Identification of endothelial Weibel-Palade bodies by single organelle flow cytometry

Abstract

Abstract Vascular endothelial cells contain typical, elongated vesicles called Weibel-Palade bodies (WPBs), which serve as a storage compartment for biologically active factors involved in hemostasis and inflammation. While the content of WPBs is dominated by the protein von Willebrand factor (VWF), it also contains subsets of other proteins including P-selectin, CD63, interleukin-8 (IL-8) and endothelin. However, many additional proteins are contained in WPBs, but our knowledge about the WPB proteome is scarce. Although several protocols are available for generation of WPB-containing fractions from cell homogenates, isolation of highly purified WPBs for analytical or preparative purposes has not been described. By density gradient centrifugation of human umbilical vein endothelial cell (HUVEC) homogenates, a particle was identified that contained high concentrations of VWF-antigen. These particles resembled WPBs by being quite dense (peaking at 1.11-1.12 g/ml in Nycodenz gradients), and VWF was released by Triton treatment, suggesting that the isolated particle consisted of membrane enclosed VWF. Furthermore, these particles were of shape similar to WPB and are VWF-positive. Moreover, HUVECs were retrovirally transduced with a vector encoding a VWF-green fluorescent protein chimera (VWF-GFP) to fluorescently label WPBs, and by single organelle flow analysis (SOFA), we found a population of GFP+ particles that exhibited a similar density distribution. This method may serve as a basis for fluorescence-activated organelle sorting (FAOS), and could provide a new tool for understanding the biology of WPBs.