Evaluation of a disintegrin-like and metalloprotease with thrombospondin type 1 repeat motifs 13 (ADAMTS13) activity enzyme-linked immunosorbent assay for measuring plasma ADAMTS13 activity in dogs

Abstract

A disintegrin-like and metalloprotease with thrombospondin type 1 repeat motifs 13 (ADAMTS13) is a von Willebrand factor (vWF)-cleaving protease. Deficiencies in ADAMTS13 activity are known to cause thrombotic diseases in human beings. The present study evaluated whether the human ADAMTS13 activity enzyme-linked immunosorbent assay (ELISA) kit containing human vWF73 (a minimal substrate) and anti-N10 antibody (which specifically recognizes the decapeptide of the C-terminal edge of cleaved vWF by human ADAMTS13) is applicable to the measurement of canine plasma ADAMTS13 activity. Human vWF73 fused with a GST-tag and a His-tag (GST-hvWF73-His) was reacted with recombinant canine (rc)ADAMTS13, canine plasma, and human plasma, and then used in Western blotting using anti-N10 antibody. Linearity and intra- and interassay reproducibility of the human ADAMTS13 activity ELISA kit in canine plasma were further evaluated. Finally, plasma ADAMTS13 activity was measured in 13 healthy dogs and 6 dogs with bacteremia using the human ADAMTS13 activity ELISA kit. Cleaved products with a 28-kDa GST-hvWF73-His were detected specifically in rcADAMTS13 as well as in human ADAMTS13, and also in canine plasma by anti-N10 antibody, showing excellent linearity. Intra-assay coefficient of variation (CV) was 3.0–12.4%, and interassay CV was 11.5–12.5%. The ADAMTS13 activity was significantly lower in dogs with bacteremia than in healthy dogs ( P = 0.0025). The current study revealed that the human ADAMTS13 activity ELISA kit is applicable for measurement of canine plasma ADAMTS13 activity to elucidate the pathology of thrombotic diseases in dogs.