A murine model to characterize the antithrombotic effect of molecules targeting human von Willebrand factor.

von Willebrand factor (VWF) is a promising target for developing antithrombotic drugs. The absence of accessible animal models impedes the study of specific human VWF (huVWF) targeting molecules in thrombosis. huVWF is not functional in the mouse because of a lack of interaction between huVWF and murine glycoprotein Ib. Using site-directed mutagenesis, we have replaced single or multiple amino acids in huVWF with their murine counterparts to eliminate species incompatibility. Using hydrodynamic injection, we have expressed the different chimeric VWF constructs into VWF(-/-) mice. Only huVWF with a complete murine A1 domain insertion was able to correct bleeding in vivo and form occlusive thrombi in mesenteric vessels after FeCl(3) treatment. Using this model, we tested the antithrombotic effect of monoclonal antibodies against huVWF, blocking its interaction with collagens (mAbs 203 and 505) or with glycoprotein IIbIIIa (mAb 9). The 3 mAbs inhibited the thrombotic process in arterioles of VWF(-/-) mice expressing huVWFmuA1. Inhibiting VWF-interaction with collagens was more potent, emphasizing the potential of such a target as an antithrombotic tool. Our results validate our murine model as a simple in vivo tool to evaluate anti-huVWF agents.

Mutation and ADAMTS13-dependent modulation of disease severity in a mouse model for von Willebrand disease type 2B.

Von Willebrand disease (VWD)-type 2B originates from a gain-of-function mutation in von Willebrand factor (VWF), resulting in enhanced platelet binding. Clinical manifestations include increased bleeding tendency, loss of large multimers, thrombocytopenia, and circulating platelet aggregates. We developed a mouse model to study phenotypic consequences of VWD-type 2B mutations in murine VWF: mVWF/R1306Q and mVWF/V1316M. Both mutations allow normal multimerization but are associated with enhanced ristocetin-induced platelet aggregation, typical for VWD-type 2B. In vivo expression resulted in thrombocytopenia and circulating aggregates, both of which were more pronounced for mVWF/V1316M. Furthermore, both mutants did not support correction of bleeding time or arterial vessel occlusion in a thrombosis model. They further displayed a 2- to 3-fold reduced half-life and induced a 3- to 6-fold increase in number of giant platelets compared with wild-type VWF. Loss of large multimers was observed in 50% of the mice. The role of ADAMTS13 was investigated by expressing both mutants in VWF/ADAMTS13 double-deficient mice. ADAMTS13 deficiency resulted in more and larger circulating platelet aggregates for both mutants, whereas the full multimer range remained present in all mice. Thus, we established a mouse model for VWD-type 2B and found that phenotype depends on mutation and ADAMTS13.

Altered thrombus formation in von Willebrand factor-deficient mice expressing von Willebrand factor variants with defective binding to collagen or GPIIbIIIa.

The role of von Willebrand factor (VWF) in thrombosis involves its binding to a number of ligands. To investigate the relative importance of these particular interactions in the thrombosis process, we have introduced mutations into murine VWF (mVWF) cDNA inhibiting VWF binding to glycoprotein (Gp) Ib, GPIIbIIIa, or to fibrillar collagen. These VWF mutants were expressed in VWF-deficient mice (VWF(-/-)) by using an hydrodynamic injection approach, and the mice were studied in the ferric chloride-induced injury model. Expression of the collagen and the GPIIbIIIa VWF-binding mutants in VWF(-/-) mice resulted in delayed thrombus growth and significantly increased vessel occlusion times compared with mice expressing wild-type (WT) mVWF (30 +/- 3 minutes and 38 +/- 4 minutes for the collagen and GPIIbIIIa mutants, respectively, vs 19 +/- 3 minutes for WT mVWF). Interestingly, these mutants were able to correct bleeding time as efficiently as WT mVWF. In contrast, VWF(-/-) mice expressing the GPIb binding mutant failed to restore thrombus formation and were bleeding for as long as they were observed, confirming the critical importance of the VWF-GPIb interaction. Our observations suggest that targeting the VWF-collagen or VWF-GPIIbIIIa interactions could be an interesting alternative for new antithrombotic strategies.

Correction of bleeding symptoms in von Willebrand factor-deficient mice by liver-expressed von Willebrand factor mutants.

OBJECTIVE: von Willebrand Factor (vWF) structure-function relationship has been studied only in vitro. To investigate the physiological importance of particular vWF domains, we have introduced mutations into murine vWF (mvWF) cDNA inhibiting vWF binding to glycoprotein (Gp) Ib, GpIIbIIIa, and to fibrillar collagen. METHODS AND RESULTS: We delivered wild-type (WT) or mutant mvWF cDNA into vWF-deficient (Vwf-/-) mice using hydrodynamic injection and assessed whether hemorrhagic symptoms could be corrected. Hydrodynamic gene transfer resulted in high expression of plasma mvWF 24 hours after injection (438+/-63% for 50 microg of cDNA). Factor VIII activity was normalized in Vwf-/- mice injected with mvWF cDNA and multimerization was achieved. Bleeding time was corrected after injection of WT mvWF cDNA in Vwf-/- mice whereas noninjected mice did not stop bleeding. Injection of the GpIIbIIIa and the collagen binding mutants in Vwf-/- mice also resulted in a correction of bleeding time whereas mice injected with the GpIb binding mutant were bleeding for as long they were observed, although blood loss was decreased compared with noninjected mice (61+/-21 microL versus 232+/-63 microL). CONCLUSIONS: Our model allows the rapid in vivo evaluation of specific mutations on plasma vWF function.

Role of von Willebrand factor in tumor metastasis.

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that plays a critical role in primary hemostasis, allowing the adhesion of platelets to the exposed subendothelium. The key role played by VWF in platelet adhesion suggests a potential implication in various pathologies where this process is involved. In cancer metastasis development, tumor cells interact with platelets and the vessel wall to extravasate from the circulation. A number of potential receptors for VWF have been identified on tumor cells such as glycoprotein Ib or the alpha(IIb)beta(3) and alpha(v)beta(3) integrins and direct interactions between VWF and tumor cells have been reported. To address the role of VWF in an experimental metastasis model, we compared the formation of pulmonary metastatic foci in C57BL/6J wild-type and VWF-null mice following I.V. injection of murine melanoma B16-BL6 cells or Lewis lung carcinoma cells. Surprisingly we found a significant increase in the number of pulmonary metastatic foci in VWF-null mice. Restoration of VWF plasma levels by co-injection of VWF with the tumor cells led to the correction of this pro-metastatic phenotype. In vitro analysis revealed that VWF did not influence tumor cell proliferation or invasion but induced cellular death. This result was confirmed in vivo where analysis of the early survival of tumor cells in the lungs revealed that the presence of VWF led to a decreased survival of these cells during the first 24 hours after injection. Our results suggest that VWF plays a role in tumor metastasis, independently of its role in hemostasis.