An Increase in Factor VIII Levels with Increasing Age in a Subgroup of Individuals with Mild Hemophilia A

Abstract Abstract 2278 Background: Hemophilia A is an X-linked hereditary disorder resulting in absent or reduced levels of functional factor VIII (FVIII:C). The baseline FVIII:C levels are important in the determination of treatment and management options in individuals with mild hemophilia who by definition exhibit FVIII:C levels between 6–40 U/dL. In individuals without hemophilia, FVIII:C increases with physiological age and this trend has not been well characterized in mild hemophilia. This is important to consider in mild hemophilia because treatment with factor concentrate or DDAVP may paradoxically increase the risk of venous or arterial thrombosis in older patients. Objectives: To describe the changes in baseline FVIII:C levels with time in a cohort of pediatric and adult subjects with mild hemophilia registered in the British Columbia Provincial and Southern Alberta Bleeding Disorder programs. Methods: All medical records for subjects with FVIII:C levels 6–40 U/dL registered with the BC Provincial and Southern Alberta Bleeding Disorder programs were reviewed for eligibility. Male subjects with a minimum of 2 FVIII:C level measurements at least 5 years apart were included in the analysis. Retrospective data was extracted from database/medical records, including age, gender, blood group, FVIII mutation, historical FVIII:C levels, historical DDAVP response (>3 fold and/or >50 U/dL), and co-infection (Hepatitis C, B and HIV) status. Linear mixed effects regression models were used to examine time trend of FVIII:C levels and subjects were stratified into 3 groups based on baseline FVIII:C (FVIII:C<15 U/dL, 15–24 U/dL, and ≥25 U/dL). Results: 198 records were reviewed and 116 subjects excluded from the analysis (chart unavailable, n=28; inadequate FVIII:C data, n=79; female, n=9) leaving 82 subjects who met eligibility criteria. The mean age at first FVIII:C measurement was 24.0 years (SD 19.6; Range 0.0–77.8). The mean follow-up time was 16.5 years (SD 19.6; Range 4.8–44.8). There was no observable trend of FVIII:C with time in the primary analysis of the whole cohort (p=0.667). However, for subjects with baseline FVIII:C<15 U/dL, who were ≥25 years of age (n=31), an increase in baseline FVIII:C levels of 0.11 percent per year (p<0.05) was observed. Subjects with baseline FVIII:C<15 U/dL (n=40) also showed less FVIII:C variability between measurements than subjects with FVIII:C≥15 U/dL (n=42). Subjects with baseline FVIII:C≥25 U/dL (n=18) demonstrated a decreasing trend of −0.15 percent per year (p<0.05), however also demonstrated the greatest variability between FVIII:C measurements. Co-infections were present in 24% (n=20) of subjects and included Hepatitis C (n=18), Hepatitis B (n=1), HIV (n=1). 73 subjects had known DDAVP response; >3-fold but ≤50 U/dL (n=11), ≤3-fold but >50 U/dL (n=15), >3-fold & >50 U/dL (n=23), ≤3-fold & ≤50 U/dL (n=24). Conclusion: This study suggests that there is an increase in FVIII:C with time in the subgroup of individuals with mild hemophilia ≥25 years of age and baseline FVIII:C levels <15 U/dL. Further long-term data particularly in the older cohort of subjects is needed to confirm this preliminary finding and further characterize this trend in subgroups with different baseline FVIII:C. Disclosures: No relevant conflicts of interest to declare.

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Tratamento de Hemofílicos A Grave Atendidos no Hospital de Hematologia da Fundação HEMOPE, com Inibidor de Fator VIII Através da Imunotolerância

Ensaios e Ciência C Biológicas Agrárias e da Saúde ◽

10.17921/1415-6938.2021v25n2p252-258 ◽

2021 ◽

Vol 25 (2) ◽

pp. 252-258

Author(s):

Amanda Oliveira Marinho ◽

Abdênego Rodrigues da Silva ◽

Robson Raion de Vasconcelos Alves ◽

Magaly do Bom Parto Lopes Vieira Lima ◽

Antônio Sérgio Alves de Almeida Junior

Keyword(s):

Factor Viii ◽

Medical Records ◽

Neutralizing Antibodies ◽

Hemophilia A ◽

Treatment Success ◽

Neutralizing Antibody ◽

Severe Hemophilia ◽

Total Treatment ◽

The Mean ◽

Hemofilia A

No Brasil, mais de 80% dos casos de hemofilia são do tipo A. A infusão do concentrado do Fator VIII (FVIII) pode desenvolver anticorpos neutralizantes. O tratamento de escolha para indivíduos que desenvolveram inibidor é a imunotolerância (IT). Este estudo objetivou avaliar a conduta e eficácia do tratamento da IT em pacientes com hemofilia A grave na Fundação de Hematologia e Hemoterapia de Pernambuco (HEMOPE), no período de 2012 a 2017. O estudo foi do tipo descritivo com uma abordagem quantitativa com análise dos prontuários de pacientes com hemofilia A grave. No total de 17 pacientes hemofílicos A grave, 47% ainda continuam na IT, 17,7% tiveram insucesso, 17,7% foram excluídos do tratamento, 11,7% conseguiram sucesso parcial e 5,9% obtiveram sucesso total. A média de idade de detecção do inibidor foi de 4 anos a partir do início do tratamento de infusões, variando de 1 a 14 anos; 7 indivíduos apresentaram o primeiro título de baixa resposta (< 5 UB/mL) e os demais título de alta resposta (> 5 UB/ mL); cerca de 70,58% conseguiram começar a IT com um período de detecção < 5 anos. A falta de uma conduta padrão diante do tratamento resulta em uma eficácia não definida, apesar de todas as limitações do presente estudo já relatadas, um pequeno percentual de pacientes obteve sucesso parcial e total do tratamento, sendo um indicativo de que a IT realizada, atualmente, nos pacientes hemofílicos A grave da Fundação HEMOPE (iniciada há apenas 6 anos) apresenta resultados promissores. Palavras-chave: Fator VIII. Anticorpo Neutralizante. Eficácia. Abstract In Brazil, more than 80% of hemophilia cases are type A. Infusion of Factor VIII concentrate (FVIII) can develop neutralizing antibodies. The treatment of choice for individuals who have developed an inhibitor is immunotolerance (IT). This study aimed to evaluate the conduct and effectiveness of treating IT in patients with severe hemophilia A at Fundação de Hematologia e Hemoterapia de Pernambuco (HEMOPE), from 2012 to 2017. The study was the descriptive type with a quantitative approach with analysis of the patients’ medical records with severe hemophilia A. In the total of 17 hemophiliac A severe patients, 47% are still on IT, 17.7% were unsuccessful, 17.7% were excluded from treatment, 11.7% were partially successful and 5.9% were totally successful. The mean detection age of the inhibitor was 4 years from the beginning of the infusions treatment, ranging from 1 to 14 years; 7 individuals had the first low response titer (<5 UB/mL) and the remaining high response titer (> 5 UB/mL); about 70.58% managed to start IT with a detection period <5 years. The lack of a standard approach to treatment results in an undefined efficacy, although all the limitations of the present study already reported, a small percentage of patients achieved partial and total treatment success, being an indication that the IT currently performed on severe hemophiliac A patients from Fundação HEMOPE (started only 6 years ago) shows promising results. Keywords: Factor VIII. Neutralizing Antibody. Effectiveness

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Differentiation of BM-HSCs Into FVIII Producing Hepatocytes: Approach to Haemophilia Treatment

Blood ◽

10.1182/blood.v120.21.4388.4388 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4388-4388

Author(s):

Amal M. El-beshlawy ◽

Hala Gabr ◽

Rania Zayed ◽

Laila Hegaz ◽

Rania Fawzy ◽

...

Keyword(s):

Factor Viii ◽

Hemophilia A ◽

Conflicts Of Interest ◽

Alpha Fetoprotein ◽

Control Group ◽

P Value ◽

Rt Pcr ◽

Significant Difference ◽

The Mean

Abstract Abstract 4388 Background: Hemophilia is caused by a single-gene defect in which a small increase in gene products could transform a severe form of hemophilia into a mild one. Hemophilia treatments are readily available in developed countries, however In Egypt, Most hemophilia patients are treated with plasma or cryoprecipitate, where the treatment is associated with a high risk of blood-borne diseases. Liver transplantation in human and canine hemophilia A results in an increase in factor VIII levels to normal. Studies showed that BMCs not only differentiated into hepatic and liver cells, but they did express the intact gene of the FVIII A3 domain. Objective: In this work we studied the ability of bone marrow derived stem cells from hemophilia patients' relatives (carrier or normal) to be differentiated into hepatocytes expressing FVIII m-RNA in vitro as a step towards transplantation in haemophilia patients. It was necessary to prove that the applied culture conditions were successful not only to obtain hepaotocyte morphology but also hepatocyte ability to produce FVIII. Methods: The study was conducted on family relatives of five hemophilia A patients attending the hematology clinic, Cairo University hospitals. From each family, one hemophilia A carrier and one healthy person were subjected to the study. Informed consent was obtained from the participants. BM-HSCs were cultured in liquid culture containing HGF for 6 days. Differentiation into hepatocytes was evaluated by alpha-fetoprotein (AFP) expression using immunohistochemistry, albumin synthesis in culture supernatant using microalbumin assay kit, factor VIII activity by one stage clotting assay and expression of FVIII mRNA by RT-PCR. Results: Cell morphology changed after 6 days culture; round or polygonal-shaped cells with moderate cytoplasm and a medium-sized nucleus were observed. Morphologic confirmation of hepatocyte differentiation was done by immunocytochemistry; human alpha fetoprotein positive cells were detected in the culture. The positive cells appeared round or pear shaped, most of them contained one nucleus. However, few cells were binucleated with brown stained cytoplasm and bluish nuclei (Figure 1 A, B). By image analysis, the mean number of alpha fetoprotein positive cells estimated in10 random high power fields was 11 ± 1.6, 11 ±1.8 cells/HPF in the carriers and controls respectively. Immunophenotyping after culture; the percentage of CD 34+ve cells for the carrier group ranged from 0.5 to 2.5 with the mean of 1.2 ± 0.8 and from 0.7 to 2.1 with the mean of 1.5 ± 0.7 for the control group. There was no statistically significant difference between the two groups (p > 0.05) and the percentage of CD 90+ve cells for the carrier group ranged from 11.1 to 14.2 with the mean of 12.7 ± 1.2 and from 12.6 to 13.8 with the mean of 13.3 ± 0.6 for the control group. There was no statistically significant difference between the two groups (p > 0.05). On comparison between immunophenotyping before and after culture in both groups, statistical analysis showed highly significant decrease in CD34 positivity (p value 0.002 and 0.001) in the carriers and controls respectively associated with highly significant increase in the percentage of CD90 positive cells (p value 0.000) in the two groups. Albumin secretion was detected in the culture supernate at the 6th day culture, the mean albumin level was 0.52 mg/L ± 0.32 and 0.6 mg/L ± 0.4 in the carriers and controls respectively. F VIII activity was estimated; with the mean of 0.14%±0.021% and 0.5%±0.4% in the carriers and controls respectively. Transcription of FVIII m-RNA was detected by qualitative RT-PCR in 2 carriers and all controls (Figure 2). Conclusion: BM derived hepatocytes showed positive AFP expression. Functional tests performed showed their ability to produce albumin and perform FVIII activity. Also FVIII mRNA expression was detected. Induction of HSCs differentiation by in vitro manipulation may become a valuable tool to provide a cell source for liver transplant procedures and treatment of haemophilia patients. Disclosures: No relevant conflicts of interest to declare.

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A Laser Scalpel for Operations on Hemophiliacs

10.1055/s-0039-1682523 ◽

1977 ◽

Cited By ~ 1

Author(s):

H. Horoszowski ◽

U. Seligsohn ◽

M. Heim ◽

I. Farin

Keyword(s):

Factor Viii ◽

Replacement Therapy ◽

Hemophilia A ◽

Operation Time ◽

Early Ambulation ◽

Fourth Patient ◽

Laser Scalpel ◽

The Mean ◽

Low Levels ◽

Hip Contracture

The CO2 laser scalpel has an excellent hemostatic effect producing a minimal parainci-sional necrotic zone. Four severe hemophilia A patients were operated with the laser scalpel. Three underwent knee synovectomy 2 of whom without placing a tourniquet. The fourth patient underwent an operation for hip contracture. Factor VIII levels at the beginning of the operations were gradually reduced from the first operation to the fourth as follows: 100%, 80%, 14.5% and 1.4% respectively. No significant bleeding was observed during any operation. The 3 patients who underwent synovectomy were ambulated as early as 3 days after operation. They were discharged after 20 - 26 days, which is considerably shorter than the mean stay of 39 days in the hospital in 8 patients who had been synovectomized previously by the conventional method. An attempt to stop replacement therapy in the fourth patient on the 6th postoperative day resulted in bleeding. Thus, the advantages of the laser scalpel are: Excellent local hemostasis, tourniquet unecessary, low levels of factor VIII sufficient at operation, early ambulation and discharge. It is not as yet clear whether the amount of postoperative replacement therapy can be reduced. The disadvantages are: Longer operation time (by about a third); smoke produced at theatre; the price of the instrument.

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Increased Prevalence of Inhibitors in Mexican-Hispanic Patients with Severe Hemophilia A Enrolled in the Universal Data Collection Project.

Blood ◽

10.1182/blood.v114.22.3488.3488 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3488-3488 ◽

Cited By ~ 2

Author(s):

Shannon Carpenter ◽

J. Michael Soucie ◽

Sophia Sterner ◽

Rodney J Presley

Keyword(s):

African Americans ◽

Data Collection ◽

Factor Viii ◽

Hemophilia A ◽

Conflicts Of Interest ◽

Treatment Center ◽

Hispanic Population ◽

Severe Hemophilia ◽

Inhibitor Development ◽

Insurance Type

Abstract Abstract 3488 Poster Board III-425 Neutralizing inhibitor formation occurs in up to 20-30% of patients with severe factor VIII deficiency, leading to significantly increased morbidity in affected individuals. It has been well-established that patients of African descent have a higher prevalence of inhibitor development. [Oldenburg, J et al. Semin Hematol, 2004] The Hispanic population also has been assumed to have an increase in inhibitor development when compared with Caucasians. The study presented here is the first to definitively demonstrate an increased prevalence of inhibitors in the Hispanic population. We compared inhibitor prevalence among various racial and ethnic groups in a cross-sectional analysis of 6198 males with severe hemophilia A that participated in the Universal Data Collection project sponsored by the Centers for Disease Control and Prevention. We used logistic regression analysis to control for potential confounding variables including age, insurance type (as a proxy for access to care and socio-economic status), age at first bleed, age at diagnosis and use of prophylaxis. The included table shows those variables that were determined to be independently predictive of inhibitors. We assigned Mexican derivation to participants who labeled themselves as Hispanic and who were born either in Mexico, in states bordering Mexico or in states with large Mexican populations as established by Census data. The prevalence of high titer inhibitors in the Mexican-Hispanic population was 26.3% compared to 16.4% for Caucasian patients [OR 1.5, 95% CI 1.1, 1.9], and 26.8% for African-Americans. The underlying cause of increased inhibitor prevalence in these populations is still unknown, though a recent study in African-Americans demonstrated wild-type factors unique from commercially available product. [Viel KR, et al. Inhibitor of Factor VIII in Black Patients with Hemophilia. N Engl J Med, 2009] Further investigation of this phenomenon in the Mexican-Hispanic population, as well as the potential impact of differing immune responses, is warranted. Multivariate analysis of ethnicity and other variables found to be independently predictive of a prevalent inhibitor Characteristic Odds Ratio 95% CI Race/Ethnicity African-American 1.5 1.2 - 1.9 Mexican Hispanic 1.5 1.1 - 1.9 Hispanic 1.2 0.9 - 1.7 Other 1.2 0.9 - 1.6 White Ref Age* (years) <2 4.2 3.0 - 5.9 2-5 6.4 5.1 - 8.0 6-10 2.8 2.2 - 3.5 11-18 1.7 1.4 – 2.1 >18 Ref Insurance type Medicare 1.8 1.4 - 2.3 Medicaid 1.3 1.1 - 1.5 State program 1.1 0.6 - 1.9 TRICARE 1.0 0.4 - 2.1 Other 0.8 0.6 - 1.2 Uninsured 1.6 1.0 - 2.4 Commercial Ref Prophylaxis Yes 0.6 0.5 - 0.7 No Ref * Age with inhibitor or last UDC visit if no inhibitor The authors wish to acknowledge the contributions of the Hemophilia Treatment Center Network Investigators in the completion of this study. Disclosures: No relevant conflicts of interest to declare.

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Inhibitor Development In Patients with Mild and Moderate Hemophilia A: Results From a Single Centre.

Blood ◽

10.1182/blood.v116.21.1407.1407 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1407-1407 ◽

Cited By ~ 1

Author(s):

Yohann Repesse ◽

Philippe Gautier ◽

Annie Borel-Derlon

Keyword(s):

Factor Viii ◽

Hemophilia A ◽

Conflicts Of Interest ◽

Direct Sequencing ◽

Immunosuppressive Treatment ◽

Gene Promoter ◽

Missense Mutations ◽

Bleeding Events ◽

Inhibitor Development ◽

Treatment Centre

Abstract Abstract 1407 The development of factor VIII (FVIII) inhibitors is usually considered uncommon among patients with mild and moderate hemophilia A (HA) and less frequent than in patients with severe HA. We report here the prevalence of FVIII inhibitors and their caracteristics in 167 patients with mild and moderate HA followed in Caen Hemophilia Treatment Centre (Table). FVIII molecular defects were identified by direct sequencing in 167 patients including 30 and 137 with mild and moderate HA, respectively. Following FVIII concentrates infusions, FVIII inhibitors occured in 7.8% of patients (13/167). Fifteen percent (2/13) were low-responding inhibitors. The risk of inhibitor development appeared to be associated with high-risk FVIII genotypes clustered in the A2 and C2 domains, especially p.Arg2150His (50%) and p.Arg593Cys mutations. Interestingly, we described inhibitor development associated with novel missense-mutations (p.Tyr1786Ser, p.Asp115Tyr and -219C>T substitutions in FVIII gene promoter). In addition, high regimen infusion of FVIII concentrates appeared as risk factor for FVIII inhibitors development. Indeed, 60% (8/13) developped FVIII inhibitors following massive infusion of FVIII concentrates associated with FVIII:C levels above 1.2 UI/dL. Inhibitors in mild HA usually cross-react with endogenous factor VIII reducing the circulating basal FVIII:C level and are associated with more bleeding events. Similarly, we observed the evolution of bleeding patterns in our cohort to severe phenotypes. Bleedings were treated with FVIII concentrates and bypassing therapies (activated FVII and activated-prothrombin complex). About 25% (3/13) of these inhibitors disappeared spontaneously. Induction of Immune Tolerance (ITI) protocoles with high doses of FVIII were initiated for 7 high-responding patients with a success rate of 85 % (6/7). However, inhibitors persisted long-term and remained troublesome in 1 of these patients despite of ITI protocole. For two patients, immunosuppressive treatment with corticosteroids was started. Inhibitors disappeared and the levels of FVIII:C became detectable within 6 months. Development of FVIII inhibitors, their disappearance and the efficacy of ITI regimen seem to be different from our experience between patients with mild or moderate HA and severe HA. Disclosures: No relevant conflicts of interest to declare.

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Pharmacokinetics of Continuous Infusion Therapy of Factor VIII Concentrates in Hemophilia A Patients with Inhibitors,

Blood ◽

10.1182/blood.v118.21.3320.3320 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3320-3320

Author(s):

Katsumi Nishiya ◽

Ichiro Tanaka ◽

Keiji Nogami ◽

Kenichi Ogiwara ◽

Koji Yada ◽

...

Keyword(s):

Factor Viii ◽

Continuous Infusion ◽

Infusion Rate ◽

Hemophilia A ◽

Conflicts Of Interest ◽

High Titer ◽

Infusion Therapy ◽

Target Level ◽

Ci Therapy ◽

The Difference

Abstract Abstract 3320 Continuous infusion (CI) of factor VIII (FVIII) concentrates is aimed at maintaining a steady hemostatic level of FVIII activity (FVIII:C) in hemophilia A patients during various surgeries. However, there are few reports that mentioned the difference of pharmacokinetics of CI therapy in hemophilia A patients with inhibitors. We investigated the relationship between the FVIII:C levels and the rate of CI, and the difference of clearance (CL) and volume of distribution (Vd) of FVIII in hemophilia A patients with/without inhibitors. 8 severe hemophilia A patients without inhibitors (arthroscopic synovectomy; 4 cases, total knee arthroplasty; 2 cases, total nephrectomy; 1 case, partial hepatectomy; 1 case), 3 patients with low-titer (2.0–2.9 BU) inhibitors and 3 patients with high-titer (6.0–9.0 BU) inhibitors (insertion or removal of a central venous access device), were enrolled in this study between 2005 and 2010. According to the Japanese guideline for hemophilia treatment, we should do CI therapy to keep target level 80–100% for 5–10 days for joint surgery and other major surgeries. An initial bolus infusion (BI) of FVIII concentrates was administered to achieve this level prior to CI. In addition, we have to neutralize the inhibitors by FVIII concentrates in case of the patients with inhibitors. FVIII:C was measured using one-stage clotting assays and FVIII inhibitor assays were performed using the Bethesda method. All therapy was conducted after obtaining fully informed consent. The median FVIII:C level after BI was 120.2% (range: 90–150) in the patients without inhibitors, 72.0% (range: 68–160) with low-titer inhibitors, and 20.0% (range: 9.4–30) with high-titer inhibitors, respectively. The target level of FVIII:C was adjusted to approximately 100%. The initial infusion rate was 3.7 U/kg/hr (range: 2.2–5.0), 8.3 U/kg/hr (range: 8.0–8.5) and 18.5 U/kg/hr (range: 15–22), respectively. After adjustment for the target level, the final infusion rate decreased to 2.6 U/kg/hr (range: 1.5–5.4), 4.7 U/kg/hr (range: 3.0–5.6) and 8.0 U/kg/hr (range: 7.0–9.0), respectively. CL was 2.3 ml/hr/kg (range: 1.5–3.9), 4.0 ml/hr/kg (range: 2.3–5.1) and 9.3 ml/hr/kg (range: 9.0–9.6), respectively. Vd was 0.04 L/kg (range: 0.031–0.047), 0.18 L/kg (range: 0.12–0.29) and 1.54 L/kg (range: 0.95–2.43), respectively. No unexpected safety concerns associated with CI, such as thrombosis, was identified during the study. On CI therapy, we could keep target level of the patients without inhibitors and with low-titer inhibitors easier than those with high-titer inhibitors. One of the reason is that CL and Vd in patients with inhibitors are higher than those in patients without inhibitors. CI with appropriate monitoring of FVIII:C level and concerning CL and Vd forms a safe method for perioperative care in hemophilia A patients with inhibitors. Disclosures: No relevant conflicts of interest to declare.

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Source and Purity of Factor VIII Products As Risk Factors for Inhibitor Development In Previously Untreated Patients with Severe Hemophilia A

Blood ◽

10.1182/blood.v118.21.25.25 ◽

2011 ◽

Vol 118 (21) ◽

pp. 25-25

Author(s):

Maria Elisa Mancuso ◽

Pier Mannuccio Mannucci ◽

Angiola Rocino ◽

Isabella Garagiola ◽

Annarita Tagliaferri ◽

...

Keyword(s):

Risk Factors ◽

Factor Viii ◽

High Purity ◽

Hemophilia A ◽

Conflicts Of Interest ◽

Specific Activity ◽

Sensitivity Analyses ◽

Severe Hemophilia ◽

Inhibitor Development ◽

Treatment Data

Abstract Abstract 25 Background: Inhibitor development is influenced by several genetic and environmental factors and the type of factor VIII (FVIII) products may play a role. Methods: We designed a cohort study whose novelty resides in the classification of products not only according to the plasmatic (pdFVIII) or recombinant (rFVIII) source of FVIII but also to the degree of purity expressed as FVIII specific activity per mg of protein. The role of FVIII product as risk factor for inhibitor development was evaluated in a multivariate model adjusting for potential confounders (i.e. age at first FVIII exposure, intensive treatment and prophylaxis). Cumulative incidences of all and high-responding inhibitors were calculated for the whole cohort of 721 patients with severe and moderate hemophilia A followed-up in 3 Italian Hemophilia Centers. Detailed treatment data from the first FVIII infusion up to inhibitor development or 150 exposure days were available for 377 patients and in this group of patients risk factors for inhibitor development including the type of FVIII product and its degree of purity (i.e. low/intermediate-, high-purity pdFVIII and rFVIII) were analysed. Results: The overall cumulative incidence of inhibitors was 22% (n=160; 130 high-responders, 18%) and it was lower in patients first treated with pdFVIII (107/586, 18%) than in those treated with rFVIII (53/135, 39%). Similar results were obtained by evaluating only high-responding inhibitors and patients with severe hemophilia. The adjusted hazard ratio of inhibitor development was 4.9 with rFVIII and 2.0 with high-purity pdFVIII (95%CI: 2.9–8.3 and 1.1–4.0), taking as reference low/intermediate-purity pdFVIII. There was no difference in the frequency of inhibitor testing between treatment groups. Sensitivity analyses - in patients who never switched product type, previously untreated patients, those treated on-demand and those with high-risk F8 mutations - confirmed an increased inhibitor risk in patients first treated with rFVIII or high-purity pdFVIII than in those treated with low/intermediate-purity pdFVIII. In fact, in all the aforementioned subgroups by multivariate analysis the risk of inhibitor development was invariably 3- to 6-fold higher in patients first treated with rFVIII than in those first treated with pdFVIII, and similar results were obtained for both all inhibitors and high-responding inhibitors. Conclusions: This study shows that the degree of purity of FVIII products influences inhibitor development independently from other risk factors, and emphasizes that differences exist also within pdFVIII products. Disclosures: No relevant conflicts of interest to declare.

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Structural Studies Of The Factor VIII C2 Domain In a Ternary Complex With Two Inhibitory Antibodies Reveals Classical and Non-Classical Epitopes

Blood ◽

10.1182/blood.v122.21.2358.2358 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2358-2358

Author(s):

Justin D Walter ◽

Rachel A Werther ◽

Caileen M Brison ◽

John F. Healey ◽

Shannon L. Meeks ◽

...

Keyword(s):

Factor Viii ◽

Ternary Complex ◽

Hemophilia A ◽

Conflicts Of Interest ◽

Structural Basis ◽

C2 Domain ◽

Proteolytic Activation ◽

X Ray ◽

Binding Interface ◽

Inhibitory Antibodies

Abstract The factor VIII C2 domain is a highly immunogenic domain, whereby inhibitory antibodies develop following factor VIII replacement therapy for congenital hemophilia A patients. Inhibitory antibodies also arise spontaneously in cases of acquired hemophilia A. The structural basis for molecular recognition by two classes of anti-C2 inhibitory antibodies that bind to factor VIII simultaneously has been investigated by small angle X-ray scattering and X-ray crystallography. The C2 domain/3E6 FAB/G99 FAB stable ternary complex, both in solution and in its crystalline state, illustrates that each antibody epitope resides on opposing faces of the factor VIII C2 domain. The 3E6 epitope is a classical antibody that forms direct contacts to the C2 domain at two loops consisting of Glu2181-Ala2188 and Thr2202-Arg2215, which inhibits the binding of the C2 domain to von Willebrand Factor and phospholipid surfaces. The G99 is a non-classical antibody that prevents proteolytic activation of factor VIII, and its epitope centers on Lys2227 and also makes direct contacts with loops Gln2222-Trp2229, Leu2261-Ser2263, His2269-Val2282 and Arg2307-Gln2311. Each binding interface is highly electrostatic, with positive charges present on both C2 epitopes and complementary negative charges on each antibody. A new model of phospholipid membrane association is also presented, where the 3E6 epitope faces the negatively charged membrane surface and Arg2320 is poised at the center of the binding interface. Furthermore, a 1.7 Å X-ray crystal structure of the porcine factor VIII C2 domain has also been determined, which supports the presented model for phospholipid binding. These results illustrate the complex nature of the polyclonal immune response against the factor VIII C2 domain, and further define the epitopes for both classical and non-classical inhibitory antibodies. Disclosures: No relevant conflicts of interest to declare.

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Acquired Factor VIII Inhibitor: A Single Institution Experience With 62 Patients

Blood ◽

10.1182/blood.v122.21.3610.3610 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3610-3610

Author(s):

Ewa M. Wysokinska ◽

Ramila Mehta ◽

Diane Grill ◽

Rajiv K. Pruthi

Keyword(s):

Factor Viii ◽

Hemophilia A ◽

Conflicts Of Interest ◽

Post Partum ◽

Rare Condition ◽

Factor Viii Inhibitor ◽

Remission Rates ◽

Autoimmune Conditions ◽

Viii Inhibitor ◽

Acquired Factor Viii Inhibitor

Abstract Background Acquired Factor VIII inhibitor or autoimmune hemophilia A (AHA), has an estimated incidence of up to 1.5 cases per million/year and may result in severe hemorrhagic complications and death. Approximately 50% of cases have an underlying condition such as malignancies, autoimmune disorders and post-partum state. AHA should be suspected in any patient presenting with unexplained bleeding and an inhibited aPTT. Management consists of maintaining hemostasis and elimination of the inhibitor, however management is not standardized. We present 62 cases of AHA managed at Mayo Clinic Rochester, over the course of 36 years. We also analyzed whether aPTT at presentation correlated with the strength of inhibition measured by Bethesda Titer. Methods After IRB approval, medical records of patients with AHA were reviewed and all clinical data collected. Cumulative incidence of death was estimated by Kaplan-Meier analysis. Spearman correlation was used to calculate relation of APTT to Bethesda titer. Results Between 1976 and 2012, we identified 62 patients (male: 35), with a median age at diagnosis of AHA 69 years (mean 64, range 20-86). Clinical presentation consisted of extensive ecchymoses (n=40, 64%) in majority of cases. 29/62 (47%) patients had at least 1 identifiable predisposing condition with 12/62 (19%) patients with an underlying malignancy and 16/62 (26%) with underlying autoimmune conditions. Median Bethesda titer was 29 (range 1 to 1178). Bethesda titer was not related to the number or duration of hospitalizations. Most (69%) patients had at least one hospitalization and 12 (19%) had more than one hospitalization for bleeding complication. Inpatient therapy for bleeding consisted most commonly of FEIBA in 21 pts (34%) and rFVIIa in 6 pts (10%). Prednisone was the most common immunosuppressant used in 54 (87%) patients while Rituximab was used in 11 (18%) patients. Of 32 patients with available follow up labs most (69%) achieved remission. There was no difference in remission rates between patients treated or not treated with Rituximab (p=0.1735). Conclusion Acquired Hemophilia A is a rare condition with very heterogenous presentation. It affects mostly older male patients who present with ecchymoses and elevated APTT. The degree of APTT prolongation at the time of diagnosis does not correlate with the strength of the Bethesda titer and should not guide choice of therapy in a patient presenting with an acute bleed. Rituximab use in the 11 patients treated at Mayo did not seem to influence remission rates or survival. Disclosures: No relevant conflicts of interest to declare.

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Clotting Factor VIII Overexpression Shows Signs of ER Stress but Does Not Cause Toxicity upon Gene Transfer

Blood ◽

10.1182/blood.v126.23.3238.3238 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3238-3238

Author(s):

Irene Zolotukhin ◽

Brett Palaschak ◽

David M Markusic ◽

Roland Herzog

Keyword(s):

Stress Response ◽

Gene Transfer ◽

Factor Viii ◽

Er Stress ◽

Hemophilia A ◽

Conflicts Of Interest ◽

Clotting Factor ◽

Potent Inducer ◽

Cost Effective Treatment ◽

Human Factor Ix

Abstract Hemophilia A is the X-linked bleeding disorder resulting from the loss of functional clotting factor VIII (FVIII). Hemophilia A patients with severe disease (<1% residual FVIII activity) experience spontaneous bleeds into the joints and closed spaces with severe morbidity. Restoration of hemostasis is managed by repeated infusions (2-3 times per week) of plasma derived or recombinant FVIII protein. While a standard treatment is available for patients, life-long infusions of FVIII protein is very expensive, has a negative impact on the patient's quality of life, and FVIII protein products are not available worldwide. Hence, there is a need to develop a more robust and cost effective treatment for hemophilia A patients. Liver-directed gene therapy using adeno-associated virus (AAV) represents a promising approach to treat hemophilia A. However, previous studies have shown that overexpression of human FVIII protein in the context of hydrodynamic delivery of plasmid vectors induces ER stress mediated through the unfolded protein response (UPR). Because human FVIII protein is inefficiently secreted into circulation, high AAV vector doses will be required to obtain therapeutic expression levels. Therefore, we sought to determine if AAV-FVIII gene delivery also triggers cellular UPR in hepatocytes in vivo. To this end, we selected to use a codon-optimized FVIII cDNA, which has been shown by our lab to significantly increase FVIII protein expression, and a high vector dose of AAV8-ApoE-hAAT-cohF8, containing a hepatocyte-specific enhancer/promoter combination. We evaluated this vector at doses of 1 x 1011 vg (4x1012 vg/kg) and 1 x 1012 vg (4x1013 vg/kg) in hemophilia A mice on a 129/BL6 background. Intravenous administration of the highest vector dose completely restored hemostasis, which was sustained and achieved super-physiological levels in some animals. Importantly, none of these mice developed inhibitors against FVIII. Next, we administered the vector at the same 2 doses to C67BL/6 mice, which show higher hepatic transduction efficiency than other strains. Experimental controls consisted of mice, with no vector or 1x1012 vg of AAV8-ApoE-hAAT-F9, expressing human factor IX (FIX) protein. Injection of tunicamycin, a potent inducer of the ER stress response, served as a positive control for all assays. Vector-treated mice were studied 2 and 4 weeks after gene transfer (n=3-4 per group). First, we evaluated the status of key molecular chaperones, known to be the mediators of the UPR: Bip, p-PERK, and p-eIF2a. Western blotting performed on the liver lysates indicated modest up-regulation of all three markers compared to normal control, but that effect was neither dose nor gene dependent. In addition, we tested the splicing of Xbp1 mRNA by PCR assay and observed low level of the 26 bp spliced fragment, indicative of the UPR, at the high vector dose. Immunohistochemistry on liver sections from each of our experimental groups including H&E staining, Tunnel staining for apoptotic cells, and reactive oxygen species staining. None of the stains yielded evidence for liver damage even in the 1x1012 AAV8-cohF8 treated mice compared to untreated controls. There was also no elevation of liver enzyme levels in plasma samples. Analysis of plasma from vector injected mice showed systemic levels of human FVIII and FIX proteins at ~30 ng/ml and ~6300 ng/ml, respectively (these ELISA-based measurements likely underestimate FVIII levels due to interference by von Willebrand factor). Overall our results suggest that over-expression of coagulation factors in hepatocytes from AAV vectors causes a mild cell stress response that is not strong enough to cause liver toxicity, is not specific for FVIII, and does impact expression or immunogenicity. Disclosures No relevant conflicts of interest to declare.

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