Factor VIII Activity Levels Research Articles

Protected by Nature: Effects of Exercise In Non-Severe Haemophilia Patients

AbstractAbstract 545 Background:An increase of clotting factor VIII (FVIII) within 10 minutes after physical exercise has been reported in healthy subjects and patients with von Willebrand disease. The mechanism of FVIII increase through exercise is thought to be similar to that of desmopressin, which increases FVIII. Therefore, moderate and mild haemophilia A patients should also be able to increase their endogenous FVIII levels after exercise. A proof of principle study was performed to test this hypothesis. Methods:Changes in coagulation associated with exercise of 15 haemophilia A patients, aged 20–39 years, were measured. Eight had moderate haemophilia (FVIII 1–5 IU/dl), 7 had mild haemophilia with endogenous FVIII activity levels at the lower end of the range (FVIII 6–16 IU/dl) Patients were encouraged to cycle until volitional exhaustion, on an electronically braked cycle ergometer. Blood samples were drawn before and 10 minutes after the incremental exercise test. Blood was frozen and analyzed for FVIII, von Willebrand factor (vWF) and von Willebrand propeptide (vWpr), in one batch to avoid inter-assay variation. In mild haemophilia patients, changes in coagulation profile were compared to DDAVP tolerance tests. Results:All 15 patients had an increased FVIII level after exercise. Median relative increase was 2.5 times (interquartile range (IQR)= 1.2–4.0). The relative increase did not vary across severities (median relative increase of FVIII was 2.4 times (IQR= 1.3–4.0) for moderate haemophilia patients compared to median 2.5 times (IQR=1.2-4.0) in patients with mild haemophilia Mann-Whitney: p =0.91). However, the absolute FVIII levels were very different with a median FVIII level of only 9 IU/dl; IQR= 8–11 IU/dl) in patients with moderate haemophilia, compared with a much higher median FVIII level of 21 IU/dl; IQR=19-44 IU/dl) attained in patients with mild haemophilia. vWF and vWFpr also increased independent of severity (p=0.3-0.6) in all patients, median 50%; IQR 30–79% for vWF and median 165%; IQR 130–244% for vWFpr, reflecting acute release of coagulation factors. DDAVP response tests were available only for patients with mild haemophilia. Relative increase after DDAVP administration was median 3.5 times (IQR= 2.5–4.5), similar to increase in FVIII after exercise (Wilcoxon: p=0.14). Conclusion:Although normal levels were not achieved, the increase of FVIII activity during exercise may reduce bleeding risk and therefore help promote participation in sports and other strenuous activities for moderate and mild haemophilia A patients with modest endogenous FVIII activities who might be considered at high bleeding risk. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Influence of the type of F8 gene mutation on inhibitor development in a single centre cohort of severe haemophilia A patients

The development of neutralizing antibodies against factor VIII (FVIII) is a major complication of treatment with FVIII in patients with severe haemophilia A. This study was designed to describe the relationship between the type and location of the factor 8 (F8) gene mutation and the development of clinically relevant inhibitors in patients with severe haemophilia A. We conducted a single centre cohort study among 318 consecutive patients (baseline FVIII activity level <0.01 IU mL(-1)) born between 1934 and 2007 who were treated with FVIII on at least 50 exposure days. The primary outcome was clinically relevant inhibitor development, defined as the occurrence of at least two positive inhibitor titres and a decreased recovery. Clinically relevant inhibitors were diagnosed in 14% (43) of patients (30 high-titre). The cumulative incidence of inhibitor development was 18% (35 of 200) in high-risk gene defects (67% in patients with large deletions, 30% in patients with nonsense mutations, 15% in patients with intron 1 or 22 inversions) and 7% (8 of 118) in low-risk gene defects (7% in patients with small deletions and insertions, 6% in patients with missense mutations, 8% in patients with splice site mutations). In patients with point mutations, the cumulative risk of developing inhibitors was highest in patients with mutations in the A3 and C2 domains (13% and 17% respectively). In conclusion, in agreement with earlier observations, the type and location of the F8 gene mutation were important determinants of inhibitor development in patients with severe haemophilia A.

Analysis of low frequency bleeding data: the association of joint bleeds according to baseline FVIII activity levels

Many studies in the field of haemophilia and other coagulation deficiencies require analyses of bleeding frequencies. In haemophilia, the association of bleeding frequency with factor VIII (FVIII) activity levels is known from experience, but significant results are lacking. Bleeding frequencies in haemophilia are highly skewed count data, with large proportions of zeros. Both the skewness and the high amount of zeros pose a problem for standard (linear) modelling techniques. This study investigated the optimal analysing strategy for bleeding data by using the association of residual clotting factor level and number of joint bleeds in moderate and mild patients treated on demand as example. In total, 433 patients with moderate (27%) and mild (73%) haemophilia A treated on demand were included in this study. One year of self-reported data on joint bleed frequency and baseline clotting factor activity were analysed using Poisson, negative binomial, zero-inflated Poisson, and zero-inflated negative binomial distributions. Multivariate regression analysis using negative binomial distribution provided the optimum data analytical strategy. This model showed 18% reduction [Rate ratio (RR) 0.82; 95%confidence interval (CI) 0.77-0.86] of bleeding frequency with every IU dL(-1) increase in residual FVIII activity. The actual association is expected to be higher because of exclusion (30 out of 463 patients) of patients on prophylaxis (baseline FVIII levels 0.01-0.06 IU mL(-1)). The best way to analyse low frequency bleeding data is using a negative binomial distribution.

Haemophilia A and von Willebrand’s disease

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

THIS MONTH IN Anesthesiology

Postoperative noninvasive ventilation can help prevent postoperative hypoxemia and acute respiratory failure.Chinese anesthesiologists are poised to play a significant role in tobacco control in China.Hypercapnia may be beneficial in early sepsis, but potentially deleterious in prolonged sepsis. See the accompanying Editorial View on page 269 Dose of local anesthetic but not concentration and volume influence nerve block characteristics.Techniques on ultrasound-guided peripheral nerve blocks of the extremities and trunk are discussed.Unrecognized patient deterioration in the postoperative period may lead to increased intensive care unit (ICU) admission rates or even death. A patient surveillance system based on pulse oximetry and wireless pagers was used for nursing notification of violation of alarm limits. Data were collected on a postoperative orthopedic ward for 11 months before and 10 months after implementation of the system and compared with matched outcome data on two other postoperative units. Rescue events decreased by 2.2 per 1,000 patient discharges and ICU transfers by 1.7 per 1,000 patient days while the comparison units had no change. Rescue events and ICU transfers were decreased after implementation of this patient surveillance system on a postoperative orthopedic ward. See the accompanying Editorial View on page 272 Factor VII is affected during the early phase of warfarin therapy and high international normalized ratios (INR &gt; 1.4) are considered unsafe for epidural catheter placement or removal by the American Society of Regional Anesthesia. Data from 121 patients who took warfarin after undergoing total joint surgery and had INRs and factor VII levels determined were reviewed. Patients had INR &gt; 1.4 on postoperative days (POD) 1 (n = 11), POD 2 (n = 78), and POD 3 (n = 84) and mean factor VII activities were 60 ± 28%, 32 ± 15%, and 44 ± 19%, respectively. Predictors of INR &gt; 1.4 on POD 2 were warfarin dose on POD 1 and factor VII activity on POD 2. The range of factor VII activities in the patients with INR &gt; 1.4 within 12 h of warfarin therapy was compatible with adequate hemostasis. Based on factor VII activity levels, the authors found no evidence that epidural catheters should not be removed even with INR up to 1.9, the highest INR on POD 1 noted in their study. See the accompanying Editorial View on page 277 Intubation efficiency varies depending on the type of laryngoscope blade used. In a multicenter cluster randomized trial, 1,072 adult patients undergoing general anesthesia under emergency conditions and requiring rapid sequence induction were randomly assigned on a weekly basis to either single-use or reusable metal blades. The rate of failed intubation was significantly decreased with metal single-use blade at the first attempt compared with the reusable blade (2.8% vs. 5.4%). The proportion of grades 3 and 4 in Cormack and Lehane score were also significantly decreased with single-use metal blade (6% vs. 10%). The global complication rate tended to be lower in the single-use group (6.8% vs. 11.5%). The single-use metal blade was more efficient than a reusable metal blade in rapid sequence induction of anesthesia for emergency surgery.

Asymptomatic factor VII deficiency: gene analysis and structure–function relationships

Factor VII Padua is a variant form of factor VII deficiency characterized by a prolongation of the prothrombin time (PT), when the assay is performed using rabbit brain thromboplastin. The PT is normal when performed using either human or ox brain thromboplastin reagents, or a recombinant human tissue factor-based thromboplastin. We report a case of an African-American woman with asymptomatic factor VII deficiency, who had a prolonged PT and factor VII activity levels of 5-8% using rabbit brain thromboplastin, but a normal PT and factor VII activity levels when measured using recombinant human brain thromboplastin or tissue factor. The amino acid substitution (R304Q), which gives rise to factor VII Padua, was found in our patient, making this only the fourth African-American case described to date with this mutation. Our report emphasizes the importance of identifying this benign form of factor VII deficiency in order to avoid unnecessary exposure of patients to treatment with either plasma-derived products or recombinant activated factor VII.

In vitro and in vivo stability of diluted recombinant factor VIII for continuous infusion use in haemophilia A

Factor VIII (FVIII) replacement by continuous infusion (CI) is used postoperatively or after significant bleeding. For young paediatric patients, CI may require FVIII dilution. Variable stabilities of diluted full-length recombinant FVIII Kogenate FS (KG-FS) have been reported under different storage conditions. We investigated the recovery and stability of diluted KG-FS in vitro and in vivo. Kogenate FS was diluted to 50-120 U mL(-1) and its recovery and stability in glass vials or polypropylene syringes was determined. Furthermore, stability of KG-FS diluted to 80 U mL(-1)'administered' via single- and double-pump mock CI systems was tested. Finally, the in vivo stability of KG-FS diluted to approximately 60 U mL(-1) and administered postsurgically by CI with the double-pump to a paediatric patient with severe haemophilia A undergoing implantable venous access device placement was investigated. Initial KG-FS dilution resulted in a 10-20% FVIII loss; a further 25-30% loss occurred over 72 h in vials or syringes. With the double-pump, 1 h recovery was 35%, increasing to 80% by 24 h; the initial losses were because of the Y-infusion of a 10-fold larger volume of saline concomitantly with the FVIII. In vivo, CI resulted in stable FVIII activity levels within the target range. These in vitro results are important for the generation of CI guidelines for diluted KG-FS in the paediatric haemophilic population. That FVIII losses occur upon dilution and with the double-pump does not preclude use of diluted KG-FS. Indeed, stable FVIII levels were maintained when diluted KG-FS was administered by CI with the double-pump to a paediatric patient postsurgically.

Acquired hemophilia A may be associated with itraconazole

Acquired hemophilia A is a rare disorder, with an annual occurrence rate of 1.48 cases per million [1], caused by autoantibodies against coagulation factor VIII (FVIII) [2].These autoantibodies mainly affect elderly patients and are often present without any underlying disease, but may occur in various conditions such as pregnancy, autoimmune disorders, solid tumors, and lymphoproliferative syndromes. Several drugs, such as penicillin and its derivatives, phenytoin, and sulfa drugs, have also been reported to be associated with antibodies to FVIII [3]. Collins et al. [1] reported the results of a 2-year national study of acquired hemophilia A performed by the UK Haemophilia Centre Doctors’ Organisation; the results of this study indicated that diagnosis of underlying disorders associated with acquired hemophilia A was very difficult, especially in old age (patients between 60 and 79 years, 42%; those aged 80 or over, 23%). Here, we report a 74year-old case of acquired hemophilia A associated with itraconazole. A 74-year-old woman was admitted to our hospital with a 3-week history of peripheral edema, palpable purpura, and arthritis. There was no previous personal or family history of bleeding. She also had no past history of illness until 4 weeks before admission, but she took 200 mg/day of itraconazole because of tinea pedis for 4 weeks before admission. On examination, the results of laboratory work-up were as follows: hemoglobin level, 5.7 g/dl; platelet count, 324 9 10 ml; white blood cell count, 9.3 9 10 ml; prothrombin time, 91% (normal 80–120%); activated partial thromboplastin time (APTT), 80.3 s (normal 25–39 s). Tests for lupus anticoagulant, anticardiolipin antibody, antinuclear factor, double stranded DNA, and rheumatoid factor were negative. Fiberscopy examination revealed esophageal ulcer as the reason for the anemia. Remarkably prolonged APTT (166.5 s) and huge iliopsoas hematoma appeared 15 days after admission. The levels of FVIII activity (FVIII:C) and FVIII-inhibitory activity on admission were \1% and 23 Bethesda units, respectively, thus confirming the presence of acquired FVIII inhibitor. The levels of von Willebrand factor (VWF) antigen (VWF:Ag) and VWF-inhibitory activity were prepared to be examined when the levels of FVIII:C were low and FVIII-inhibitory activity was negative. A diagnosis of acquired hemophilia A was made and the patient was treated with activated prothrombin complex concentrate (APCC); 1500 units every 12 h in addition to 50 mg/day of cyclophosphamide and 1 mg/kg of prednisolone. Her iliopsoas hematoma was effectively controlled with no subsequent recurrence. APCC was discontinued after 3 days. Cyclophosphamide and prednisolone were continued with slow tapering, and FVIII inhibitor became undetectable after 10 weeks, and FVIII:C level got normalized after 18 weeks. In spite of additional examination of another underlying disease, none of autoimmune disorders, solid tumors, and lymphoproliferative syndromes were detected. A possible link between autoimmune acquired hemophilia and itraconazole has not been previously reported. Itraconazole has been associated with neutropenia [4], suggesting other possible immune mediated adverse events. This patient could be cured because of the rapid diagnosis. Investigation of FVIII:C and FVIII-inhibitory H. Tamai S. Tanosaki Department of Hematology, The Fraternity Memorial Hospital, Tokyo, Japan

Tissue Factor in Brain Is Not Saturated With Factor VIIa

The recent commentary by Dr Torbey on “Intracerebral Hemorrhage: What’s Next?”1 has prompted us to share some data and thoughts on the use of activated factor VII (FVIIa, NovoSeven, Novo Nordisk A/S) in the management of intracerebral hemorrhage. FVIIa is approved to promote hemostasis in hemophiliacs with inhibitors. Studies have also suggested that it promotes hemostasis in nonhemophilic patients with bleeding attributable to trauma2 or with intracranial hemorrhage.3 The commercial FVIIa product is the activated form of the naturally occurring human protein. The precursor, FVII, is readily activated by a number of coagulation and noncoagulation proteases once it has bound to its cofactor, tissue factor (TF). FVIIa has little proteolytic activity in solution. Binding to a suitable phospholipid surface enhances its activity somewhat, but binding to TF greatly enhances proteolytic activity toward its substrates, FIX and FX. Plasma levels of FVIIa are normally around 100 pmol/L.4 Because of the very low activity exhibited by FVIIa in the absence of TF and the very high affinity of TF for FVII, it is thought that FVIIa only exhibits significant activity in vivo when complexed to TF. Exogenous FVIIa was originally thought to enhance hemostasis by increasing activation of FX by the TF pathway. However, very high levels of FVIIa are required to promote hemostasis in hemophilia: up to 1000× the normal …

Most Factor VIII B Domain Missense Mutations Are Unlikely to Be Causative Mutations for Hemophilia A: Implications for Factor VIII Genetic Analysis

Hemophilia A results from the quantitative or qualitative deficiency of coagulation factor VIII (FVIII). FVIII is synthesized as a single-chain polypeptide of approximately 280 kDa with the domain structure A1-A2-B-A3-C1-C2. Whereas the A and C domains exhibit ~40% amino acid identity to each other and to the A and C domains of coagulation factor V, the B domain is not homologous to any known protein and is dispensable for FVIII cofactor activity. Missense mutations in the FVIII B domain have been described in patients with variable phenotypes of hemophilia A. According to the NCBI SNPs (single nucleotide polymorphism) database, 22 SNPs are reported within FVIII, 11 of which occur within the B domain. FVIII B domain variant D1241E has been reported as a missense mutation associated with mild or severe hemophilia A, yet this mutation is also present in the NCBI SNPs database. We hypothesize that D1241E and most other reported B domain missense mutations are not the causative mutation for hemophilia A in these patients but represent SNPs or otherwise non-pathologic mutations. To investigate this, we analyzed 7 B domain missense mutations that were previously found in hemophilia A patients (T751S, V993L, H1047Y, D1241E, T1353A, P1641L and S1669L). Comparative analysis showed that the amino acids at these positions are not conserved in all species and in some cases, the amino acid substitution reported in hemophilia patients is represented in the native sequence in other species. Analysis with PolyPhen Software showed that only H1047Y mutation was considered as “possibly damaging”, while the others were considered as “benign”. To investigate this further, we constructed seven plasmid vectors containing these B domain missense mutations. The synthesis and secretion of FVIII wild-type (WT) and these seven mutants were compared after transient DNA transfection into COS-1 monkey cells in vitro. Analysis of the FVIII clotting activity and antigen levels in the conditioned medium demonstrated that all mutants had FVIII activity and antigen levels similar to FVIII WT. Further, FVIII WT, H1047Y and D1241E mutants were introduced into a FVIII exon 16 knock-out mouse model of hemophilia A by hydrodynamic tailvein injection in vivo. The mouse plasma was analyzed at 24 hrs for activity and antigen expression. Mutants H1047Y and D1241E expressed at 211 mU/mL and 224 mU/mL activity with FVIII antigen levels of 97 ng/mL and 118 ng/mL, respectively, similar to FVIII WT. These results suggested that H1047Y and D1241E mutants did not lead to impairments in secretion or functional activity. 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. Investigators should search for additional potentially causative mutations elsewhere within the FVIII gene when B domain missense mutations are identified.

New assays for monitoring haemophilia treatment

Precise measurements of factor VIII (FVIII) or factor IX (FIX) activity are believed to be essential for clinical management in haemophilia, although discrepancies between factor levels and clinical severity have been recognized. Clot wave form analysis has demonstrated that different wave form patterns may be evident in severe haemophilia A patients with levels of FVIII activity <1 IU dL(-1), and this might explain, in part, the phenotypic heterogeneity seen in these patients. In addition, the relatively new technique of computer-assisted thrombelastography (TEG), in which coagulation is initiated by tissue factor, has revealed a considerable degree of variability in different patients in the presence FVIII levels, which are sufficient to normalize TEG parameters. In contrast, a global thrombin generation test (TGT) has been proposed as a sensitive and reliable method for assessing overall clotting function in haemophilia patients. Several studies have demonstrated a significant correlation between TGT and FVIII/FIX levels, and these measurements also appear to correlate with the clinical phenotype. The TGT may be very useful, therefore, for evaluating overall haemostasis in different clinical situations, although substantial inter-assay and inter-individual variations have been reported. Both the TEG and TGT have been found to be particularly helpful for monitoring haemostatic therapy with bypassing agents or conventional FVIII or FIX concentrates in patients with inhibitors. These global tests enable the selection of appropriate therapeutic agents in individual circumstances and offer the opportunity to tailor the most effective haemostatic treatment even during severe bleeding or major surgery.

Compound heterozygous mutations in severe factor VII deficiency including a novel nonsense mutation

A 26-year-old man who had a history of recurrent spontaneous nasal bleeding was admitted to our hospital with uncontrolled traumatic subdural hemorrhage. His plasma factor VII activity was less than 10% of normal. DNA sequence analysis revealed that the proband had a compound heterozygote for a novel nonsense mutation (F7 NM_000131.2 c.345C > A; p.Cys115X) in exon 4 encoding the EGF1 domain and a known missense mutation (F7 NM_000131.2 c.1027G > A; p.Gly343Ser) in exon 8 encoding the serine protease domain of F7. The same F7 Gly343Ser mutation was present in the asymptomatic father, who exhibited a modest reduction in the plasma level of factor VII activity (48%).

Comparison of coagulant activity of factor VII and activated factor VII activity assays when used for determination of recombinant activated factor VII levels in plasma

Two assays [coagulant activity of factor VII (FVII:C) and activated factor VII (FVIIa) activity] are currently available for the assessment of factor VII and FVIIa pharmacokinetics. This article presents the results of a comparison of the two assays when applied both in vitro as well as during clinical pharmacokinetic trials of recombinant FVIIa (rFVIIa) administered to healthy individuals and haemophilia patients. The in-vitro data showed that, for the FVII:C assay, plasma samples do not dilute in parallel. For the FVIIa activity assay, dilutions of samples are both parallel and linear with different dilutions of the calibrator. Moreover, intra-assay variation was found to be smaller for the FVIIa activity assay than for the FVII:C assay. When adding different amounts of rFVIIa (0-6 microg/ml) to normal plasma, a mean specific activity of rFVIIa of 48.6 U/mug was observed on applying the FVII:C assay; however, the specific activity decreased with increasing levels of rFVIIa. For the FVIIa activity assay, the mean specific activity was 45.4 IU/mug. Direct comparison of the two activity assays showed that no simple conversion between FVII:C and FVIIa activity measurements are possible. When applying the two assays for pharmacokinetic assessments in two clinical trials, statistically significant different estimates for the area under the curve, half-life, clearance and volume of distribution were obtained. In conclusion, for evaluation of rFVIIa pharmacokinetic properties, activity should be measured with the FVIIa activity assay - which is a more specific and reliable assay of the two available factor VII activity assays, especially when assessing low activity levels.

ACQUIRED HEMOPHILIA IN OLDER PEOPLE: A POOR PROGNOSIS DESPITE INTENSIVE CARE

ACQUIRED HEMOPHILIA IN OLDER PEOPLE: A POOR PROGNOSIS DESPITE INTENSIVE CARE

A sequence variation scan of the coagulation factor VIII (FVIII) structural gene and associations with plasma FVIII activity levels

AbstractPlasma factor VIII coagulant activity (FVIII:C) level is a highly heritable quantitative trait that is strongly correlated with thrombosis risk. Polymorphisms within only 1 gene, the ABO blood-group locus, have been unequivocally demonstrated to contribute to the broad population variability observed for this trait. Because less than 2.5% of the structural FVIII gene (F8) has been examined previously, we resequenced all known functional regions in 222 potentially distinct alleles from 137 unrelated nonhemophilic individuals representing 7 racial groups. Eighteen of the 47 variants identified, including 17 single-nucleotide polymorphisms (SNPs), were previously unknown. As the degree of linkage disequilibrium across F8 was weak overall, we used measured-genotype association analysis to evaluate the influence of each polymorphism on the FVIII:C levels in 398 subjects from 21 pedigrees known as the Genetic Analysis of Idiopathic Thrombophilia project (GAIT). Our results suggested that 92714C>G, a nonsynonymous SNP encoding the B-domain substitution D1241E, was significantly associated with FVIII:C level. After accounting for important covariates, including age and ABO genotype, the association persisted with each C-allele additively increasing the FVIII:C level by 14.3 IU dL−1 (P = .016). Nevertheless, because the alleles of 56010G>A, a SNP within the 3′ splice junction of intron 7, are strongly associated with 92714C>G in GAIT, additional studies are required to determine whether D1241E is itself a functional variant.

Multiyear therapeutic benefit of AAV serotypes 2, 6, and 8 delivering factor VIII to hemophilia A mice and dogs

Hemophilia A, a deficiency of functional coagulation factor VIII (FVIII), is treated via protein replacement therapy. Restoring 1% to 5% of normal blood FVIII activity prevents spontaneous bleeding, making the disease an attractive gene therapy target. Previously, we have demonstrated short-term activity of a liver-specific AAV2 vector expressing canine B-domain-deleted FVIII (cFVIII) in a hemophilia canine model. Here, we report the long-term efficacy and safety of AAV-cFVIII vectors of serotypes 2, 5, 6, and 8 in both hemophilia A mice and dogs. AAV6-cFVIII and AAV8-cFVIII restored physiologic levels of plasma FVIII activity in hemophilia A mice. The improved efficacy is attributed to more efficient gene transfer in liver compared with AAV2 and AAV5. However, supraphysiologic cFVIII levels correlated with the formation of cFVIII-neutralizing antibodies in these mice. Of importance, hemophilia A dogs that received AAV2-cFVIII, AAV6-cFVIII, and AAV8-cFVIII have persistently expressed therapeutic levels of FVIII, without antibody formation or other toxicities, for more than 3 years. However, liver transduction efficiencies are similar between AAV2, AAV6, and AAV8 serotypes in hemophilia A dogs, in contrast to mice. In summary, this is the first report demonstrating multiyear therapeutic efficacy and safety of multiple AAV-cFVIII vectors in hemophilia A dogs and provides the basis for human clinical studies.

The significance of published polymorphisms in 14 cases of mild factor VII deficiency

Factor VII (FVII) plays a critical role in the initiation of blood coagulation, and patients with dysfunctional or reduced levels of this protein are susceptible to mucosal bleeding. There is poor correlation between the clinical presentation and the phenotypic data; and in cases of a mild bleeding tendency, mild to moderate reductions in both FVII antigen and activity may be overlooked. The prevalence of FVII deficiency may therefore be underestimated. Polymorphic differences throughout the FVII gene are associated with variations in plasma FVII antigen and activity levels. This study highlights the significance of mild FVII deficiency, and examines the importance of seven previously published polymorphisms in such patients.

Phenotype Correction of Hemophilia A Mice with Adeno-Associated Virus (AAV) Vectors Carrying the B Domain Deleted Canine Factor VIII Gene.

Hemophilia A is a life-threatening bleeding disorder caused by mutations in the factor VIII (FVIII) gene that lead to deficiency of FVIII. Gene therapy is respected to provide an alternative to current FVIII supplemental therapy. Among a variety of vectors, AAV vectors are thought to be ideal for transfer of therapeutic genes since they are derived from non-pathogenic virus and have been demonstrated to provide sustained transgene expression in non-dividing cells with little toxicity, though, delivery of the FVIII gene using AAV vectors are limited by its small packaging capacity. To overcome the packaging capacity limit, we developed AAV1 vectors and AAV8 vectors carrying the B domain deleted canine FVIII gene (BDD cFVIII) utilizing a minimum promoter (150b) and expressed canine FVIII in hemophilia A mice. Previous reports suggested AAV1 serotype is suitable for transduction of skeletal muscles and AAV8 serotype is superior to other AAV serotypes for transduction of the liver, thus, AAV1 vectors carrying the BDD cFVIII gene (AAV1 cFVIII) were injected to the skeletal muscles of hemophilia A mice. Since the liver could be transduced with intravenously injected AAV8 vectors carrying the Lac Z gene as efficiently as intraportally injected vectors, AAV8 vectors carrying the BDD cFVIII gene (AAV8 cFVIII) were injected to hemophilia A mice intravenously. FVIII clotting activities measured by the APTT method increased in mice injected with AAV1cFVIII in a dose dependent manner. The FVIII activity level in peripheral blood increased to 2.9±1.0% in hemophilia A mice with the AAV1cFVIII dose at 1x1012 gc/body, suggesting partial correction of phenotype with AAV1cFVIII vectors. The FVIII clotting activity levels in hemophilia A mice injected with AAV8cFVIII increased also in a dose dependent manner, achieving supernormal FVIII levels (436±219 %) in hemophilia A mice with the AAV8 cFVIII dose at 1x1012 gc/body. It is apparent that transduction of the liver with AAV8cFVIII is superior to transduction of skeletal muscles with AAV1cFVIII regarding FVIII production, however, AAV1 vectors have advantages of removing the transgenes in case of inconvenience and of less systemic distribution of vectors. These data suggested that both AAV1 and AAV8 vectors carrying the FVIII gene utilizing a minimum promoter have a potential for hemophilia A gene therapy.

Confirmation of a New Statistical Method for Comprehensively Dissecting QTLs Using In Vitro Transcription, the Model Factor VII Gene and a Novel Modification of the G-Free Cassette.

Thrombosis is a genetically complex disorder that plays a prominent pathogenic role in myocardial infarction, stroke and pulmonary embolism. Because these distinct endpoints of cardiovascular disease are the most common cause of death worldwide, the goal of investigators in several countries is to comprehensively catalogue human DNA polymorphisms influencing thrombosis susceptibility. It is now generally accepted that accomplishing this goal will require the use of linkage analysis and individuals from extended-pedigrees, even though previous investigations -- which predominantly were based on association analyses and case/control candidate gene studies -- resulted in several notable discoveries including the factor V Leiden mutation. In this regard, we recently completed the first genome linkage screen for DNA regions affecting normal variance of factor VII activity (fVII:C) levels in the 21 families from the Genetic Analysis of Idiopathic Thrombophilia (GAIT) Project. Only one quantitative trait locus (QTL) (LOD score=3.2; p-value=0.022) was identified and localized to the region of chr13q containing the fVII gene, F7. We performed the first comprehensive variation scan of this locus by resequencing the F7 genes in 40 unrelated GAIT subjects. The 49 polymorphisms identified were then genotyped in the full GAIT Project. Using a newly developed BQTN (Bayesian Quantitative Trait Nucleotide) method to estimate the posterior probability of a given variant being functional, we identified strong evidence for the functionality of 2 rare variants, including a novel SNP in intron 7 (C9830T) and a previously known SNP in the coding region of exon 8 (A294V). We also found 2 clusters of promoter (P) and intron 1 (I1) polymorphisms, designated C1: {A-630C, G-402A, T700C, G707A} and C2 {C-2989A, G-401T, -323ins0/10, T-122C, G73A, T835C}, within which all variants exhibit strong linkage disequilibrium (LD) and posterior probabilities of >90% for affecting fVII:C levels. In this study, we will use in vitro transcription to test the statistical findings of the BQTN analysis and, if confirmed, to specifically delineate the functional variant(s) in each cluster. We are creating G-free cassette (GfC) reporter constructs representing the natural P/I1 haplotypes statistically predicted as functional and 10 pairs of constructs where each pair represents the 2 alleles of each of the 10 variants in C1 and C2. All constructs have the same size P (0.9kb) and I1 (1kb) fragments ligated 5′ and 3′ of the GfC, respectively. Because C1 and C2 contain P and I1 variants, a new 120bp GfC was made to match the size of exon 1a and to approximate the native spacing of these polymorphisms in vivo. Once these constructs are sequenced, we will measure and compare their rate of transcriptional initiation using mouse liver nuclear extracts. Because of the strong LD among these P and I1 polymorphisms, population studies alone essentially only analyze the natural haplotypes that are present. The in vitro assays developed in this study will allow the specific influence of each polymorphism to be investigated in the context of a near native configuration of these known regulatory regions.

Influence of polymorphisms in the factor VII gene promoter on activated factor VII levels and on the risk of myocardial infarction in advanced coronary atherosclerosis.

In this study, we investigate the influence of three factor VII (FVII) gene polymorphisms on activated FVII levels (FVIIa), and also on the risk of myocardial infarction (MI) in patients with advanced coronary atherosclerotic disease (CAD). The -323A2 allele in the promoter is known to be associated with low FVII levels, and has been suggested to protect against MI in some studies. The -402GA promoter polymorphism, that in vitro has been associated with having opposite effect, is less well studied clinically. For this study, plasma FVIIa levels and three FVII gene polymorphisms were assessed in 934 subjects of both sexes, all with an angiographic documentation of coronary vessels. Our results show that two promoter polymorphisms, plasma cholesterol, and gender, were significant predictors of FVIIa levels. The -402A allele was associated to a significant increase of FVIIa levels in males (by 19.2%). In a selected clinical model including the patients with severe CAD, with or without a thrombotic complication (MI), male carriers of the -402A had an increased risk of MI (OR=1.79; 95% CI 1.15-2.80). The -323A2 allele was associated to a significant decrease in FVIIa (by 36.02% in males, and 39.7% in females). Male carriers of the -323A2 were protected from MI (OR=0.6; 95% CI 0.39-0.94), but only after correction for the confounding effect of combined heterozygosity for the promoter polymorphisms. We can conclude that FVII gene polymorphisms with an opposite effect on FVIIa levels may modulate the risk of MI in males with advanced CAD. This study highlights a "within-gene" interaction, and the need to explore polymorphisms in candidate gene(s) in detail.