Factor VIII Activity Levels Research Articles

Increased volume of distribution for recombinant activated factor VII and longer plasma-derived factor VII half-life may explain their long lasting prophylactic effect

Prophylaxis with plasma-derived or recombinant activated factor VII is beneficial in severe factor VII deficiency. To understand why prophylactic treatment with both products is efficacious, we conducted a pharmacokinetic study. Ten factor VII deficient patients were treated with either recombinant activated (20 μg/kg) or plasma-derived (25 IU/kg) factor VII in a cross-over design. Pharmacokinetic parameters were analyzed through activated factor VII activity, factor VII clotting activity, and factor VII antigen levels on depicted time points. Factor VII activity half-lifes, determined by non-compartmental and one-compartmental analysis (results in brackets), were shorter for recombinant activated (1.4h; 0.7h) than for plasma-derived factor VII (6.8h; 3.2h); both recombinant activated (5.1h; 2.1h and plasma-derived factor VII (5.8h; 3.2h) resulted in longer half-lives of factor VII antigen. Activated factor VII half-lives (based on activated factor VII activity levels) were significantly higher compared to factor VII clotting activity (1.6h; 0.9h). Volumes of distribution were significantly higher for activated factor VII (236 ml/kg; 175 ml/kg, measured by activated factor VII) as compared to plasma-derived factor VII (206 ml/kg; 64 ml/kg, measured by factor FVII activity), suggesting a plasma- and extracellular fluid distribution for recombinant activated factor VII. Recombinant activated factor VII showed significantly shorter half-lifes than plasma-derived factor VII. Volumes of distribution were significantly higher for treatment with recombinant activated factor VII. The longer half-life for plasma-derived factor VII, compared to recombinant activated factor VII, and the increased volume of distribution for recombinant activated factor VII, compared to plasma-derived factor VII may further elucidate the beneficial effect of prophylactic treatment of both products.

Double heterozygous mutations Gln100Leu and His348Gln of the F7 gene in a patient with factor VII deficiency

A 25-year-old Chinese woman who had a history of easy bruising was admitted to hospital due to uncontrolled epistaxis. She showed factor VII activity level of 2% and factor VII antigen level of 4% of the normal value. We detected a novel missense mutation g.8355 A>T (p.Gln100Leu) in the second epidermal growth factor-like (EGF) domain and a g.11482 T>G (p.His348Gln) in the catalytic domain. Although the Gln100 residue is close to the junction of EGF-2 domain with the serine protease domain, we infer that the substitution of polar negatively charged Gln residue at the position 100 with introduction of nonpolar Leu residue may be likely to perturb proper folding, resulting in decreasing factor VII activity.

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

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

Phenotypic correction and stable expression of factor VIII in hemophilia A mice by embryonic stem cell therapy

Hereditary deficiency of factor VIII (FVIII) leads to hemophilia A, a severe X-linked bleeding disorder. Current therapies include fixed-dose FVIII prophylaxis, factor replacement therapy, and most recently, gene therapy. Prophylaxis and FVIII replacement therapies are limited by incomplete efficacy, high cost, restricted availability, and development of neutralizing antibodies in chronically treated individuals. Limited success has been obtained in preclinical trials using gene therapy for the treatment of hemophilia. Therefore, new options for therapy for hemophilia A are needed. We evaluated the potential of embryonic stem cells for correcting hemophilia A in mice. FVIII-deficient mouse blastocysts were collected and injected with mouse embryonic stem cells stably expressing green-fluorescent protein (GFP) and transferred to pseudopregnant recipient mice. Expression of FVIII was measured in the liver and plasma of the 5 chimeric mice that were produced. Three of these mice were GFP-positive at the age of 6 months. The plasma FVIII activity levels were equal to those of wild-type mice. These data demonstrate that embryonic stem cell transplantation at an early embryonic stage has potential as therapy for this progressively debilitating, life-threatening bleeding disorder.

Extensive Coagulation Profile in Children with Sickle Cell Disease and Its Role in Cerebral Micro-Vasculopathy

AbstractAbstract 3247Activation of the coagulation system is present in adults with Sickle Cell Disease (SCD) who display higher levels of thrombin-antithrombin complex (TAT), prothrombin fragment F1+2, D-dimer, tissue factor and platelet activity. Whether the activation of the coagulation system is a bystander phenomenon or a main determinant of clinical complications is under investigation. Although thrombotic complications in SCD arise since infancy, the coagulation system in children has been poorly explored. We performed a comprehensive and systematic evaluation of the coagulation system in children with SCD and investigated its possible role in the development of the main clinical manifestations of SCD in childhood.The following markers of hemostatic and endothelial function were evaluated during steady state: factor VIII activity (FVIII), von Willebrand factor antigen (vWF:Ag) and collagen binding activity (vWF:CBA), PAI-1 antigen (PAI-1:Ag), t-PA antigen (t-PA:Ag), D-dimer, P-selectin, Nitric Oxide (NO), F1+2, TAT, ADAMTS-13 antigen (ADAMTS-13:Ag) and activity (ADAMTS-13:act). Markers of hemolysis (hemoglobin, reticulocytes, aptoglobin, bilirubin, LDH), inflammation (white blood cells, neutrophils, C reactive protein) were measured. Children were not in chronic transfusion.Magnetic Resonance (MRI), Angio Magnetic Resonance (MRA), Transcranial Doppler (TCD), Tricuspid Regurgitant Velocity (TRV) data and frequency of clinical complications were correlated with coagulation parameters. Continuous variables were compared using two-sided Student's t-test and Mann-Whitney non-parametric test, categorical variables using Chi-square and Fisher's exact test. Correlations between variables were evaluated by the Pearson or Spearman coefficient.Thirty-five HbS/HbS- 3 HbS/betathalassemia° (20 M and 18 F, mean age 6.49 years), and 6 HbS/HbC patients (3 M and 3 F, mean age 11.2 years) were evaluated. 60% of HbS/HbS and HbS/betathalassemia° children presented higher level of FVIII, D-dimer, F1+2 and lower level of NO, revealing coagulation activation since early age in HbS/HbS and HbS/betathalassemia° but not in HbS/HbC (Table 1). Coagulation variables correlated positively with markers of inflammation, hemolysis, endothelial activation demonstrating a broad involvement of the coagulation system in these processes, while correlated negatively with HbF (Table 2). No correlation was seen between coagulation variables and cerebral large vessel vasculopathy investigated by TCD and MRA nor TRV (p >0.05). Patients with Silent Infarcts on MRI (n.9) showed significant decrease in t-PA (4.53 vs 7.67, p 0.03) and ADAMTS-13 Ag (1.15 vs 1.50, p 0.05) -suggesting the presence of endothelial dysfunction- and a tendency toward higher D-dimer (2879.01 vs 1569.51), F1+2 (319 vs 231), TAT (17.17 vs 10.46) -suggesting a trend towards enhanced clotting activation compared to patients without Silent infarcts (n.21). Increase in D-dimer was associated with a Relative risk of 6 (p <0.05) to develop Silent Infarcts. No correlation was found between coagulation activation and Acute Chest Syndrome or Vaso-occlusive crisis.Table 1Coagulation profile in HbS/HbS-HbS/Betathalassemia° and HbS/HbC patientsHbS/HbS+HbS/Betathalassemia°HbS/HbCpVariableMeanFVIII:C %184.96119.220.003VWF:Ag %150.05100.630.013VWF:CBA %135.38107.360.124PAI:Ag ng/ml8.736.850.833t-PA ng/ml6.914.520.062D-dimer ng/ml1906.39366.480.000NO μmol/L7.188.70.336P-selectin ng/ml47.7629.700.010F1+2 pmol/l254.26118.500.037TAT ng/ml12.345.140.359ADAMTS-13:Ag μg/ml1.371.460.653ADAMTS-13:act %79.4278.940.801ADAMTS-13:act/VWF:Ag0.520.780.003Table 2Correlations between hematologic and coagulation parameters in HbS/HbS- HbS/Betathalassemia° (first value is Pearson or Spearman coefficient, second is p-value)White Blood CellsNeutrophilsPlateletsFetal HemoglobinReticulocytesLDHFVIII:C−0.3740.021VWF:Ag−0.3220.049VWF:CBA0.3680.023PAI:Ag0.4490.3690.3330.3520.0050.0250.0440.033t-PA0.3440.034D-dimer0.5590.438−0.3930.3890.4210.0010.0110.0210.0230.013P-selectin0.4610.4000.520−0.4130.3810.0040.0160.0010.0110.020F1+20.4000.5120.3370.0160.0010.044TAT0.3970.3810.3760.0170.0220.024ADAMTS-13 Ag0.4710.004 Disclosures:No relevant conflicts of interest to declare.

Recombinant B-Domain Deleted Porcine Factor VIII (OBI-1) Safety and Efficacy in the Treatment of Acquired Hemophilia A: Interim Results.

AbstractAbstract 2224 IntroductionOBI-1 is an investigational B-domain deleted recombinant porcine factor VIII (FVIII) with low cross-reactivity to anti-human FVIII antibodies. Acquired hemophilia A (AHA) is caused by autoantibodies (inhibitors) against human FVIII. Patients are predominantly elderly and have co-morbidities. Current pharmacologic treatment of bleeds is guided by clinical assessment alone as there is no laboratory surrogate for efficacy. Importantly, OBI-1 efficacy can be monitored by FVIII levels in addition to clinical assessment. MethodsAccur8 Auto-antibody trial (NCT01178294) is a prospective, open label, Phase 2/3 study. The primary objective is to evaluate efficacy of OBI-1 treatment for serious (life- or limb-threatening) bleeds in patients ≥18 years with AHA. FVIII levels are obtained before and within 10–20 min following initial OBI-1 dose (200U/kg) and at 2–3 h. Additional OBI-1 doses (≤400U/kg every 2–3 h) are administered to achieve target FVIII levels. The primary efficacy outcome is the control of bleeding 24 h after starting OBI-1. ResultsAs of July, 2012, fifteen patients with severe bleeds were entered into the trial along with one individual treated under compassionate use and all had successful control of hemorrhage at 24 h and subsequent resolution of the bleed. Therapeutic FVIII activity levels were achieved and maintained with intermittentOBI-1 administration based on FVIII levels. Six serious adverse events were reported including four deaths after treatment was discontinued, all being unrelated to OBI-1 as determined by the investigators. Antibodies to OBI-1 developed in two subjects indicated with an * in the table below. However, both responded toOBI-1.Sex (Age)Primary bleed siteAnti-human FVIII titer-pre-dose (BU)Anti-OBI-1 titer-pre-dose (BU)FVIII activity (%)24 h clinical outcome (hemostasis)Pre-first infusion20 min post-first infusion2-3 h post-first infusionM (72)1Surgical incision23Not detected9361186EffectiveF (82)Right knee/arm21Not detected1258230EffectiveM (43)Right arm (compartment syndrome)12Not detected18524601EffectiveM (91)Right deltoid (compartment syndrome)321013812EffectiveM (69)Left upper extremity swelling27Not detected22270233EffectiveM (66)*Interossei-dorsal of hand294Not detected1224164EffectiveF (73)Right thigh (surgical incision)124Not done0.37648EffectiveM (66)Hematoma of right upper extremity850.7111942EffectiveM (86)Upper limbs28Not detected9417278EffectiveF (79)Retroperitoneal hemorrhage651Not detected17720EffectiveF (81)Right iliacus muscle29210205EffectiveM (61)Surgical hemicolectomy36Not detected7296174EffectiveM (51)*Fundus of stomach16Not detected7240Not doneEffectiveF (64)Subcutaneous hematoma of left forearm11Not detected3288197EffectiveF (79)Left knee hemarthosis3Not detected14439331EffectiveF (82)Surgical tracheotomy290.9620982Effective1ompaidual treated under nate use individual1Compassionate use patient ConclusionsThese interim results provide support for the safety and efficacy of OBI-1 in the treatment of serious bleeding episodes in AHA. Additional confirming data could establish OBI-1 as a useful treatment option for AHA. Disclosures:St. Louis:Inspiration Biopharmaceuticals Inc: Research Funding. Kruse-Jarres:Inspiration Biopharmaceiticals Inc: Research Funding. Greist:Inspiration Biopharmaceuticals Inc: Research Funding. Shapiro:Inspiration Biopharmaceuticals Inc: Research Funding. Smith:Inspiration Biopharmaceuticals Inc: Research Funding. Drebes:Inspiration Biopharmaceuticals Inc: Research Funding. Gomperts:Inspiration Biopharmaceuticals Inc: Consultancy.

Recombinant factor VIIa treatment for asymptomatic factor VII deficient patients going through major surgery

Factor VII deficiency is the most common among the rare autosomal recessive coagulation disorders worldwide. In factor VII deficient patients, the severity and clinical manifestations cannot be reliably determined by factor VII levels. Severe bleeding tends to occur in individuals with factor VII activity levels of 2% or less of normal. Patients with 2-10% factor VII vary between asymptomatic to severe life threatening haemorrhages behaviour. Recombinant factor VIIa (rFVIIa) is the most common replacement therapy for congenital factor VII deficiency. However, unlike haemophilia patients for whom treatment protocols are straight forward, in asymptomatic factor VII deficiency patients it is still debatable. In this study, we demonstrate that a single and very low dose of recombinant factor VIIa enabled asymptomatic patients with factor VII deficiency to go through major surgery safely. This suggestion was also supported by thrombin generation, as well as by thromboelastometry.

Relationship between ABO blood groups and von Willebrand factor, ADAMTS13 and factor VIII in patients undergoing hemodialysis

Several studies have demonstrated that non-O blood groups subjects present an increased VTE risk as compared to those carrying O blood group. The aim of this study was to investigate the ABO blood groups influence on factor VIII (FVIII) activity, von Willebrand factor (VWF), and ADAMTS13 plasma levels in patients undergoing hemodialysis (HD). Patients undergoing HD (N=195) and 80 healthy subjects (control group) were eligible for this cross-sectional study. The ABO blood group phenotyping was performed by the reverse technique. FVIII activity was measured through coagulometric method, and VWF and ADAMTS13 antigens were assessed by ELISA. FVIII activity and VWF levels were significantly higher and ADAMTS13 levels was decreased in HD patients, as compared to healthy subjects (P<0.001, in three cases). HD patients carrying non-O blood groups showed a significant increase in FVIII activity (P=0.001) and VWF levels (P<0.001) when compared to carriers of O blood group. However, no significant difference was observed in ADAMTS13 levels (P=0.767). In the control group, increased in FVIII activity (P=0.001) and VWF levels (P=0.002) and decreased in ADAMTS13 levels (P=0.005) were observed in subjects carrying non-O blood groups as compared to carriers of O blood group.Our data confirmed that ABO blood group is an important risk factor for increased procoagulant factors in plasma, as FVIII and VWF. Admitting the possible role of kidneys in ADAMTS13 synthesis or on its metabolism, HD patients were not able to increase ADAMTS13 levels in order to compensate the increase of VWF levels mediated by ABO blood groups. Considering that non-O blood groups constitute a risk factor for thrombosis, it is reasonable to admit that A, B and AB HD patients need a careful and continuous follow-up in order to minimize thrombotic events.

Interaction between oral estrogen plus progestogen therapy and ABO blood groups on coagulation activation in postmenopausal women

The aim of this study was to investigate the effect of postmenopausal hormone therapy on coagulation and whether this effect differs according to ABO blood groups. This was a prospective observational study to evaluate factor VIII (FVIII) activity, factor von Willebrand (vWF), and D-dimer (D-Di) levels and ABO blood groups in 61 postmenopausal women using oral estrogen plus progestogen therapy (EPT; 2 mg estradiol + 1 mg norethisterone acetate) for 3 months and in 101 women not using EPT. After 3 months, all eligible women who had completed the treatment scheme proposed for the EPT group or those who opted to participate but had not undergone EPT had a blood sample collected for analysis. Significant differences were observed in FVIII activity and vWF levels in the control group between those carrying group O and non-group O blood. For EPT users, significant differences were observed for FVIII activity, vWF, and D-Di levels. After a multivariate regression analysis, FVIII activity and ABO blood groups were independently associated with vWF levels, whereas interaction between ABO blood groups and EPT were independently associated with FVIII activity. Besides diabetes, the ABO × EPT interaction was also noted to be independently associated with D-Di levels. These findings suggest an interactive effect between oral EPT and non-O blood groups, contributing to the mechanism by which estrogen triggers the hypercoagulability state and increased risk for venous thrombosis in women undergoing oral EPT.

Managing incidentally diagnosed isolated factor VII deficiency perioperatively: a brief expert consensus report

While isolated factor VII (FVII) deficiency is being more frequently diagnosed owing to improved preoperative screening procedures, there is no specific guideline for perioperative management of such patients. To complicate the issue, FVII activity levels seem to correlate less well with the risk of hemorrhage than the patient’s past and family bleeding history do. We have devised expert consensus recommendations for managing such patients perioperatively, taking into consideration the personal and family bleeding history, the FVII activity level and the inherent bleeding risk of the procedure itself. We hope that clinicians will find this a useful tool in the decision-making process, thereby limiting the use of recombinant factor VIIa to those who need it most, and preventing possible thrombotic complications in those without a strong indication for its use.

Surgery in patients with congenital factor VII deficiency: A single center experience

BackgroundCongenital factor VII (FVII) deficiency is a rare hemorrhagic disorder that can cause excessive bleeding during and after surgery in affected patients. The recombinant form of activated factor VII (rFVIIa, NovoSeven® from Novo Nordisk, Bagsvaerd, Denmark), which was developed as a second-generation bypassing agent, has recently been used in the management of bleeding for patients with congenital FVII deficiency.MethodsWe reviewed the results of 8 surgical procedures in 5 patients with congenital FVII deficiency at the Kyung Hee University Hospital, Gangdong, Seoul, Korea, between January 2008 and June 2010. We administrated rFVIIa preoperatively in six patients and postoperatively in five patients.ResultsBetween January 2008 and June 2010 at our center, 8 operations were performed successfully and no complications were observed in the 5 patients with congenital FVII deficiency. The median level of FVII activity was 2% (range, 0.6-7%). Four orthopedic procedures, 1 tonsillectomy, and 3 dental extractions were performed. The median duration of hospitalization was 8.5 days (range, 0-15 days). rFVIIa was administered at all procedures, except the dental extraction that was performed using only antifibrinolytic agents without any replacement. No bleeding or thrombogenic complications were observed in any case.ConclusionPatients with congenital FVII deficiency who require surgery can be treated efficiently and safely with rFVIIa or antifibrinolytic agents. rFVIIa was well tolerated and maintained effective hemostasis and showed good clinical outcome after the major surgery.

A Spectrum of Clinical and Laboratory Findings and Treatment Modalities in Patients with Factor VII Deficiency- A Single Medical Center Experience,

Abstract 3306▪▪This icon denotes a clinically relevant abstract Introduction:Factor VII, a vitamin K dependent glycoprotein serine protease secreted by the liver, has a fundamental role in the activation of the extrinsic coagulation pathway. It is found in the plasma as a zymogen, and is activated by tissue factor and calcium. Hereditary factor VII deficiency is a rare coagulation disorder with autosomal recessive inheritance found approximately at a prevalence of 1:500,000. In Israel, factor VII deficiency is found mostly in Sephardic Jews of Iranian, Moroccan and Tunisian origin as well as in the Druze population. Bleeding symptoms usually occur in homozygotes or compound heterozygotes. In symptomatic patients, the clinical severity of bleeding ranges from mild to severe (such as intracranial bleeding) and does not always correlate with plasma levels of factor VII activity. However, patients with factor VII activity of less than 1% may present with symptoms similar to severe hemophilia. Patients with factor VII activity of 5% or more usually present with milder bleeding such as epistaxis, gingival bleeding, menorrhagia or bruising. Therapy for factor VII deficiency depends on severity and type of the bleeding. Treatment includes fresh frozen plasma (FFP), tranexamic acid and recombinant factor VIIa (Novoseven). Aims:The study goal was to find correlation between level of factor VII activity, bleeding complications and treatment modalities that were used in symptomatic patients or in those undergoing surgical procedures. Patients and methods:We performed a retrospective study of 125 patients with decreased levels of factor VII, referred to our department between 1990–2010. 56 patients with isolated deficiency of factor VII were included in the study. Patients with combined deficiencies or liver disease were excluded. Demographics, clinical symptoms and their correlation to levels of factor VII, as well as treatment modalities were investigated. A factor VII activity level below 50 % was considered as decreased. The data collection was performed after approval of hospital Helsinki committee. Results:Of 56 patients with isolated factor VII deficiency there were 29 males and 27 females. 31/56 (55.4%) of the patients were Jews of Sephardic origin. 18/56 (32%) of the patients had relatives with known factor VII deficiency. The activity of factor VII ranged from <1%-49%. There was no significant difference in factor VII activity levels between the symptomatic (mean = 20.87, SD = 17.9) and the asymptomatic patients (mean = 23.13, SD = 17.4). 11/56 (19.6%) of the patients had clinical symptoms of bleeding, 6 of them had factor VII level<10 %. No life threatening bleeding was reported. Most episodes of bleeding related to factor VII deficiency were epistaxis and menorrhagia. Less common bleeding included hematoma and gingival bleeding. Only 9% of the symptomatic patients required treatment with FFP or tranexamic acid. Recommendation for prophylactic treatment such as FFP and/or tranexamic acid prior to surgical procedure was given in 19 patients. The post operative period was uneventful in all the patients. 5 patients did not receive any prophylactic treatment for different surgical procedures and no significant post operative bleeding was observed. Conclusions:Though factor VII deficiency had been described in the literature as a rare disease, we collected and analyzed a large cohort of 56 patients with this disorder in a single medical center. Most of our patients were Sephardic Jews. There had been several reports of excessive bleeding which complicated surgery, especially of the oropharynx and urogenital tract in patients with factor VII deficiency. Levels of 15–25% are recommended for surgery, but the plasma factor VII level required for surgery has been not formally established. We did not find statistically significant differences in factor VII activity levels between the symptomatic patients and the asymptomatic ones (P value<0.36). No life threatening bleeding occurred; only one patient required acute treatment to stop the bleeding. There was no uniform protocol used for prophylactic treatment, which was usually based on “old” drugs such as FFP and tranexamic acid. There was no need for Novoseven. We conclude that treatment guidelines based on the family, personal, surgical history and laboratory results are required for optimal treatment of patients with inherited factor VII deficiency. Disclosures:No relevant conflicts of interest to declare.

At Disease Presentation, Severity of the Bleeding Symptom Predicts the Following Bleeds in Patients with FVII Deficiency

Abstract 30▪▪This icon denotes a clinically relevant abstract Background:Inherited Factor VII (FVII) deficiency is the most common of the “rare” autosomal recessive bleeding disorders. Affected individuals display a wide range of clinical phenotypes and treatment demand may vary from prophylaxis to the need of rare or no replacement at all. As a matter of fact, it would be important to individualize the optimal and safest management with reference to the risk of bleeding. Objective:In a large number of subjects with FVII deficiency, we evaluated whether the type of symptom at disease presentation could help to predict further symptoms during the “Observation Period” (OP). Methods:Appropriate information in subjects with FVII deficiency, collected in the multicentre STER and IF7SG Registries, were employed. OP= time elapsed between the date of disease-presentation symptom and that of enrolment into the registry. Data for this analysis were complete for 687 individuals. Results:At diagnosis, among the 687 subjects (356 [51.8%] females, 331 [48.2%] males, mean age 29.6±19.63 yrs), 272 (39.6%) were asymptomatic; of the symptomatic individuals, 338 (49.2%) displayed a mild “Platelet- Like” (PL) bleeding defect (i.e. muco-cutaneous bleedings) and 77 (11.2%) had a severe, “Hemophilia-Like” (HL), phenotype. Mean age at diagnosis for the asymptomatic individuals was 23.8±18.8; mean ages at disease presentation were 10.54 ±11.8 for the PL, and 5.5 ±12.3 for the HL individuals ( p for trend= 0.000). FVII activity (FVIIc) was: females 17.53±19.68%, males 18.58±17.50% (p=0.460), overall: 18.04 ±18,66%.Of the 338 individuals with a PL-disorder, 268 (79.3%) had new muco-cutaneous bleedings; 51 (15.1%) developed severe bleedings, and 12 (4%) did not develop any subsequent bleed. Among the patients (n=77) with a HL-disorder, 38 (49.4%) experienced new severe bleeds, 34 (44.2%) developed mucosal bleedings, and only 5 (6.5%) had no further bleeds. Among those asymptomatic at diagnosis (n=272), 237 (87.1%) remained asymptomatic, while 35 (12.9%) developed only muco-cutaneous bleedings. Thus, 87.1% of the asymptomatic individual at diagnosis remained so, 79.3% of those with a platelet-like disorder at diagnosis displayed the same type of bleeding and, finally, 50% of those with severe bleedings at diagnosis had, again, severe bleeds.FVIIc helped to refine clinical information. Asymptomatic individuals at diagnosis that subsequently developed muco-cutaneous bleedings, had FVIIc similar to that of individuals with the same mild bleeds at onset (∼15–20%). FVIIc of individuals with PL-disorder at diagnosis who subsequently experienced a life- and limb-threatening bleeding episode, were as low (<5%) as those of individuals that had a severe phenotype at disease presentation. Regardless whether documented at diagnosis or during the OP, severe bleeding was invariably associated with FVIIc <5%. At diagnosis, the latter levels were found in 88.3% of individuals with severe symptoms, in 57.8% of those with muco-cutaneous bleedings and in 14.7% of asymptomatic individuals. Conclusions:In Factor VII deficiency, three clinical phenotypes can be identified: 1. the most prevalent cohort experience muco-cutaneous bleeding (PL-disorder); 2. Ten–15% of patients exhibit potentially life- or limb-threatening hemorrhages, such as hemartroses, central nervous system (CNS) or gastrointestinal (GI), a phenotype that may be as severe or more severe than that in hemophilia, and 3. about 1/3 of cases are asymptomatic and tend to remain so. Bleeding symptom at disease presentation predicts the subsequently developed bleeding phenotype. FVIIc levels refine such clinical prediction and help to identify individuals that call for early prophylaxis or on demand treatment. Disclosures:No relevant conflicts of interest to declare.

Elucidation of the Roles of Individual Asparagine-Linked Glycans Outside of the B Domain on Factor VIII Secretion

Abstract 2238Post-translational modifications play vital roles in the secretion, function, intermolecular interactions and degradation of most secreted and transmembrane proteins. Factor VIII (FVIII) is a heavily glycosylated protein with up to 25 asparagine (Asn)-linked glycans, the bulk of which are present within its B domain. However, deletion of the B domain is not deleterious to FVIII expression and function. In addition, FVIII has several potential Asn-linked glycosylation sequons in its other domains, of which four have been experimentally deduced to be glycosylated: Asn41 and Asn239 in the A1 domain, Asn1810 in the A3 domain and Asn2118 in the C1 domain. Of these, Asn239 and Asn2118 have been determined to comprise complex oligomannose structures. Such complex oligomannose structures have been proposed to play a role in mediating interaction with immunomodulatory cells (i.e. dendritic cells). The present study was aimed at delineating the role(s) of these four Asn-linked glycans in the expression of FVIII in vitro and in vivo and to identify possible bioengineering targets to influence FVIII expression, clearance and processing by immunomodulatory cells. Individual Asn residues were mutated to glutamine (Gln) to create single and multiple glycosylation mutants in both full length (FVIII-WT) and B domain-deleted (BDD)–FVIII, by site-directed mutagenesis. A variant of BDD-FVIII completely devoid of Asn-linked glycans, designated as Degly-BDD-FVIII, was also generated. Transient transfections of the mutants were carried out in COS-1 and CHO cells and their secretion and function were analyzed and compared to that of the respective native FVIII proteins. Antigen and activity assays revealed that the secretion and function of Asn41Gln and Asn1810Gln mutants were only modestly affected (85–90% of WT) but a more significant reduction was observed in the case of Asn239Gln mutant (35–50% of WT). Interestingly, there was no significant difference in secretion or function for Asn2118Gln in either FVIII-WT or BDD-FVIII protein backbones. The double mutants, Asn41/239Gln and Asn239/2118Gln behaved similarly to that of Asn239Gln mutant (30–45% of WT). The triple mutants, Asn41/239/2118Gln and Asn239/1810/2118Gln showed a further decline in secretion (∼30-40% of WT) while Degly-BDD-FVIII demonstrated secretion of only about 15–20% of BDD-FVIII. The FVIII specific activity of each of these glycosylation mutants was similar to the native FVIII proteins. An ELISA-based Von Willebrand Factor (VWF) binding assay revealed no significant differences between immunoaffinity-purified FVIII-WT and Asn2118Gln mutant in their ability to bind VWF. Findings from in vivo expression (via hydrodynamic tail vein injection of plasmid DNA) of these glycosylation mutants in a F8−/− (exon 16 knock-out) hemophilia A mouse model were similar to the in vitro results in the cell lines. Plasma FVIII activity levels were measured 24 hrs post-injection via orbital bleed. While Asn2118Gln (5.2 – 6 U/mL) did not exhibit any difference from BDD-FVIII (4.8 – 5.9 U/ml), Asn239Gln (1.9 – 2.4 U/ml) was expressed at less than 50% of BDD-FVIII levels. The expression of Degly-BDD-FVIII (0.4 – 0.7 U/ml) was further reduced to ∼10% of BDD-FVIII levels. Taken together, these results indicate that of the four Asn-linked glycans, Asn239 was the most crucial for proper secretion of FVIII whereas, Asn2118 did not contribute to the efficiency of FVIII expression. The oligosaccharide structure on Asn239 is positioned at the A1-A2 interface and likely contributes to proper protein folding. However, the sugar moieties on Asn2118 have been shown to be positioned at the A3-C1 domain interface and postulated to participate in packing and stabilization (Shen et al, 2008). This would have suggested that disruption of this residue within the C1 domain might have a deleterious effect on protein secretion or function. Our results with Asn2118Gln in both FVIII-WT and BDD-FVIII protein backbones suggest that this Asn-linked glycosylation can be eliminated without any impact on FVIII expression or function including no impact on FVIII-VWF interaction. This Asn-linked glycan, therefore, could be targeted in bioengineering strategies to determine if eliminating this particular oligomannose structure might impact mannose-receptor mediated uptake of FVIII by dendritic cells. Disclosures:No relevant conflicts of interest to declare.

A novel missense mutation close to the charge-stabilizing system in a patient with congenital factor VII deficiency

Congenital factor VII (FVII) deficiency is a rare autosomal recessive bleeding disorder. Its clinical manifestation and mutational spectrum are highly variable. The purpose of this study was to identify and characterize the mutation causing the FVII deficiency in a Chinese patient and his family. The FVII gene was analyzed by genomic DNA sequencing, and the FVII levels in patient's plasma were measured with an enzyme-linked immunoabsorbent assay (ELISA) and one-stage prothrombin time based method. In addition, the FVII-Phe190 mutant identified in the pedigree was expressed in the HEK293 cells, and the subcellular localization experiments in the Chinese hamster ovary (CHO) cells were performed. The patient had a prolonged prothrombin time and low levels of both FVII antigen and activity, and two heterozygous mutations were identified in F7 gene (NG-009262.1): a g.15975 G>A in the splice receptor site of intron 6 and a novel g.16750 C>T in exon 8 resulting in Ser190 to Phe190 replacement. In expression experiments, the reduced antigen and activity levels of FVII-Phe190 in the culture medium were found, whereas an ELISA and Western blotting analysis of FVII revealed that mutant FVII-Phe190 was synthesized in the cells as the wild-type FVII-Ser190. And FVII-Phe190 was found in endoplasmic reticulum and Golgi apparatus. Compound heterozygous mutations in F7 gene should be responsible for the FVII deficiency in this patient. The FVII-Phe190 can normally be synthesized and transported from endoplasmic reticulum to Golgi apparatus, but degraded or inefficiently secreted.

Most factor VIII B domain missense mutations are unlikely to be causative mutations for severe hemophilia A: implications for genotyping

The factor VIII (FVIII) B domain shares very little amino acid homology with other known proteins and is not directly necessary for procoagulant activity. Despite this, missense mutations within the B domain have been reported in patients with hemophilia A. Given that the B domain is dispensable for secretion and function of FVIII, we hypothesized that these mutations should not be causative of hemophilia A in these patients. Plasmid vectors containing B domain missense mutations that were reported to be associated with moderate/severe hemophilia A (T751S, D826E, V993L, H1047Y, T1353A, N1441K, L1462P, E1579D, A1591S, P1641L and S1669L) were analyzed for their effect on synthesis and secretion compared with FVIII wild-type (WT) following transient transfection into COS-1 and CHO cells in vitro. Further, H1047Y, N1441K and E1579D mutants were expressed in vivo in a hemophilia A mouse model by hydrodynamic tail-vein injection. FVIII activity and antigen levels for all mutants expressed into the conditioned media of COS-1 and CHO cells were similar to FVIII WT. Also, plasma expression of these mutants was similar to FVIII WT in hemophilia A mice. An in vivo tail clip bleeding assay also demonstrated that blood loss from hemophilia A mice expressing FVIII WT, H1047Y, N1441K and E1579D was similar. We conclude that most missense mutations within the FVIII B domain would be unlikely to lead to severe hemophilia A and that the majority of such missense mutations represent polymorphisms or non-pathologic mutations.

Efficacy and Safety of Long-term Prophylaxis in Severe Hemophilia A Dogs Following Liver Gene Therapy Using AAV Vectors

Developing adeno-associated viral (AAV)-mediated gene therapy for hemophilia A (HA) has been challenging due to the large size of the factor VIII (FVIII) complementary DNA and the concern for the development of inhibitory antibodies to FVIII in HA patients. Here, we perform a systematic study in HA dogs by delivering a canine FVIII (cFVIII) transgene either as a single chain or two chains in an AAV vector. An optimized cFVIII single chain delivered using AAV serotype 8 (AAV8) by peripheral vein injection resulted in a dose-response with sustained expression of FVIII up to 7% (n = 4). Five HA dogs administered two-chain delivery using either AAV8 or AAV9 via the portal vein expressed long-term, vector dose-dependent levels of FVIII activity (up to 10%). In the two-chain approach, circulating cFVIII antigen levels were more than fivefold higher than activity. Notably, no long-term immune response to FVIII was observed in any of the dogs (1/9 dogs had a transient inhibitor). Long-term follow-up of the dogs showed a remarkable reduction (>90%) of bleeding episodes in a combined total of 24 years of observation. These data demonstrate that both approaches are safe and achieve dose-dependent therapeutic levels of FVIII expression, which supports translational studies of AAV-mediated delivery for HA.

Recombinant factor VIIa affects anastomotic patency of vascular grafts in a rabbit model

Recombinant factor VIIa can decrease postoperative bleeding after cardiac surgery. However, the potential for recombinant factor VIIa to cause early vascular graft occlusion at the site of new vascular anastomoses has not been fully explored. We hypothesized that recombinant factor VIIa would cause a dose-dependent reduction in vascular graft patency in rabbits. Reversed end-to-end interpositional vein grafts were sutured into the carotid artery of heparinized rabbits, and then recombinant factor VIIa (300 μg/kg, 90 μg/kg, or 20 μg/kg intravenously) or placebo was administered (n=16/group). Graft patency was assessed at 24 hours using a vascular ultrasound probe. Factor VII activity levels were measured using a prothrombin time-based assay. In different rabbits, the patency of venous end-to-side anastomoses and simple carotid arterial repairs was assessed (recombinant factor VIIa, 300 μg/kg vs placebo, n=8/group). Data were analyzed using Fisher's exact test, t tests, or analysis of variance. Physiologic variables (activated clotting time, hemoglobin, pH, Pao(2)) and vessel diameter were not different between groups. Vein graft patency was reduced (93.8%, 81.2%, 13.8%, and 6.3%) as factor VII activity levels increased (1.8 ± 0.4, 4.4 ± 2.1, 11.8 ± 4.7, and 23.6 ± 16.9 U/mL, respectively) with increasing doses of recombinant factor VIIa administered (0, 20, 90, and 300 μg/kg, respectively, P<.05). Patency in the arterial repairs and end-to-side venous grafts was also reduced in recombinant factor VIIa-treated rabbits (P<.05 for both). This study suggests that recombinant factor VIIa is associated with a dose-dependent increase in fresh vascular graft occlusion. Higher doses of recombinant factor VIIa may be associated with increased thrombotic outcomes.

Imaging and Intervention of Paraneoplastic Effect of a Right Atrial Myxoma on Factor VII Activity Levels

We present the case of a 64-year-old Pakistani man with right atrial myxoma, recently diagnosed with acquired severe factor VII (FVII) deficiency. The patient presented with a history of chronic hiccups and weight loss. Initial evaluation revealed an isolated prolonged prothrombin time, severely reduced FVII activity level, and a giant right atrial myxoma protruding into the right ventricle on computed tomographic thorax and echocardiography. After surgical resection, the patient maintained normal prothrombin time with increased FVII activity level in the immediate 24 hours postoperatively, and a dramatically high level of FVII activity at the 2-month follow-up. We believe that the paraneoplastic effect of myxoma on the FVII activity levels is previously unreported. In addition, we believe that hiccups as a presenting symptom for a myxoma with an atypical origin from the lateral wall of the right atrium has not been reported.

Cerebellar hemorrhage as a first presentation of acquired Hemophilia A

Acquired hemophilia A (AHA) is an uncommon coagulation disorder caused by the development of autoantibodies against coagulation factor VIII (FVIII). While intracranial hemorrhage is a known complication of AHA, intracranial hemorrhage as the presenting manifestation of AHA has only been described in three previous case reports. We report a case of an 86-year-old woman with no previously reported history of coagulopathy presenting with an acute intraparenchymal cerebellar hemorrhage and laboratory studies demonstrating an isolated prolonged activated partial thromboplastin time (aPTT). We discuss an approach to the prolonged aPTT, and review the literature concerning the diagnosis and treatment of AHA. Occipital decompressive craniectomy with evacuation of the hemorrhage was performed. Eight hours following the procedure, the patient's status acutely declined with demonstration of a reoccurrence of the cerebellar hemorrhage and new right frontal lobe hemorrhage. After discussion with the patient's family, life-sustaining support measures were withdrawn. Postmortem analysis revealed a low FVIII activity level and the presence of FVIII inhibitor. The presentation of intracranial hemorrhage with an isolated prolonged aPTT is concerning for an acquired hemophilia with FVIII deficiency. Other causes of isolated prolonged aPTT such as a lupus anticoagulant must also be considered. Preoperative identification and work-up of the coagulation abnormality is essential to guide initial treatment.