Hagfish Vwf processing, plasma multimers, ADAMTS13 cleavage, and platelet aggregation. (A) Western blot (WB) of heart lysates from mouse or hagfish using polyclonal anti-human VWF antibody (Dako) or monoclonal anti-hagfish VWF antibody (B10), and of hagfish plasma using either monoclonal anti-hagfish Vwf antibody (B10) or polyclonal anti-hagfish Vwf antibody. Shown are bands whose sizes are consistent with mouse pro-VWF (∼300 kDa) and mature VWF (∼250 kDa), or hagfish pro-VWF (∼240 kDa) and mature VWF (∼165 kDa). (B) Human, mouse, and hagfish plasma proteins were separated on a nonreducing 1.5% agarose gel. Western blotting was carried out with polyclonal anti-human VWF antibody (Dako) alongside molecular weight marking samples of reduced and nonreduced recombinant human VWF (rhVWF) and recombinant human ADAMTS13 (rhADAMTS13). (C) Samples of hagfish Vwf from expressing CHO-cell lysates in 1.5 M urea buffer were incubated for up to 10 hours with 70 nM of full-length recombinant human ADAMTS13 with (+) or without (−) 15 mM EDTA. Note the significant reduction of the ∼240-kDa pro-Vwf band in the presence of ADAMTS13 and the appearance of a C-terminal cleavage product (∼61 kDa) detected with monoclonal anti-hagfish B10 antibody raised against C-terminal Vwf sequence. (D) Thrombocyte-rich and thrombocyte-poor plasma was prepared from ∼12 hagfish animals and measured for thrombocyte aggregation as measured by light transmission over time. Thrombocytes resuspended in thrombocyte-poor plasma were equilibrated for 2 minutes at 37°C with stirring and then incubated with varying concentrations of botrocetin (B jararaca snake venom). Thrombocyte agglutination was monitored over time (reported as minutes following addition of botrocetin) using a dual-channel optical aggregometer. Data represent the mean and SD from 3 independent experiments. Statistical analysis was carried out using paired Student t test (*P ≤ .5; **P ≤ .005). polyAB, polyclonal antibody; TBS, Tris-buffered saline; WB, western blot.