Trafficking of VWF to proplatelet extensions. (A) VWF distribution (green) during proplatelet formation. Tubulin was used to stain the cytoskeleton (red). Confocal immunofluorescence images (left panels) were analyzed with ImarisCell, an analytical tool by Bitplane that quantifies cellular morphology. Different steps in image analysis are shown here (middle and right panels). Scale bar, 30 μm. (B) Quantification of the number of VWF-containing vesicles from confocal immunofluorescence images by the use of ImarisCell. Results are shown as number of vesicles per mm² of proplatelet area (n = 30 MKs imaged from 3 mice per genotype). Mean ± SEM; ***P < .001. (C) Suggested model for the function of VPS33B homologs in platelet granule biogenesis. Early endosomes are formed by endocytosis of cargo and following maturation they lead to MVB I (green arrows). MVB I communicate with the Golgi apparatus receiving vesicles with newly synthesized cargo (purple arrow). MVB I undergo further maturation to MVB II that may receive additional cargo for sorting (dotted green and purple arrows). VPS33A and its interacting partner VPS16A are required for sorting of proteins from endosomes into maturing MVB II leading to the formation of δ-granules. On the other hand, VPS33B in complex with VIPAR is likely to be responsible for sorting of cargo from the trans-Golgi network to α-granule–destined MVBs and subsequently promoting α-granule formation. VPS33B deficiency results in a defect in trafficking of some cargo proteins to MVB II (dotted red arrow) resulting in abnormal MVB maturation and defective α-granule biogenesis (red arrow). Accumulation of large vacuolar structures and the presence of small granules are characteristics of those MKs. A possible role of VPS33B in the sorting of some δ-granule proteins cannot be ruled out.