Immunolocalization of von Willebrand protein in Weibel-Palade bodies of human endothelial cells

Abstract

Immunofluorescence staining of cultured human umbilical vein endothelial cells has shown the presence of von Willebrand protein in the perinuclear region, in small rodlike structures through the cytoplasm, and on filaments of the extracellular matrix. Nonendothelial cells showed no staining with anti-von Willebrand protein antiserum. At the light microscope level, immunoperoxidase treatment of endothelial cells revealed the same pattern and antibody specificity as the fluorescence staining. Thin sections of the peroxidase-stained cells showed decorated filaments close to the substratum and also specific deposits in the endoplasmic reticulum and WeibeI-Palade bodies. Control antisera against other selected proteins in endothelial cells failed to stain the WeibeI-Palade bodies. These data suggest that the WeibeI-Palade bodies of endothelial cells are storage and/or processing organelles for von Willebrand protein.Von Willebrand protein is a large glycoprotein of complex multimeric structure (1, 2) that mediates attachment ofplatelets to the subendothelium after vascular injury (3). It is synthesized by megakaryocytes (4), which assure its presence in platelets in granulelike storage compartments (5, 6). After activation, platelets bind both von Willebrand protein released from internal storage sites and protein recruited from plasma to their surface membrane (7). Endothelial cells also synthesize von Willebrand protein (8) and the low concentration present in plasma and the subendothelium is probably derived from this source. We studied the distribution of von Willebrand protein in endothelial cells, in an attempt to detect and identify a storage compartment which would allow rapid release of this protein upon appropriate stimulus or physiologic demand. Using indirect immunofluorescence and immunoelectron microscopy, we determined that von Willebrand protein is concentrated in Weibel-Palade bodies. These are membrane-bound, elongate vesicles of 0.1 x 2-3 #In size that contain regularly spaced tubular structures aligned parallel to the longitudinal axis (9). Our data suggest that Weibel-Palade bodies serve as storage and/or processing vesicles for this protein and provide the first demonstration of unique function for these endothelial cell-specific organelles. MATERIALS AND METHODS Cells and Culture ConditionsEndothelial cells were obtained from human umbilical vein using a modification of the method described by Gimbrone et al.(10). Mild proteolytic digestion was carried out with 5 mg/ml of pronase (Calbiochem-Behring,

Gray platelet syndrome. Dissociation between abnormal sorting in megakaryocyte alpha-granules and normal sorting in Weibel-Palade bodies of endothelial cells.

“…The soluble protein content of a-granules was shown to be synthesized normally but improperly packaged and lost in the demarcation membrane system early during megakaryocyte maturation (4,28). We describe the presence oftypical WPB, the specific storage organelles for vWF ( 16), in endothelial cells from two patients with GPS with normal ultrastructure and normal internal distribution of both vWF and P-selectin.…”

“…In endothelial cells, the vWF is stored in Weibel-Palade bodies (WPB), in its highest molecular weight form (16)(17)(18)(19), whereas in megakaryocytes and platelets, vWF is localized in a-granules, along with many other hemostatic proteins (20,21). Both WPB and a-granules are lined by a single membrane that contains P-selectin (8,22,23), a glycoprotein involved in monocyte and neutrophil recognition (24)(25)(26)(27).…”

The VE‐cadherin binding domain of fibrinogen induces endothelial barrier permeability and enhances transendothelial migration of malignant breast epithelial cells

“…14,[26][27][28] Control experiments indicated that Fg added to breast cancer or endothelial cells was not processed further by protease degradation or crosslinking to higher ordered structures (data not shown). Synthetic peptides corresponding to the Fg b-chain primary structure were custom synthesized as previously described, 11 and include b , b [15][16][17][18][19][20][21][22][23][24][25][26][27] , b 18-31 and b [24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42] (Table I). Fibrinopeptide A (Bachem) was used as a nonspecific peptide for permeability studies.…”

“…24 EC used for permeability and TEM assays were cultured in phenol red-free McCoy's 5A with 5% fetal bovine serum without ECGS and heparin. The HMEC-1 human dermal microvascular endothelial cell line was cultured in MCDB-131 (Invitrogen) with 10% fetal bovine serum, 10 ng/ml recombinant human EGF and 1 lg/ml hydrocortisone (Sigma).…”

Gene therapy for hemophilia

“…Studies in vitro have suggested that this stabilizing effect is enhanced when F.VIII and vWF are co-expressed in the same cell, rather than simply adding vWF to the media (219,228). However, while vWF is believed to be produced in endothelial cells, platelets, and megakaryocytes, the site of F.VIII synthesis is somewhat controversial (229)(230)(231). The liver has been implicated as a major site of F.VIII synthesis due to the ability of liver transplantation to cure hemophilia A in canine models as well as human patients (232)(233)(234).…”