Low Factor VIII Levels Research Articles

Retrospective Audit of a Factor VIII Continuous Infusion Protocol to Examine Inhibitor Formation, Complications and Product Use.

Continuous infusion (CI) of factor concentrates has been suggested to decrease both the risk of bleeding and cost in the treatment of bleeding disorders. However, concerns have been raised with regards to the stability and sterility of products administered by CI and the risk of local thrombophlebitis. Most recently, there has been a concern about a potential association between the use of CI and the development of an inhibitor in mild hemophilia. A retrospective chart review was conducted to determine the effectiveness, safety, product use and risk of inhibitor formation of a continuous infusion protocol in the management of hemophilia A. Currently 248 patients are registered with the Provincial Hemophilia Program for Newfoundland and Labrador. These patients include 104 with mild hemophilia A, 3 with moderate hemophilia A and 5 with severe hemophilia A. The charts of twelve hemophilia A patients who received factor VIII (FVIII) by CI a total of 18 times between April 1998 and September 2003 were reviewed. All patients were affected by hemophilia A; 8 patients were diagnosed as mild, 1 as moderate and 3 as severe. The mean age of the cohort was 36.1 years and the patients' ages ranged from 4 months to 75 years. Continuous infusion was used as treatment for severe bleeds in 12 cases and as prophylaxis for surgical procedures in 6 cases. All patients received recombinant FVIII. Recommended devices such as a syringe pump and mini-pump were unavailable therefore all infusions were carried out by diluting the reconstituted concentrate in saline and delivering via IV infusion. The CI protocol followed required a bolus of 50 U/kg of FVIII followed by CI at an initial rate of 4 U/kg/hr. Based on daily FVIII levels, this maintenance dose could be adjusted, with an initial targeted FVIII level of 1.0 U/L. The maximum initial infusion rate was 6.7 U/kg/hr and the minimum was 3.2 U/kg/hr (mean 4.3 U/kg/hr). The infusion rate was reduced in 8 cases to a minimum of 1.2 U/kg/hr. Adequate FVIII levels were maintained after the reduction of the infusion rate in all cases. The mean low and high FVIII levels were 0.79 U/L and 1.23 U/L, respectively (range 0.05 to 1.82 U/L). Excluding the patient who developed an inhibitor, the FVIII level remained above the minimum acceptable level of 0.3 U/L for the duration of CI in all but one case (low 0.29 U/L). Patients required an average dose of 53,163 units (range 2651–299,137 units) of factor concentrate delivered by continuous infusion over a mean period of 8.1 days (range 2–21 days). FVIII infusion was well tolerated in all cases; there were no documented cases of significant bleeding, adverse reactions, thrombophlebitis or infection. One patient was documented to have a minor bleed from an NG tube despite adequate FVIII levels throughout the period of infusion. One patient with mild hemophilia developed a low titer inhibitor (0.8 BU) approximately 2 years after receiving FVIII by continuous infusion for 8 days. Another mild hemophilia patient was discovered to have a low titer inhibitor (1.3 BU) on the eighth day after the third infusion had begun. It is possible that CI contributed to the development of an inhibitor in this patient. CI was not found to significantly reduce product use. More rapid downward adjustment of infusion rates in response to high FVIII levels might improve product utilization.

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.

Recombinant factor VIII expression in hematopoietic cells following lentiviral transduction.

Autologous transplantation of gene-modified hematopoietic stem cells may provide a therapeutic strategy for several monogeneic disorders. In previous studies, retroviral gene transfer of coagulation factor VIII (FVIII) into FVIII(-/-) mouse bone marrow (BM) cells did not result in detectable plasma FVIII levels. However, specific immune tolerance was achieved against neo-antigenic FVIII. Here, we used lentiviral vectors to study the ability of various hematopoietic cell types to synthesize and secrete recombinant FVIII. Several myeloid, monocytic and megakaryocytic cell lines (K-562, TF-1, Monomac-1, Mutz-3, Meg-01) expressed FVIII at 2-12 mU/10(4) cells. In contrast, two lymphatic cell lines, BV-173 and Molt-4, were less-efficiently transduced and did not express detectable FVIII. Similarly, peripheral blood-derived primary monocytes were transduced efficiently and expressed up to 20 mU/10(4) cells, whereas primary lymphocytes did not express FVIII. Although human and canine CD34(+) cells were transduced efficiently, the cells expressed very low levels of FVIII (up to 0.8 mU/10(4) cells). Following xenotransplantation of transduced CD34(+) into NOD/SCID mice, ELISA failed to detect FVIII in the plasma of engrafted mice. However, NOD/SCID repopulating cell (SRC)-derived human monocytes isolated from BM of these mice secreted functional recombinant FVIII after culture ex vivo. Again, SRC-derived human lymphocytes did not secrete FVIII. Therefore, certain hematopoietic cell types are able to synthesize and secrete functional recombinant FVIII. Our results show for the first time that transplantation of transduced CD34(+) progenitors may give rise to differentiated hematopoietic cells secreting a nonhematopoietic recombinant protein.

Efficient production of human FVIII in hemophilic mice using lentiviral vectors

Lentiviral vectors (LV) have the ability to integrate their proviral DNA containing a therapeutic gene into the host cell's genome. Therefore, these vectors have a great potential for gene therapy especially in the treatment of hereditary diseases like hemophilia A, which require lifelong expression of the transgene. We constructed an HIV-1-based LV containing human B-domain-deleted factor VIII (FVIII) cDNA under the control of a promoter consisting of the chicken β-actin promoter, CMV enhancers, and a large synthetic intron (CAG), which is a robust transcription promoter. High levels of FVIII expression from this vector could be demonstrated in vitro in 293T cells, primary liver cells, and hematopoietic progenitor cells. To test whether this viral vector was able to correct the bleeding disorder of C57BL/6 FVIII knockout mice, we transduced these mice with the FVIII LV either by intraperitoneal injection or by transplantation with transduced syngeneic bone marrow. FVIII production was analyzed in the blood plasma for a period of 3 months; however, only low levels of FVIII (<50 mU), which were below 5% of normal FVIII levels of 1000 mU, could be detected. Further analysis revealed that the low levels of FVIII activity present in the blood plasma were due to the presence of neutralizing antibodies to FVIII and not due to lack of expression of FVIII from the viral vector. FVIII expression could be detected in the tissues of the transduced mice by Western blot analysis and in ex vivo cultures. These data demonstrate that LVs are able to produce therapeutic levels of FVIII in knockout mice when administered by ip infection or by transduced hematopoietic cells. The challenge is to overcome the immune barriers to the therapeutic gene product.

Modulation of factor VII levels by intron 7 polymorphisms: population and in vitro studies

AbstractPrevious studies have established that factor VII gene (F7) polymorphisms (5′F7 and R353Q) contribute about one-third of factor VII (FVII) level variation in plasma. However, F7 genotyping in patients with cardiovascular disease has produced conflicting results. Population and expression studies were used to investigate the role of intron 7 (IVS7 ) polymorphisms, including repeat and sequence variations, in controlling activated FVII (FVIIa) and antigen (FVIIag) levels. Genotype–phenotype studies performed in 438 Italian subjects suggested a positive relation between the IVS7 repeat number and FVII levels. The lowest values were associated with theIVS7 + 7G allele. The screening of 52 patients with mild FVII deficiency showed an 8-fold increase in frequency (8%) of this allele, and among heterozygotes for identical mutations, lower FVII levels were observed in the IVS7 + 7G carriers. This frequent genetic component participates in the phenotypic heterogeneity of FVII deficiency. The evaluation of the individual contribution of polymorphisms was assisted by the expression of each IVS7variant, as a minigene, in eukaryotic cells. The novel quantitative analysis revealed that higher numbers of repeats were associated with higher mRNA expression levels and that the IVS7 + 7Gallele, previously defined as a functionally silent polymorphism, was responsible for the lowest relative mRNA expression. Taken together, these findings indicate that the IVS7 polymorphisms contribute to the plasmatic variance of FVII levels via differential efficiency of mRNA splicing. These studies provide further elements to understand the control of FVII levels, which could be of importance to ensure the hemostatic balance under pathologic conditions.

Modulation of factor VII levels by intron 7 polymorphisms: population and in vitro studies

Previous studies have established that factor VII gene (F7) polymorphisms (5′F7 and R353Q) contribute about one-third of factor VII (FVII) level variation in plasma. However, F7 genotyping in patients with cardiovascular disease has produced conflicting results. Population and expression studies were used to investigate the role of intron 7 (IVS7 ) polymorphisms, including repeat and sequence variations, in controlling activated FVII (FVIIa) and antigen (FVIIag) levels. Genotype–phenotype studies performed in 438 Italian subjects suggested a positive relation between the IVS7 repeat number and FVII levels. The lowest values were associated with theIVS7 + 7G allele. The screening of 52 patients with mild FVII deficiency showed an 8-fold increase in frequency (8%) of this allele, and among heterozygotes for identical mutations, lower FVII levels were observed in the IVS7 + 7G carriers. This frequent genetic component participates in the phenotypic heterogeneity of FVII deficiency. The evaluation of the individual contribution of polymorphisms was assisted by the expression of each IVS7variant, as a minigene, in eukaryotic cells. The novel quantitative analysis revealed that higher numbers of repeats were associated with higher mRNA expression levels and that the IVS7 + 7Gallele, previously defined as a functionally silent polymorphism, was responsible for the lowest relative mRNA expression. Taken together, these findings indicate that the IVS7 polymorphisms contribute to the plasmatic variance of FVII levels via differential efficiency of mRNA splicing. These studies provide further elements to understand the control of FVII levels, which could be of importance to ensure the hemostatic balance under pathologic conditions.

Recombinant factor VIIa corrects prothrombin time in cirrhotic patients: A preliminary study

BACKGROUND & AIMS: Cirrhotic patients with a prolonged prothrombin time (PT) are known to have low levels of factor VII. Because the current modalities to correct this problem are not ideal, recombinant factor VIIa (rFVIIa) may be useful in correcting the prolonged PT observed in the coagulopathy of cirrhosis. The aim of this study was to evaluate the effectiveness of rFVIIa in nonbleeding volunteer patients with the coagulopathy of cirrhosis. METHODS: A preliminary, single-center, dose- escalation trial was performed. Cirrhotic patients with a PT of > 2 seconds above the upper limit of the reference value received an intramuscular injection of vitamin K. Ten patients whose PT did not correct to within 2 seconds above the control of the upper limit of the reference value were given three successive dosages of rFVIIa (5, 20, and 80 micrograms/kg) during a 3-week period. RESULTS: The mean PT transiently corrected to normal in all three dosage groups. No adverse effects were noted. There was no evidence of the induction of disseminated intravascular coagulation. CONCLUSIONS: This preliminary trial shows rFVIIa to be effective in transiently reversing the prolonged PT in a select group of nonbleeding cirrhotic patients. These preliminary observations support conducting a large-scale efficacy trial. (Gastroenterology 1997 Dec;113(6):1930-7)

Acquired von Willebrand factor deficiency during high-dose infusion of recombinant factor VIII.

Constant infusion of factor VIII (FVIII) into patients with haemophilia A after major surgery has been recommended as optimal treatment to avoid peaks and valleys in the circulating levels of FVIII and to allow the use of much lower doses of FVIII than are historically required. One of our young patients with severe (< 0.01 U/ml FVIII) haemophilia suffered a subdural haematoma for which he received treatment with 815190 recombinant FVIII (rFVIII) units over a period of 52d. 2 weeks after admission, because of low FVIII levels and the presence of FVIII inhibitors, the infusion rate was increased to > 100 U/kg/h for 14d. During this time the FVIII level fluctuated between 0.6 and 4.2 U/ml. For some period it was not possible to detect ristocetin co-factor activity in this patient's plasma and the von Willebrand factor (VWF) level and VWF multimer pattern resembled those of a patient with von Willebrand's disease. Subsequently, when the rFVIII dose was increased 2-fold, this was not reflected by the plasma level of FVIII although antibodies were not detected. The data suggest that the prolonged infusion of very high levels of rFVIII which is deficient in von Willebrand factor can result in depletion of VWF from existing stores, producing a laboratory picture which is consistent with the diagnosis of von Willebrand's disease. Further, in the absence of complexing with VWF, FVIII appears to be cleared from the circulation at an increased rate. This is expensive and potentially compromising. Therefore, when administering very high doses of FVIII concentrates devoid of VWF for prolonged periods of time, ristocetin cofactor and VWF levels should be monitored.

Characterisation of Type 2N von Willebrand Disease Using Phenotypic and Molecular Techniques

von Willebrand factor (vWF) is a multimeric glycoprotein found in plasma non covalently linked to factor VIII (FVIII). Type 2N von Willebrand disease (vWD) is caused by a mutation in the vWF gene that results in vWF with a normal multimeric pattern, but with reduced binding to FVIII. We have utilised methods for the phenotypic and genotypic detection of type 2N vWD. The binding of FVIII to vWF in 69 patients, 36 with type 1 vWD, 32 with mild haemophilia A and one possible haemophilia A carrier with low FVIII levels was studied. Of these, six were found to have reduced binding (five type 1 vWD, one possible haemophilia A carrier). DNA was extracted from these patients and exons 18-23 of the vWF gene encoding the FVIII binding region of vWF were analysed. After direct sequencing and chemical cleavage mismatch detection, a Thr28Met mutation was detected in two unrelated individuals, one of whom appears to be a compound heterozygote for the mutation and a null allele. No mutations were found in the region of the vWF gene encoding the FVIII binding region of vWF in the other four patients.

Reduced Thrombus Formation in Native Blood of Homozygous Factor VII-Deficient Patients at High Arterial Wall Shear Rate

Inhibition of thrombin formation in flowing native blood reduces thrombus formation on subendothelium, dacron, or collagen fibrils at arterial wall shear rates of 450 to 650 s-1. In the present study, we have investigated the role of low levels of factor VII (FVII) in thrombus formation on collagen fibrils at arterial wall shear rates of 650 s-1 (coronary arteries), 2,600 s-1 (mildly stenosed arteries), and 10,510 s-1 (severely stenosed arteries) in parallel-plate perfusion chambers. In the perfusion chamber with the highest wall shear rate, thrombus formation took place at the apex of an eccentric stenosis, which reduced the cross-sectional area of the blood flow channel by 80%, thus simulating thrombus formation at an atherosclerotic plaque rupture. Native blood from 21 healthy volunteers and 12 homozygous FVII-deficient patients was drawn by a pump directly from an antecubital vein over a surface of fibrillar collagen positioned in the respective perfusion chambers. The patients had FVII coagulant activities ranging from 1.3% to 4.5% and FVII antigen levels of 16% to 23% of normal. Immunoaffinity purification of the patients' FVII followed by electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) and immunoblotting showed a protein with similar molecular mass as normal FVII. In the perfusion studies, a reduction in thrombus volume of 54% of normal (P < .007) at 10,510 s-1 was observed. The deposition of fibrin on the thrombogenic surface and the plasma level of fibrinopeptide A (FPA) in blood samples collected distal to the perfusion chamber were concomitantly reduced (P < .002 and P < .04, respectively). The plasma FPA level was also reduced at 2,600 s-1 (P < .04), but not at 650 s-1. However, at the lower shear conditions, the thrombus volume and the fibrin deposition were within the ranges observed in normal blood. The platelet-collagen adhesion was not affected at any of the three shear conditions. Thus, low plasma levels of FVII result in significantly less formation of thrombin and fibrin in and around growing platelet masses at high shear condition. This may weaken the thrombus stability and reduce platelet recruitment, thereby lowering thrombus volume. In support of this theory, one patient with afibrinogenemia had an 83% reduction in thrombus volume at this high shear condition.

Reduced thrombus formation in native blood of homozygous factor VII- deficient patients at high arterial wall shear rate

Inhibition of thrombin formation in flowing native blood reduces thrombus formation on subendothelium, dacron, or collagen fibrils at arterial wall shear rates of 450 to 650 s-1. In the present study, we have investigated the role of low levels of factor VII (FVII) in thrombus formation on collagen fibrils at arterial wall shear rates of 650 s-1 (coronary arteries), 2,600 s-1 (mildly stenosed arteries), and 10,510 s-1 (severely stenosed arteries) in parallel-plate perfusion chambers. In the perfusion chamber with the highest wall shear rate, thrombus formation took place at the apex of an eccentric stenosis, which reduced the cross-sectional area of the blood flow channel by 80%, thus simulating thrombus formation at an atherosclerotic plaque rupture. Native blood from 21 healthy volunteers and 12 homozygous FVII- deficient patients was drawn by a pump directly from an antecubital vein over a surface of fibrillar collagen positioned in the respective perfusion chambers. The patients had FVII coagulant activities ranging from 1.3% to 4.5% and FVII antigen levels of 16% to 23% of normal. Immunoaffinity purification of the patients' FVII followed by electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) and immunoblotting showed a protein with similar molecular mass as normal FVII. In the perfusion studies, a reduction in thrombus volume of 54% of normal (P &lt; .007) at 10,510 s-1 was observed. The deposition of fibrin on the thrombogenic surface and the plasma level of fibrinopeptide A (FPA) in blood samples collected distal to the perfusion chamber were concomitantly reduced (P &lt; .002 and P &lt; .04, respectively). The plasma FPA level was also reduced at 2,600 s-1 (P &lt; .04), but not at 650 s-1. However, at the lower shear conditions, the thrombus volume and the fibrin deposition were within the ranges observed in normal blood. The platelet-collagen adhesion was not affected at any of the three shear conditions. Thus, low plasma levels of FVII result in significantly less formation of thrombin and fibrin in and around growing platelet masses at high shear condition. This may weaken the thrombus stability and reduce platelet recruitment, thereby lowering thrombus volume. In support of this theory, one patient with afibrinogenemia had an 83% reduction in thrombus volume at this high shear condition.

Natural antibodies to factor VIII (anti-hemophilic factor) in healthy individuals.

Spontaneous inhibitors of factor VIII (FVIII) are pathogenic IgG autoantibodies of restricted isotypic heterogeneity found in the plasma of patients presenting with bleeding episodes and low levels of FVIII. We now report the presence of a natural FVIII-neutralizing activity in 85 of 500 plasma samples (17%) from healthy donors. FVIII-inhibitory activity was present in F(ab')2 fragments of purified IgG and was dose-dependent. The titer of anti-FVIII antibodies in normal plasma ranged between 0.4 (threshold of detection) and 2.0 Bethesda units. Anti-FVIII IgG was also detected in normal plasma by using an ELISA. Anti-FVIII antibodies from healthy individuals did not exhibit restricted isotypic heterogeneity. Mean levels of FVIII activity did not differ significantly between individuals with and without detectable anti-FVIII antibodies in plasma. Natural anti-FVIII IgG inhibited FVIII activity in pools of normal plasma and in plasma of certain donors in the pool but did not inhibit FVIII activity in autologous plasma. These observations demonstrate that polyclonal IgG antibodies against procoagulant FVIII are present in healthy individuals. The antibodies are natural IgG autoantibodies and/or antibodies directed against epitopes associated with a so far unidentified allotypic polymorphism of the human FVIII molecule.

The use of porcine factor viii in the treatment of patients with acquired hemophilia: The United Kingdom experience

Data have been collected by questionnaire on 15 acute bleeding episodes in 12 patients with acquired hemophilia, treated with porcine factor VIII (FVIII). The median initial anti-human FVIII inhibitor level was 40 Bethesda Units (BU)/ml, whereas that against porcine was 1 BU/ml. The mean initial dose of porcine FVIII infused was 54 micrograms/kg, which resulted in a rise of 0.57 IU/mL in the FVIII concentration. Therapy was continued for a mean of 8.5 days, during which time the average number of infusions was 11. Clinical response was rated as good or excellent in 82% of recipients. Side effects were uncommon; only one patient experienced a severe reaction. Following therapy, no increase in antihuman antibody levels was noted; increased levels of antiporcine antibody was detected in only two patients. One patient bled on three further occasions and was successfully retreated with porcine FVIII. Porcine FVIII is a safe and clinically effective treatment for bleeding episodes in acquired hemophilia and should be considered as first-line therapy for patients with low antiporcine FVIII levels.

Factor VII and ischaemic heart disease: epidemiological evidence.

High levels of factor VII are associated with an increased risk of death from cardiovascular disease, especially ischaemic heart disease (IHD). Theoretical considerations and experimental evidence, the latter including the results of clinical trials, suggest that the association may be one of cause and effect. The general epidemiology of factor VII also supports this view. Thus, characteristics such as increasing age, diabetes, obesity, the use of oral contraceptives and the occurrence of the menopause are each associated with raised factor VII levels as well as with an increased risk of IHD. Black ethnic group and vegetarianism, both apparently protective against IHD, are associated with low factor VII levels.