein injection and pulse-train acoustic exposure produced transiently localized liver damages however the damages were repaired and the liver recovered rapidly. Phenotypic correction of HA mice was further examined by tail clip assay. Blood loss of US/MB treated mice was significantly reduced compared with naive HA mice. Furthermore, a novel plasmid encoding a B domain-deleted FVIII variant containing mutations of 10 amino acids in the A1 domain (BDDFVIII-X10, a kind gift from Weidong Xiao) was constructed. Preliminary results from ongoing study showed that the gene transfer efficiency could be further improved with better plasmid and more efficient immune modulation. Together all the results indicate that US/MB mediated gene transfer is highly promising for efficient and safe gene therapy of hemophilia A. Disclosures No relevant conflicts of interest to declare.

Is Hemophilia B Less Severe Than Hemophilia A? Results Of Global Coagulation Assays

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2352-2352 ◽  
Author(s):  
Maria Rosaria Fasulo ◽  
Maria Elisa Mancuso ◽  
Veena Chantarangkul ◽  
Antonino Cannavò ◽  
Marigrazia Clerici ◽  
...  
Keyword(s):  
Hemophilia A ◽  
Phenotypic Variability ◽  
Gene Mutations ◽  
Factor Ix ◽  
Hemophilia B ◽  
Bleeding Tendency ◽  
Severe Hemophilia ◽  
Coagulation Assays ◽  
One Stage ◽  
Research Grant

Abstract Introduction Some observations suggest that severe hemophilia B (HB) may exhibit a milder bleeding tendency than severe hemophilia A (HA), however possible differences in the coagulation profile of severe HB and HA that may account for such phenotypic variability have not been extensively investigated. The aim of this study was to compare the clinical and laboratory phenotype of patients with severe HB (cases) with those with severe HA (controls) in order to ascertain potential determinants for a milder bleeding phenotype. Methods: patients with severe HB and HA (FIX and FVIII <1 IU/dL) of any age without inhibitor history followed up at a single center were asked to undergo blood sampling after a minimum wash-out period of 5 days from the last factor IX (FIX) or factor VIII (FVIII) infusion in order to perform coagulation assays including global testing by thrombin generation assay (TGA) and thromboelastography (TEG). Data on medical history, annual bleeding frequency, factor consumption, treatment regimen, orthopaedic status and FIX/FVIII gene mutations were collected from patients’ files. TGA was performed in platelet-rich plasma (PRP) with the addition of corn trypsin inhibitor (CTI) and TEG in whole citrated blood by means of the 4 channel ROTEM Gamma equipment. FIX and FVIII were measured by one-stage clotting and chromogenic assays. Results are available from the first 33 consecutive patients with severe hemophilia (16 HB and 17 HA) who agreed on and were able to maintain the required wash-out period. Age at enrolment (median: 40.8 and 41.2 years, interquartile range: 36-54 and 37-48, in HB and HA, respectively) and the proportion of patients with target joints (75% in HB and 82% in HA) were similar in the two groups. The proportion of patients on regular prophylaxis was higher in HA (41% vs 19% in HB) however not statistically different. HB patients had an older age at first bleed (median 2.8 vs 1.4 yrs in HA, p=0.05), lower prevalence of null gene mutations (13% vs 59% in HA, p<0.01) and lower rate of orthopaedic surgery (19% vs 71% in HA, p<0.01). Considering only patients treated on demand (10 HA and 13 HB), HB patients had less joint bleeds/year (median 1.4 vs 11 in HA, p=0.05), lower concentrate consumption (median 320 vs 1448 IU/kg/yr in HA, p=0.01) and a lower Hemophilia Joint Health Score (HJHS, median 5 vs 31 in HA, p<0.01). Baseline levels of FIX and FVIII were confirmed <1 IU/dL by both one-stage and chromogenic assays. The thrombin peak detected by TGA in HB was higher than in HA patients (median 30.4 vs 18.4 nM, p=0.05) but ETP values were similar. TEG testing showed that HB patients had a shorter Clotting Time (median 310 vs 598 sec in HA, p<0.01), a shorter Clotting Formation Time (median 93 vs 133 sec in HA, p<0.01) and a wider alfa-Angle (median 72 vs 65 degrees in HA, p<0.01) while Maximum Clot Firmness was similar in HA and HB patients. Conclusions: our results indicate that patients with severe HB may have a milder bleeding phenotype as compared with severe HA patients. Global coagulation assays such as TGA and TEG have the potential to reveal different coagulation profiles and to investigate correlations between clinical and laboratory phenotype in hemophilia. Further studies are warranted in order to explore the biological mechanisms that may enhance coagulation activation in hemophilia irrespective of FIX/FVIII activity in plasma. Disclosures: Fasulo: Pfizer: Unrestricted Research Grant Other. Santagostino:Pfizer: Unrestricted Research Grant Other.

Genetics and Hemostatic Potential in Persons with Mild to Moderate Hemophilia A with a Discrepancy between One-Stage and Chromogenic FVIII Assays

2020 ◽  
Author(s):  
Annelie Strålfors ◽  
Danijela Mikovic ◽  
David Schmidt ◽  
Liselotte Onelöv ◽  
Nida Mahmoud Hourani Soutari ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Genetic Alterations ◽  
Plasma Samples ◽  
Background Factor ◽  
Novel Mutations ◽  
Endogenous Thrombin Potential ◽  
One Stage ◽  
Fviii Activity ◽  
A Domains

Abstract Background Factor VIII (FVIII) activity (FVIII:C) can be measured by different methods including one-stage clotting assays (OSAs) and chromogenic assays (CSAs). Discrepancy between FVIII:C assays is known and associated with genetic variations causing mild and moderate hemophilia A (HA). We aimed to study the discrepancy phenomenon and to identify associated genetic alterations. Further, we investigated if hemostatic global assays could discriminate the group with discrepant FVIII:C from them. Methods The study contained plasma samples from 45 patients with HA (PwHA) from Hemophilia Centers in Stockholm, Sweden, and Belgrade, Serbia. We measured FVIII:C with OSA and CSA, sequenced the F8 gene, and performed two global hemostatic assays; endogenous thrombin potential and overall hemostatic potential. Results Nineteen of 45 PwHA had a more than twofold higher FVIII:C using OSA compared to CSA and were considered discrepant. Thirty-four causal mutations were detected, where of five had not previously been associated with assay discrepancy. These novel mutations were p.Tyr25Cys, p.Phe698Leu, p.Met699Leu, p.Ile1698Thr, and Ala2070Val. We found no difference between discrepant and nondiscrepant cases with either of the global assays. Conclusion There was a discrepancy between FVIII:C assays in almost half of the PwHA, which for some could lead to missed HA diagnoses or misclassification of severity. Genotyping confirmed that mutations associated with FVIII:C discrepancy cluster in the A domains of F8, and five mutations not previously associated with FVIII:C discrepancy was identified. Global hemostatic assays did not contribute to distinguish assay discrepancy in PwHA.

Hemophilia A Mutations within the Factor VIII A2-A3 Subunit Interface Destabilize Factor VIIIa and Cause One-stage/ Two-stage Activity Discrepancy

2002 ◽  
Vol 88 (11) ◽  
pp. 781-787 ◽  
Author(s):  
William Hakeos ◽  
Hongzhi Miao ◽  
Nongnuch Sirachainan ◽  
Geoffrey Kemball-Cook ◽  
Evgueni Saenko ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Homology Model ◽  
Hydrophobic Core ◽  
Two Stage ◽  
Threefold Axis ◽  
Subunit Dissociation ◽  
One Stage ◽  
Fviii Activity ◽  
A Domains

SummaryThrombin-activated factor VIII (FVIIIa) is a heterotrimer with the A2 subunit in a weak ionic interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2 subunit correlates with inactivation of FVIIIa. A homology model (Blood 89:2413, 1997) of the triplicated A domains of factor VIII (FVIII) predicts a pseudo-threefold axis at the tightly packed hydrophobic core with several interdomain interactions. These lie at the interface of A1-A2, A2-A3 and A1-A3. We have previously demonstrated that hemophilia A mutations (R531H, A284E, S289L) within the predicted A1-A2 and A1-A3 interface disrupt potential intersubunit hydrogen bonds and have the molecular phenotype of increased rate of inactivation of FVIIIa due to increased rate of A2 subunit dissociation. Patients with these mutations exhibit a clinical phenotype where the FVIII activity by one-stage(1-st) assay is at least two-fold higher than by two-stage(2-st) assay. We have now also explored mutations within the predicted A2-A3 interface (N694I, R698W and R698L) that also have the phenotype of 1-st/2-st activity discrepancy. These mutations exhibit the same molecular mechanism of increased instability of FVIIIa as those mutations described along the A1-A2 and A1-A3 interfaces. This suggests that the entire tightly packed hydrophobic core within the predicted pseudo-threefold axis contributes to stabilization of FVIIIa.

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