Demonstration of disulfide bond formation-mediated interaction of ...
Demonstration of disulfide bond formation-mediated interaction of ADAMTS13 with VWF under shear stress H. Rottensteiner, S. Skalicky, B. Plaimauer, F. Scheiflinger Baxter Innovations GmbH, Vienna, Austria
Introduction ADAMTS13 is the key regulator of VWF activity. In addition to its established proteolytic function involving the metalloprotease domain and adjacent regions up to the spacer domain, a free thiol-dependent interaction of the enzyme’s C-terminal part with VWF is hypothesized to regulate the lateral association of VWF multimers under shear stress. Since this latter activity has been associated with the overall antithrombotic function of ADAMTS13, we attempted to demonstrate such an interaction in vitro using purified recombinant components.
The kinetics of complex formation was studied by keeping a mixture of rADAMTS13 and rVWF under shear stress for up to 72 h. A similar mixture but containing 20 mM EDTA to inhibit the proteolytic activity of ADAMTS13 was studied in parallel. 40 µg/mL rA13 + 100 µg/mL rVWF
0
3
6
24
48 72 0 24 timepoints in h
40 µg/mL rA13 + 100 µg/mL rVWF
Results
40 µg/mL rA13 + 100 µg/mL rVWF
40 µg/mL rA13 100 µg/mL rVWF rA13
72
0
24
0
72
3
6
24
40 µg/mL rA13 100 µg/mL rVWF rA13
48 72 0 24 timepoints in h
40 µg/mL rA13 + 100 µg/mL rVWF
100 µg/mL rVWF rVWF
72
0
100 µg/mL rVWF rVWF
To see whether ADAMTS13 can form a covalent, disulfide bond-mediated complex with VWF, full-length rADAMTS13 was mixed with rVWF under static conditions and under shear stress. Adducts formed were detected by SDS agarose gel electrophoresis under non-reducing conditions.
40µg/mL rA13 + 0 475 237 119 59 30 µg/mL rVWF
rA13 475 237 119 59 30 µg/mL rVWF
40µg/mL rA13 + 475 237 119
59
µg/mL rVWF
30
40µg/mL rA13 + 0
rVWF 475 237 119
59
µg/ml rVWF
Static conditions
30
rA13
475 237 119 59 30 µg/mL rVWF
475 237 119 59 30 µg/mL rVWF
40µg/mL rA13 + 475
237 119
59
µg/mL rVWF
30
rVWF 475 237 119
59
µg/ml rVWF
Shear stress
30
Figure 1. Covalent complex formation of ADAMTS13 with VWF under static conditions and under shear stress. Fulllength rADAMTS13 was mixed with increasing concentrations of recombinant VWF and incubated for 24 h at room temperature under static conditions (A), under shear stress (B), and at 37°C under static conditions (C). The samples were then separated by 2.5% agarose gel electrophoresis under nonreducing conditions and stained for ADAMTS13 (left panels) and VWF (right panels) by Western blot analysis. For comparison, non incubated preparations of ADAMTS13 (rA13) and VWF (rVWF) were loaded.
ADAMTS13-VWF adduct formation was clearly discernible and was greatly stimulated under shear stress
0
3
6
24
48 72
0
24
72
0
timepoints in h
3
6
24
48
24
72
0
24
72
Figure 2. Kinetics of rADAMTS13rVWF complex formation under shear stress in the presence or absence of 20 mM EDTA. Full-length rADAMTS13 (40 µg/mL) was mixed with rVWF (100 µg/mL) and incubated for the indicated time points under shear stress in the (A) absence or (B) presence of 20 mM EDTA. Samples were then separated by 2.5% agarose gel electrophoresis under nonreducing conditions and stained for ADAMTS13 (upper panel) and VWF (lower panel) by Western blot analysis.
VWF complex formation under shear stress was also tested with MDTCS, a C-terminally truncated fragment of ADAMTS13. Due to the low sensitivity of the antibody, a higher concentration of MDTCS was used. FIX was used as a negative control.
0
24
48
96
200 µg/mL rA13 MDTCS
0 24 96 timepoints in h
200 µg/mL rVWF
0
24
rA13 MDTCS
96
Shear stress
12 µg/mL rFIX + 100 µg/mL rVWF
0
24
48
72
12 µg/mL rFIX
0 24 72 0 timepoints in h
100 µg/mL rVWF
24
rFIX
72
Shear stress, EDTA
Figure 3. Test for VWF adduct formation under shear stress with MDTCS and rFIX. rVWF (100 µg/mL) was mixed with (A) MDTCS, a C-terminally truncated rADAMTS13 (200 µg/mL) and (B) rFIX (12 µg/mL) and incubated for the indicated time points at RT under shear stress. Samples were then separated by 2.5% agarose gel electrophoresis under non-reducing conditions and stained for ADAMTS13 by Western blot analysis.
No or only very weak binding to rVWF was noted for the MDTCS fragment even at an almost 10-fold molar excess compared to full-length ADAMTS13 No evidence for homo-oligomerization of MDTCS
Presented at the Bari International Conference (8th BIC) 2014 in Bari, Italy • Oct 3-5, 2014 Conflicts of interest: The authors of this presentation make the following disclosure of financial or personal relationships with commercial entities that may have a direct or indirect interest in the subject matter of this presentation: All authors are full-time employees of Baxter Innovations GmbH, Vienna, Austria.
---- NEM
IAA ----
NEM IAA
Shear stress
40 µg/mL rA13 C1275 S
----
----
NEM
IAA
rA13 C1275S
NEM IAA
Shear stress
Figure 4. Impact of NEM and IAA on rADAMTS13-rVWF complex formation.. Full-length rADAMTS13 or rADAMTS13 C1275S (40 µg/mL) was mixed with rVWF (100 µg/mL) and incubated with 3.3 mM NEM or 10 mM IAA or without any agent (-) for 24 h under shear stress. Samples were separated by 2.5% agarose gel electrophoresis and immunoblotted with anti-ADAMTS13 antibodies in comparison to a non-incubated preparation (rA13).
ADAMTS13 adduct formation was prevented by NEM and partially by IAA The C1275S variant did not influence adduct formation
Methods
Shear stress, EDTA
ADAMTS13 adduct formation was slow and was not quantitative ADAMTS13 homo-oligomerization could be also observed The proteolytic activity of ADAMTS13 was not required for adduct formation
200 µg/mL rA13 MDTCS + 200 µg/mL rVWF
40 µg/mL rA13 + 100 µg/mL rVWF 40 µg/mL rA13 rA13
40 µg/mL rA13 C1275S + 100 µg/mL rVWF
72
timepoints in h
Shear stress
To prove that the observed complex formation occurs through disulfide bond formation, the thiol-reactive compounds NEM and IAA were added to the ADAMTS13VWF mixture during incubation. The ADAMTS13 C1275S variant thought to be important for covalent VWF complex formation was included in the experiment.
Recombinant VWF, full-length rADAMTS13, rMDTCS, a C-terminally truncated version of ADAMTS13, and rADAMTS13 C1275S, a mutant described to partially block the thiolmediated interaction with VWF, were purified from CHO cells. Full-length rADAMTS13 and variants were incubated with rVWF at room temperature (RT) under static conditions and under shear stress. As control, mixtures were incubated in the presence of N-ethylmaleimide (NEM) or iodoacetamide (IAA). Covalent interaction between ADAMTS13 and VWF was assayed by agarose gel electrophoresis under non-reducing conditions and immunoblot analysis using VWF- and ADAMTS13-specific antibodies.
Conclusions • We demonstrate covalent binding of the C-terminal part of ADAMTS13 to VWF especially under shear stress • Binding also occurred in the presence of EDTA, where VWF is not cleaved by ADAMTS13 • Addition of NEM prevented interaction of ADAMTS13 with VWF • Binding was not affected by the ADAMTS13 C1275S variant • Our data support the hypothesis of thiol-dependent regulation of VWF activity by ADAMTS13
Introduction ADAMTS13 is the key regulator of VWF activity. In addition to its established proteolytic function involving the metalloprotease domain and adjacent regions up to the spacer domain, a free thiol-dependent interaction of the enzyme’s C-terminal part with VWF is hypothesized to regulate the lateral association of VWF multimers under shear stress. Since this latter activity has been associated with the overall antithrombotic function of ADAMTS13, we attempted to demonstrate such an interaction in vitro using purified recombinant components.
The kinetics of complex formation was studied by keeping a mixture of rADAMTS13 and rVWF under shear stress for up to 72 h. A similar mixture but containing 20 mM EDTA to inhibit the proteolytic activity of ADAMTS13 was studied in parallel. 40 µg/mL rA13 + 100 µg/mL rVWF
0
3
6
24
48 72 0 24 timepoints in h
40 µg/mL rA13 + 100 µg/mL rVWF
Results
40 µg/mL rA13 + 100 µg/mL rVWF
40 µg/mL rA13 100 µg/mL rVWF rA13
72
0
24
0
72
3
6
24
40 µg/mL rA13 100 µg/mL rVWF rA13
48 72 0 24 timepoints in h
40 µg/mL rA13 + 100 µg/mL rVWF
100 µg/mL rVWF rVWF
72
0
100 µg/mL rVWF rVWF
To see whether ADAMTS13 can form a covalent, disulfide bond-mediated complex with VWF, full-length rADAMTS13 was mixed with rVWF under static conditions and under shear stress. Adducts formed were detected by SDS agarose gel electrophoresis under non-reducing conditions.
40µg/mL rA13 + 0 475 237 119 59 30 µg/mL rVWF
rA13 475 237 119 59 30 µg/mL rVWF
40µg/mL rA13 + 475 237 119
59
µg/mL rVWF
30
40µg/mL rA13 + 0
rVWF 475 237 119
59
µg/ml rVWF
Static conditions
30
rA13
475 237 119 59 30 µg/mL rVWF
475 237 119 59 30 µg/mL rVWF
40µg/mL rA13 + 475
237 119
59
µg/mL rVWF
30
rVWF 475 237 119
59
µg/ml rVWF
Shear stress
30
Figure 1. Covalent complex formation of ADAMTS13 with VWF under static conditions and under shear stress. Fulllength rADAMTS13 was mixed with increasing concentrations of recombinant VWF and incubated for 24 h at room temperature under static conditions (A), under shear stress (B), and at 37°C under static conditions (C). The samples were then separated by 2.5% agarose gel electrophoresis under nonreducing conditions and stained for ADAMTS13 (left panels) and VWF (right panels) by Western blot analysis. For comparison, non incubated preparations of ADAMTS13 (rA13) and VWF (rVWF) were loaded.
ADAMTS13-VWF adduct formation was clearly discernible and was greatly stimulated under shear stress
0
3
6
24
48 72
0
24
72
0
timepoints in h
3
6
24
48
24
72
0
24
72
Figure 2. Kinetics of rADAMTS13rVWF complex formation under shear stress in the presence or absence of 20 mM EDTA. Full-length rADAMTS13 (40 µg/mL) was mixed with rVWF (100 µg/mL) and incubated for the indicated time points under shear stress in the (A) absence or (B) presence of 20 mM EDTA. Samples were then separated by 2.5% agarose gel electrophoresis under nonreducing conditions and stained for ADAMTS13 (upper panel) and VWF (lower panel) by Western blot analysis.
VWF complex formation under shear stress was also tested with MDTCS, a C-terminally truncated fragment of ADAMTS13. Due to the low sensitivity of the antibody, a higher concentration of MDTCS was used. FIX was used as a negative control.
0
24
48
96
200 µg/mL rA13 MDTCS
0 24 96 timepoints in h
200 µg/mL rVWF
0
24
rA13 MDTCS
96
Shear stress
12 µg/mL rFIX + 100 µg/mL rVWF
0
24
48
72
12 µg/mL rFIX
0 24 72 0 timepoints in h
100 µg/mL rVWF
24
rFIX
72
Shear stress, EDTA
Figure 3. Test for VWF adduct formation under shear stress with MDTCS and rFIX. rVWF (100 µg/mL) was mixed with (A) MDTCS, a C-terminally truncated rADAMTS13 (200 µg/mL) and (B) rFIX (12 µg/mL) and incubated for the indicated time points at RT under shear stress. Samples were then separated by 2.5% agarose gel electrophoresis under non-reducing conditions and stained for ADAMTS13 by Western blot analysis.
No or only very weak binding to rVWF was noted for the MDTCS fragment even at an almost 10-fold molar excess compared to full-length ADAMTS13 No evidence for homo-oligomerization of MDTCS
Presented at the Bari International Conference (8th BIC) 2014 in Bari, Italy • Oct 3-5, 2014 Conflicts of interest: The authors of this presentation make the following disclosure of financial or personal relationships with commercial entities that may have a direct or indirect interest in the subject matter of this presentation: All authors are full-time employees of Baxter Innovations GmbH, Vienna, Austria.
---- NEM
IAA ----
NEM IAA
Shear stress
40 µg/mL rA13 C1275 S
----
----
NEM
IAA
rA13 C1275S
NEM IAA
Shear stress
Figure 4. Impact of NEM and IAA on rADAMTS13-rVWF complex formation.. Full-length rADAMTS13 or rADAMTS13 C1275S (40 µg/mL) was mixed with rVWF (100 µg/mL) and incubated with 3.3 mM NEM or 10 mM IAA or without any agent (-) for 24 h under shear stress. Samples were separated by 2.5% agarose gel electrophoresis and immunoblotted with anti-ADAMTS13 antibodies in comparison to a non-incubated preparation (rA13).
ADAMTS13 adduct formation was prevented by NEM and partially by IAA The C1275S variant did not influence adduct formation
Methods
Shear stress, EDTA
ADAMTS13 adduct formation was slow and was not quantitative ADAMTS13 homo-oligomerization could be also observed The proteolytic activity of ADAMTS13 was not required for adduct formation
200 µg/mL rA13 MDTCS + 200 µg/mL rVWF
40 µg/mL rA13 + 100 µg/mL rVWF 40 µg/mL rA13 rA13
40 µg/mL rA13 C1275S + 100 µg/mL rVWF
72
timepoints in h
Shear stress
To prove that the observed complex formation occurs through disulfide bond formation, the thiol-reactive compounds NEM and IAA were added to the ADAMTS13VWF mixture during incubation. The ADAMTS13 C1275S variant thought to be important for covalent VWF complex formation was included in the experiment.
Recombinant VWF, full-length rADAMTS13, rMDTCS, a C-terminally truncated version of ADAMTS13, and rADAMTS13 C1275S, a mutant described to partially block the thiolmediated interaction with VWF, were purified from CHO cells. Full-length rADAMTS13 and variants were incubated with rVWF at room temperature (RT) under static conditions and under shear stress. As control, mixtures were incubated in the presence of N-ethylmaleimide (NEM) or iodoacetamide (IAA). Covalent interaction between ADAMTS13 and VWF was assayed by agarose gel electrophoresis under non-reducing conditions and immunoblot analysis using VWF- and ADAMTS13-specific antibodies.
Conclusions • We demonstrate covalent binding of the C-terminal part of ADAMTS13 to VWF especially under shear stress • Binding also occurred in the presence of EDTA, where VWF is not cleaved by ADAMTS13 • Addition of NEM prevented interaction of ADAMTS13 with VWF • Binding was not affected by the ADAMTS13 C1275S variant • Our data support the hypothesis of thiol-dependent regulation of VWF activity by ADAMTS13
