The sorting signal for von Willebrand factor (vWF) granular storage lies within the first 140 amino acids of the D2 domain of the VWF propeptide (VWFpp)

Abstract number: OC401

Haberichter^* S. L., Fahs† S. A., Jozwiak† M. A., Retzlaff† K. L., Montgomery^* R. R.

†Blood Research Institute, USA ^*Department of Pediatrics, Medical College of Wisconsin, USA;

VWF is synthesized in endothelial cells and megakaryocytes/platelets where it is stored in Weibel–Palade bodies and a-granules, respectively. Our previous studies demonstrated that VWF is routed to storage granules by virtue of an association with its propeptide, VWFpp. VWFpp independently traffics to storage granules in AtT-20 cells that contain an intact storage pathway where VWFpp colocalizes with the endogenous hormone, ACTH and undergoes agonist-induced secretion. We sought to identify the minimal sequence within VWFpp required for sorting to storage. Several C- or N-terminal truncated VWFpp were constructed, expressed in AtT-20 cells, and the intracellular localization of protein determined by immunostaining and confocal microscopy. While some truncated VWFpp demonstrated a diffuse staining pattern, five truncated VWFpp were stored in ACTH-containing granules. The smallest N-terminal truncated VWFpp that was stored in granules contained signal peptide followed by the D2 domain, amino acids (aa) 386–763. The minimal C-terminal truncated VWFpp with granular storage consisted of aa 1–567. The D1 domain alone (aa 1–385) was not stored in granules. To further localize the storage signal, we constructed C-terminal truncations of the D2 domain that were tagged with GFP to circumvent loss of antibody recognition. Two such truncated VWFpp were routed to storage in AtT-20 cells: VWFpp-386–651 that contained 265 aa of D2 domain and VWFpp-386–523 that contained only 137 aa of D2. We investigated whether the N- or C-terminal truncated VWFpp could associate with coexpressed propeptide-deleted VWF (d pro) to route it to storage granules. Only one truncated VWFpp trafficked coexpressed d pro to storage granules, VWFpp-1–745. While the D2 domain alone was not sufficient for association/storage of co-expressed d pro, we asked whether replacing the D1 domain of VWFpp with a D2 domain would maintain proper alignment of VWFpp necessary for association with d pro. A D2D2-VWFpp was co-expressed with d pro in AtT-20 cells and both the D2D2-VWFpp and VWF were co-localized in granules. Swapping the position of the D1 and D2 domains resulted in a loss of VWF association and storage although storage of D2D1-VWFpp was observed. Thus, the signal for VWF storage resides within the first 137 residues of the D2 domain. Maintenance of proper VWFpp alignment may be necessary for association with VWF that results in VWF storage.