Figure 5. 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).