The Effect of von Willebrand Factor (VWF) on Site-Specific Factor VIII (FVIII) Expression in Hemophilia A Mice.

von Willebrand factor (VWF) is a carrier protein for factor VIII (FVIII) and protects plasma FVIII from protease degradation. Our laboratory has had a longstanding interest in the association of FVIII with VWF both in vitro and in vivo. Our in vitro studies have demonstrated that FVIII stores together with VWF in both endothelial cells and megakaryocytes if FVIII is made in these cells. Furthermore, we demonstrated that FVIII and VWF are both releasable by agonist stimulation. To investigate the association of VWF and FVIII in vivo, we generated two lines of transgenic mice that express FVIII either in endothelial cells or in platelets using either the endothelial cell-specific Tie2 promoter or the platelet-specific αIIb promoter, respectively. When the platelet-specific FVIII (2bF8) transgene is bred into the FVIII^null mouse, FVIII can only be detected in platelets, with a level of 0.76 ± 0.27 mU/10⁸ platelets in heterozygous and 1.53 ± 0.14 mU/10⁸ platelets in homozygous 2bF8 mice. When the endothelial cell-specific FVIII (Tie2F8) transgene is bred into the FVIII^null mouse, homozygous Tie2F8 mice maintained normal plasma FVIII levels (1.15 ± 0.16 U/ml) and 50% levels in heterozygous mice (0.56 ± 0.16 U/ml). Both 2bF8^trans and Tie2F8^trans phenotypes effectively abrogate the bleeding diathesis in hemophilic mice. When 2bF8 transgene was bred into a FVIII and VWF double knockout background, the level of platelet-FVIII significantly decreased, but this platelet-derived FVIII was still stored in a-granules and still maintained clinical efficacy. In contrast, when the Tie2F8 transgene was bred into the double knockout background, plasma FVIII dropped to undetectable levels. This is in contrast to the situation in VWF^null mice in which normal endogenous murine FVIII is synthesized with about 10% of normal FVIII activity persisting in plasma. This could be due to a difference in survival between human FVIII and murine FVIII. All Tie2F8^trans/FVIII^nullVWF^null mice (n=15) survived tail clipping even though there is no FVIII:C detected in the plasma. To investigate the effect of murine VWF on the levels of plasma FVIII, plasma from FVIII^null mice was infused into Tie2F8^trans/FVIII^nullVWF^null mice to restore VWF levels to 25% of normal. As expected, the endothelial cell-derived plasma FVIII was stabilized by the infused VWF and was detected within 1 hour after infusion, with a peak (25% level) at 4 hours. The level of plasma FVIII at 24 hours was still about 20% of normal while the level of remaining VWF was only 5% of normal. These results demonstrate that VWF is important for site-specific FVIII expression. Co-expression with VWF in platelets is important for optimal platelet-specific FVIII expression and endothelial cell-derived plasma FVIII is VWF-dependent.