Figure 3.
Figure 3. Expression of human or canine VWF proteins in canine VWD aortic endothelial cells. (A-F) Wild-type VWF. Canine VWD aortic endothelial cells were transiently transfected with either human VWF (A-C) or canine VWF (D-F) by nucleofection. Transfected cells were fixed, immunolabeled, and examined by confocal microscopy as described in “Materials and methods.” Cells were immunolabeled with a mix of several mouse monoclonal anti-VWFpp antibodies (A), a rabbit anti-VWF (B), AVW-1 mouse monoclonal anti-VWF (D), or rabbit anti–P-selectin (E). Immunostaining of cells expressing human VWF demonstrated a granular distribution of VWFpp and VWF (A-B). Merging the 2 images revealed colocalization of the proteins (C). Dual staining for VWF and P-selectin revealed that the canine VWF (D-F) colocalized with P-selectin in granules. Expression of canine VWF reestablishes the Weibel-Palade body distribution of endogenous P-selectin. (G-L) Dimeric VWF. Cells were transiently transfected with either a dimeric, propeptide-deleted VWF (Δpro) or dimeric, mutant Y87S-VWF. Cells were immunostained using the monoclonal anti-VWF, AVW-1 (G), rabbit anti–P-selectin (H), a mix of several mouse monoclonal anti-VWFpp antibodies (J) or polyclonal anti-VWF (K). Cells expressing human Δpro (G-I) showed diffuse VWF staining with no apparent VWF-containing granules (G). P-selectin distribution was unchanged (H), and the 2 proteins did not appear to colocalize (I). A dimeric, Y87S-VWF mutant was also transiently expressed in the canine VWD aortic endothelial cells (J-L). The mutant Y87S VWFpp and VWF showed a granular staining pattern in addition to diffuse staining and were found to colocalize (J-L). VWF multimerization is not necessary for Weibel-Palade body formation. (M-O) Coexpression of VWFpp and Δpro. Cells were transiently transfected with Δpro and VWFpp in trans as 2 separate constructs. Cells were immunostained using AVW-1 monoclonal anti-VWF (M) and rabbit anti–P-selectin (N). In cells expressing VWF, P-selectin was found to colocalize with VWF in granules (O). Coexpression of VWFpp and Δpro generates Weibel-Palade bodies in canine VWD aortic endothelial cells.

Expression of human or canine VWF proteins in canine VWD aortic endothelial cells. (A-F) Wild-type VWF. Canine VWD aortic endothelial cells were transiently transfected with either human VWF (A-C) or canine VWF (D-F) by nucleofection. Transfected cells were fixed, immunolabeled, and examined by confocal microscopy as described in “Materials and methods.” Cells were immunolabeled with a mix of several mouse monoclonal anti-VWFpp antibodies (A), a rabbit anti-VWF (B), AVW-1 mouse monoclonal anti-VWF (D), or rabbit anti–P-selectin (E). Immunostaining of cells expressing human VWF demonstrated a granular distribution of VWFpp and VWF (A-B). Merging the 2 images revealed colocalization of the proteins (C). Dual staining for VWF and P-selectin revealed that the canine VWF (D-F) colocalized with P-selectin in granules. Expression of canine VWF reestablishes the Weibel-Palade body distribution of endogenous P-selectin. (G-L) Dimeric VWF. Cells were transiently transfected with either a dimeric, propeptide-deleted VWF (Δpro) or dimeric, mutant Y87S-VWF. Cells were immunostained using the monoclonal anti-VWF, AVW-1 (G), rabbit anti–P-selectin (H), a mix of several mouse monoclonal anti-VWFpp antibodies (J) or polyclonal anti-VWF (K). Cells expressing human Δpro (G-I) showed diffuse VWF staining with no apparent VWF-containing granules (G). P-selectin distribution was unchanged (H), and the 2 proteins did not appear to colocalize (I). A dimeric, Y87S-VWF mutant was also transiently expressed in the canine VWD aortic endothelial cells (J-L). The mutant Y87S VWFpp and VWF showed a granular staining pattern in addition to diffuse staining and were found to colocalize (J-L). VWF multimerization is not necessary for Weibel-Palade body formation. (M-O) Coexpression of VWFpp and Δpro. Cells were transiently transfected with Δpro and VWFpp in trans as 2 separate constructs. Cells were immunostained using AVW-1 monoclonal anti-VWF (M) and rabbit anti–P-selectin (N). In cells expressing VWF, P-selectin was found to colocalize with VWF in granules (O). Coexpression of VWFpp and Δpro generates Weibel-Palade bodies in canine VWD aortic endothelial cells.

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