Quantification and morphometric assessment of pseudo-WPBs
Image restoration and 3-dimensional (3D) analysis of pilled-up z-stack images (least eight fields counting a minimum of 100 cells expressing VWF for each wt and mutants) were executed by the arivis Vision4D 3.2 software (arivis AG, Imaging Science, Germany). The cells were highlighted based on edge detection algorithms followed by a smoothing preprocessing using Arivis’s proprietary algorithm Blob Finder. The cells expressing VWF (green signal) were elected. Subsequently, the pseudo-WPBs were detected on the green signal after applying enhancement preprocessing tasks. The average number of pseudo-WPBs per cell was calculated by dividing the global count of the WPBs in each acquired image arena by the cells encompassing at least one WPB. The pseudo-WPBs features including, length, depth, and width (provided by measuring the longest, middle, and shortest sides of the 3D oriented bounds, respectively) as well as the shape (characterized by Sphericity factor, Ψ) and mean intensities, were computed for whole WPBs-like organelles population, including 1105 of pseudo-WPBs for wt and between 237-355 for heterozygous variants. Sphericity (Ψ) describes the roundness of the 3D segment represented as a value between 0 and 1, where 1 is an ideal sphere. Mean intensities show the average intensity signal value for every single pseudo-WPBs.