Two children from a Turkish consanguineous family suffering from significant mucocutaneous bleeding episodes were diagnosed with von Willebrand disease type 2A. The relative reduction of large plasma von Willebrand factor (VWF) multimers together with the absent triplet structure was consistent with type 2A (phenotype IIC) VWD. Surprisingly, platelet VWF was completely deficient of multimers beyond the VWF protomer, suggesting defective α-granular storage of larger multimers. The two patients were nearly unresponsive to DDAVP, consistent with a lack of regulated VWF release from endothelial cell Weibel-Palade bodies, suggesting defective storage in endothelial cells as well as in platelets. We identified the homozygous mutation in these patients, N528S, in the D2 domain of the VWF propeptide (VWFpp). To examine the effect of the N528S mutation on VWF regulated storage, a VWF expression vector containing the mutation was constructed and expressed in AtT-20 cells that contain an intact regulated storage pathway. Transfected cells were immunofluorescently labeled and the intracellular localization of VWFpp and VWF were examined by confocal microscopy. In cells expressing N528S-VWF, VWF was not stored in granules but was localized to the endoplasmic reticulum (ER). Our previous studies have shown that VWF granular storage is initiated by VWFpp: VWF is trafficked to storage granules through its non-covalent association with VWFpp. To determine if the loss of granular storage of N528S-VWF was due to defective VWFpp trafficking, an N528S-VWFpp expression vector was constructed and expressed in AtT-20 cells. The N528S-VWFpp was found to be colocalized with endogenous ACTH-containing storage granules, demonstrating normal granular trafficking of the variant VWFpp. The N528S-VWFpp was co-expressed with propeptide-deleted, mature VWF (Δpro) to examine regulated storage when the two proteins are expressed in trans. In the majority of transfected AtT-20 cells, N528S-VWFpp was trafficked to granules while the coexpressed Δpro appeared to be ER-localized, consistent with a loss of non-covalent association between VWFpp and VWF. In a subset of cells neither VWFpp nor VWF were stored in granules. In contrast, when N528S-VWF was expressed as a full-length construct (in cis), VWFpp granular storage was observed only in a subset of cells and the majority of transfected cells demonstrated ER-localization of both proteins, suggesting the loss of regulated storage of N528S-VWF is not due to defective VWFpp sorting, but rather an aberrant interaction of VWFpp with the mature VWF protein. Together these data indicate that the phenotype observed in patients with N528S variant VWF is result of defective multimerization, storage, and secretion due to an aberrant VWFpp/VWF interaction.

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