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Correction of a murine model of von Willebrand disease by gene transfer.

ABSTRACT: von Willebrand disease (VWD), the most common inherited bleeding disorder in the U.S. population, is caused by defects in the expression and processing of von Willebrand factor (VWF), a blood glycoprotein required for normal hemostasis that mediates the adhesion of platelets to sites of vascular damage by binding to specific platelet glycoproteins and to constituents of exposed connective tissue. To assess whether VWF deficiency can be corrected by gene transfer, a plasmid expressing the intact 8.4-kb murine VWF coding sequence, directed by the cyto-megalovirus immediate/early promoter/enhancer, was delivered through hydrodynamic tail vein injection into VWF knockout mice (VWF(-/-)) that exhibit defects in hemostasis, including highly prolonged bleeding time and spontaneous bleeding events, closely mimicking severe human VWD. VWF antigen levels in plasma from animals receiving VWF cDNA, but not control animals, revealed normalized levels of circulating VWF that persisted for at least 1 week after injection. Western blot analysis of plasma from animals receiving VWF cDNA, but not control animals, revealed high molecular-weight multimers with patterns similar to those observed in wild-type mice. Reverse transcription-polymerase chain reaction (RT-PCR) on RNA isolated from the livers of animals receiving VWF cDNA, but not control animals, demonstrated that VWF was expressed in the liver, and immunohistochemical analysis of the livers of treated VWF(-/-) mice revealed VWF-specific staining throughout the liver parenchyma but not in endothelial cells. Plasma from treated VWF(-/-) mice, but not control VWF(-/-) mice, supported the hypothesis that murine platelets aggregate in the presence of botrocetin. Although levels of circulating factor VIII in untreated VWF(-/-) mice were less than 10% those in wild-type mice, levels of factor VIII in VWF(-/-) animals treated with VWF cDNA, but not in control animals, were normalized to values in wild-type mice, indicating the restoration of factor VIII carrier function for VWF in treated mice that persisted for at least 1 week at higher doses of VWF cDNA. Most important, bleeding time was normalized by 48 hours after the delivery of VWF cDNA, but not by the control plasmid. These data suggest that with the use of gene transfer of VWF cDNA, VWF protein can be expressed, processed, and secreted in a physiologically active form; thus, it may be possible to correct VWD using gene transfer.

SUBMITTER: Pergolizzi RG

PROVIDER: S-EPMC1895849 | biostudies-literature | 2006 Aug

REPOSITORIES: biostudies-literature

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Correction of a murine model of von Willebrand disease by gene transfer.

Pergolizzi Robert G RG Jin Guangchun G Chan Diane D Pierre Lorraine L Bussel James J Ferris Barbara B Leopold Philip L PL Crystal Ronald G RG

Blood 20060425 3

von Willebrand disease (VWD), the most common inherited bleeding disorder in the U.S. population, is caused by defects in the expression and processing of von Willebrand factor (VWF), a blood glycoprotein required for normal hemostasis that mediates the adhesion of platelets to sites of vascular damage by binding to specific platelet glycoproteins and to constituents of exposed connective tissue. To assess whether VWF deficiency can be corrected by gene transfer, a plasmid expressing the intact ...[more]

PMID: 16638935

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Project description:Essentials Defective binding to collagen IV has been seen in von Willebrand factor (VWF) A1 domain variants. We developed a murine model of defective VWF-collagen IV interactions with VWF variant p.R1399H. p.1399HH homozygous mice had decreased binding to collagen IV and increased bleeding times. p.1399HH homozygous mice had increased time to thrombosis and decreased platelet adhesion. SUMMARY: Background von Willebrand factor (VWF) binding to type IV collagen occurs via the VWF A1 domain, with p.R1399H being the most common VWF variant affecting this interaction. Objectives We generated a murine model of 1399H VWF to investigate its in vivo effects. Methods Mice expressing the murine 1399H variant were generated via gene targeting in embryonic stem cells. VWF antigen and VWF collagen binding were measured with ELISA. Tail bleeding time assays were performed by clipping a 3-mm segment. Ferric chloride-induced thrombosis was measured via ultrasound in the carotid artery. Platelet aggregation in response to collagens I and IV was measured. VWF-dependent platelet adhesion to collagen IV was measured under flow. Results Breeding of heterozygous p.R1399H and homozygous p.1399HH mice was observed to follow normal Mendelian ratios. No spontaneous bleeding was observed for any of the offspring. VWF expression was normal, but VWF binding to collagen IV was decreased in both heterozygous and homozygous offspring. Blood loss following tail resection was increased for p.1399HH mice, and occlusion times following ferric chloride-induced thrombosis were prolonged. Platelet aggregation was unaffected, but platelet adhesion to collagen IV under flow was diminished for p.1399HH mice. Conclusions These results show that a decrease in the ability of 1399H VWF to bind collagen IV under static conditions corresponds to a decrease in binding under flow conditions, an increased bleeding time, and a prolonged time to thrombosis. This study supports the potential for a bleeding phenotype in patients with aberrant VWF-collagen IV binding.

Project description:Essentials von Willebrand factor (VWF) is synthesized in endothelial cells and platelet precursors. Type 3 patients with Pro2808Leufs*24 have lower bleeding scores than other type 3s. The Pro2808Leufs*24 variant was examined in patient platelets and endothelial cells. Type 3s with this variant contain releaseable VWF, possibly reducing bleeding. SUMMARY:Background A novel variant, p.Pro2808Leufs*24, in the von Willebrand factor (VWF) gene was previously identified in the Canadian von Willebrand disease (VWD) patient population. Clinical observations of type 3 VWD patients with this variant indicate a milder bleeding phenotype compared with other type 3 patients. Objective To assess the effect of the Pro2808Leufs*24 variant on the molecular pathogenesis of VWD and correlate this with the phenotype observed in patients. Patients/Methods Phenotypic data from individuals in the Canadian type 3 VWD study were analyzed. VWF expression in platelets and plasma was assessed via immunoblotting. Cellular expression of VWF in platelets and blood outgrowth endothelial cells (BOEC) was examined via immunofluorescence microscopy and biochemical analysis in a type 3 index case and family member with Pro2808Leufs*24. Results Twenty-six individuals with the Pro2808Leufs*24 variant (16 type 3 VWD homozygous or compound heterozygous and 10 heterozygous family members) were studied. Bleeding scores were lower in type 3 patients with Pro2808Leufs*24 compared with type 3 patients with other variants, confirming a milder bleeding phenotype. Immunoblotting of platelet lysates detected VWF in the platelets of type 3 patients with Pro2808Leufs*24. Examination of an index case detected VWF within platelets via immunofluorescence microscopy, and in vitro experiments showed that this VWF was released upon platelet activation. Patient BOECs showed decreased VWF synthesis and secretion, although some VWF-containing granules were observed. Conclusion Type 3 VWD patients with the Pro2808Leufs*24 have bioavailable platelet-derived VWF that may produce a milder bleeding phenotype than other type 3s.

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Correction of a murine model of von Willebrand disease by gene transfer.

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Correction of a murine model of von Willebrand disease by gene transfer.

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