Mouse Models of the Common, Recurring 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 antigen (VWF:Ag is 5–50% of normal levels) and a mild bleeding phenotype. The common recurrent 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%) and accelerated clearance. Although well described in patients and through in vitro studies, extensive controlled in vivo investigation of these mutations has yet to be performed. In this study, we compared both Y1584C and R1205H to wild type VWF using hydrodynamic gene delivery of mouse VWF and ADAMTS13 transgenes in the VWF knockout mouse to determine the pathological mechanisms associated with these variants.

Hydrodynamic injections were performed using 100 μ g wild type (WT) or mutant mouse Vwf cDNA in Ringer's solution in 7–9 week old C57Bl6 VWF knockout mice, replacing plasma VWF. Co-injections with mouse Adamts13 cDNA were also performed. Mice were sampled at days 2, 5, 8, and then weekly. Mouse plasma was analyzed for complete blood counts, VWF:Ag, VWF propeptide, and VWF multimer structure. Thrombotic injury was induced using ferric chloride injury to the arterioles of the cremaster in VWF knockout mice expressing VWF:Ag levels from 0.5–2 U/ml. Platelets were labeled with Rhodamine-6G to evaluate platelet accumulation. Time to stable vessel occlusion and platelet accumulation by relative fluorescence intensity were compared.

Hydrodynamic injection caused no adverse events in any animals. Complete blood count values were unaffected for both variants compared to WT. Initial high VWF:Ag values at day 2 were similar for WT VWF (25.4 ± 2.5 U/ml, n= 12, mean U/ml±SEM, n) and Y1584C (26.8 ± 5.5, n= 10), but R1205H levels were 36% lower (16.3 ± 2.1, n= 10). Lower VWF:Ag levels were demonstrated in both “homozygous” and “heterozygous” forms for both type 1 mutations from days 14–42, when VWF expression plateaus. R1205H VWF:Ag was 34.3 ± 5.9% of WT (P < 0.001) and “heterozygous” 1:1 ratio R1205H/WT co-delivery was 27.5 ± 4.7% (p < 0.001). Y1584C was 29.4 ± 7.5% of WT (P < 0.001), and Y1584C/WT was 51.1 ± 4.6% (p < 0.001). VWF propeptide to VWF:Ag ratios (days 2–42) demonstrate that R1205H mouse VWF had an increased clearance rate (165.4 ± 13.5%, p < 0.001), while Y1584C was normal (97.1 ± 6.8 %, P > 0.05) compared to WT (100.0 ± 10.0%). The R1205H mutation showed no significant difference in multimer structure by mean multimer band numbers (days 2 to 42, 93 ± 16%, n = 4, P > 0.05) to wild type VWF (100 ± 12%, n = 4). In contrast, Y1584C had a significant decrease (66 ± 18%, n = 4, P < 0.001). This effect was exaggerated by co-delivery of mouse ADAMTS13 for Y1584C, but not R1205H. Y1584C showed reduced thrombus formation in a ferric chloride injury model while R1205H demonstrated similar thrombogenic activity to wild type VWF. Mean occlusion times were WT = 29.9 ± 2.1 minutes, n = 8, R1205H = 29.1 ± 4.0, n = 8 (p > 0.05), and Y1584C = 38.7 ± 1.1, n = 9 (p = 0.001). Total platelet accumulation was decreased for Y1584C (83.6 ± 6.3%, p = 0.043), but was similar for R1205H (103 ± 6.3, P = 0.72) and WT (100 ± 5.3%).

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 and evidence of accelerated clearance with R1205H. However, there is no alteration in multimer structure and apparently normal participation in a thrombosis model. Y1584C, in contrast, shows a loss of high molecular weight multimers that is exacerbated by the additional expression of ADAMTS13, indicating that ADAMTS13 cleavage is increased. Y1584C also has an initially high VWF:Ag level that was less than WT levels from day 14 onward, but shows no alteration in clearance, suggesting that there is a biosynthetic defect. Y1584C shows a significant defect in the arteriolar thrombosis model, presenting a Type 2A VWD-like phenotype in the mouse model, which is more severe than the human phenotype. This study has elucidated several novel mechanistic details for these two mutations and highlights that the pathogenic aspects of type 1 VWD can be recapitulated in the VWF knockout hydrodynamic injection model.

No relevant conflicts of interest to declare.