VWF granule biogenesis in HEK293 cells transfected with mutant and wild-type VWF. (A) K9-VWD-AECs were transiently transfected with N528S-VWF, wild-type VWF, or pCIneo (Mock). Cells were immunostained for VWF (green; i, iv, vii) and P-selectin/CD62P (red; ii, v, viii) and intracellular localization examined by confocal microscopy. The merged image is shown in the last column with colocalization shown in yellow (iii, vi, ix). Wild-type VWF formed granules and recruited CD62P, whereas N528S-VWF failed to form granules. (B) HEK293 cells were transiently transfected with N528S-VWF, wild-type VWF-myc-his, or heterozygous N528S-VWF/wild-type VWF-myc-his. Cells were immunostained using monoclonal AVW-5 that does not recognize wild-type VWF-myc-his (green; x, xiii, xvi, xix) and rabbit antimyc that does not recognize the N528S-VWF lacking a myc-his tag (red; xi, xiv, xvii, xx). Intracellular localization was determined by confocal microscopy and the merged image shown in panels xii, xv, xviii, and xxi. When expressed alone, wild-type VWF formed granules, whereas N528S-VWF did not. When coexpressed, both N528S-VWF and wild-type VWF are localized in granules.