Inhibition of rADAMTS13 activity by TTP patient plasma containing antibodies neutralizing VWF-cp activity.

VWF-cp assay mixtures were loaded onto an SDS-1% agarose gel, and the VWF multimer pattern was detected by immunostaining. (A) Inhibitory TTP patient plasma was added (+) to the samples before dilution (1:20) activation and incubation with VWF. Buffer was used instead of TTP plasma in some samples (−). Samples used were pooled NHP (lanes 2 and 3), conditioned medium derived from transfections using the vector control (lanes 4 and 6), and VWF-cp expression vector (lanes 5 and 7). VWF-substrate is shown in lane 1. (B) Purified IgG derived from pooled NHP or acquired TTP patient plasma and buffer as control was mixed with conditioned medium containing rADAMTS13 either concentrated (lanes 1, 5, 9) or diluted 1:4 (lanes 2, 6, 10), 1:16 (lanes 3, 7, 11), or 1:64 (lanes 4, 8, 12). (C) NHP was incubated directly (lanes 1, 3, 5) or diluted 1:2 (lanes 2, 4, 6) with purified IgG from NHP or inhibiting TTP patient plasma and buffer.