Intracellular distribution of canine vW AgII and vWF.

Canine constructs were transiently expressed in AtT-20 cells. Cells were fixed, permeabilized, dual stained with monoclonal antibodies to vW AgII and a polyclonal antibody to vWF that cross-react with canine and are detected with Texas Red-conjugated donkey antimouse IgG and FITC-conjugated donkey antirabbit IgG by confocal microscopy. (A-D) Cells stained for vW AgII. (E-H) vWF staining. Merges of vW AgII stain and vWF stain are shown in I to L; colocalization of vW AgII and vWF is shown in yellow. Cells expressing full-length canine vWF (A, E, I) exhibited granular storage of both vW AgII and vWF, which were colocalized as shown in I. Cells expressing canine vW AgII alone (B, F, J) displayed granular storage of vW AgII, whereas no vWF was detected. Expression of canine mature vWF resulted in a cytoplasmic staining pattern of canine vWF; no granular storage was observed (C, G, K). Co-expression of canine vW AgII and canine mature vWF (D, H, L) resulted in colocalized granular storage of both vW AgII and vWF. These results demonstrate that vW AgII chaperones vWF to regulated storage in dogs as in humans. Total magnification, 1410×.