PB0836 - Functional analysis of four novel Von Willebrand Disease causing mutations

Background: Von Willebrand Factor (VWF) is a plasma glycoprotein essential to primary haemostasis. Its main function is to initiate platelet capture at sites of vascular injury. Quantitative or qualitative deficiency of VWF results in Von Willebrand's Disease (VWD), the most common inherited bleeding disorder. Understanding how different causative mutative mutations affect VWF function is essential to help our understanding of VWF biology and to help guide treatment.

Aims: The study investigates the pathogenic nature of 4 recently reported VWD mutations and how they interfere with the production, secretion, and function of VWF.

Methods: Site-directed mutagenesis was used to generate four uncharacterised point mutations associated with VWD (Gln499Pro, Asp1277Tyr, Asp1277His and Lys1491Glu). These were transiently expressed in HEK293T cells, followed by quantification of protein expression levels. In addition, co-transfections were performed with wtVWF to mimic the heterozygous state and determine the rescue of poorly expressed variants. Finally, the physiological function of recombinant VWF was assessed using static and shear-based assays.

Results: Lys1491Glu showed normal expression levels relative to the wtVWF, while Asp1277Tyr and Asp1277His showed significantly reduced protein expression ( < 20%), which could be rescued in co-transfection experiments. In contrast, the Gln499Pro variant was undetectable in the media and was shown to be retained intracellularly in the ER, and expression could not be rescued by co-transfection with wtVWF. All the secreted proteins showed a normal multimer profile and collagen binding; interestingly, Lys1491Glu was proteolysed faster by ADAMTS13 and had apparent increased platelet capture function under shear stress.

Conclusion(s): The data generated in this study highlights the heterogeneous effect of VWD mutations on VWF function. VWD causing mutations can either impair VWF secretion, leading to intracellular retention or interfere with protein haemostatic function, affecting its ability to bind exposed collagen and capture platelets at sites of vascular injury.