PB0800 - Quantitative von Willebrand factor multimer analysis in von Willebrand disease patients enrolled in the Zimmerman Program study.

Background: The diagnosis of von Willebrand disease (VWD) requires many assays including VWF multimer structure analysis. Accurate VWD subtype classification relies on a correct assessment of these multimers and their molecular weight distribution.

Aims: To assess VWF multimer structure in patients with VWD using a precise, quantitative multimer assay.

Methods: VWF multimers were analyzed with lithium dodecyl sulfate (LiDS) agarose gels and subsequent commercial VWF polyclonal antibody western blots for healthy controls and VWD subjects (index cases and family members with well‐defined genotypes and phenotypes) recruited through the Zimmerman Program. Densitometry was performed and area‐under‐the‐curve (AUC) calculated using Multi Gauge software. Percentage of low molecular weight (LMW, bands 1–5), intermediate molecular weight (IMW, bands 6–10) and high molecular weight (HWM) multimers (bands >10) was calculated for each sample.

Results: We defined multimer distribution ranges for healthy individuals and for each VWD subtype. Subjects with low VWF and type 2N VWD displayed normal multimers. Although type 1 and 2M VWD subjects had essentially normal multimers, there was often a subtle shift from HMW to LMW species (HMW AUC of 20%). Most type 1C subjects showed a more pronounced shift (HMW AUC of 15%). Type 2A and 2B VWD subjects demonstrated a loss of HMW multimers as expected (HMW AUC of 6-7%). Additionally, we correlated recurrent sequence variants (SVs), and their positions within VWF, with abnormal multimers in type 1 and 2 VWD (Table 1). Sixteen SVs spread across the D’D3, A1, and A2 domains coincided with abnormal multimers.

Conclusion(s): Using a detailed quantitative VWF multimer analysis method we were able to detect subtle distribution differences, as in type 1C VWD, as well as clear differences like those seen in type 2A/2B VWD. Additionally, we correlated causative SVs with abnormal multimers, paving the way for a more in-depth analysis of these genetic variants.