Figure 1.
Characterization of novel ADAMTS13 variants with constitutive activity. (A) Three-dimensional model of the “open” conformation of ADAMTS13 was constructed using the disintegrin-like domain, thrombospondin type-1 motif, cysteine–rich domain, and spacer domain (DTCS) crystal structure (PDB 3GHM38), homology modeling of the MP-Dis domains, homology modeling of the CUB domains, and the thrombospondin-1 type 1 repeat crystal structure (PDB 1LSL39). Modeling of the linker-3 regions in wtADAMTS13 and Ala1144Val ADAMTS13 was performed using Pymol 2.4. (B) Conformation-dependent immunoprecipitation of ADAMTS13-Myc/His6 proteins. Equal volumes of starting material (IN), the unbound fraction (FT), and immunoprecipitated protein (E) were loaded for comparison. (C) The proteolytic activities of each ADAMTS13 variant were determined using the FRETS-VWF73 assay, performed in the absence (−) and presence (+) of the VWF D4-CK domain fragment. All activities were normalized to that of wtADAMTS13 and are presented as mean plus or minus standard deviation (n = 3-4). (D) The extent of VWF-mediated platelet capture under parallel flow was performed in the presence of increasing concentrations of ADAMTS13 (WT, GoF, or Ala1144Val). Results are presented as mean plus or minus standard deviation (n = 3-5). In each plot, ADAMTS13− (n = 6) and VWF− (n = 3) controls are included for comparison. (E) Fibrinolysis curves were generated by measuring the absorbance of normal human plasma following the addition of thrombin and t-PA, alone or in combination with wtADAMTS13, Ala1144Val ADAMTS13, or NAC. Each timepoint represents the mean ± standard deviation of 6 independent experiments. Individual plots were used to determine lysis times which were compared by ordinary 1-way analysis of variance (ANOVA) with Dunnett’s test of multiple comparisons. *P < .05, ****P < .0001. (F-G) The fibrinolysis assay was adapted to allow thrombin-induced fibrin formation to occur before the addition of t-PA and ADAMTS13. This was performed in a plasma-based assay (F) and purified fibrinogen-based assay (G). Curves are presented as the mean plus or minus standard deviation of 3 independent experiments.

Characterization of novel ADAMTS13 variants with constitutive activity. (A) Three-dimensional model of the “open” conformation of ADAMTS13 was constructed using the disintegrin-like domain, thrombospondin type-1 motif, cysteine–rich domain, and spacer domain (DTCS) crystal structure (PDB 3GHM38), homology modeling of the MP-Dis domains, homology modeling of the CUB domains, and the thrombospondin-1 type 1 repeat crystal structure (PDB 1LSL39). Modeling of the linker-3 regions in wtADAMTS13 and Ala1144Val ADAMTS13 was performed using Pymol 2.4. (B) Conformation-dependent immunoprecipitation of ADAMTS13-Myc/His6 proteins. Equal volumes of starting material (IN), the unbound fraction (FT), and immunoprecipitated protein (E) were loaded for comparison. (C) The proteolytic activities of each ADAMTS13 variant were determined using the FRETS-VWF73 assay, performed in the absence (−) and presence (+) of the VWF D4-CK domain fragment. All activities were normalized to that of wtADAMTS13 and are presented as mean plus or minus standard deviation (n = 3-4). (D) The extent of VWF-mediated platelet capture under parallel flow was performed in the presence of increasing concentrations of ADAMTS13 (WT, GoF, or Ala1144Val). Results are presented as mean plus or minus standard deviation (n = 3-5). In each plot, ADAMTS13− (n = 6) and VWF− (n = 3) controls are included for comparison. (E) Fibrinolysis curves were generated by measuring the absorbance of normal human plasma following the addition of thrombin and t-PA, alone or in combination with wtADAMTS13, Ala1144Val ADAMTS13, or NAC. Each timepoint represents the mean ± standard deviation of 6 independent experiments. Individual plots were used to determine lysis times which were compared by ordinary 1-way analysis of variance (ANOVA) with Dunnett’s test of multiple comparisons. *P < .05, ****P < .0001. (F-G) The fibrinolysis assay was adapted to allow thrombin-induced fibrin formation to occur before the addition of t-PA and ADAMTS13. This was performed in a plasma-based assay (F) and purified fibrinogen-based assay (G). Curves are presented as the mean plus or minus standard deviation of 3 independent experiments.

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