The genetics of Canadian type 3 von Willebrand disease: further evidence for co-dominant inheritance of mutant alleles.

BACKGROUND: Type 3 von Willebrand disease (VWD) is the most severe form of the disease and is classically inherited in an autosomal recessive fashion. OBJECTIVES: The aim of the current study was to investigate the molecular pathogenesis of a Canadian cohort of type 3 VWD patients. PATIENTS AND METHODS: Thirty-four families comprised of 100 individuals were investigated. Phenotypic data, including bleeding scores (BS), von Willebrand factor (VWF) laboratory values and anti-VWF inhibitor status were included as well as sequence analysis. RESULTS: We identified 31 different mutations (20 novel): 8 frameshift, 5 splice site, 9 nonsense, 1 gene conversion, 6 missense and 2 partial gene deletion mutations. The majority of mutations identified were in the propeptide (42%); index cases (IC) with these mutations exhibited more severe bleeding (BS = 22) than those with mutations elsewhere in VWF (BS = 13). Sixty-two out of 68 (91%) mutant alleles were identified. Twenty-nine IC (85%) had a VWF null genotype identified; 17 homozygous, 12 compound heterozygous. In five IC (15%), two mutant VWF alleles were not identified to explain the type 3 VWD phenotype. In four ICs only one mutant VWF allele was identified and in one IC no mutant VWF alleles were identified. CONCLUSIONS: We have investigated the molecular pathogenesis of a Canadian cohort of type 3 VWD patients. Obligate carriers are not phenotypically silent in the Canadian population; 48% have been diagnosed with type 1 VWD. In approximately 50% of families in this study the inheritance pattern for type 3 VWD is co-dominant and not recessive.

Genetic linkage and association analysis in type 1 von Willebrand disease: results from the Canadian type 1 VWD study.

BACKGROUND: von Willebrand disease (VWD) is the most common bleeding disorder known in humans, with type 1 VWD representing the majority of cases. Unlike the other variant forms of VWD, type 1 disease represents a complex genetic trait, influenced by both genetic and environmental factors. AIM: To evaluate the contribution of the von Willebrand factor (VWF) and ABO blood group loci to the type 1 VWD phenotype, and to assess the potential for locus heterogeneity in this condition, we have performed genetic linkage and association studies on a large, unselected type 1 VWD population. METHOD: We initially collected samples from 194 Canadian type 1 VWD families for analysis. After the exclusion of families found to have either type 2 or type 3 VWD, and pedigrees with samples from single generations, linkage and association analysis was performed on 155 type 1 VWD families. RESULTS AND CONCLUSION: The linkage study has shown a low heterogeneity LOD score of 2.13 with the proportion of families linked to the VWF gene estimated to be 0.41. Linkage was not detected to the ABO locus in this type 1 VWD population. In the family-based association test, significant association was found between the type 1 VWD phenotype, the quantitative traits, VWF:Ag, VWF:RCo, and FVIII:C and the ABO 'O' and 'A' alleles and the VWF codon 1584 variant. There was also weak association with the -1185 promoter polymorphism and VWF:Ag, VWF:RCo, and FVIII:C plasma levels. These studies provide further evidence to support the role for genetic loci other than VWF and ABO in the pathogenesis of type 1 VWD.

Factors influencing therapeutic efficacy and the host immune response to helper-dependent adenoviral gene therapy in hemophilia A mice.

BACKGROUND: Adenoviral-based methods of gene therapy have been ineffective at providing sustained factor (F)VIII expression in outbred populations of large animal hemophilic models primarily due to the immunogenicity of these vectors. Improvements have been made in vector design leading to the development of the helper-dependent adenoviral (HD) system. Unfortunately, it remains unclear whether these modifications are sufficient to circumvent the induction of inhibitor formation associated with adenoviral gene transfer. OBJECTIVE: To develop an HD vector capable of mediating sustained FVIII expression and to determine the variables that influence inhibitor development. METHODS: HD vectors were constructed encoding the canine FVIII B-domain deleted transgene under the control of either the cytomegalovirus (CMV) promoter or a tissue-restricted hybrid element consisting of five HNF-1 binding sites, located upstream of the human FVIII proximal promoter. Inbred and outbred populations of hemophilic mice were treated, and monitored for vector-induced toxicity, therapeutic efficacy, and inhibitor formation. RESULTS: When HD vectors utilizing the CMV promoter were administered, all hemophilic mice developed high levels of FVIII inhibitors. In contrast, vectors under the control of the HNF/FVIII element were capable of achieving sustained elevations of FVIII for over 6 months. Strain-specific differences were also observed, with outbred animals showing a greater propensity towards inhibitor development in response to treatment. CONCLUSIONS: HD vectors can be used to provide long-term FVIII expression in hemophilic animals, but treatment outcome and the induction of inhibitors is dependent on a number of variables including the transgene promoter, the vector dose, and the genetic background of the host.

Systemic delivery of an adenoviral vector encoding canine factor VIII results in short-term phenotypic correction, inhibitor development, and biphasic liver toxicity in hemophilia A dogs.

Canine hemophilia A closely mimics the human disease and has been used previously in the development of factor VIII (FVIII) protein replacement products. FVIII-deficient dogs were studied to evaluate an in vivo gene therapy approach using an E1/E2a/E3-deficient adenoviral vector encoding canine FVIII. Results demonstrated a high level of expression of the canine protein and complete phenotypic correction of the coagulation defect in all 4 treated animals. However, FVIII expression was short-term, lasting 5 to 10 days following vector infusion. All 4 dogs displayed a biphasic liver toxicity, a transient drop in platelets, and development of anticanine FVIII antibody. Canine FVIII inhibitor development was transient in 2 of the 4 treated animals. These data demonstrate that systemic delivery of attenuated adenoviral vectors resulted in liver toxicity and hematologic changes. Therefore, the development of further attenuated adenoviral vectors encoding canine FVIII will be required to improve vector safety and reduce the risk of immunologic sequelae, and may allow achievement of sustained phenotypic correction of canine hemophilia A.

The Factor VIII acute phase response requires the participation of NFkappaB and C/EBP.

Coagulation Factor VIII is an acute phase protein in humans that has recently been shown to be transcriptionally responsive to interleukin-6. In this study, we have demonstrated that the human Factor VIII promoter is activated in cultured hepatocytes exposed to bacterial lipopolysaccharide (LPS). Deletion analysis has narrowed the LPS-responsive element of the Factor VIII promoter to a small region which contains two C/EBP binding sites and an adjacent NFkappaB binding site. Mutation of the downstream C/EBP site reduces LPS-responsiveness by approximately 50%, while mutation of the NFkappaB binding site completely eliminates LPS-responsiveness. While binding of C/EBPbeta and NFkappaB is still observed in gel retardation studies using acute phase nuclear extracts and a probe containing mutations to the downstream C/EBP site, neither NFkappaB nor C/EBP appear to bind to a probe in which the NFkappaB site has been mutated. Conservation of this region of the Factor VIII promoter in species which exhibit an increase in Factor VIII levels in response to inflammatory stimuli suggests that these transcription factor binding sites are important for normal regulation of the Factor VIII gene under conditions of stress.

In vivo evaluation of an adenoviral vector encoding canine factor VIII: high-level, sustained expression in hemophiliac mice.

Hemophilia A is the most common severe hereditary coagulation disorder and is caused by a deficiency in blood clotting factor VIII (FVIII). Canine hemophilia A represents an excellent large animal model that closely mimicks the human disease. In previous studies, treatment of hemophiliac dogs with an adenoviral vector encoding human FVIII resulted in complete correction of the coagulation defect and high-level FVIII expression [Connelly et al. (1996). Blood 88, 3846]. However, FVIII expression was short term, limited by a strong antibody response directed against the human protein. Human FVIII is highly immunogenic in dogs, whereas the canine protein is significantly less immunogenic. Therefore, sustained phenotypic correction of canine hemophilia A may require the expression of the canine protein. In this work, we have isolated the canine FVIII cDNA and generated an adenoviral vector encoding canine FVIII. We demonstrate expression of canine FVIII in hemophiliac mice at levels 10-fold higher than those of the human protein expressed from an analogous vector. Canine FVIII expression was sustained above human therapeutic levels (50 mU/ml) for at least 1 year in hemophiliac mice.

Changes in the pattern of distribution of von Willebrand factor in rat aortic endothelial cells following thrombin generation in vivo.

The pattern of distribution of von Willebrand factor (VWF) in relatively large sheets of rat aortic endothelial cells (EC) obtained by the Häutchen technique were analysed by immunocytochemistry and light microscopy. EC were examined pre and post administration of a procoagulant mixture of factor Xa (F.Xa) and phosphotidylcholine/phosphotidylserine (PCPS) vesicles which was demonstrated to result in the selective loss of high molecular weight multimers (HMWM) of plasma VWF in the rat. In placebo animals the pattern was heterogenous both in overall distribution and in individual cells which showed both a diffuse and granular pattern. Groups of intensely stained EC were oriented parallel to the longitudinal axis of the aorta and staining was particularly prominent around the orifices of the intercostal arteries, implicating shear-stress as a possible factor in VWF expression by EC. Changes in the pattern of distribution of staining were observed at various time points post-infusion of F.Xa/PCPS, suggesting the immediate release of VWF from EC stores followed by the recruitment of EC to synthesize and store VWF. These changes are consistent with the decrease in EC Weibel-Palade Body (WPB) content observed by EM in previously reported studies using this model.

Morphological alterations in endothelial cells associated with the release of von Willebrand factor after thrombin generation in vivo.

von Willebrand factor (vWF) is synthesized by endothelial cells and stored in endothelium-specific granules, the Weibel-Palade (WP) bodies. The release of vWF from endothelial cells in vitro in response to secretagogues such as thrombin is considered to result in the loss of WP bodies through the fusion of the WP bodies with the plasma membrane. Biochemical and morphological techniques, including transmission (TEM) and scanning (SEM) electron microscopy, were used to examine the plasma profile of vWF in parallel with morphological alterations in endothelial cells associated with the generation of thrombin in vivo. There was a rapid loss of high-molecular-weight multimers of the circulating vWF, with full recovery within 1 hour. Simultaneously, TEM demonstrated that the endothelial cells lost WP bodies and became severely vacuolated; this was associated with the appearance of craters in the endothelial surface on SEM. Release of stored vWF in WP bodies seemed to follow the fusion of multiple rather than individual WP bodies, with the resulting vacuole fusing and rupturing through the plasmatic membrane. Within 1 hour there was increased morphological evidence of metabolic organelle activity associated with replacement of WP bodies, presumably due to de novo synthesis of the basic protomer and its packaging in high-molecular-weight multimeric form in the storage organelles.

The development of homologous (canine/anti-canine) antibodies in dogs with haemophilia A (factor VIII deficiency): a ten-year longitudinal study.

The development of inhibitors to factor VIII in patients with haemophilia A remains as a serious complication of replacement therapy. An apparently analogous condition has been described in a canine model of haemophilia A (Giles et al., Blood 1984; 63: 451). These animals and their relatives have now been followed for 10 years. The observation that the propensity for inhibitor development was not related to the ancestral factor VIII gene has been confirmed by the demonstration of vertical transmission through three generations of the segment of the family related to a normal (non-carrier) female that was introduced for breeding purposes. Haemophilic animals unrelated to this animal have not developed functionally significant factor VIII inhibitors despite intensive factor VIII replacement. Two animals have shown occasional laboratory evidence of factor VIII inhibition but this has not been translated into clinical significant inhibition in vivo as assessed by clinical response and F.VIII recovery and survival characteristics. Substantial heterogeneity of inhibitor expression both in vitro and in vivo has been observed between animals and in individual animals over time. Spontaneous loss of inhibitors has been observed without any therapies designed to induce tolerance, etc., being instituted. There is also phenotypic evidence of polyclonality of the immune response with variable expression over time in a given animal. These observations may have relevance to the human condition both in determining the pathogenetic factors involved in this condition and in highlighting the heterogeneity of its expression which suggests the need for caution in the interpretation of the outcome of interventions designed to modulate inhibitor activity.

In vivo characterization of recombinant factor VIII in a canine model of hemophilia A (factor VIII deficiency).

Infusion studies of recombinant factor VIII were performed in hemophilic (factor VIII-deficient) animals. Functional activity was determined using a standardized model of bleeding and kinetic characteristics charted by performing assays of factor VIII functional and antigenic activities over time. To obtain an adequate comparison with plasma-derived factor VIII, a crossover study in two animals was performed in which each animal received either recombinant factor VIII or a highly purified plasma-derived factor VIII on day 1 and the alternative three days later (day 4). Both factor VIII preparations were functionally effective with complete correction of the cuticle bleeding time occurring one hour after infusion. The observed recovery was full and close to predicted for both preparations. The survival curves obtained for both functional and antigenic activities for both preparations were virtually identical and within the anticipated range determined from previous experiments using infusions of conventional factor VIII preparations. The plasma half-disappearance time (T 1/2) for recombinant factor VIII and plasma-derived factor VIII was 9.2 and 7.9 hours, respectively. Plasma samples obtained following infusion were subjected to chromatography on Sepharose 4B. The elution profile of factor VIII antigen activity was compared with that obtained with the infusates. A clear shift in profile was observed with the plasma samples, suggesting complexing of the infused factor VIII material with circulating canine von Willebrand factor (vWF). The elution profile of vWF antigen was superimposable, thus providing further evidence in support of this assumption. The study provided evidence that recombinant factor VIII possesses full functional activity in vivo, binds to circulating vWF, and exhibits normal recovery and survival characteristics.

In vivo studies of the role of factor VII in hemostasis.

The effect of both congenital and acquired factor VII deficiency on the cuticle bleeding time (CBT) was evaluated in dogs. The CBT has been previously documented to be a sensitive indicator of factor VIII:C deficiency in hemophilic dogs. Serial CBT determinations were made on normal dogs treated with high-dose warfarin. At 48 hours post-treatment, the CBT was normal, although the factor VII level was less than 1%, whereas the levels of factors II, IX, and X were 44%, 25%, and 17%, respectively. At 120 hours the CBT became abnormal when all vitamin K-dependent clotting factors had dropped to less than 18%. Administration of a plasma concentrate of factors II, IX, and X corrected the CBT, despite the factor VII level remaining at less than 1%. Similar studies in a congenitally factor VII-deficient dog (factor VII less than 2%) confirmed that this deficiency state was not associated with an abnormality of the CBT. Administration of heparin to both normal and factor VII-deficient animals was associated with prolongation of the CBT, but the heparin dose required in the normal animals was substantially higher than in the factor VII-deficient animals. These data do not suggest that factor VII/VIIa has an exclusive role in generating factor Xa, either directly or indirectly, by way of factor IXa generation, in vivo. However, the increase in heparin sensitivity of the factor VII-deficient animals does suggest that factor VII/VIIa may, in some circumstances, present a significant alternative pathway of factor X activation, although the activation pathway involved cannot be determined from the studies performed.

A platelet release defect induced by aspirin or penicillin G does not increase gastrointestinal blood loss in thrombocytopenic rabbits.

Gastrointestinal blood loss was compared in groups of normal and thrombocytopenic animals treated with medications known to induce qualitative platelet dysfunction. Thrombocytopenia was induced in rabbits by the intraperitoneal injection of busulphan dissolved in polyethylene glycol (PEG) at a dose of 60 mg/kg. Control animals received PEG alone; each group subsequently received daily intravenous injections of penicillin G, aspirin, sodium salicylate or isotonic saline. Mean daily gastrointestinal blood loss was determined by monitoring the appearance of 51Cr radioactivity in the faeces following the administration of 51Cr-labelled erythrocytes prior to the administration of the test and control therapies. The administration of penicillin G was not associated with increased gastrointestinal blood loss in the thrombocytopenic animals as compared with the saline treated thrombocytopenic controls. Platelet aggregation studies confirmed the presence of a mild but significant defect in platelet aggregation. Aspirin produced a more pronounced defect in platelet aggregation but did not induce increased bleeding in the normal animals as compared with the controls, nor did it exacerbate the bleeding in thrombocytopenic animals. Sodium salicylate did not produce an aggregation defect and did not significantly modify gastrointestinal blood loss. It was concluded that drug-induced qualitative platelet dysfunction does not necessarily increase bleeding through intact vessels despite previous evidence of a significant effect on platelet plug formation as monitored by the bleeding time.

Development of factor VIII:C antibodies in dogs with hemophilia A (factor VIII:C deficiency).

Classic hemophilia A (factor VIII:C deficiency) was diagnosed in a miniature Schnauzer dog and a breeding program established. Inbreeding and crossbreeding produced 16 hemophilic animals. All were initially treated with canine cryoprecipitate, as required, for sporadic hemorrhagic events. Five animals developed potent antibodies to canine factor VIII:C. All were the offspring of obligate carriers, resulting from the mating of a hemophilic purebred miniature Schnauzer male to a normal female Brittany spaniel. The mean age at first treatment and factor VIII exposure at the time of inhibitor development was 10.3 wk and 286.3 U, respectively. The remaining hemophilic animals have not developed antibodies, despite receiving a mean factor VIII dosage of 1.5 X 10(3) U. This group includes animals derived from a mating between the same purebred miniature Schnauzer hemophilic male and a purebred miniature Schnauzer carrier female. In each case, the antibodies recognize both canine and human but not porcine VIII:C. They are non-precipitating IgG immunoglobulins. Following inhibitor development, infusion of canine cryoprecipitate was hemostatically ineffective and factor VIII:C recovery at 30 min was negligible. Infusion of a concentrate of porcine factor VIII resulted in a correction of the hemostatic defect and optimal factor VIII:C recovery. All animals receiving porcine factor VIII:C subsequently developed antibodies to this protein. The chance occurrence of this complication should facilitate further studies directed at elucidating the pathogenesis and management of hemophilia complicated by the development of antibodies to factor VIII:C.

A canine model of hemophilic (factor VIII:C deficiency) bleeding.

A model of bleeding due to clotting factor deficiency has been developed in dogs. Normal and hemophilic (factor VIII:C deficient) animals were used. Bleeding was induced in lightly anesthetized animals by severing the apex of the nail cuticle using a guillotine device. In normal animals, bleeding usually ceased spontaneously after 2-8 min. In contrast, in hemophilic animals, bleeding continued for up to 20 min and necessitated either cauterization or the application of topical thrombin to achieve hemostasis. Pretreatment of the hemophilic animals with canine cryoprecipitate corrected the cuticle bleeding time to within the range noted for normal animals. The method is simple and reproducible and has the advantage that a number of observations can be made sequentially on the same animal. Rebleeding of the cauterized cuticle of the hemophilic animals did not usually occur. This model has considerable potential for the preclinical testing of products considered to bypass or replace factor VIII:C in patients with acquired inhibitors of factor VIII:C and may be adapted to the study of other mechanisms involved in normal and abnormal hemostasis.