Differential effects of enzymatic treatments on the storage and secretion of von Willebrand factor by human endothelial cells

Enzymatic treatment used for passaging of endothelial cells may induce rele ase of von Willebrand factor (vWF). Deceased ability to replenish intracell ular stores results in decreased secretion of vWF in later passages of cell s. Since both trypsin. and pancreatin complex have been used for passaging endothelial cells, we analyzed the effects of successive passaging with the se two enzyme preparations on the storage and secretion of VWF by human umb ilical vein endothelial cells (HUVECs). Measurements were performed after t he second to fifth passages. Cytoplasmic vWF was analyzed by indirect immun ofluorescence and secreted vWF was measured in the supernatant of cultured HUVECs by ELISA. In trypsin-passaged cells, secreted vWF decreased progress ively from passages 2 to 5. Respective concentrations were 355.0 +/- 30.4, 201.0 +/- 84.5, 150.0 +/- 1.4 and 120.5 +/- 38.9 ng vWF/10(5) cells. Compar atively, pancreatin-passaged cells secreted even less vWF protein (P = .001 ) at passages 4 and 5 (108.5 +/- 12.0 and 100.0 +/- 4.2 ng/10(5) cells, res pectively) and had less vWF-positive cytoplasmic granules per cell. Thus, i n experiments involving measurements. of endothelial vWF, the use of low pa ssage cells is recommendable and passaging with a pure trypsin preparation appears to be more appropriate. (C) 2001 Elsevier Science Ltd. All rights r eserved.