In Vitro and In Vivo Mouse Models of the Type 1 Von Willebrand Disease Mutations Y1584C and R1205H.

Type 1 von Willebrand disease (VWD) is caused by mutations that result in moderate decreases in VWF (5-50% of normal levels) and a mild bleeding phenotype. The VWF missense mutation Y1584C is associated with mildly decreased VWF:Ag levels, increased ADAMTS13 cleavage, as well as a possible increase in clearance. The Vicenza mutation, R1205H, exhibits a more severe phenotype (VWF:Ag ∼10%), as well as accelerated clearance. However, extensive controlled in vitro and in vivo investigation of these mutations has yet to be described. In this study, we examine both Y1584C and R1205H in comparison to wild type VWF using in vitro and in vivo strategies employing both human and mouse VWF.

Recombinant murine and human VWF and ADAMTS13 were produced via transient transfection in HEK293T cells in serum free OPTIMEM for 72 hours. Full length ADAMTS13 digests were performed in a Tris-Urea system and analyzed via multimer pattern. VWF115 digests were performed in an ELISA based assay. Hydrodynamic injections were performed using 100 μg wild type (WT) or mutant ET-mVWF plasmid DNA in Ringer's solution in 7-9 week old C57Bl6 VWF knockout mice. Mice were sampled at days 2, 5, 8, and then weekly. Mouse plasma was analyzed for CBCs, VWF:Ag, and VWF multimer structure.

In the HEK293T transient transfection system, secreted mutant protein was similar to that of wild type recombinant protein, with high molecular weight material present. ADAMTS13 digestion of full length recombinant Y1584C versus wild type showed no statistical difference: 50% cleavage for hVWF WT 1.78U hADAMTS13, hVWF Y1584C 1.67 U, P=0.58; mVWF WT 0.32 U mADAMTS13; Y1584C 0.42 U, P=0.11. In contrast, the Y1584C substitution in the hVWF115 construct required 40% less hADAMTS13 to effect equivalent cleavage (WT 0.087±0.014, 4; Y1584C 0.052±0.005, 8, mean U/ml ADAMTS13±SEM, N, P=0.013). Mouse ADAMTS13 cleavage of mVWF115 was also increased 20% for Y1584C (WT: 0.20±0.06, 4; Y1584C: 0.16±0.01, 4, P=0.014). Hydrodynamic injection caused no adverse events in any animals. CBC values were not statistically significantly different between wild type and mutants. Initial high VWF:Ag values were similar for wild type VWF (25.6±2.9, 15, mean U/ml±SEM, n) and Y1584C (27.2±5.0, 11), but R1205H levels were 36% lower (16.3±2.1, 10). At 14 days, WT VWF:Ag was 5.33±1.13, 15, with R1205H (1.73±0.44, 12) and Y1584C (1.84±0.59, 11) VWF:Ag levels being 68% and 65% lower, respectively. R1205H continued to remain approximately 40% of WT values for the next three weeks, while Y1584C continued to decrease, dropping to 15% on day 21 and 8% on day 28, compared to the wild type values at these time points. The R1205H mutation showed no significant difference in multimer structure defined by observed number of bands (days 2 to 42, mean difference -0.49, P>0.05) to wild type VWF. In contrast, Y1584C had a significant decrease in band number (3.38, P<0.001).

This study demonstrates that these two type 1 VWD mutations have a strong observable effect in the VWF knockout mouse model. R1205H exhibits a large decrease in VWF:Ag levels at all measured time points, but no alteration in multimer structure. Y1584C, in contrast, shows a loss of high molecular weight material, and an initially high VWF:Ag level that rapidly decays from day 14 onward, suggesting increased ADAMTS13 cleavage and increased clearance. In addition, in vitro ADAMTS13 testing shows that Y1584C responds differently in the two assay systems with only the VWF115 assay showing significant increases in ADAMTS13-mediated cleavage.

No relevant conflicts of interest to declare.