Cleavage of von Willebrand factor requires the spacer domain of the metalloprotease ADAMTS13

Abstract

ADAMTS13 consists of a reprolysin-type metalloprotease domain followed by a disintegrin domain, a thrombospondin type 1 motif (TSP1), Cys-rich and spacer domains, seven more TSP1 motifs, and two CUB domains. ADAMTS13 limits platelet accumulation in microvascular thrombi by cleaving the Tyr1605-Met1606 bond in von Willebrand factor, and ADAMTS13 deficiency causes a lethal syndrome, thrombotic thrombocytopenic purpura. ADAMTS13 domains required for substrate recognition were localized by the characterization of recombinant deletion mutants. Constructs with C-terminal His6 and V5 epitopes were expressed by transient transfection of COS-7 cells or in a baculovirus system. No association with extracellular matrix or cell surface was detected for any ADAMTS13 variant by immunofluorescence microscopy or chemical modification. Both plasma and recombinant full-length ADAMTS13 cleaved von Willebrand factor subunits into two fragments of 176 kDa and 140 kDa. Recombinant ADAMTS13 was divalent metal ion-dependent and was inhibited by IgG from a patient with idiopathic thrombotic thrombocytopenic purpura. ADAMTS13 that was truncated after the metalloprotease domain, the disintegrin domain, the first TSP1 repeat, or the Cys-rich domain was not able to cleave von Willebrand factor, whereas addition of the spacer region restored protease activity. Therefore, the spacer region is necessary for normal ADAMTS13 activity toward von Willebrand factor, and the more C-terminal TSP1 and CUB domains are dispensable in vitro.

Fig. 1.. ADAMTS13 constructs.

Secreted ADAMTS13 consists of a metalloprotease domain, a disintegrin domain, 8 thrombospondin-1 (TSP1) repeats, a Cys-rich and spacer domain, and two CUB domains. The signal peptide and propeptide that occur in the primary translation product (residues 1–74) are not part of mature ADAMTS13 and are not shown. Truncation mutants were constructed by inserting a V5 epitope, His₆ tag, and termination codon after the following amino acid residues: full-length (FL) after Thr¹⁴²⁷; del1 after Ala¹¹⁹¹; del2 after Ala⁶⁸⁵; del3 after Cys⁵⁵⁵; del4 after Glu⁴³⁹; del5 after Gly³⁸⁵; and del6 after Gln²⁸⁹. The domains present in each construct are shown schematically.

Fig. 2.. Immunofluorescence localization of ADAMTS13.

COS-7 cells expressing full-length ADAMTS13 (FL) or the indicated deletion mutants described in Fig. 1 were fixed with ethanol:acetic acid and incubated with anti-V5 IgG and Cy3-conjugated anti-mouse IgG (in red). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (in blue).

Fig. 3.. Surface biotinylation does not detect secreted cell-surface ADAMTS13.

Full-length ADAMTS13 was expressed in COS-7 cells. Cell surface proteins were biotinylated and collected by adsorption on streptavidin-agarose. Samples of the initial cell lysate, the streptavidin flow-through fraction (Unbound), the streptavidin eluate (Bound), and conditioned medium (Medium) were digested with endoglycosidase H (EndoH). Products were separated by SDS-PAGE and visualized by Western blotting with anti-V5 antibody. The position of molecular mass standards is indicated at the left.

Fig. 4.. Secreted ADAMTS13 is undetectable in extracellular matrix.

Full-length ADAMTS13 was expressed in RFL-6 cells. Cell lysate, conditioned medium, and extracellular matrix were prepared, and samples were digested with endoglycosidase H (EndoH) as described under “Experimental Procedures.” Products were separated by SDS-PAGE and visualized by Western blotting with anti-V5 antibody. The position of molecular mass standards is indicated at the left.

Fig. 5.. ADAMTS13 variants expressed in COS-7 cells.

A, COS-7 cells were transiently transfected with plasmids encoding the indicated construct that is described under Fig. 1 or with vector only (Control). Conditioned medium was concentrated 5-fold, samples (50 μl) were analyzed by SDS-PAGE on a 5–15% gradient gel, and Western blotting was done with anti-V5 antibody. B, the ability of recombinant ADAMTS13 proteins to cleave plasma VWF was measured in a two-stage assay as described under “Experimental Procedures.” The relative concentration of recombinant ADAMTS13 proteins was determined by Western blotting with anti-V5 antibody and densitometry. Samples containing equivalent amounts of recombinant ADAMTS13 ~5 μl of concentrated conditioned medium) were incubated for 16 h with VWF substrate in buffer containing 5 mM Tris-HCl, pH 8.0, 3 mM BaCl_2, and 1.5 m urea, and the integrity of the remaining VWF multimers was assessed in a collagen binding assay. The results were normalized to the values obtained with full-length ADAMTS13 (FL). The bars represent the S.E. of four independent assays.

Fig. 6.. Cleavage of VWF subunits by ADAMTS13 variants.

VWF (10 μg/ml) was incubated with the indicated recombinant ADAMTS13 protein expressed in COS-7 cells or with normal human plasma (Plasma) in buffer containing 5 mM Tris-HCl, pH 8.0, 3 mM BaCl₂ and 1.5 m urea as described under “Experimental Procedures.” Control reactions containing full-length recombinant ADAMTS13 (FL) were performed in the presence of IgG (2.4 mg/ml) from a patient with autoimmune TTP (lane 1) or 10 mM EDTA (lane 2). Products were analyzed by SDS-PAGE under reducing conditions on a 5% gel and Western blotting with polyclonal anti-VWF antibody. The mass of the intact VWF subunit and the cleavage products generated by plasma ADAMTS13 are indicated at the left.

Fig. 7.. Comparison of ADAMTS13 variants expressed in COS-7 and Sf9 cells.

The indicated ADAMTS13 constructs were expressed by transient transfection of COS-7 cells or by baculovirus infection of Sf9 cells. Samples of concentrated conditioned medium were assayed for VWF cleaving activity in a collagen-binding assay. COS-7 cells: full-length (FL), 1.4 units/ml; del2, 0.73 units/ml, del6, 0 units/ml. Sf9 cells: full-length, 0.67 units/ml; del2, 1.42 units/ml; del6, 0 units/ml. The amount of recombinant protein was determined by SDS-PAGE, Western blotting with anti-V5 antibody and densitometry as described under “Experimental Procedures.” COS-7 cells: full-length, 12.9 nmol/liter; del2, 9.0 nmol/liter; del6, 12.9 nmol/liter. Sf9 cells: full-length, 6.4 nmol/liter; del2, 13.1 nmol/liter; del6, 13.4 nmol/liter. The signals obtained for the standard protein (Positope, Invitrogen) are shown at the right. Its apparent mass is 53 kDa, and its mass calculated from amino acid sequence is 47.7 kDa.