von Willebrand Factor Is Secreted and Distributed in Two Sides of Thrombin-treated Endothelial Cells in vitro

The secretion of von Willebrand factor (vWF) in both constitutive and regulated pathways mediated through vesicles or Weibel-Palade bodies (WPb) respectively are described in cultured human umbilical vein endothelial cells (HUVEC). Competitive enzyme-linked immunosorbent assay (ELISA) analyses demonstrated that release of vWF into the culture medium and subendothelial matrix from thrombin-treated cells was 3.8 and 1.4 times of that from controls. In order to reveal the vWF secretion from cells after thrombin treatment, the immunoelectron microscopy was applied and the HUVEC cultured on coverslips were treated with thrombin. Cells on coverslips were fixed before immunostained with antibodies conjugated with peroxidase or colloidal gold. Subsequently, cells were flat-embedded in resin. The tangential and cross sections were both prepared. The peroxidase-labeled vWF was localized in the vesicles close to the cell surfaces, in WPb-like vacuoles, and on the extracellular matrix. Protrusions of vWF-positive vesicles into the cell processes, fusion vWF-positive vesicles with cytoplasmic filaments and cell membranes were observed. Accumulation of vWF on the extracellular matrix was detected by immunoperoxidase and immunogold labelings. These results suggested that both the constitutive and regulated pathways of the secretion of vWF from HUVEC treated with thrombin are involved.