Identification of cysteine thiol-based linkages in ADAMTS13 in support of a non-proteolytic regulation of von Willebrand factor

Abstract

Background: ADAMTS13, a plasma metalloprotease, cleaves von Willebrand factor (VWF) to regulate its function. Additionally, ADAMTS13 is thought to regulate lateral association of VWF multimers to form fibrillar structures through its free thiols.

Objective: The purpose of the present study is to obtain direct evidence for ADAMTS13 to engage in thiol/disulfide exchange reactions.

Methods: Covalent complexes between ADAMTS13 and VWF were determined by agarose gel electrophoresis under nonreducing conditions. Free thiols in ADAMST13 were identified by a reversed phase high-performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry system.

Results: We demonstrate formation of covalent linkage between ADAMTS13 and VWF, which is time, concentration, temperature, and shear dependent. This interaction is independent of proteolytic activity of ADAMTS13 but depends on the C-terminal domains comprising the fifth through eighth thrombospondin type 1 repeats and C1r/C1s, Uegf, Bmp1 (CUB) domains. The interaction can be blocked by thiol-reactive agents, indicating that association is accomplished through disulfide bridge formation. Several partially reduced free thiols are identified in ADAMTS13, with cysteines 1254 and 1275 being the most prominent, although a point mutation (C1275S) in ADAMTS13 does not alter its ability to form covalent linkages with VWF. This suggests functionally relevant disulfide plasticity in ADAMTS13. Interestingly, ADAMTS13 also forms homo-oligomers under the same conditions as required for the generation of hetero-oligomeric complexes of ADAMTS13 and VWF.

Conclusions: Our results suggest that a dynamic network of free thiols in ADAMTS13 undergoing intra- and inter-molecular redox reactions may add another layer of regulation to VWF function under various conditions.

Keywords: ADAMTS13; catalysis-independent regulation; free thiols; homo-oligomerization; von Willebrand factor.

Figure 1

Covalent complex formation of ADAMTS13 with von Willebrand factor (VWF). ADAMTS13 (40 μg/mL) was mixed with VWF (100 μg/mL) and incubated for 24 hours at room temperature (RT) under static conditions and shear stress, as well as under static conditions at +37°C. As controls, the single components were incubated under otherwise identical conditions (A13, VWF). In addition, freshly thawed aliquots of the single components were loaded on the gel (A13 untreated, VWF untreated). Samples were separated by 2.5% agarose gel electrophoresis and subjected to immunoblot analysis using (A) anti‐ADAMTS13 and (B) anti‐VWF antibodies. Note the appearance of oligomeric high molecular weight bands for the samples containing ADAMTS13 and VWF

Figure 2

Covalent complex formation of ADAMTS13 and von Willebrand factor (VWF) does not require the proteolytic activity of ADAMTS13. Influence of ethylenediaminetetraacetic acid (EDTA) on complex formation. VWF (100 μg/mL) was mixed with ADAMTS13 (40 μg/mL) and incubated in the presence or absence of 20 mmol/L EDTA (A13 + VWF + EDTA and A13 + VWF, respectively) under shear stress conditions at room temperature for up to 72 hours. Samples were separated by 2.5% agarose gel electrophoresis and immunoblotted with anti‐ADAMTS13 antibodies and compared with a nonincubated preparation (A13 untreated). High molecular weight bands were clearly discernible under both conditions

Figure 3

The C‐terminal region of ADAMTS13 is responsible for covalent complex formation with von Willebrand factor (VWF). (A) Schematic drawing of the ADAMTS13 fragments used. Full‐length ADAMTS13 (40 μg/mL) or equimolar quantities of the ADAMTS13 fragments MDTCS (18.3 μg/mL), T2C (21.7 μg/mL), and T5C (15.8 μg/mL) were mixed with VWF (100 μg/mL) and incubated for 24 hours (B) under static conditions at +37°C and (C) at room temperature under shear stress. As controls, ADAMTS13 and the respective fragments were incubated as single components under otherwise identical conditions (A13, MDTCS, T2C, T5C). Freshly thawed aliquots of these components were also loaded on the gel (untreated). Samples were separated by 2.5% agarose gel electrophoresis and immunoblotted with anti‐ADAMTS13 antibodies. High molecular weight bands were discernible for full‐length ADAMTS13, T2C, and T5C, but not for MDTCS, even after extended imaging of the blot. CUB, C1r/C1s, Uegf, Bmp1; D, disintegrin; MP, metalloprotease; T1, thrombospondin type 1

Figure 4

Impact of thiol‐sensitive reagents on complex formation of ADAMTS13 with von Willebrand factor (VWF). ADAMTS13 (40 μg/mL) was mixed with VWF (100 μg/mL) and incubated for 24 hours with 3.3 mmol/L N‐ethylmaleimide (NEM), 10 mmol/L iodoacetamide (IAA), or without any agent (—) under static conditions at +37°C or under shear stress conditions at room temperature. As controls, the single components were incubated under otherwise identical conditions (A13, VWF). Samples were separated by 2.5% agarose gel electrophoresis and immunoblotted with (A) anti‐ADAMTS13 antibodies and (B) anti‐VWF antibodies in comparison with nonincubated preparations. A13 untreated and VWF untreated in (A) and (B), respectively. NEM and IAA interfered with the complex formation of ADAMTS13 and VWF, but did not affect the oligomeric profile of VWF and its proteolytic degradation by ADAMTS13

Figure 5

Impact of the thiol‐sensitive agent glutathione on ADAMTS13 structure and function. ADAMTS13 (230 μg/mL) was treated with 2 or 8 mmol/L of reduced (GSH) or oxidized (GSSG) glutathione and incubated for 3 hours at room temperature (RT) under static (−) or shear stress (+) conditions. As control, ADAMTS13 was incubated in bulk drug substance buffer. Samples were separated by 2.5% agarose gel electrophoresis under nonreducing conditions and ADAMTS13 was detected by immunoblot analysis with anti‐ADAMTS13 antibodies (A). For comparison, nonincubated ADAMTS13 was also loaded (A13 untreated). An increase in homo‐oligomers was specifically detected under shear stress in the presence of GSH but not GSSG. ADAMTS13 homo‐oligomerization was quantified by size exclusion chromatography, for which ADAMTS13 was similarly incubated for 3 hours under static conditions with increasing concentrations of GSH (2–25 mmol/L) (B). The resulting chromatograms were evaluated for the relative content (%) of ADAMTS13 monomers, dimers, and oligomers. The same samples were assayed for ADAMTS13 proteolytic activity using the FRETS‐VWF73 assay, with activities being presented in percentage of the control incubation without GSH (defined as 100%) (B). To assess the concentration of free cysteine thiols in glutathione‐treated ADAMTS13, the protein was incubated with 8 or 25 mmol/L GSH or GSSG for 3 hours at RT and, following dialysis, incubated with maleimide‐polyethylene glycol 2‐biotin (MBP) for 15 minutes to label free cysteine thiols (C). Unbound MBP was removed by dialysis and bound MBP visualized by immunoblot analysis using horseradish peroxide–coupled streptavidin. A freshly thawed aliquot of ADAMTS13 was also loaded on the gel (A13 untreated). The amount of free cysteine thiols was substantially higher in the GSH‐treated samples

Figure 6

Impact of mutating cysteine 1275 on complex formation with von Willebrand factor (VWF) in ADAMTS13. The point‐mutated variant ADAMTS13 C1275S (40 μg/mL) was mixed with VWF (100 μg/mL) and incubated for 24 h with 3.3 mmol/L N‐ethylmaleimide (NEM), 10 mmol/L iodoacetamide (IAA), or without any agent (—) under static conditions at +37°C or under shear stress conditions at room temperature. As controls, the single components were incubated under otherwise identical conditions (A13 C1275S, VWF). Samples were separated by 2.5% agarose gel electrophoresis and immunoblotted with (A) anti‐ADAMTS13 antibodies and (B) anti‐VWF antibodies in comparison with nonincubated preparations (A13 C1275S untreated and VWF untreated in (A) and (B), respectively). The NEM‐sensitive appearance of oligomeric high molecular weight bands indicated that ADAMTS13 C1275S retained the ability to form covalent complexes with VWF