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.
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 murinevWF (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.
Authors: Lan Wang; Jonathan B Rosenberg; Bishnu P De; Barbara Ferris; Rui Wang; Stefano Rivella; Stephen M Kaminsky; Ronald G Crystal Journal: Hum Gene Ther Date: 2012-06-25 Impact factor: 5.695
Authors: Yasuaki Shida; Natalia Rydz; David Stegner; Christine Brown; Jeffrey Mewburn; Kate Sponagle; Ozge Danisment; Bredon Crawford; Barbara Vidal; Carol A Hegadorn; Cynthia M Pruss; Bernhard Nieswandt; David Lillicrap Journal: Blood Date: 2014-07-22 Impact factor: 22.113