Figure 1
Recombinant VWF and ADAMTS13 fragments. (A) A first series of recombinant VWF fragments (VWFD′D3, VWFA2, VWFA1A2A3, and VWFA1CK) were expressed in mammalian cells and purified as described. They all had a polyhistidine epitope tag (C-terminal for VWFD'D3 and VWFA2; N-terminal for VWFA1A2A3 and VWFA1CK) to facilitate purification. The fragments are represented as labeled arrows underneath the diagram of VWF domains. The VWF numbering for each fragment is given. (B) An additional series of recombinant VWF C-terminal fragments (VWFA3CK, VWFD4CK, VWFD4, and VWFB1CK) were expressed in mammalian cells and purified as described. They all had a C-terminal polyHis epitope tag, with the exception of VWFD4 in which no tag was present. (C) Full-length ADAMTS13 consists of a metalloprotease domain (M), a disintegrin-like domain (D), a thrombospondin type 1 repeat, (1) a Cys-rich and spacer domain (Cys Spacer), 7 additional thrombospondin type 1 repeats (2-8), and 2 complement Clr/Cls sea urchin epidermal growth and bone morphogenic protein 1 or CUB domains (Cub). ADAMTS13 variants truncated after the spacer domain, MDTCS, and after the fourth thrombospondin repeat, del(TSP5-CUB), were also generated and are represented as labeled arrows underneath the diagram of ADAMTS13 domains. (D) The VWF fragments depicted in panel A were purified and analyzed by SDS-PAGE followed by Coomassie staining, under both nonreducing (NR) and reducing (R) conditions. VWFD′D3 and VWFA1CK were expressed as dimers, which were reduced to their expected monomeric size under reducing conditions. (E) The wild-type ADAMTS13 and its truncated variants were analyzed by SDS-PAGE followed by Coomassie staining, under reducing conditions. ADAMTS13, del(TSP5-CUB), and MDTCS proteins were visualized on the gel as single bands of approximately 195, 120, and 95 kDa, respectively. (F) The VWF fragments depicted in panel B were purified and analyzed by SDS-PAGE followed by Coomassie staining, under both nonreducing (NR) and reducing (R) conditions. VWFD4 was visualized as a band of the expected molecular weight of approximately 40 kDa. VWFA3CK, VWFD4CK, and VWFB1CK were expressed as dimers (∼ 254, 200, and 132 kDa, respectively) since they all included the CK region.

Recombinant VWF and ADAMTS13 fragments. (A) A first series of recombinant VWF fragments (VWFD′D3, VWFA2, VWFA1A2A3, and VWFA1CK) were expressed in mammalian cells and purified as described. They all had a polyhistidine epitope tag (C-terminal for VWFD'D3 and VWFA2; N-terminal for VWFA1A2A3 and VWFA1CK) to facilitate purification. The fragments are represented as labeled arrows underneath the diagram of VWF domains. The VWF numbering for each fragment is given. (B) An additional series of recombinant VWF C-terminal fragments (VWFA3CK, VWFD4CK, VWFD4, and VWFB1CK) were expressed in mammalian cells and purified as described. They all had a C-terminal polyHis epitope tag, with the exception of VWFD4 in which no tag was present. (C) Full-length ADAMTS13 consists of a metalloprotease domain (M), a disintegrin-like domain (D), a thrombospondin type 1 repeat, (1) a Cys-rich and spacer domain (Cys Spacer), 7 additional thrombospondin type 1 repeats (2-8), and 2 complement Clr/Cls sea urchin epidermal growth and bone morphogenic protein 1 or CUB domains (Cub). ADAMTS13 variants truncated after the spacer domain, MDTCS, and after the fourth thrombospondin repeat, del(TSP5-CUB), were also generated and are represented as labeled arrows underneath the diagram of ADAMTS13 domains. (D) The VWF fragments depicted in panel A were purified and analyzed by SDS-PAGE followed by Coomassie staining, under both nonreducing (NR) and reducing (R) conditions. VWFD′D3 and VWFA1CK were expressed as dimers, which were reduced to their expected monomeric size under reducing conditions. (E) The wild-type ADAMTS13 and its truncated variants were analyzed by SDS-PAGE followed by Coomassie staining, under reducing conditions. ADAMTS13, del(TSP5-CUB), and MDTCS proteins were visualized on the gel as single bands of approximately 195, 120, and 95 kDa, respectively. (F) The VWF fragments depicted in panel B were purified and analyzed by SDS-PAGE followed by Coomassie staining, under both nonreducing (NR) and reducing (R) conditions. VWFD4 was visualized as a band of the expected molecular weight of approximately 40 kDa. VWFA3CK, VWFD4CK, and VWFB1CK were expressed as dimers (∼ 254, 200, and 132 kDa, respectively) since they all included the CK region.

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