PB1057 - VWF type 2N variant markedly enhances FVIII levels when both proteins are expressed in platelets

Background: In blood circulation, FVIII binds VWF, forming a VWF/FVIII complex. Type-2N-VWF variants are mutations that occur in the FVIII binding site of the VWF gene, resulting in VWD with low plasma-FVIII levels. How 2N-VWF impacts intracellular FVIII levels in vivo is unclear. We recently developed a novel Type-2N VWD mouse model in which VWF is incapable of binding FVIII in neutral pH but is otherwise fully functional.

Aims: To investigate how 2N-VWF affects intracellular FVIII using platelet-targeted FVIII expression (2bF8Tg) models.

Methods: VWF2N/2N was crossed onto 2bF8Tg mice to establish 2bF8tg+/+F8-/-VWF2N/2N (2bF8TgVWF2N). Bone marrow transplantation (BMT) was carried out to further study the impact of platelet- and plasma-2NVWF on platelet-FVIII (plt-F8) levels. Functional plt-F8 levels were determined by chromogenic assay, and VWF antigen levels were determined by ELISA.

Results: Plt-F8 level in the 2bF8TgVWF2N group was 12.14±2.06 mU/108plts, which was significantly higher than in the 2bF8Tg group (8.57±1.27 mU/108plts). Interestingly, plt-VWF levels in the 2bF8TgVWF2N group were significantly lower than in the 2bF8Tg group (111±14 vs. 146±19 mU/108plts, respectively). Similarly, plt-VWF levels in VWF2N mice were significantly lower than in wild-type controls. Plasma-VWF levels were comparable between the two groups. To exclude the impact of plasma-derived VWF, we performed BMT from 2bF8TgVWF2N and 2bF8Tg to VWFnullF8null mice to establish models with plt-F8 together with 2NVWF (2bF8Tg-pltVWF2N) or normal VWF in platelets (2bF8Tg-pltVWF+), without plasma-VWF. The plt-F8 level in the 2bF8Tg-pltVWF2N group was 11.73±1.19 mU/108plts, which was significantly higher than in the 2bF8Tg-pltVWF+ group (6.99±0.54 mU/108plts). When BM cells from 2bF8TgVWF2N mice were transplanted into VWF2NF8null and F8null mice, plt-F8 levels in the 2bF8TgVWF2N/VWF2NF8null group were slightly, but significantly lower than in the 2bF8TgVWF2N/F8null group (11.86±0.8 and 12.46±2.54 mU/108plts, respectively).

Conclusion(s): Type-2N-VWF variant reduces VWF expression in platelets but dramatically enhances FVIII levels when both proteins are expressed in platelets.