VPS33B regulates protein sorting into and maturation of α-granule progenitor organelles in mouse megakaryocytes by Danai Bem, Holly Smith, Blerida Banushi, Jemima J. Burden, Ian J. White, Joanna Hanley, Nadia Jeremiah, Frédéric Rieux-Laucat, Ruth Bettels, Gema Ariceta, Andrew D. Mumford, Steven G. Thomas, Steve P. Watson, and Paul Gissen Blood Volume 126(2):133-143 July 9, 2015 ©2015 by American Society of Hematology

Characterization of the Vps33bfl/fl-ERT2 mice. Characterization of the Vps33bfl/fl-ERT2 mice. (A) Analysis of Vps33b messenger RNA in BM-derived MKs of Vps33bfl/fl and Vps33bfl/fl-ERT2 mice. (B) Spleen to body weight ratio was analyzed in 11-week-old mice 5 weeks post-induction (n = 12 to 16 mice per genotype). (C) Extramedullary hematopoiesis in Vps33bfl/fl-ERT2 mice. Representative images of hematoxylin and eosin-stained spleen (top panels) and femur BM (bottom panels) sections from littermate controls (left top and bottom panels) and Vps33bfl/fl-ERT2 mice (right top and bottom panels). MKs are indicated by yellow arrowheads (n = 3 mice per genotype). Bright field images were obtained using a Zeiss Axiovert 200 inverted high-end microscope with a 20× objective. Scale bar, 50 μm. (D-E) Determination of MK numbers per visual field (294 × 221 μm, 36 fields of view from 3 mice per genotype) in hematoxylin and eosin-stained spleen (D) and BM (E) sections of Vps33bfl/fl vs Vps33bfl/fl-ERT2 mice. Mean ± SEM. All values are mean ± standard deviation (SD) unless otherwise indicated. ***P < .001. Danai Bem et al. Blood 2015;126:133-143 ©2015 by American Society of Hematology

Ultrastructural analysis of Vps33bfl/fl-ERT2 platelets reveals an α-granule deficiency. Ultrastructural analysis of Vps33bfl/fl-ERT2 platelets reveals an α-granule deficiency. (A) Representative transmission electron micrographs of platelets from Vps33bfl/fl (left panels) and Vps33bfl/fl-ERT2 (right panels) mice at low (scale bar, 1 μm) and high magnification (scale bar, 0.5 μm). Images were obtained using a Tecnai G2 Spirit TEM. (B) Percentage of α-granule containing platelets per ultrathin section (70 to 90 nm) in Vps33bfl/fl and Vps33bfl/fl-ERT2 mice (n = 200 platelets per genotype). (C) Platelets containing small granules were observed in TEM from Vps33bfl/fl-ERT2 mice. Those platelets were devoid of α-granules. Scale bar, 0.5 μm. (D) Representative transmission electron micrographs of an ARC patient with a VIPAS39 mutation (p.Arg270*, top right panel) and 2 ARC patients with VPS33B mutations (p.Pro412Argfs*7 or p.Pro147Argfs*4, bottom left and right panel respectively). Small α-granule–like structures are shown in white arrowheads. Scale bar, 1 μm. (E) Immunogold labeling (IEM) using the Tokuyasu method for the presence of VWF (10 nm gold) in platelets from Vps33bfl/fl (left panel) and Vps33bfl/fl-ERT2 (right panel) mice. Scale bar, 200 nm. All values are mean ± SD. ***P < .001. α, α-granule; δ, δ-granule; G, α-granule-like structure; M, mitochondrion. Danai Bem et al. Blood 2015;126:133-143 ©2015 by American Society of Hematology

Characterization of Vps33bfl/fl-ERT2 platelet function. Characterization of Vps33bfl/fl-ERT2 platelet function. (A) VWF and P-selectin levels were measured in platelets by immunoblot analysis. β-Actin was used as a loading control. Densitometric analysis showed a reduction in VWF and P-selectin content in Vps33bfl/fl-ERT2 platelets (n = 3 mice) in comparison with Vps33bfl/fl platelets (n = 5 mice). (B) Thrombin (0.1 U/mL) induced P-selectin expression on the surface of Vps33bfl/fl-ERT2 (n = 15 mice) platelets in comparison with Vps33bfl/fl (n = 19 mice). Histogram (left) shows an IgG control (purple), P-selectin expression in controls (green and pink), and Vps33bfl/fl-ERT2 platelets (blue). (C) Representative aggregation responses to thrombin (0.05 U/mL), collagen (3 μg/mL), and ADP (3 μM) were similar in washed platelets from control and Vps33bfl/fl-ERT2 mice (n = 12 mice per genotype). (D) δ-Granule secretion was measured in the lumi-aggregometer using a luciferase assay (ATP). ATP secretion in Vps33bfl/fl and Vps33bfl/fl-ERT2 platelets (n = 12 mice per genotype) in response to 0.05 U/mL thrombin. (E) Reduced ATP secretion was observed in Vps33bfl/fl-ERT2 vs Vps33bfl/fl platelets (n = 12 mice per genotype) upon collagen stimulation. (F) ATP secretion measured by lummi-aggregometry after stimulation of washed platelets with the divalent calcium ionophore A23187 (10 μM) (n = 5 mice per genotype). (G) Tail-bleeding assay (n = 15 to 17 mice per genotype). Open circles (○) represent individual mice. Horizontal lines represent means. (H) Representative fluorescence images (DiOC6) at 2 and 4 minutes of blood perfusion (left and middle panels). Representative phase-contrast images (BF) at the end of the perfusion period (right panel). Images were obtained using a Zeiss Axiovert 200 inverted high-end microscope with a 40× objective. Scale bar, 10 µm. All values are mean ± SEM. *P < .05; **P < .01; ***P < .001. BF, bright field; MFI, mean fluorescence intensity; ns, not significant. Danai Bem et al. Blood 2015;126:133-143 ©2015 by American Society of Hematology

Abnormal ultrastructure of femoral BM Vps33bfl/fl-ERT2 MKs Abnormal ultrastructure of femoral BM Vps33bfl/fl-ERT2 MKs. (A) Representative transmission electron micrographs of femoral BM sections showing the classification of MKs in 3 main maturation stages. Abnormal ultrastructure of femoral BM Vps33bfl/fl-ERT2 MKs. (A) Representative transmission electron micrographs of femoral BM sections showing the classification of MKs in 3 main maturation stages. Scale bars, main image 5 μm; inset 1 μm. (B) Quantification of the percentage of MKs present per maturation stage in Vps33bfl/fl and Vps33bfl/fl-ERT2 mice (n = 32 to 44 MKs analyzed in 3 mice per genotype). (C) Vps33bfl/fl MKs showing nice distribution of granules in maturation stages II and III (top panels), whereas Vps33bfl/fl-ERT2 MKs were devoid of morphologically distinct α-granules but were abundant of small α-granule–like structures (bottom panels). Clusters of lamellar structures were also evident in Vps33bfl/fl-ERT2 MKs. Scale bar, 0.5 μm. (D-E) Quantification of α-granules and immature granules at maturation stage II (D) and III (E) reveals a marked decrease in their numbers in Vps33bfl/fl-ERT2 mice when compared with controls (n = 32 to 44 MKs analyzed in 3 mice per genotype). All values are mean ± SEM. **P < .01; ***P < .001. α, α-granule; δ, δ-granule; G, α-granule-like structure; im, immature granule; Lm, lamellar structure; M, mitochondrion. Danai Bem et al. Blood 2015;126:133-143 ©2015 by American Society of Hematology

Characterization of Vps33bfl/fl-ERT2 MKs in primary culture. Characterization of Vps33bfl/fl-ERT2 MKs in primary culture. (A) Distribution of Vps33bfl/fl and Vps33bfl/fl-ERT2 BM-derived MKs ploidy after 5 days in culture. The percentage of cells with 2N to 128N ploidy was quantified by propidium iodide staining and flow cytometry (n = 5 mice per genotype). Mean ± SD. (B) Proplatelet formation was unaltered in Vps33bfl/fl-ERT2 MKs (n = 3 mice per genotype). Mean ± SD. (C) Transmission electron micrographs of BM-derived MKs showing representative images of MVB I (left) and MVB II (right) in control MKs. Scale bar, 0.5 μm. (D) Representative transmission electron micrographs of BM-derived MKs. Vps33bfl/fl MKs had normal α- and δ-granules, whereas MBV I and MBV II were also present (left panels). Note the presence of large vacuolar structures in Vps33bfl/fl-ERT2 MKs (right panels). Scale bar, 0.5 μm. (E-F) Quantification of organelles present in MK sections. Number of MVB I, α-, and δ-granules (E), and number of classical and atypical MVB II and vacuoles (F) per MK section. Twenty to 27 MKs imaged per genotype, 4 to 5 fields of view (4.98 × 3.32 μm) per MK taken at a magnification of ×30 000. Mean ± SEM. *P < .05. A, atypical MVB II; α, α-granule; δ, δ-granule; M, mitochondrion; ns, not significant; V, empty vacuole. Danai Bem et al. Blood 2015;126:133-143 ©2015 by American Society of Hematology

Abnormal trafficking of VWF in Vps33bfl/fl-ERT2 mice. Abnormal trafficking of VWF in Vps33bfl/fl-ERT2 mice. (A) VWF levels were measured in MKs by immunoblot (left panel) and densitometry (right panel) using a rabbit polyclonal antibody anti-VWF (H-300) (1:1000), and a goat anti-rabbit and a goat anti-mouse horseradish peroxidase-conjugate secondary antibody (1:1000). β-Actin was used as a loading control (n = 3 mice per genotype). (B) Immunogold labeling (IEM) using the Tokuyasu method for double labeling of VWF and CD63 (i-iv) in MKs from Vps33bfl/fl and Vps33bfl/fl-ERT2 mice. Black arrowheads, VWF 10 nm gold particles; black arrows, CD63 15 nm gold particles. Scale bar, 250 nm. A total of 15 MKs were imaged per genotype, 4 to 5 fields of view (4.98 × 3.32 μm) per MK taken at a magnification of ×30 000. (C) Fibrinogen uptake in cultured MKs after incubation with 488-fibrinogen for 2 hours. Mean ± SEM. A, atypical MVB II, MFI, mean fluorescence intensity; ns, not significant. Danai Bem et al. Blood 2015;126:133-143 ©2015 by American Society of Hematology

Trafficking of VWF to proplatelet extensions. 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. Danai Bem et al. Blood 2015;126:133-143 ©2015 by American Society of Hematology