Figure 3.
Figure 3. A novel assay for ADAMTS13. / (A) Structures of substrates A–E. The domain structure of human preproVWF is depicted. Five recombinant proteins flanked with GST- and H-tags were expressed for the ADAMTS13 assay. Substrates D and E were designated as GST-VWF73-H and GST-VWF64-H, respectively. / (B) Cleavage of recombinant proteins. The recombinant proteins were incubated with normal plasma at 37°C for 1 hour. The reaction products were run by SDS-polyacrylamide gel electrophoresis and the products were detected by anti-GST antibodies. The product bands are indicated by asterisks. The nonspecific bands of approximately 50 kDa are plasma albumin. / (C) Amino acid sequence of VWF73. Nine amino acid residues indispensable for cleavage are underlined. / (D) Effect of urea and ion concentration on cleavage. GST-VWF73-H was incubated with plasma for the indicated time in reaction buffer (5 mM Tris-HCl, 10 mM BaCl2, pH 8.0) or in the same buffer supplemented with 1.5 M urea or 150 mM NaCl. The products are marked by an asterisk. / (E) Cleavage of GST-VWF73-H by patients’ plasma. GST-VWF73-H was incubated with serially diluted normal plasma (0%–100%) or with plasma from patients with Upshaw-Schulman syndrome (USS), ticlopidine-associated thrombotic thrombocytopenic purpura (Tc-TTP), idiopathic TTP, or hemolytic uremic syndrome (HUS), and the products were detected by anti-GST antibodies. / A part of these figures were originally published in Blood. Kokame K, Matsumoto M, Fujimura Y, Miyata T. VWF73, a region from D1596 to R1668 of von Willebrand factor, provides a minimal substrate for ADAMTS-13. Blood. 2004:103:607–612.

A novel assay for ADAMTS13.

(A) Structures of substrates A–E. The domain structure of human preproVWF is depicted. Five recombinant proteins flanked with GST- and H-tags were expressed for the ADAMTS13 assay. Substrates D and E were designated as GST-VWF73-H and GST-VWF64-H, respectively.

(B) Cleavage of recombinant proteins. The recombinant proteins were incubated with normal plasma at 37°C for 1 hour. The reaction products were run by SDS-polyacrylamide gel electrophoresis and the products were detected by anti-GST antibodies. The product bands are indicated by asterisks. The nonspecific bands of approximately 50 kDa are plasma albumin.

(C) Amino acid sequence of VWF73. Nine amino acid residues indispensable for cleavage are underlined.

(D) Effect of urea and ion concentration on cleavage. GST-VWF73-H was incubated with plasma for the indicated time in reaction buffer (5 mM Tris-HCl, 10 mM BaCl2, pH 8.0) or in the same buffer supplemented with 1.5 M urea or 150 mM NaCl. The products are marked by an asterisk.

(E) Cleavage of GST-VWF73-H by patients’ plasma. GST-VWF73-H was incubated with serially diluted normal plasma (0%–100%) or with plasma from patients with Upshaw-Schulman syndrome (USS), ticlopidine-associated thrombotic thrombocytopenic purpura (Tc-TTP), idiopathic TTP, or hemolytic uremic syndrome (HUS), and the products were detected by anti-GST antibodies.

A part of these figures were originally published in Blood. Kokame K, Matsumoto M, Fujimura Y, Miyata T. VWF73, a region from D1596 to R1668 of von Willebrand factor, provides a minimal substrate for ADAMTS-13. Blood. 2004:103:607–612.

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