Requirement of VPS33B, a member of the Sec1/Munc18 protein family, in megakaryocyte and platelet α-granule biogenesis by Bryan Lo, Ling Li, Paul Gissen, Hilary Christensen, Patrick J. McKiernan, Charles Ye, Mohamed Abdelhaleem, Jason A. Hayes, Michael D. Williams, David Chitayat, and Walter H. A. Kahr Blood Volume 106(13):4159-4166 December 15, 2005 ©2005 by American Society of Hematology

Abnormal platelets observed in blood films from patients with ARC syndrome. Abnormal platelets observed in blood films from patients with ARC syndrome. Romanowsky (Wright-Giemsa)–stained blood films from 3 patients with ARC syndrome (A-C) and a neonatal control (D) visualized using oil-immersion 63×/1.32-0.6 objective lenses). Large homogeneous, nongranular gray-appearing platelets (arrows) are observed in the fetal (A), the related neonatal (B), and unrelated neonatal (C) patients with ARC syndrome (A). Inset (C) shows magnified view of a typical large platelet compared with an erythrocyte taken from the top right corner of the underlying image. Normal neonatal control platelets (D) are typically smaller, granular, and stained dark blue (arrows). Bryan Lo et al. Blood 2005;106:4159-4166 ©2005 by American Society of Hematology

Absent α-granules and increased δ-granules in platelets from patients containing mutations in VPS33B. Absent α-granules and increased δ-granules in platelets from patients containing mutations in VPS33B. Thin-section transmission electron micrographs of representative platelets from related ARC fetal (A) and neonatal (B) blood, control fetal (C), and unrelated ARC neonatal (D) blood. Magnifications are ×30 000 (A-C) and ×25 000 (D). The black bar represents 500 nm. Multiple α-granules (white arrows) are seen in the control platelet (C), and are absent in ARC platelets (A, B, D). The dark-staining structures seen in ARC platelets (A, B, D) contain distinct internal membranes and may represent mitochondria. Prominent Golgi structures are also visible in ARC platelets (eg, near the center of the platelet in panel B). Multiple dark densities encircled by a lighter halo representing δ-granules (arrows) are visualized in a single thin-section EM image of a neonatal ARC platelet (D), where one would expect to see less than the average of 0.4 δ-granules per platelet thin section observed in adults.34 Bryan Lo et al. Blood 2005;106:4159-4166 ©2005 by American Society of Hematology

α-granule protein deficiencies in ARC megakaryocytes and platelets. α-granule protein deficiencies in ARC megakaryocytes and platelets. Immunoblots comparing megakaryocyte (MK) and platelet (PLT) whole-cell lysates or plasma from ARC and normal (N) fetal, neonatal, and adult sources as indicated for each lane. (A) Markedly reduced PF4 content in ARC fetal megakaryocytes (lane 1) compared with control fetal megakaryocytes (lanes 2, 3), control adult platelets (lane 4), and megakaryocytes (lane 5) were observed (reduced 10% SDS-PAGE). Lysate from equivalent numbers of megakaryocytes (CD41+-labeled cells) was loaded for lanes 1 to 3. Equivalent protein loading is indicated by visualizing actin on the same blot. (B) Significantly decreased PF4 was also observed in ARC neonatal platelets (lane 1) compared with normal neonatal (lanes 2, 3) and adult (lane 4) platelets (reduced 15% SDS-PAGE; PF4 and actin were probed and developed simultaneously). (C) Platelet VWF is undetectable in ARC platelets (lane 1) compared with normal neonatal (lanes 2, 3) and adult (lane 4) platelets (reduced 8% SDS-PAGE). (D) In contrast, VWF was present at roughly equivalent quantities (0.5 μL plasma/lane) in plasma from patients with ARC syndrome (lane 1) compared with normal neonatal (lanes 2, 3) and adult (lane 4) control plasma (reduced 8% SDS-PAGE). Bryan Lo et al. Blood 2005;106:4159-4166 ©2005 by American Society of Hematology

Soluble and membrane-bound α-granule protein deficiencies in ARC platelets and the presence of VPS33B in normal fibroblasts and megakaryocytes. Soluble and membrane-bound α-granule protein deficiencies in ARC platelets and the presence of VPS33B in normal fibroblasts and megakaryocytes. Immunoblots comparing megakaryocyte (MK) and platelet (PLT) or fibroblast whole-cell lysates from ARC and normal (N) neonatal and adult sources as indicated for each lane. Actin was visualized as a protein concentration loading control. (A) Significantly decreased β-TG was observed in ARC neonatal platelets (lane 1) compared with normal neonatal (lanes 2, 3) and adult (lane 4) platelets (reduced 15% SDS-PAGE; β-TG and actin were probed and developed simultaneously). (B) ARC platelets contained undetectable amounts of TSP-1 (lane 1) compared with normal neonatal platelets (lane 2, reduced 10% SDS-PAGE). (C) Fibrinogen was undetectable in ARC platelets (lane 1) despite its normal presence in neonatal control platelets (lane 2, nonreduced 9% SDS-PAGE) and ARC plasma (not shown). (D) The α-granule membrane-containing protein P-selectin was virtually undetectable in ARC platelets (lane 1) when compared with normal neonatal (lanes 2, 3) and adult (lane 4) platelets (reduced 8% SDS-PAGE, stripped VWF blot from Figure 3C). (E) Affinity-purified polyclonal anti–human VPS33B detected VPS33B in normal neonatal fibroblasts (lane 2) and normal adult megakaryocytes (lane 3), but not in ARC neonatal fibroblasts (lane 1) or normal adult control platelets (lane 4). Actin immunostaining is shown on the same blot. Bryan Lo et al. Blood 2005;106:4159-4166 ©2005 by American Society of Hematology

Immunofluorescent localization of VPS33B with α-granule, late endosome/lysosome, δ-granule/lysosome, and Golgi complex markers in normal human megakaryocytes. Immunofluorescent localization of VPS33B with α-granule, late endosome/lysosome, δ-granule/lysosome, and Golgi complex markers in normal human megakaryocytes. Human cultured megakaryocytes were cytocentrifuged onto glass slides, fixed, permeabilized, and labeled with polyclonal rabbit anti-VPS33B antibodies together with monoclonal antibodies to VWF (A), LAMP-1 (B), CD63 (C), and GM130 (D), followed by labeling with secondary Alexa Fluor 488 goat antirabbit and Cy3 donkey antimouse antibodies. Stained megakaryocytes were visualized by confocal microscopy where multilobed nuclei (unique to megakaryocytes) could be recognized readily using the differential interference contrast mode. Staining was typically granular. Representative images are shown and the white bar corresponds to 10 μm. (A) The merged images revealed significant partial colocalization (yellow) of VPS33B with VWF. (B) Although partial colocalization of VPS33B with LAMP-1 was observed (merged), it was less than with VWF. (C) VPS33B colocalized minimally with CD63 (merged). (D) No colocalization of VPS33B with GM130 was detected (merged). Bryan Lo et al. Blood 2005;106:4159-4166 ©2005 by American Society of Hematology