Fig 2: ARF1 and ARF4 and Their GEF GBF1 Are Involved in WPB Biogenesis(A) Immunofluorescence images showing HUVECs treated with the indicated siRNAs. Representative images acquired using an Opera confocal and used for image quantification, stained for pro-VWF, green; TGN46, red; and DAPI, blue. Scale bars, 20 µm.(B) Mean number of WPB per cell in control and ARF siRNA treated cells. n = 5 wells, where for each well the mean for each of the 9 fields of view were analyzed, SEM., one-way ANOVA with Dunnett’s multiple comparisons test, *p < 0.5, **p < 0.05, ***p < 0.005, ****p < 0.0005.(C) Proportion of secreted VWF from total VWF in control and ARF siRNA treated cells upon 30 min histamine stimulation. Results standardized to the amount secreted by control cells in each experiment. n = 3 independent experiments, SEM., one-way ANOVA with Dunnett’s multiple comparisons test, *p < 0.5, **p < 0.05, ***p < 0.005, ****p < 0.0005.(D) Mean number of WPB per cell in control and ARF GEF siRNA treated cells. n = 5 wells, where for each well the mean for each of the 9 fields of view were analyzed, SEM., one-way ANOVA with Dunnett’s multiple comparisons test, *p < 0.5, **p < 0.05, ***p < 0.005, ****p < 0.0005.(E) Proportion of secreted VWF from total VWF in control and ARF GEF siRNA treated cells upon 30 min histamine stimulation. Results standardized to the amount secreted by control cells in each experiment. n = 3 independent experiments, SEM., one-way ANOVA with Dunnett’s multiple comparisons test, *p < 0.5, **p < 0.05, ***p < 0.005, ****p < 0.0005.(F) Mean number of WPB per cell in control and ARF1 and ARF4 and GBF1 siRNA treated cells. n = 5 wells, where for each well the mean for each of the 9 fields of view were analyzed, SEM., one-way ANOVA with Dunnett’s multiple comparisons test, *p < 0.5, **p < 0.05, ***p < 0.005, ****p < 0.0005.(G) Proportion of secreted VWF from total VWF in control and ARF1 and ARF4 and GBF1 siRNA treated cells upon 30 min histamine stimulation. Results standardized to the amount secreted by control cells in each experiment. n = 3 independent experiments, SEM., one-way ANOVA with Dunnett’s multiple comparisons test, *p < 0.5, **p < 0.05, ***p < 0.005, ****p < 0.0005.(H) GBF1 depletion in endothelial cells does not induce gross morphological Golgi defects but causes dispersion of the ERGIC. Immunofluorescence confocal images of control and GBF1-siRNA-treated HUVECs for various ER-Golgi proteins. Scale bars, 10 µm.(I) Super-resolution structured illumination microscopy (SR-SIM) reconstruction of control and GBF1-siRNA-treated cells stained for TGN46, green and GM130, red. Scale bars, 5 µm.See also Figures S1 and S2.

Fig 3: GBF1 Depletion Causes an Accumulation of Unprocessed VWF in Endothelial Cells(A) Amount of VWF mRNA (relative to mock cells). n = 5 independent experiments, SEM, unpaired t test, p = 0.002.(B) Total amount of VWF protein measured by western blot (WB) (relative to mock cells). n = 4 independent experiments, SEM, unpaired t test, n.s. = not significant.(C) Representative western blot showing GBF1 protein depletion and VWF processing. “pro-VWF,” processed VWF and “unp-VWF,” unprocessed VWF.(D) Percentage of pro-VWF from total VWF (quantification from WB). n = 4 independent experiments, SEM, unpaired t test, p < 0.0001.(E) Immunofluorescence images of HUVECs stained with antibodies targeting unp-VWF, green and pro-VWF, red. Scale bars, 10 µm.(F) Immunofluorescence images of HUVECs stained for the ER-transmembrane protein calnexin, green and unp-VWF, red. Scale bars, 10 µm.(G) Immunofluorescence images of HUVECs stained for the ER-luminal protein calreticulin, green and unp-VWF, red. Scale bars, 10 µm.(H) Immunofluorescence images of HUVECs stained for the ER-luminal protein PDI, green and unp-VWF, red. Scale bars, 10 µm.See also Figure S3.

Fig 4: WPB size modulates the recruitment of plasma vWF to the endothelial surface.(a) HUVECs were subjected to flow assays as indicated. When WPB exocytosis was stimulated, histamine (100 µmol/L) was supplemented to buffer and human normal pooled plasma. (b) At the end of the assays, cells were fixed and the extracellular vWF immuno-stained and imaged. Scale bar: 25 µm. (c) Diagram of the procedure in panels (a) and (b) illustrates the assay and what it measures. (d) vWF associated with the endothelial surface following the assays described in panel (a). The area of the vWF signal on the endothelial surface was measured from 36 fields of view (the data points displayed) per condition. Bars: medians and interquartile ranges; histamine stimulation in buffer and histamine stimulation coupled to plasma were compared to the unstimulated plus plasma condition; ****P < 0.0001 (Mann-Whitney test). (e) Luciferase- or vWF-siRNA treated HUVECs were subjected to the indicated flow assay (left) and vWF deposition on endothelial cells measured (right); ****P < 0.0001 (Mann-Whitney test). (f) vWF plasma recruitment after a brief treatment with recombinant ADAMTS13 (rADAMTS13) at the indicated concentrations; *P < 0.05; ***P < 0.001 (Mann-Whitney test). (g) HUVECs treated for 24 h with vehicle (DMSO, control), medium at pH 6.4 and supplemented with acetate or nocodazole (1 µg/mL) were processed for immunofluorescence. Scale bar: 5 µm. (h) HTM analysis of HUVECS treated as described in (g). Left, Cumulative frequency (C.F.) plots of the total number of WPBs as a function of the organelles’ length. N ~ 105 per condition. £, P < 10-15 for the indicated pairwise comparisons (Mann-Whitney test). Right; long WPBs (>2 µm). Bars: mean ± range for two replicate HTM experiments. (i) HUVECs treated as described and subjected to the indicated flow assay (left) to measure vWF deposition on endothelial cells (right). Quantifications were as described above. ****P < 0.0001 (Mann-Whitney test). (j) vWF deposition on HUVECs treated with vehicle (DMSO), 0.5 or 2.5 µmol/L simvastatin for 24 h and perfused as described (left). ****P < 0.0001 (Mann-Whitney test).

Fig 5: GBF1 Protein Levels and Phosphorylation Control its Function in VWF Golgi Trafficking(A) Representative blot showing the amount of GBF1 protein in cells treated with varying levels of siRNA targeting GBF1 (applies to experiments shown in (B)–(I)).(B) Quantification of the amount of GBF1 protein in cells treated with varying levels of siRNA targeting GBF1 (quantification of WB). n = 3 independent experiments, one-way ANOVA with Dunnett’s multiple comparisons test, ****p < 0.0001.(C) Mean number of WPB per cell. n = 8 wells, where for each well the mean for each of the 9 fields of view were analyzed, SEM, one-way ANOVA with Dunnett’s multiple comparisons test, ****p < 0.0001.(D) Mean number of VWF quanta per cell. n = 8 wells, where for each well the mean for each of the 9 fields of view were analyzed, SEM, one-way ANOVA with Dunnett’s multiple comparisons test, **p < 0.01, ****p < 0.0001.(E) Proportion of WPBs longer than 2 µm in the entire WPB population. n = 8 wells, where for each well the mean for each of the 9 fields of view were analyzed, SEM, one-way ANOVA with Dunnett’s multiple comparisons test, ***p < 0.001, ****p < 0.0001.(F) Amount of unprocessed VWF, quantified by image analysis (see STAR Methods). n = 8 wells, where for each well the mean for each of the 9 fields of view were analyzed, SEM, one-way ANOVA with Dunnett’s multiple comparisons test, **p < 0.01, ****p < 0.0001.(G) Mean number of VWF exocytic sites per cell upon 15 min of histamine stimulation. n = 8 wells, where for each well the mean for each of the 9 fields of view were analyzed, SEM, one-way ANOVA with Dunnett’s multiple comparisons test, ****p < 0.0001.(H) Proportion of secreted VWF from total VWF upon 30 min of histamine stimulation, relative to mock cells. n = 3 independent experiments, SEM, one-way ANOVA with Dunnett’s multiple comparisons test, ***p < 0.001, ****p < 0.0001.(I) rtPCR showing changes in mRNA levels for the indicated transcript relative to mock samples.(J) Representative blot showing the varying amounts of GBF1Thr1337 phosphorylation in HUVECs treated with varying concentrations of AICAR for 24 h.(K) Quantification of the amount of GBF1Thr1337 relative to loading control (quantification of WB). n = 2 independent experiments, SD, one-way ANOVA with Dunnett’s multiple comparisons test, *p = 0.038, **p = 0.0021, ***p = 0.0003, n.s. = not significant.(L) Quantification of the total amount of GBF1 relative to loading control (quantification of WB). n = 2 independent experiments, SD, one-way ANOVA with Dunnett’s multiple comparisons test, n.s. = not significant.(M) Proportion of WPBs longer than 2 µm in the entire WPB population after 24 h AICAR treatment. n = 5 wells, where for each well the mean for each of the 9 fields of view were analyzed, SEM, one-way ANOVA with Dunnett’s multiple comparisons test, *p = 0.0110, ****p < 0.0001.(N) Control and GBF1-siRNA-treated cells were treated with 2 mM AICAR for 24 h. Proportion of WPBs longer than 2 µm in the entire WPB population. n = 7 wells, where for each well the mean for each of the 9 fields of view were analyzed, SEM, two-way ANOVA with a Sidak's multiple comparisons test, ****p = 0.0001, ***p = 0.001.(O) The change in WPB size after AICAR treatment in control and GBF1-siRNA-treated cells. n = 7 independent experiments, median (with minimum and maximum), unpaired t test, *** p = 0.0006.(P) Proportion of WPBs longer than 2 µm in the entire WPB population after 24 h of varying glucose treatment. n = 8 wells, where for each well the mean for each of the 9 fields of view were analyzed, SEM, one-way ANOVA with Dunnett’s multiple comparisons test, ****p < 0.0001.(Q) Control and GBF1-siRNA-treated cells were treated with 5 nM (“+”) or 0.1 mM glucose (“-”) for 24 h. Proportion of WPBs longer than 2 µm in the entire WPB population. n = 8 wells, where for each well the mean for each of the 9 fields of view were analyzed, SEM, two-way ANOVA with a Sidak's multiple comparisons test, ****p = 0.0001, n.s. = not significant.(R) Model of the effect of GBF1 depletion in retrograde membrane retrieval and anterograde cargo trafficking. The levels of GBF1-dependent COPI-mediated Golgi membrane retrieval control the rates of anterograde cargo traffic through the ER-Golgi. Low levels of GBF1 in cells, reduce the amount of COPI vesicles, reducing the rate of Golgi membrane retrieval and hence the rate of Golgi maturation and anterograde trafficking. Phosphorylation of GBF1 via AICAR, 2DG, or glucose starvation increase GBF1 activity resulting in an increase in anterograde trafficking.See also Figure S4.