Fig. 2.

Allosteric activation of ADAMTS13. (A) Monoclonal anti-ADAMTS13 antibodies were added to ADAMTS13 assays with pooled normal plasma (PNP). Reaction rates were normalized to the rate with PNP only. The location of epitopes is indicated. Activation values >1.5× control (orange bars) are significantly different from the PNP reference condition (P < 0.05). (B) Recombinant ADAMTS13 and MT8 were immunoprecipitated with antibodies in PNP or BCW49 plasma, and the recovered enzyme was assayed with substrate VWF71. (C and D) MDTCS (blue bars) and ADAMTS13 (orange bars) were assayed with the indicated fluorogenic substrates (Fig. 1B). (C) Bars indicate the ratio of reaction rates at pH 6 and pH 7.4 (Left), or the ratio of reaction rates with and without activating monoclonal antibodies 7C4, 19H4, and 12D4 (Right). (D) Plasma VWF multimers (0.45 µM) or VWF D4 monomers (40 µM) increased the activity of ADAMTS13 toward VWF71. SPI (2 µM), SPII (2 µM), and SPIII (0.54 µM) fragments of VWF, which are cleaved within the D4 domain by V8 protease, had little or no effect on ADAMTS13 activity. Error bars indicate 95% confidence intervals.