Multifaceted Pathomolecular Mechanism of a VWF Large Deletion Involved in the Pathogenesis of Severe VWD

An in-frame heterozygous large deletion of exons 4-34 of the von Willebrand factor (VWF) gene was identified in an index patient (IP) with type 3 von Willebrand disease (VWD), as the only mutation. The IP exhibited severe bleeding episodes despite prophylaxis treatment, with a short VWF half-life after infusion of VWF/FVIII concentrates. This study intends to elucidate the causal molecular mechanism of this large deletion. Transcript analysis confirmed transcription of normal VWF mRNA besides an aberrant deleted transcript. The amount of secreted VWF from blood outgrowth endothelial cells (BOECs) isolated from the IP was not significantly different from that of controls. However, IP-BOECs exhibited a deficiency in the assembly of VWF multimers and biogenesis of the Weibel-Palade bodies (WPBs). Furthermore, immunostaining of IP-BOECs demonstrated subcellular mislocalization of WPBs pro-inflammatory cargos angiopoietin-2 (Ang2) and P-selectin. Additionally, whole-transcriptome RNA-sequencing of the BOECs and subsequent Ingenuity Pathway Analysis indicated the significant alterations of canonical pathways in IP-BOECs related to inflammatory responses, cell adhesion, extracellular organization, and angiogenesis (e.g. granulocyte adhesion and Rho-related signaling pathways), which are known downstream signaling pathways induced by Ang2. Accordingly, the IP-BOECs exhibited an increased adhesiveness to leukocytes which may contribute to accelerated VWF clearance. In conclusion, deleted VWF has a dominant-negative impact on the elongation of multimers and biogenesis of WPBs. Aberrant WPBs lead to the alternative trafficking of its cargos in a specific way, which, in turn, may cause distinctive perturbations in cellular signaling pathways, resulting in the exceptional phenotypes in the current patient. Overall design: 6 samples